(19)
(11)EP 3 122 773 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
22.07.2020 Bulletin 2020/30

(21)Application number: 15727063.8

(22)Date of filing:  27.03.2015
(51)International Patent Classification (IPC): 
C07K 14/335(2006.01)
C07K 16/12(2006.01)
C07K 16/00(2006.01)
(86)International application number:
PCT/IB2015/000407
(87)International publication number:
WO 2015/145250 (01.10.2015 Gazette  2015/39)

(54)

EXPRESSION OF A SINGLE CHAIN ANTIBODY AGAINST SALMONELLA IN LACTOBACILLUS

EXPRESSION EINES EINKETTIGEN ANTIKÖRPERS GEGEN SALMONELLEN IN LACTOBACILLUS

EXPRESSION D'UN ANTICORPS À CHAÎNE UNIQUE CONTRE LA SALMONELLE DANS UN LACTOBACILLUS


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 27.03.2014 IN 1100MU2014

(43)Date of publication of application:
01.02.2017 Bulletin 2017/05

(73)Proprietor: Ghosh, Sambuddha
Pune 411060 (IN)

(72)Inventors:
  • BANERJEE, Sanjiban Kumar
    Pune 411033 (IN)
  • SABNIS, Manisha Pravin
    Pune 411015 (IN)
  • GHOSH, Sambuddha
    Pune 411060 (IN)

(74)Representative: Lavoix 
2, place d'Estienne d'Orves
75441 Paris Cedex 09
75441 Paris Cedex 09 (FR)


(56)References cited: : 
WO-A1-2010/029293
  
  • EL KHATTABI MOHAMED ET AL: "Llama single-chain antibody that blocks lipopolysaccharide binding and signaling: Prospects for therapeutic applications", CLINICAL AND VACCINE IMMUNOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC, US, vol. 13, no. 10, 1 October 2006 (2006-10-01), pages 1079-1086, XP002510699, ISSN: 1556-6811, DOI: 10.1128/CVI.00107.06 [retrieved on 2006-08-23]
  • EL KHATTABI MOHAMED ET AL: "Llama single-chain antibody that blocks lipopolysaccharide binding and signaling: Prospects for therapeutic applications", CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 13, no. 10, 1 October 2006 (2006-10-01), pages 1079-1086, XP002510699, ISSN: 1556-6811, DOI: 10.1128/CVI.00107.06 [retrieved on 2006-08-23]
  • DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; January 2011 (2011-01), MEYER TORSTEN ET AL: "Isolation of scFv fragments specific to OmpD of Salmonella Typhimurium", Database accession no. PREV201100131900 & VETERINARY MICROBIOLOGY, vol. 147, no. 1-2, January 2011 (2011-01), pages 162-169, ISSN: 0378-1135, DOI: 10.1016/J.VETMIC.2010.06.023
  • DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; January 2011 (2011-01), MEYER TORSTEN ET AL: "Isolation of scFv fragments specific to OmpD of Salmonella Typhimurium", Database accession no. PREV201100131900 & VETERINARY MICROBIOLOGY, vol. 147, no. 1-2, January 2011 (2011-01), pages 162-169, ISSN: 0378-1135, DOI: 10.1016/J.VETMIC.2010.06.023
  • JANUSZ WESOLOWSKI ET AL: "Single domain antibodies: promising experimental and therapeutic tools in infection and immunity", MEDICAL MICROBIOLOGY AND IMMUNOLOGY, SPRINGER, BERLIN, DE, vol. 198, no. 3, 16 June 2009 (2009-06-16) , pages 157-174, XP019740594, ISSN: 1432-1831, DOI: 10.1007/S00430-009-0116-7
  • GIL-CRUZ CRISTINA ET AL: "The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response", PNAS, NATIONAL ACADEMY OF SCIENCES, US, vol. 106, no. 24, 16 June 2009 (2009-06-16), pages 9803-9808, XP002564409, ISSN: 0027-8424, DOI: 10.1073/PNAS.0812431106 [retrieved on 2009-06-01]
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

FIELD OF INVENTION



[0001] The current disclosure relates to the field of microbiology and genetic engineering. The current disclosure provides recombinant chimeric proteins and antibodies directed against salmonella.

BACKGROUND OF THE INVENTION



[0002] Fermented food products have been developed and used by mankind with the aid of lactic acid bacteria (LAB), which have been classified as probiotics and are categorized as generally recognized as safe (GRAS) by the United states Food & Drug Administration (USFDA). In addition to be considered as the powerhouses for the food industry, LAB continue to be the focus of considerable interest as probiotic organisms, since they have also been conferred with specific health promoting functions which they execute by modulating the gut environment of the host.

[0003] Their ability to adhere to certain areas of the gastrointestinal tract has created interests to tap the potential of such microbes as vehicles for the delivery of biologically active compounds & vaccines (Pouwels et al., Int J Food Microbiol., 1998, 41, 155-157).

[0004] Most infectious organisms gain entry at the mucosal surfaces, there is a great deal of interest in developing vaccines that elicit effective mucosal immune responses against various pathogens. LAB, which are safe and nonpathogenic, are excellent mucosal delivery vehicles for heterologous antigens and therapeutic proteins. Many LABs produce extracellular polysaccharides and these have been extensively studied in terms of their biosynthesis, structure & function and engineering , including the importance of these molecules in host microbe interactions (Leeber et al., Microbial Biotechnology, 2011, 4(3), 368-374).

[0005] Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding and could be delivered on mucosal surfaces by the lactic acid bacteria for various therapeutic interventions. The unique physicochemical and pharmacological properties of these camelid heavy chain antibody (VHH) fragments have led to its prospective use as new generation therapeutic agents. The remarkable preference of VHH fragments to bind clefts and cavities on protein surfaces offers the possibility to develop selective therapeutics (Paalanen et al., Eur J Pharm Sci., 2011, 42(4),332-9) by activity modulation of cell surface proteins, such as receptors, ion channels involved in various biological activities (Wei et al., PLoS ONE, 2011, 6(12). Moreover, VHH fragment molecules recognize cryptic epitopes hidden deeply in clefts of various pathogens (Forsman et al., J. Virol., 2008, 82(24), 12069-12081) and have high structural stability and solubility (Muyldermans et al., Biochem Sci., 2001, 26, 230-235; Philipp et al., Nat. Biotechnol., 2005,23(9), 1126-1136).

[0006] Salmonellosis is the most common food borne disease and gastrointestinal infection across the world. Salmonella is the second major cause of food borne diseases in U.S, Europe & in the world causing as many as 1.3 billion cases of diseases annually. In addition to the health consequences, Salmonella species with about 2600 existing serovars are being identified belonging to six subspecies (Coburn et al., Immunology and Cell Biology, 2007, 85, 112-118; Ochman et al., EXS, 1994, 69, 479-493). Sub species are further sub divided into serovars that are differentiated by their flagellar, carbohydrate and lipopolysaccharide (LPS) structures. S. enteric species are typically orally acquired pathogens that cause one of the four major syndromes, Enteric fever (typhoid) enterocolitis/diarrhea, bacteremia and chronic asymptomatic carriage. The disease manifestation depends on both host susceptibility and the infectious. S. enteric serovar (Fierer et al., J Clin Invest., 2001, 107,775-780). Prominent inflammatory disease outcomes are a common feature of typhoid & enterocolitis. The various patho-biological outcomes of infection are mainly due to the interaction of the salmonella species with host defence mechanisms at various tissues in different stages of infection. This results in significant host immunopathology, morbidity and mortality.

[0007] Salmonella is a significant pathogen for food producing animals and these animals are the primary source of salmonellosis. It is one of the most commonly isolated food borne pathogens associated with poultry, raw meats, eggs, milk and dairy products, fresh farm produce like fruits & vegetables etc. In recent years, the incidence of food borne outbreaks caused by the contamination of fresh fruits and vegetables has increased and become a great concern in industrialized countries.

[0008] The major types of vaccines used to control salmonellosis are the killed bacteria vaccine, subunit vaccines and live attenuated vaccines. Comparative analysis of live and killed vaccines revealed that killed vaccines are usually less effective as they comprise of surface antigens that give rise to inadequate protective immune response, they fail to elicit secretory immune response at the mucosal surfaces which is critical in inhibiting the colonization of the pathogens at the mucosal surface. Attempts to overcome all these shortcomings by the use of various adjuvants has led to only partial success (Smith, J Hyg., 1956, 54, 419-432; Singh et al., Haryana Vet., 2005, 44, 1-12; Baljer et al., J Med Vet., 1986, 33, 206-212).

[0009] The utility of live vaccines in eradication of salmonellosis is limited, as there are multiple serovars of salmonella and vaccines made from any one serovar do not confer cross protection against another serovar. The organisms are capable to adapt in different animal species whilst still maintaining their zoonotic and interspecies transfer potential. Moreover, effective vaccines against some host adapted and common serovars in the primary source of host have been developed but their use has led to the emergence of other serovars. This has been further compounded by the international trade and movement of animal and farm products which has led various serovars to cross continental boundaries. Thus, there is a need in the art to develop anti-salmonella biological and it is desirable to develop and provide an alternative means for the control and management of enteropathogenic salmonella, by therapy and/or prophylaxis.

[0010] EP1066375B1 relates to use of transformed Lactobacillus species as vaccine delivery vehicles.

[0011] US2008/0206233 A1 relates to heavy chain immunoglobulins or fragments thereof of the VHH or VNAR type or domain antibodies (dAbs) suitable for use in the management of infections, particularly of the gastrointestinal tract.

[0012] US2009/0226418 A1 relates to food products or pharmaceutical preparations comprising antibodies or antibody fragments which are active in the gut and probiotic microorganisms independent from their antibodies or antibody fragments.

[0013] Single chain antibodies directed against Salmonela lipopolysaccharide were studied (El Khattabi et al., 2006, Clinical and Diagnostic Laboratory Immunology, American Society for Microbiology, 13(10): 1079-1086). Single-chain variable fragments specific to OmpD of Salmonella Typhimurium were also described (Torsten et al., 2011, Vetenary microbiology, 147(1-2)) .Single domain antibodies (sdAb) were also used to target toxins of Salmonella (Wesolowski et al., Med. Microbiol. Immunol., 2009, 198:157-174).

SUMMARY OF THE INVENTION



[0014] An aspect of the present disclosure relates to a single chain antibody or a fragment thereof against salmonella surface proteins, comprising of 3 complementarity determining regions.

[0015] An aspect of the present disclosure relates to a recombinant host cell expressing on the surface one or more chimeric proteins, wherein the chimeric protein comprises of (a) at least one single chain antibody or a fragment thereof against salmonella surface proteins, comprising of 3 complementarity determining regions, and (b) at least one protein that is expressed on the surface of the recombinant host cell, wherein the surface protein expressed in the recombinant host cell is MuB or CnBP.

[0016] An aspect of the present disclosure relates to a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against salmonella surface proteins, comprising of three complementarity determining regions.

[0017] An aspect of the present disclosure relates to a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions.

[0018] An aspect of the present disclosure relates to a recombinant host cell comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions.

[0019] An aspect of the present disclosure relates to a recombinant host cell comprising a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions.

[0020] An aspect of the present disclosure relates to a chimeric protein comprising amino acid sequence selected from the group consisting of SEQ ID NO:93, 95, 97, 99, 101, 130, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227 , 229, 231, 233, 235, 237, 239, 241, and 243.

[0021] An aspect of the present disclosure relates to a food product comprising a recombinant host cell comprising of a single chain antibody or a fragment thereof against salmonella surface proteins, comprising of 3 complementarity determining regions.

[0022] An aspect of the present disclosure relates to a food product comprising a recombinant host cell comprising a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against salmonella surface proteins, comprising of three complementarity determining regions.

[0023] An aspect of the present disclosure relates to a food product comprising a recombinant host cell comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against salmonella surface proteins, comprising of three complementarity determining regions.

[0024] An aspect of the following disclosure relates to a formulation comprising a single chain antibody or a fragment thereof against salmonella surface proteins, comprising of 3 complementarity determining regions, additionally consisting of a diluent, excipient or a carrier.

[0025] An aspect of the present disclosure relates to a method of inhibiting the growth of salmonella, said method comprising contacting a food product comprising a single chain antibody or a fragment thereof with a sample containing salmonella.

[0026] An aspect of the present disclosure relates to a method of inhibiting activity of salmonella, said method comprising contacting a food product comprising a single chain antibody or a fragment thereof with sample containing salmonella.

[0027] An aspect of the present disclosure relates to an isolated lactobacillus strain, Lactobacillus reuteri 1LB7 deposited with Microbial Type Culture Collection and Gene Bank (MTCC) having accession number 5894 for management of enteric Salmonella population in animal husbandry.

[0028] This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in determining or limiting the scope of the claimed subject matter. The invention is defined by the claims.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS



[0029] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1 depicts the effect of secreted anti-salmonella camelid VHH antibody fragments in a milk based formulation on growth of salmonella, in accordance with an embodiment of the present disclosure.

Figure 2 depicts the effect of secreted anti-salmonella camelid VHH antibody fragments in an egg based formulation on growth of salmonella, in accordance with an embodiment of the present disclosure.

Figure 3 depicts the effect of heat inactivated modified Lactobacillus reuteri expressing on its surface anti-salmonella camelid VHH antibody fragment on growth of salmonella, in accordance with an embodiment of the present disclosure.

Figure 4 depicts the effect of secreted anti-salmonella camelid VHH antibody fragments on growth of Salmonella typhimurium, in accordance with an embodiment of the present disclosure.

Figure 5 depicts the effect of secreted anti-salmonella camelid VHH antibody fragments on growth of Salmonella gallinarium, in accordance with an embodiment of the present disclosure.

Figure 6 depicts the effect of secreted anti-salmonella camelid VHH antibody fragments on growth of Salmonella newport, in accordance with an embodiment of the present disclosure.

Figure 7 depicts the effect of secreted anti-salmonella camelid VHH antibody fragments on growth of Salmonella abony, in accordance with an embodiment of the present disclosure.

Figure 8 depicts the vector map used to clone, in accordance with an embodiment of the present disclosure.


DETAILED DESCRIPTION OF THE INVENTION


Definitions



[0030] For convenience, before further description of the present invention, certain terms employed in the specification, examples are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

[0031] As used in the specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0032] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only

[0033] Functionally-equivalent processes and methods are clearly within the scope of the disclosure, as described herein.

Brief description of sequences



[0034] 

SEQ ID NO: 1 shows the CDR1 amino acid sequence of antibody A, I, J, K, and L.

SEQ ID NO: 2 shows the CDR2 amino acid sequence of antibody A.

SEQ ID NO: 3 shows the CDR3 amino acid sequence of antibody A, B, I, J, K, and L.

SEQ ID NO: 4 shows the CDR1 amino acid sequence of antibody B.

SEQ ID NO: 5 shows the CDR2 amino acid sequence of antibody B.

SEQ ID NO: 6 shows the CDR1 amino acid sequence of antibody C.

SEQ ID NO: 7 shows the CDR2 amino acid sequence of antibody C, E, F, and G.

SEQ ID NO: 8 shows the CDR3 amino acid sequence of antibody C, E, F, and G.

SEQ ID NO: 9 shows the CDR1 amino acid sequence of antibody D.

SEQ ID NO: 10 shows the CDR2 amino acid sequence of antibody D.

SEQ ID NO: 11 shows the CDR3 amino acid sequence of antibody D.

SEQ ID NO: 12 shows the CDR1 amino acid sequence of antibody E, F, and G.

SEQ ID NO: 13 shows the CDR1 amino acid sequence of antibody H, N, and O.

SEQ ID NO: 14 shows the CDR2 amino acid sequence of antibody H, N, and O.

SEQ ID NO: 15 shows the CDR3 amino acid sequence of antibody H, N, and O.

SEQ ID NO: 16 shows the CDR2 amino acid sequence of antibody I.

SEQ ID NO: 17 shows the CDR2 amino acid sequence of antibody J, and L.

SEQ ID NO: 18 shows the CDR2 amino acid sequence of antibody K.

SEQ ID NO: 19 shows the CDR1 amino acid sequence of antibody M.

SEQ ID NO: 20 shows the CDR2 amino acid sequence of antibody M.

SEQ ID NO: 21 shows the CDR3 amino acid sequence of antibody M.

SEQ ID NO: 22 shows the CDR1 amino acid sequence of antibody P.

SEQ ID NO: 23 shows the CDR2 amino acid sequence of antibody P.

SEQ ID NO: 24 shows the CDR3 amino acid sequence of antibody P.

SEQ ID NO: 25 shows the CDR1 amino acid sequence of antibody Q, R, and S.

SEQ ID NO: 26 shows the CDR2 amino acid sequence of antibody Q, R, and S.

SEQ ID NO: 27 shows the CDR3 amino acid sequence of antibody Q, R, and S.

SEQ ID NO: 28 shows the CDR1 nucleotide sequence of antibody A, I, J, K, and L.

SEQ ID NO: 29 shows the CDR2 nucleotide sequence of antibody A.

SEQ ID NO: 30 shows the CDR3 nucleotide sequence of antibody A, B, I, J, K, and L.

SEQ ID NO: 31 shows the CDR1 nucleotide sequence of antibody B.

SEQ ID NO: 32 shows the CDR2 nucleotide sequence of antibody B.

SEQ ID NO: 33 shows the CDR1 nucleotide sequence of antibody C.

SEQ ID NO: 34 shows the CDR2 nucleotide sequence of antibody C, E, F, and G.

SEQ ID NO: 35 shows the CDR3 nucleotide sequence of antibody C, E, F, and G.

SEQ ID NO: 36 shows the CDR1 nucleotide sequence of antibody D.

SEQ ID NO: 37 shows the CDR2 nucleotide sequence of antibody D.

SEQ ID NO: 38 shows the CDR3 nucleotide sequence of antibody D.

SEQ ID NO: 39 shows the CDR1 nucleotide sequence of antibody E, F, and G.

SEQ ID NO: 40 shows the CDR1 nucleotide sequence of antibody H, N, and O.

SEQ ID NO: 41 shows the CDR2 nucleotide sequence of antibody H, N, and O.

SEQ ID NO: 42 shows the CDR3 nucleotide sequence of antibody H, N, and O.

SEQ ID NO: 43 shows the CDR2 nucleotide sequence of antibody I.

SEQ ID NO: 44 shows the CDR2 nucleotide sequence of antibody J, and L.

SEQ ID NO: 45 shows the CDR2 nucleotide sequence of antibody K.

SEQ ID NO: 46 shows the CDR1 nucleotide sequence of antibody M.

SEQ ID NO: 47 shows the CDR2 nucleotide sequence of antibody M.

SEQ ID NO: 48 shows the CDR3 nucleotide sequence of antibody M.

SEQ ID NO: 49 shows the CDR1 nucleotide sequence of antibody P.

SEQ ID NO: 50 shows the CDR2 nucleotide sequence of antibody P.

SEQ ID NO: 51 shows the CDR3 nucleotide sequence of antibody P.

SEQ ID NO: 52 shows the CDR1 nucleotide sequence of antibody Q, R, and S.

SEQ ID NO: 53 shows the CDR2 nucleotide sequence of antibody Q, R, and S.

SEQ ID NO: 54 shows the CDR3 nucleotide sequence of antibody Q, R, and S.

SEQ ID NO: 55 shows the amino acid sequence of antibody A.

SEQ ID NO: 56 shows the nucleotide sequence of antibody A.

SEQ ID NO: 57 shows the amino acid sequence of antibody B.

SEQ ID NO: 58 shows the nucleotide sequence of antibody B.

SEQ ID NO: 59 shows the amino acid sequence of antibody C.

SEQ ID NO: 60 shows the nucleotide sequence of antibody C.

SEQ ID NO: 61 shows the amino acid sequence of antibody D.

SEQ ID NO: 62 shows the nucleotide sequence of antibody D.

SEQ ID NO: 63 shows the amino acid sequence of antibody E.

SEQ ID NO: 64 shows the nucleotide sequence of antibody E.

SEQ ID NO: 65 shows the amino acid sequence of antibody F.

SEQ ID NO: 66 shows the nucleotide sequence of antibody F.

SEQ ID NO: 67 shows the amino acid sequence of antibody G.

SEQ ID NO: 68 shows the nucleotide sequence of antibody G.

SEQ ID NO: 69 shows the amino acid sequence of antibody H.

SEQ ID NO: 70 shows the nucleotide sequence of antibody H.

SEQ ID NO: 71 shows the amino acid sequence of antibody I.

SEQ ID NO: 72 shows the nucleotide sequence of antibody I.

SEQ ID NO: 73 shows the amino acid sequence of antibody J.

SEQ ID NO: 74 shows the nucleotide sequence of antibody J.

SEQ ID NO: 75 shows the amino acid sequence of antibody K.

SEQ ID NO: 76 shows the nucleotide sequence of antibody K.

SEQ ID NO: 77 shows the amino acid sequence of antibody L.

SEQ ID NO: 78 shows the nucleotide sequence of antibody L.

SEQ ID NO: 79 shows the amino acid sequence of antibody M.

SEQ ID NO: 80 shows the nucleotide sequence of antibody M.

SEQ ID NO: 81 shows the amino acid sequence of antibody N.

SEQ ID NO: 82 shows the nucleotide sequence of antibody N.

SEQ ID NO: 83 shows the amino acid sequence of antibody O.

SEQ ID NO: 84 shows the nucleotide sequence of antibody O.

SEQ ID NO: 85 shows the amino acid sequence of antibody P.

SEQ ID NO: 86 shows the nucleotide sequence of antibody P.

SEQ ID NO: 87 shows the amino acid sequence of antibody Q.

SEQ ID NO: 88 shows the nucleotide sequence of antibody Q.

SEQ ID NO: 89 shows the amino acid sequence of antibody R.

SEQ ID NO: 90 shows the nucleotide sequence of antibody R.

SEQ ID NO: 91 shows the amino acid sequence of antibody S.

SEQ ID NO: 92 shows the nucleotide sequence of antibody S.

SEQ ID NO: 93, 131, and 169 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody A and MuB.

SEQ ID NO: 94, 132, and 170 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody A and MuB.

SEQ ID NO: 95, 133, and 171 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody B and MuB.

SEQ ID NO: 96, 134, and 172 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody B and MuB.

SEQ ID NO: 97, 135, and 173 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody C and MuB.

SEQ ID NO: 98, 136, and 174 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody C and MuB.

SEQ ID NO: 99, 137, and 175 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody D and MuB.

SEQ ID NO: 100, 138, and 176 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody D and MuB.

SEQ ID NO: 101, 139, and 177 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody E and MuB.

SEQ ID NO: 102, 140 and 178 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody E and MuB.

SEQ ID NO: 103, 141, and 179 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody F and MuB.

SEQ ID NO: 104, 142, and 180 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody F and MuB.

SEQ ID NO: 105, 143, and 181 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody G and MuB.

SEQ ID NO: 106, 144, and 182 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody G and MuB.

SEQ ID NO: 107, 145, and 183 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody H and MuB.

SEQ ID NO: 108, 146, and 184 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody H and MuB.

SEQ ID NO: 109, 147, and 185 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody I and MuB.

SEQ ID NO: 110, 148, and 186 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody I and MuB.

SEQ ID NO: 111, 149, and 187 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody J and MuB.

SEQ ID NO: 112, 150, and 188 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody J and MuB.

SEQ ID NO: 113, 151, and 189 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody K and MuB.

SEQ ID NO: 114, 152, and 190 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody K and MuB.

SEQ ID NO: 115, 153, and 191 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody L and MuB.

SEQ ID NO: 116, 154, and 192 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody L and MuB.

SEQ ID NO: 117, 155, and 193 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody M and MuB.

SEQ ID NO: 118, 156, and 194 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody M and MuB.

SEQ ID NO: 119, 157, and 195 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody N and MuB.

SEQ ID NO: 120, 158, and 196 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody N and MuB.

SEQ ID NO: 121, 159, and 197 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody O and MuB.

SEQ ID NO: 122, 160, and 198 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody O and MuB.

SEQ ID NO: 123, 161, and 199 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody P and MuB.

SEQ ID NO: 124, 162, and 200 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody P and MuB.

SEQ ID NO: 125, 163, and 201 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody Q and MuB.

SEQ ID NO: 126, 164, and 202 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody Q and MuB.

SEQ ID NO: 127, 165, and 203 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody R and MuB.

SEQ ID NO: 128, 166, and 204 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody R and MuB.

SEQ ID NO: 129, 167, and 205 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody S and MuB.

SEQ ID NO: 130, 168, and 206 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody S and MuB.

SEQ ID NO: 207 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody A and CnBP.

SEQ ID NO: 208 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody A and CnBP.

SEQ ID NO: 209 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody B and CnBP.

SEQ ID NO: 210 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody B and CnBP.

SEQ ID NO: 211 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody C and CnBP.

SEQ ID NO: 212 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody C and CnBP.

SEQ ID NO: 213 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody D and CnBP.

SEQ ID NO: 214 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody D and CnBP.

SEQ ID NO: 215 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody E and CnBP.

SEQ ID NO: 216 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody E and CnBP.

SEQ ID NO: 217 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody F and CnBP.

SEQ ID NO: 218 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody F and CnBP.

SEQ ID NO: 219 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody G and CnBP.

SEQ ID NO: 220 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody G and CnBP.

SEQ ID NO: 221 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody H and CnBP.

SEQ ID NO: 222 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody H and CnBP.

SEQ ID NO: 223 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody I and CnBP.

SEQ ID NO: 224 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody I and CnBP.

SEQ ID NO: 225 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody J and CnBP.

SEQ ID NO: 226 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody J and CnBP.

SEQ ID NO: 227 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody K and CnBP.

SEQ ID NO: 228 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody K and CnBP.

SEQ ID NO: 229 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody L and CnBP.

SEQ ID NO: 230 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody L and CnBP.

SEQ ID NO: 231 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody M and CnBP.

SEQ ID NO: 232 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody M and CnBP.

SEQ ID NO: 233 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody N and CnBP.

SEQ ID NO: 234 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody N and CnBP.

SEQ ID NO: 235 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody O and CnBP.

SEQ ID NO: 236 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody O and CnBP.

SEQ ID NO: 237 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody P and CnBP.

SEQ ID NO: 237 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody P and CnBP.

SEQ ID NO: 239 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody Q and CnBP.

SEQ ID NO: 240 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody Q and CnBP.

SEQ ID NO: 241 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody R and CnBP.

SEQ ID NO: 242 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody R and CnBP.

SEQ ID NO: 243 shows the contiguous amino acid sequence within the chimeric protein comprising of antibody S and CnBP.

SEQ ID NO: 244 shows the contiguous nucleotide sequence within the nucleotide sequence encoding the chimeric protein comprising of antibody S and CnBP.

SEQ ID NO: 245 shows the forward primer sequence for identification of lactobacillus.

SEQ ID NO: 246 shows the reverse primer sequence for identification of lactobacillus.

SEQ ID NO: 247 shows the forward primer sequence for identification of Lactobacillus reuteri.

SEQ ID NO: 248 shows the reverse primer sequence for identification of Lactobacillus reuteri.

SEQ ID NO: 249 shows the forward primer sequence for amplification of a 1.7kb partial MuB gene fragment.

SEQ ID NO: 250 shows the reverse primer sequence for amplification of a 1.7kb partial MuB gene fragment.

SEQ ID NO: 251 shows the forward primer sequence for amplification of the complete 1.08kb CnBP gene.

SEQ ID NO: 252 shows the reverse primer sequence for amplification of the complete 1.08kb CnBP gene.

SEQ ID NO: 253 shows the forward primer sequence for amplification of the 900bp VHH large insert.

SEQ ID NO: 254 shows the reverse primer sequence for amplification of the 900bp VHH large insert.

SEQ ID NO: 255 shows the forward primer sequence for the 4.7kb MuB gene inverse PCR product.

SEQ ID NO: 256 shows the reverse primer sequence for the 4.7kb MuB gene inverse PCR product.

SEQ ID NO: 257 shows the forward primer sequence for 400bp VHH insert in to the MuB gene.

SEQ ID NO: 258 shows the reverse primer sequence for 400bp VHH insert in to the MuB gene.

SEQ ID NO: 259 shows the forward primer sequence for the 1.7kb L.reuteri MuB gene fragment without restriction sites.

SEQ ID NO: 260 shows the reverse primer sequence for the 1.7kb L.reuteri MuB gene fragment without restriction sites.

SEQ ID NO: 261 shows the forward primer sequence for the 4.1kb CnBP gene inverse PCR product.

SEQ ID NO: 262 shows the reverse primer sequence for the 4.1kb CnBP gene inverse PCR product.

SEQ ID NO: 263 shows the forward primer sequence for the 400bp VHH insert in to the CnBP gene.

SEQ ID NO: 264 shows the reverse primer sequence for the 400bp VHH insert in to the CnBP gene.

SEQ ID NO: 265 shows the phosphoryalted forward primer sequence for the 1.4kb CnBP gene.

SEQ ID NO: 266 shows the phosphoryalted reverse primer sequence for the 1.4kb CnBP gene.

SEQ ID NO: 267 shows the forward primer sequence for the 1kb nucleotide fragment encoding salmonella FimH protein.

SEQ ID NO: 268 shows the reverse primer sequence for the 1kb nucleotide fragment sequence encoding salmonella FimH protein.

SEQ ID NO: 269 shows the forward primer sequence for the 1.1kb nucleotide fragment encoding salmonella OmPD protein.

SEQ ID NO: 270 shows the reverse primer sequence for the 1.1kb nucleotide fragment encoding salmonella OmPD protein.

SEQ ID NO: 271 shows the nucleotide sequence of the amplicon generated by primers as set forth in SEQ ID NO: 267 and 268.

SEQ ID NO: 272 shows the nucleotide sequence of the amplicon generated by primers as set forth in SEQ ID NO: 269 and 270.



[0035] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of 3 complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: : 1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (l)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0036] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of 3 complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (l)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0037] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody A, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 55 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 56.

[0038] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody B, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 57 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 58.

[0039] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody C, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 59 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 60.

[0040] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody D, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 61 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 62.

[0041] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody E, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 63 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 64.

[0042] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody E, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 65 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 66.

[0043] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody F, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 67 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 68.

[0044] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody G, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 69 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 70.

[0045] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody H, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 71 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 72.

[0046] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody I, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 73 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 74.

[0047] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody J, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 75 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 76.

[0048] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody K, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 77 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 78.

[0049] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody L, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 79 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 80.

[0050] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody M, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 81 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 82.

[0051] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody N, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 83 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 84.

[0052] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody O, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 85 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 86.

[0053] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody P, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 87 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 88.

[0054] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody Q, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 89 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 90.

[0055] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody R, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 91 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 92.

[0056] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, designated antibody S, against whole cell salmonella, having amino acid sequence as set forth in SEQ ID NO: 93 encoded by a polynucleotide sequence as set forth in SEQ ID NO: 94.

[0057] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 55, 57, 59, 63, 65, 67, 71, 73, 75, 77, or 79 that binds to FimH protein of salmonella.

[0058] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof encoded by a nucleotide sequence as set forth in SEQ ID NO: 56, 58, 60, 64, 66, 68, 72, 74, 76, 78, or 80 that binds to FimH protein of salmonella.

[0059] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 61, 69, 81, 83, or 85 that binds to OmPD protein of salmonella.

[0060] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof encoded by a nucleotide sequence as set forth in SEQ ID NO: 62, 70, 82, 84 or 86 that binds to OmPD protein of salmonella.

[0061] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof having amino acid sequence as set forth in SEQ ID NO: 87, 89, or 91 that bind to whole cell salmonella.

[0062] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof encoded by a nucleotide sequence as set forth in SEQ ID NO: 88, 90, or 92 that binds to whole cell salmonella.

[0063] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence selected from the group consisting of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and 91.

[0064] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, and 92.

[0065] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 55, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 1 for CDR1, SEQ ID NO: 2 for CDR2, and SEQ ID NO:3 for CDR3.

[0066] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 57, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3.

[0067] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 59, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3.

[0068] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 61, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3.

[0069] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 63, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 12 for CDR1, SEQ ID NO: 7 for CDR2, and SEQ ID NO:8 for CDR3.

[0070] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 65, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3.

[0071] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 67, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 12 for CDR1, SEQ ID NO: 7 for CDR2, and SEQ ID NO:8 for CDR3.

[0072] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO: 69, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3.

[0073] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:71, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3.

[0074] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:73, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3.

[0075] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:75, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3.

[0076] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:77, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3.

[0077] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:79, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3.

[0078] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:81, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3.

[0079] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:83, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3.

[0080] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:85, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3.

[0081] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:87, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0082] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:89, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0083] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, having amino acid sequence as set forth in SEQ ID NO:91, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0084] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:56, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3.

[0085] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:58, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3.

[0086] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:60, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3.

[0087] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:62, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3.

[0088] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:64, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3.

[0089] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:66, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3.

[0090] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:68, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3.

[0091] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:70, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3.

[0092] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:72, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3.

[0093] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:74, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3.

[0094] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:76, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3.

[0095] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:78, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3.

[0096] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:80, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3.

[0097] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:82, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3.

[0098] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:84, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3.

[0099] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:86, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3.

[0100] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:88, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0101] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:90, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0102] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against Salmonella surface proteins, encoded by a polynucleotide sequence as set forth in SEQ ID NO:92, wherein the single chain antibody or a fragment thereof has 3 complementarity determining regions encoded by a polynucleotide sequence as set forth in SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0103] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof that binds to Salmonella.

[0104] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof that binds to FimH protein in Salmonella.

[0105] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof having amino acid sequence selected from the group consisting of SEQ ID NO:55, 57, 59, 63, 65, 67, 71, 73, 75, 77, and 79, wherein said single chain antibody or a fragment thereof binds to FimH protein in Salmonella.

[0106] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:56, 58, 60, 64, 66, 68, 72, 76, 78, and 80, wherein said single chain antibody or a fragment thereof binds to FimH protein in Salmonella.

[0107] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof that binds to OmPD protein in Salmonella.

[0108] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof having amino acid sequence selected from the group consisting of SEQ ID NO:61, 69, 81, 83, and 85, wherein said single chain antibody or a fragment thereof binds to OmPD protein in Salmonella.

[0109] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof encoded by a polypeptide sequence selected from the group consisting of SEQ ID NO: 62, 70, 82, 84, and 86, wherein said single chain antibody or a fragment thereof binds to OmPD protein in Salmonella.

[0110] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof that binds to a surface protein in Salmonella.

[0111] In an embodiment of the present disclosure, there is provided a recombinant host cell expressing on the surface one or more chimeric proteins, said chimeric protein comprising of: (a) at least one single chain antibody or a fragment thereof against Salmonella surface protein comprising of 3 complementarity determining regions, and (b) at least one surface protein that is expressed on the surface of the recombinant host cell, wherein the surface protein expressed in the recombinant host cell is MuB or CnBP.

[0112] In an embodiment of the present disclosure, the chimeric protein as described herein is encoded within the host genome.

[0113] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, further comprising one or more exogenous nucleic acid sequences encoding another antibody or a fragment thereof against Salmonella surface proteins.

[0114] In an embodiment of the present disclosure, the protein expressed on the surface of the recombinant host cell is a chimeric protein comprising MuB, and a antibody or a fragment thereof as described herein.

[0115] In an embodiment of the present disclosure, the protein expressed on the surface of the recombinant host cell is a chimeric protein comprising CnBP, and an antibody or a fragment thereof as described herein.

[0116] In an embodiment of the present disclosure, the proteins expressed on the surface of the recombinant host cell are two different chimeric proteins, each comprising CnBP or MuB, and an antibody or a fragment thereof as described herein.

[0117] In an embodiment of the present disclosure, the recombinant host cell expressing on the surface one or more chimeric proteins is a member of the genera Lactobacillus.

[0118] In an embodiment of the present disclosure, the recombinant host cell expressing on the surface one or more chimeric proteins is selected from the group not limited to: Lactobacillus acidophilus, Lactobacillus acidophilus LAFTI L10, Lactobacillus casei, Lactobacillus casei LAFTI L26, Lactobacillus acidophilus DDS-1, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus CD 1285, Lactobacillus casei 431, Lactobacillus casei F19, Lactobacillus casei Shirota, Lactobacillus paracasei, Lactobacillus paracasei St11, Lactobacillus johnsonii, Lactobacillus johnsonii La1, Lactobacillus lactis, Lactobacillus lactis L1A, Lactobacillus plantarum, Lactobacillus plantarum 299v, Lactobacillus reuteri, Lactobacillus reuteri ATCC55730, Lactobacillus rhamnosus, Lactobacillus rhamnosus ATCC53013, Lactobacillus rhamnosus LB21, Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14, Lactobacillus rhamnosus R011, Lactobacillus helveticus, and Lactobacillus helveticus R0052.

[0119] In a preferred embodiment of the present disclosure, there is provided a recombinant host cell expressing on the surface one or more chimeric proteins, said recombinant host cell is Lactobacillus reuteri.

[0120] In an embodiment of the present disclosure, there is provided a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: : 1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (l)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0121] In an embodiment of the present disclosure, there is provided a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (l)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0122] In an embodiment of the present disclosure, there is provided a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (l)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0123] In an embodiment of the present disclosure, there is provided a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (l)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0124] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (l)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0125] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (l)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0126] In an embodiment of the present disclosure, the recombinant host cell comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions is selected from the group not limited to: Lactobacillus acidophilus, Lactobacillus acidophilus LAFTI L10, Lactobacillus casei, Lactobacillus casei LAFTI L26, Lactobacillus acidophilus DDS-1, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus CD 1285, Lactobacillus casei 431, Lactobacillus casei F19, Lactobacillus casei Shirota, Lactobacillus paracasei, Lactobacillus paracasei St11, Lactobacillus johnsonii, Lactobacillus johnsonii La1, Lactobacillus lactis, Lactobacillus lactis L1A, Lactobacillus plantarum, Lactobacillus plantarum 299v, Lactobacillus reuteri, Lactobacillus reuteri ATCC55730, Lactobacillus rhamnosus, Lactobacillus rhamnosus ATCC53013, Lactobacillus rhamnosus LB21, Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14, Lactobacillus rhamnosus R011, Lactobacillus helveticus, and Lactobacillus helveticus R0052.

[0127] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (l)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0128] In an embodiment of the present disclosure, there is provided a recombinant host cell comprising a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (l)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0129] In an embodiment of the present disclosure, the recombinant host cell comprising a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions is selected from the group not limited to: Lactobacillus acidophilus, Lactobacillus acidophilus LAFTI L10, Lactobacillus casei, Lactobacillus casei LAFTI L26, Lactobacillus acidophilus DDS-1, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus CD 1285, Lactobacillus casei 431, Lactobacillus casei F19, Lactobacillus casei Shirota, Lactobacillus paracasei, Lactobacillus paracasei St11, Lactobacillus johnsonii, Lactobacillus johnsonii La1, Lactobacillus lactis, Lactobacillus lactis L1A, Lactobacillus plantarum, Lactobacillus plantarum 299v, Lactobacillus reuteri, Lactobacillus reuteri ATCC55730, Lactobacillus rhamnosus, Lactobacillus rhamnosus ATCC53013, Lactobacillus rhamnosus LB21, Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14, Lactobacillus rhamnosus R011, Lactobacillus helveticus, and Lactobacillus helveticus R0052.

[0130] In a preferred embodiment of the present disclosure, there is provided a recombinant host cell secreting a single chain antibody or a fragment thereof having amino acid sequence selected from the group consisting of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and 91, wherein the recombinant host cell is Bacillus subtilis.

[0131] In an embodiment of the present disclosure, there is provided a recombinant host cell expressing a single chain antibody or a fragment thereof encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, and 92, wherein the recombinant host cell secretes the said single chain antibody or a fragment thereof extracellularly.

[0132] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof, said single chain antibody or a fragment thereof is a camelid antibody.

[0133] In an embodiment of the present disclosure, there is provided a chimeric protein having at least a contiguous amino acid sequence as set forth in SEQ ID NO: 93, 95, 97, 99, 101, 130, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227 , 229, 231, 233, 235, 237, 239, 241, and 243.

[0134] In an embodiment of the present disclosure, there is provided a chimeric protein having at least a contiguous polynucleotide sequence as set forth in SEQ ID NO: 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 , 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, and 244.

[0135] In an embodiment of the present disclosure, there is provided a chimeric protein comprising of a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (l)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0136] In an embodiment of the present disclosure, there is provided a chimeric protein comprising of a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (1)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0137] In an embodiment of the present disclosure, there is provided a food product comprising a recombinant host cell comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of:

(a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3;

(b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3;

(c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3;

(d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3;

(e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3;

(f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3;

(g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3;

(h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3;

(i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3;

(j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3;

(k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and

(1)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.



[0138] In an embodiment of the present disclosure, there is provided a food product comprising a recombinant host cell comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (1)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0139] In an embodiment of the present disclosure, there is provided a food product comprising a recombinant host cell comprising a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (1)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0140] In an embodiment of the present disclosure, there is provided a food product comprising a recombinant host cell comprising a recombinant DNA vector comprising a recombinant DNA construct comprising a polynucleotide sequence encoding a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (1)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0141] In an embodiment of the present disclosure, there is provided a food product comprising a single chain antibody or a fragment thereof having amino acid sequence selected from the group consisting of SEQ ID NO:55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and 91.

[0142] In an embodiment of the present disclosure, there is provided a food product comprising a single chain antibody or a fragment thereof encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, and 92.

[0143] In an embodiment of the present disclosure, there is provided a food product comprising a chimeric protein having at least a contiguous amino acid sequence selected from the group consisting of SEQ ID NO:93, 95, 97, 99, 101, 130, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227 , 229, 231, 233, 235, 237, 239, 241, and 243.

[0144] In an embodiment of the present disclosure, there is provided a food product comprising a chimeric protein having at least a contiguous polynucleotide sequence selected from the group consisting of SEQ ID NO: 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198 , 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, and 244.

[0145] In an embodiment of the present disclosure, there is provided a food product comprising of a chimeric protein comprising of a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of:

(a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3;

(b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3;

(c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3;

(d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3;

(e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3;

(f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3;

(g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3;

(h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3;

(i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3;

(j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3;

(k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and

(1)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.



[0146] In an embodiment of the present disclosure, there is provided a food product comprising of a chimeric protein comprising of a single chain antibody or a fragment thereof against Salmonella surface proteins, comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (1)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0147] In an embodiment of the present disclosure, there is provided a food product comprising a chimeric protein, further comprising a carrier selected from the group consisting of a lubricant, a surfactant, solvent, emulsifier, wetting agent, animal feed, dye or oral solution.

[0148] In an embodiment of the present disclosure, there is provided a food product comprising a single chain antibody or a fragment thereof, further comprising a carrier selected from the group consisting of a lubricant, a surfactant, solvent, emulsifier, wetting agent, animal feed, dye or oral solution.

[0149] In an embodiment of the present disclosure, there is provided a formulation comprising a single chain antibody or a fragment thereof having amino acid sequence selected from the group consisting of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and 91.

[0150] In an embodiment of the present disclosure, there is provided a formulation comprising a single chain antibody or a fragment thereof encoded by polynucleotide sequence selected from the group consisting of SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, and 92.

[0151] In an embodiment of the present disclosure, there is provided a formulation comprising a single chain antibody or a fragment thereof comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (1)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0152] In an embodiment of the present disclosure, there is provided a formulation comprising a single chain antibody or a fragment thereof comprising of three complementarity determining regions encoded by a polynucleotide sequence selected from the group consisting of: (a)SEQ ID NO:28 for CDR1, SEQ ID NO:29 for CDR2, and SEQ ID NO:30 for CDR3; (b)SEQ ID NO:31 for CDR1, SEQ ID NO:32 for CDR2, and SEQ ID NO:30 for CDR3; (c)SEQ ID NO:33 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (d)SEQ ID NO:36 for CDR1, SEQ ID NO:37 for CDR2, and SEQ ID NO:38 for CDR3; (e)SEQ ID NO:39 for CDR1, SEQ ID NO:34 for CDR2, and SEQ ID NO:35 for CDR3; (f)SEQ ID NO:40 for CDR1, SEQ ID NO:41 for CDR2, and SEQ ID NO:42 for CDR3; (g)SEQ ID NO:28 for CDR1, SEQ ID NO:43 for CDR2, and SEQ ID NO:30 for CDR3; (h)SEQ ID NO:28 for CDR1, SEQ ID NO:44 for CDR2, and SEQ ID NO:30 for CDR3; (i)SEQ ID NO:28 for CDR1, SEQ ID NO:45 for CDR2, and SEQ ID NO:30 for CDR3; (j)SEQ ID NO:46 for CDR1, SEQ ID NO:47 for CDR2, and SEQ ID NO:48 for CDR3; (k)SEQ ID NO:49 for CDR1, SEQ ID NO:50 for CDR2, and SEQ ID NO:51 for CDR3; and (1)SEQ ID NO:52 for CDR1, SEQ ID NO:53 for CDR2, and SEQ ID NO:54 for CDR3.

[0153] In an embodiment of the present disclosure, there is provided a formulation comprising a single chain antibody or a fragment thereof, further consisting of a diluent or an excipient or a carrier.

[0154] In an embodiment of the present disclosure, there is provided a method of inhibiting growth of Salmonella, said method comprising contacting a sample containing Salmonella with a food product comprising a single chain antibody or a fragment thereof comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (1)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0155] In an embodiment of the present disclosure, there is provided a method of inhibiting growth of Salmonella, said method comprising contacting a sample containing Salmonella with a food product comprising a chimeric protein with at least a contiguous amino acid sequence selected from the group consisting of SEQ ID NO: 93, 95, 97, 99, 101, 130, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227 , 229, 231, 233, 235, 237, 239, 241, and 243.

[0156] In an embodiment of the present disclosure, there is provided a method of inhibiting activity of Salmonella, said method comprising contacting a sample containing Salmonella with a food product comprising a single chain antibody or a fragment thereof comprising of three complementarity determining regions having amino acid sequence selected from the group consisting of: (a)SEQ ID NO: :1 for CDR1, SEQ ID NO:2 for CDR2, and SEQ ID NO:3 for CDR3; (b)SEQ ID NO:4 for CDR1, SEQ ID NO:5 for CDR2, and SEQ ID NO:3 for CDR3; (c)SEQ ID NO:6 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (d)SEQ ID NO:9 for CDR1, SEQ ID NO:10 for CDR2, and SEQ ID NO:11 for CDR3; (e)SEQ ID NO:12 for CDR1, SEQ ID NO:7 for CDR2, and SEQ ID NO:8 for CDR3; (f)SEQ ID NO:13 for CDR1, SEQ ID NO:14 for CDR2, and SEQ ID NO:15 for CDR3; (g)SEQ ID NO:1 for CDR1, SEQ ID NO:16 for CDR2, and SEQ ID NO:3 for CDR3; (h)SEQ ID NO:1 for CDR1, SEQ ID NO:17 for CDR2, and SEQ ID NO:3 for CDR3; (i)SEQ ID NO:1 for CDR1, SEQ ID NO:18 for CDR2, and SEQ ID NO:3 for CDR3; (j)SEQ ID NO:19 for CDR1, SEQ ID NO:20 for CDR2, and SEQ ID NO:21 for CDR3; (k)S EQ ID NO:22 for CDR1, SEQ ID NO:23 for CDR2, and SEQ ID NO:24 for CDR3; and (1)SEQ ID NO:25 for CDR1, SEQ ID NO:26 for CDR2, and SEQ ID NO:27 for CDR3.

[0157] In an embodiment of the present disclosure, there is provided a method of inhibiting activity of Salmonella, said method comprising contacting a sample containing Salmonella with a food product comprising a chimeric protein with at least a contiguous amino acid sequence selected from the group consisting of SEQ ID NO: 93, 95, 97, 99, 101, 130, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227 , 229, 231, 233, 235, 237, 239, 241, and 243.

[0158] In an embodiment of the present disclosure, there is provided a method of inhibiting activity of Salmonella in-ovo, said method comprising contacting a single chain antibody or a fragment thereof having amino acid sequence selected from the group consisting of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and 91 with Salmonella present in-ovo.

[0159] In an embodiment of the present disclosure, there is provided a method of inhibiting growth of Salmonella in-ovo, said method comprising contacting a single chain antibody or a fragment thereof having amino acid sequence selected from the group consisting of SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, and 91 with Salmonella present in-ovo.

[0160] In an embodiment of the present disclosure, there is provided a single chain antibody A or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 1 for CDR1, SEQ ID NO: 2 for CDR2, and SEQ ID NO: 3 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 28 for CDR1, SEQ ID NO: 29 for CDR2, and SEQ ID NO: 30 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 55, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 56.

[0161] In an embodiment of the present disclosure, there is provided a single chain antibody B or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 4 for CDR1, SEQ ID NO: 5 for CDR2, and SEQ ID NO: 3 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 31 for CDR1, SEQ ID NO: 32 for CDR2, and SEQ ID NO: 30 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 57, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 58.

[0162] In an embodiment of the present disclosure, there is provided a single chain antibody C or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 6 for CDR1, SEQ ID NO: 7 for CDR2, and SEQ ID NO: 8 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 33 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 35 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 59, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 60.

[0163] In an embodiment of the present disclosure, there is provided a single chain antibody D or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 9 for CDR1, SEQ ID NO: 10 for CDR2, and SEQ ID NO: 11 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 36 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 38 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 61, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 62.

[0164] In an embodiment of the present disclosure, there is provided a single chain antibody E or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 12 for CDR1, SEQ ID NO: 7 for CDR2, and SEQ ID NO: 8 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 39 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 35 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 63, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 64.

[0165] In an embodiment of the present disclosure, there is provided a single chain antibody F or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 12 for CDR1, SEQ ID NO: 7 for CDR2, and SEQ ID NO: 8 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 39 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 35 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 65, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 66.

[0166] In an embodiment of the present disclosure, there is provided a single chain antibody G or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 12 for CDR1, SEQ ID NO: 7 for CDR2, and SEQ ID NO: 8 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 39 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 35 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 67, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 68.

[0167] In an embodiment of the present disclosure, there is provided a single chain antibody H or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 13 for CDR1, SEQ ID NO: 14 for CDR2, and SEQ ID NO: 15 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 40 for CDR1, SEQ ID NO: 41 for CDR2, and SEQ ID NO: 42 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 69, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 70.

[0168] In an embodiment of the present disclosure, there is provided a single chain antibody I or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 1 for CDR1, SEQ ID NO: 16 for CDR2, and SEQ ID NO: 3 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 28 for CDR1, SEQ ID NO: 43 for CDR2, and SEQ ID NO: 30 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 71, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 72.

[0169] In an embodiment of the present disclosure, there is provided a single chain antibody J or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 1 for CDR1, SEQ ID NO: 17 for CDR2, and SEQ ID NO: 3 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 28 for CDR1, SEQ ID NO: 44 for CDR2, and SEQ ID NO: 30 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 73, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 74.

[0170] In an embodiment of the present disclosure, there is provided a single chain antibody K or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 1 for CDR1, SEQ ID NO: 18 for CDR2, and SEQ ID NO: 3 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 28 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 30 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 75, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 76.

[0171] In an embodiment of the present disclosure, there is provided a single chain antibody L or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 1 for CDR1, SEQ ID NO: 17 for CDR2, and SEQ ID NO: 3 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 28 for CDR1, SEQ ID NO: 44 for CDR2, and SEQ ID NO: 30 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 77, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 78.

[0172] In an embodiment of the present disclosure, there is provided a single chain antibody M or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 19 for CDR1, SEQ ID NO: 20 for CDR2, and SEQ ID NO: 21 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 46 for CDR1, SEQ ID NO: 47 for CDR2, and SEQ ID NO: 48 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 79, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 80.

[0173] In an embodiment of the present disclosure, there is provided a single chain antibody N or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 13 for CDR1, SEQ ID NO: 14 for CDR2, and SEQ ID NO: 15 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 40 for CDR1, SEQ ID NO: 41 for CDR2, and SEQ ID NO: 42 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 81, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 82.

[0174] In an embodiment of the present disclosure, there is provided a single chain antibody O or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 13 for CDR1, SEQ ID NO: 14 for CDR2, and SEQ ID NO: 15 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 40 for CDR1, SEQ ID NO: 41 for CDR2, and SEQ ID NO: 42 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 83, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 84.

[0175] In an embodiment of the present disclosure, there is provided a single chain antibody P or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 22 for CDR1, SEQ ID NO: 23 for CDR2, and SEQ ID NO: 24 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 49 for CDR1, SEQ ID NO: 50 for CDR2, and SEQ ID NO: 51 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 85, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 86.

[0176] In an embodiment of the present disclosure, there is provided a single chain antibody Q or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 25 for CDR1, SEQ ID NO: 26 for CDR2, and SEQ ID NO: 27 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 52 for CDR1, SEQ ID NO: 53 for CDR2, and SEQ ID NO: 54 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 87, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 88.

[0177] In an embodiment of the present disclosure, there is provided a single chain antibody R or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 25 for CDR1, SEQ ID NO: 26 for CDR2, and SEQ ID NO: 27 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 52 for CDR1, SEQ ID NO: 53 for CDR2, and SEQ ID NO: 54 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 89, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 90.

[0178] In an embodiment of the present disclosure, there is provided a single chain antibody S or a fragment thereof comprising of 3 complementarity determining regions having amino acid sequence as set forth in SEQ ID NO: 25 for CDR1, SEQ ID NO: 26 for CDR2, and SEQ ID NO: 27 for CDR3, wherein the nucleotide sequence encoding the CDRs is as set forth in SEQ ID NO: 52 for CDR1, SEQ ID NO: 53 for CDR2, and SEQ ID NO: 54 for CDR3, wherein the amino acid sequence of the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 91, and the nucleotide sequence encoding the single chain antibody A or a fragment thereof is as set forth in SEQ ID NO: 92.

[0179] In an embodiment of the present disclosure, there is provided an isolated lactobacillus strain, Lactobacillus reuteri 1LB7 deposited with Microbial Type Culture Collection and Gene Bank (MTCC) having accession number (5894) for management of enteric Salmonella population in animal husbandry.

[0180] In an embodiment of the present disclosure, there is provided a food formulation comprising anti-salmonella VHH antibodies or fragments thereof as described herein that inhibit Salmonella growth.

[0181] In an embodiment of the present disclosure, there is provided a milk based formulation comprising anti-salmonella VHH antibodies or fragments thereof as described herein that inhibit Salmonella growth.

[0182] In an embodiment of the present disclosure, there is provided an egg yolk based formulation comprising anti-salmonella VHH antibodies or fragments thereof as described herein that inhibit Salmonella growth.

[0183] In an embodiment of the present disclosure, there is provided a modified Lactobacillus reuteri having anti-salmonella camelid VHH antibody gene insert in the MuB gene as described herein that inhibits salmonella growth upon heat inactivation.

[0184] In an embodiment of the present disclosure, there is provided a modified Lactobacillus reuteri having anti-salmonella camelid VHH antibody gene insert in the CnBP gene as described herein that inhibits salmonella growth upon heat inactivation.

[0185] In an embodiment of the present disclosure, there is provided camelid VHH antibody fragments as described herein that inhibit growth of salmonella serovars.

[0186] In an embodiment of the present disclosure, there is provided camelid VHH antibody fragments as described herein that inhibit growth of Salmonella typhimurium.

[0187] In an embodiment of the present disclosure, there is provided camelid VHH antibody fragments as described herein that inhibit growth of Salmonella gallinarum.

[0188] In an embodiment of the present disclosure, there is provided camelid VHH antibody fragments as described herein that inhibit growth of Salmonella newport.

[0189] In an embodiment of the present disclosure, there is provided camelid VHH antibody fragments as described herein that inhibit growth of Salmonella abony.

[0190] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against salmonella surface proteins as described herein, or a recombinant host cell as described herein, or a recombinant DNA construct as described herein, or a recombinant DNA vector as described herein, or a chimeric protein as described herein, or a food product as described herein, or a formulation as described herein, or an isolated lactobacillus strain as described herein, for use in inhibiting Salmonella growth or infection.

[0191] In an embodiment of the present disclosure, there is provided a single chain antibody or a fragment thereof against salmonella surface proteins as described herein, for use in inhibiting Salmonella growth or infection.

[0192] In an embodiment of the present disclosure, there is provided a recombinant host cell as described herein, for use in inhibiting Salmonella growth or infection.

[0193] In an embodiment of the present disclosure, there is provided a recombinant DNA construct as described herein, for use in inhibiting Salmonella growth or infection.

[0194] In an embodiment of the present disclosure, there is provide a recombinant DNA construct as described herein, for use in inhibiting Salmonella growth or infection.

[0195] In an embodiment of the present disclosure, there is provided a chimeric protein as described herein, for use in inhibiting Salmonella growth or infection.

[0196] In an embodiment of the present disclosure, there is provided a food product as described herein, for use in inhibiting Salmonella growth or infection.

[0197] In an embodiment of the present disclosure, there is provided a formulation as described herein, for use in inhibiting Salmonella growth or infection.

[0198] In an embodiment of the present disclosure, there is provided an isolated lactobacillus strain as described herein, for use in inhibiting Salmonella growth or infection.

EXAMPLES



[0199] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

Example 1


Isolation and characterization of lactobacillus



[0200] Various organs (trachea, crop, gizzard, small intestine, large intestine, and ceacum) were collected from backyard poultry birds. Lactobacillus was isolated by inoculating the field sample in lactobacillus selective broth (LSB) (HiMedia/M1166-500G) and incubated at 37°C under anaerobic conditions. Selected colonies were enriched on MRS broth (HiMedia/M369-500G). Purity of the selected colonies was checked by Gram staining (gram positive short rods). The genetic identity of lactobacillus was confirmed by carrying out a sequencing reaction of the 900bp amplicon produced by amplifying the 16S RNA gene using primers as set forth in SEQ ID NO: 245 (forward primer) and SEQ ID NO:246 (reverse primer). The genetic identity of lactobacillus reuteri was confirmed by carrying out a sequencing reaction of the 303bp amplicon produced by species specific primers as set forth in SEQ ID NO: 247 (forward primer) and SEQ ID NO: 248 (reverse primer). The Lactobacillus reuteri 1LB7 strain isolated from poultry bird crop and found in the entire gastrointestinal tract, was selected as the host strain for surface display of a camelid heavy chain antibody or a fragment thereof. The 1LB7 strain is devoid of any plasmids.

Example 2


Isolation of lactobacillus reuteri MuB, and CnBP



[0201] Genomic DNA was isolated from the 1LB7 strain by resuspending a bacterial pellet in 5ml TNE buffer containing lysozyme at a concentration of 10mg/ml. 500µl of 10% SDS and 250µl proteinase K at a concentration of 10mg/ml was added and incubated at 55°C for two hours with intermittent shaking. Genomic DNA was isolated using the phenol chloroform extraction method (Raya et al, Food Microbiology Protocols, 2001,14, 135-139) .

[0202] A partial 1.7kb region of the MuB gene was PCR amplified using primers as set forth in SEQ ID NO: 249 (forward primer) and SEQ ID NO: 250 (reverse primer). This 1.7kb region comprises the LPTQG motif. The amplicon was subsequently cloned in to pJet vector as per manufacturer's instructions (catalog number: K1231, Thermo Scientific) and sequenced.

[0203] The complete CnBP gene (1.08kb) was PCR amplified using primers as set forth in SEQ ID NO: 251 (forward primer) and SEQ ID NO: 252 (reverse primer). The amplicon was subsequently cloned in to pJet vector as per manufacturer's instructions and sequenced.

Example 3


Generation of camelid antibodies against whole cell salmonella



[0204] Immunization of camels with whole cell inactivated Salmonella enteric: Briefly, actively growing cultures of Salmonella enteric (log phase) was subjected to inactivation for 24 hours at 37°C by addition of 0.5% of formalin. The culture was kept under constant shaking at 20rpm. Subsequently the cultures were stored at 4°C and a representative sample was tested in enriched growth media for innocuity. On confirmation of the inactivation, the bacterial cultures were washed thrice in IX PBS buffer and re-suspended at a concentration of 200µg/ml. 5ml of the suspension was mixed with adjuvant (Montanide ISA 206V) to form an emulsion.

[0205] Final bleeding of immunized camels was done at 60 days post immunization. Total RNA was isolated from isolated peripheral blood lymphocytes. PCR reaction was carried out for amplification of heavy chain antibody fragments using primers as set forth in SEQ ID NO: 253 (forward primer) and SEQ ID NO: 254 (reverse primer). {Amplicon size: 900 bps (comprising of the framework and CDR regions & CHI, CH2, CH3 including the hinge region of the camelid conventional heavy chain antibody pairing with the light chain), 690bp (comprising of the framework and CDR regions & the long hinge, CH2, CH3 regions of the camelid heavy chain VHH antibody), 620bp (comprising of the framework and CDR regions & the short hinge, CH2, CH3 regions of the camelid heavy chain VHH antibody)}. PCR condition used are given below in Table 1:
Table 1
No. of cyclesTemperatureTime
1 94°C 4 mins
29 94°C 30 sec
50°C 1 min
72°C 90 sec
1 72°C 5 mins


[0206] The amplicons were subsequently cloned in to a Bacillus subtilis secretory vector, 3VE vector (Figure 8). Single colonies were isolated by limited dilution plating. Single colonies were plated on 2XYT agar plates for growth. Induction of cloned antibodies was carried out by treating 3VE bacillus vector cultures with IPTG for secretion of antibodies.

[0207] The secreted antibodies were screened for anti-salmonella activity by assaying for anti-salmonella biological activity, and salmonella cell invasion inhibition assay.

[0208] Plasmids from clones showing anti-salmonella activity were isolated and the polynucleotide encoding the heavy chain antibody fragment showing anti-salmonella activity was digested with BamHI and AatII restriction enzyme and subsequently cloned in to pJet vector. The heavy chain antibody fragment was further sequenced to identify the complementarity determining regions.

[0209] The identified heavy chain antibody fragments showing anti-salmonella activity were further used for site specific insertion in to Lactobacillus reuteri MuB and CnBP genes.

Example 4


Generation of chimeric proteins


Insertion of specific camelid heavy chain antibody (VHH) within MuB repeat R-VI of the cloned MuB gene in pJet



[0210] Inverse PCR of the MuB gene cloned in pJet was carried out to introduce the flanking enzymes NdeI and BamHI at the VHH antibody insertion sites using primers as set forth in SEQ ID NO: 255 and SEQ ID NO: 256 (amplicon size 4.7kb). PCR conditions are given below in Table 2:
Table 2
No. of cyclesTemperatureTime
1 94°C 4 mins
29 94°C 30 sec
58°C 30 sec
72°C 8 mins
1 72°C 10 mins


[0211] PCR primers with flanking restriction enzyme sites BamHI and NdeI used to pull out the selected VHH cloned in to the secretory vector 3VE are as set forth in SEQ ID NO: 257 (forward primer) and SEQ ID NO: 258 (reverse primer) (amplicon size 400bp). The VHH PCR fragment with BamHI and NdeI restriction sites was ligated to the MuB gene inverse PCR product (pJet vector) and transformed in to E.coli. Clones harboring the MuB gene with the camelid VHH engineered within the MuB gene were screened and sequenced. PCR conditions are given below in Table 3:
Table 3
No. of cyclesTemperatureTime
1 94°C 4 mins
29 94°C 30 sec
60°C 1 min
72°C 1 mins
1 72°C 10 mins


[0212] A PCR product of the MuB gene harboring the camelid VHH was obtained using primers as set forth in SEQ ID NO: 259 (forward primer) and SEQ ID NO: 260 (reverse primer) that lack the BamHI or NdeI restriction sites (amplicon size 2.1kb). PCR conditions are given below in Table 4:
Table 4
No. of cyclesTemperatureTime
1 94°C 4 mins
29 94°C 30 sec
57°C 1 min
72°C 4 mins
1 72°C 10 mins


[0213] The amplicon obtained was electroporated in to Lactobacillus reuteri strain 1LB7 for host genome integration by double-cross over.

Insertion of specific camelid heavy chain antibody (VHH) within cloned CnBP gene in pJet



[0214] Inverse PCR of the CnBP gene cloned in pJet was carried out to introduce the flanking enzymes NdeI and BamHI at the VHH antibody insertion sites using primers as set forth in SEQ ID NO: 261 and SEQ ID NO: 262 (amplicon size 4.2kb). PCR conditions are given below in Table 5:
Table 5
No. of cyclesTemperatureTime
1 94°C 4 mins
29 94°C 30 sec
52°C 30 sec
72°C 7 mins
1 72°C 10 mins


[0215] PCR primers with flanking restriction enzyme sites BamHI and NdeI used to pull out the selected VHH cloned in to the secretory vector 3VE are as set forth in SEQ ID NO: 263 (forward primer) and SEQ ID NO: 264 (reverse primer) (amplicon size 400bp). PCR conditions are given below in Table 6:
Table 6
No. of cyclesTemperatureTime
1 94°C 4 mins
29 94°C 30 sec
65°C 1 min
72°C 2 mins
1 72°C 10 mins


[0216] The VHH PCR fragment with BamHI and NdeI restriction sites was ligated to the CnBP gene inverse PCR product (pJet vector) and transformed in to E.coli. Clones harboring the CnBP gene with the camelid VHH engineered within the MuB gene were screened and sequenced.

[0217] A PCR product of the CnBP gene harboring the camelid VHH was obtained using primers as set forth in SEQ ID NO: 265 (forward primer) and SEQ ID NO: 266

[0218] (reverse primer) (phosphorylated oligos) that lack the BamHI or NdeI restriction sites to form a circular DNA product (amplicon size 1.4kb). PCR conditions are given below in Table 7:
Table 7
No. of cyclesTemperatureTime
1 94°C 4 mins
29 94°C 30 sec
50°C 1 min
72°C 3 mins
1 72°C 10 mins


[0219] The circularized DNA product (full length CnBP gene) with the VHH insert was used for electroporation in to the Lactobacillus reuteri strain 1LB7 for host genomic integration by single Campbell like cross-over.

Example 5


Identification of antigenic salmonella surface proteins



[0220] Primers as set forth in SEQ ID NO: 267 (forward primer) and SEQ ID NO: 268 (reverse primer) for amplification of salmonella FimH protein encoding polynucleotide. The polynucleotide sequence of the amplicon is as set forth in SEQ ID NO: 271.

[0221] Primers as set forth in SEQ ID NO: 269 (forward primer) and SEQ ID NO: 270 (reverse primer) for amplification of salmonella OmPD protein encoding polynucleotide. The polynucleotide sequence of the amplicon is as set forth in SEQ ID NO: 272.

Whole cell (Lactobacillus reuterii) ELISA results



[0222] Briefly, L.reuteri parental host strain was transformed and modified for surface display of anti-salmonella specific camelid heavy chain antibodies on the MuB and CnBP proteins present at the bacterial cell surface. Selection of the clones/constructs with surface display antibodies specifically against the Salmonella FimH and OmPD proteins was done on the basis of binding/attachment/baiting of the clones over the recombinant FimH and OmPD proteins immobilized on nitrocellulose membranes.

[0223] Subsequently, the positive binders were subjected to Lactobacillus whole cell ELISA, wherein the histidine tagged recombinant Salmonella FimH and OmPD was used as the cell surface displayed specific antibody tracers or binders. Subsequently, specific binding of the modified Lactobacillus reuteri cell surface displayed antibody molecules to recombinant Salmonella FimH and OmPD proteins was traced with mouse monoclonal anti-His antibodies. Table 8 and 9 shows the results of ELISA.
Table 8
RECOMBINANT SALMONELLA FimH PROTEIN TAGGED WITH HISTIDINE USED IN THE WHOLE CELL ELISA AS A TRACER MOLECULE FOR THE ANTIBODY FRAGMENTS DISPLAYED ON THE SURFACE OF THE MODIFIED L. reuteri.
Sr. NoLactobacillus reuteri bacterial sampleO.D values
Neat1:2 dilution1:4 dilution1:8 dilution1:16 dilution
1 Modified L. reuteri with surface displayed camelid antibodies specific against Salmonella 0.925 0.411 0.249 0.165 0.106
2 L. reuteri parental host control 0.79 0.232 0.124 0.088 0.069
3 Modified L. reuteri with surface displayed camelid antibodies specific against Salmonella 1.29 0.552 0.341 0.264 0.18
4 L. reuteri parental host control 1.15 0.262 0.191 0.17 0.166
Table 9
RECOMBINANT SALMONELLA OmPD PROTEIN TAGGED WITH HISTIDINE USED IN THE WHOLE CELL ELISA AS A TRACER MOLECULE FOR THE ANTIBODY FRAGMENTS DISPLAYED ON THE SURFACE OF THE MODIFIED L. reuteri.
Sr. NoLactobacillus reuteri bacterial sampleO.D values
Neat1:2 dilution1:4 dilution1:8 dilution1:16 dilution
1 Modified L. reuteri with surface displayed camelid antibodies specific against Salmonella 0.823 0.403 0.197 0.108 0.071
2 L. reuteri parental host control 0.832 0.139 0.073 0.053 0.047
3 Modified L. reuteri with surface displayed camelid antibodies specific against Salmonella 1.381 0.697 0.393 0.169 0.089
4 L. reuteri parental host Control 1.394 0.302 0.118 0.081 0.063


[0224] Based on the results in Table 8, camelid antibodies having amino acid sequence as set forth in SEQ ID NO: 55, 57, 59, 63, 65, 67, 71, 73, 75, 77, and 79 bind to salmonella FimH protein.

[0225] Based on the results in Table 9, camelid antibodies having amino acid sequence as set forth in SEQ ID NO: 61, 69, 81, 83, and 85 bind to salmonella OmPD protein.

[0226] Camelid antibodies having amino acid sequences as set forth in SEQ ID NO: 87, 89, and 91 bind to whole cell salmonella.

Example 6


Anti-salmonella biological activity



[0227] Briefly, Bacillus subtilis vector clones with the antibody gene fragment (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 61) were induced with IPTG (ImM, 12 hours at 37°C on shaker at 180RPM) and the culture supernatant was collected by centrifugation (5000RPM for 10 minutes) and filtered through 0.45um filter. Similar treatment was given to the supernatant of the induced plasmid without any camelid heavy chain antibody gene fragment insert and the 2 xYT growth media in which the Bacillus cultures was grown and these were used as controls.

[0228] Test supernatant and the two controls were subsequently challenged with Salmonella bacterium inoculums of 25,000 cells in a total test volume of 2 mL and incubated at 37° C under shaking at 180 RPM. Representative samples from the test and the two control reactions were drawn at 2, 4, 6, 8 and 24 hours of incubation and were plated on selective XLT agar media to enumerate the Salmonella colony forming units. The results are summarized in the Table 10 below.
Table 10
 Colony forming units
Test supernatant from Induced secretory Vector having Camelid Antibody Gene fragment insertTest Control supernatant from Induced secretory empty vector without Antibody Gene insert2xYT growth Media control% reduction of test supernatant over induced empty vector control% reduction of test supernatant over 2xYT growth media control
0hr 98 95 97    
2hrs 60 850 901 92.94 93.34
4hrs 66 TNTC TNTC >95 >95
6hrs 53 TNTC TNTC >95 >95
8hrs 35 Mat Mat >95 >95
24hrs 0 Mat Mat 100 100
TNTC : Colonies too numerous to count.
Mat: Complete Bacterial growth on the plate with merged colonies.

Example 7


Anti-Salmonella Biological Activity Titration



[0229] Bacillus subtilis vector clones with the antibody gene fragments (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 61) were induced with IPTG (ImM, 12 hours at 37°C on shaker at 180RPM) and the culture supernatant was collected by centrifugation (5000RPM for 10 minutes) and filtered through 0.45um filter. This was then subjected to two-fold dilution in the 2xYT bacterial growth media and 1:2 and 1:4 along with the neat supernatant were subjected to anti-Salmonella biological activity testing. Similar treatment was given to the supernatant of the induced plasmid without any camelid heavy chain antibody gene fragment insert and the 2 xYT growth media in which the Bacillus cultures was grown and these were used as controls.

[0230] Test supernatant and the two controls were subsequently challenged with Salmonella bacterium inoculums of 25,000 cells in a total test volume of 2 ml and incubated at 37°C under shaking at 180RPM. Representative samples from the test and the two control reactions were drawn at 2 hours of incubation and were plated on selective XLT agar media to enumerate the Salmonella colony forming units. The results are summarized in the Table 11 below.
Table 11
SampleColony Forming units% reduction of test supernatant over supernatant from induced empty vector control
Test Control supernatant from Induced secretory empty vector (neat) 991 -
Test Control supernatant from Induced secretory empty vector diluted (1:2) 889 -
Test Control supernatant from Induced secretory empty vector diluted (1:4) 868 -
Test supernatant from Induced secretory Vector having Camelid Antibody fragment Gene insert (neat) 94 90.51
Test supernatant from Induced secretory Vector having Camelid Antibody fragment Gene insert diluted (1:2) 223 74.91
Test supernatant from Induced secretory Vector having Camelid Antibody fragment insert in plasmid diluted (1:4) 402 53.68
2xYT growth Media control 983  

Salmonella cell invasion inhibition assay



[0231] The supernatant from the induced plasmid with camelid Heavy chain antibody gene fragment (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 61) insert along with the supernatant from induced plasmid without any antibody gene fragment insert as control as well as 2xYT bacterial growth media as control was tested for the Salmonella cell invasion inhibitory properties. Cell substratum used was INT 407 intestinal cell line.

[0232] Challenge dose of 2.5X108 Salmonella bacterium in 1 ml of MEM (Himedia cat no: AL047S was added in 1 ml of the test and control supernatant. On addition of challenge bacterium, supernatant mixtures were incubated at 37°C for 1 hour under shaking at 180rpm. Subsequently entire contents of each 2ml volume of test and control was seeded onto at least 90% confluent INT 407 cell monolayer in each of the six well culture plates and further incubated at 37°C for 2 hours to allow bacterial invasion to occur.

[0233] Upon completion of incubation, INT 407 monolayer cells in each well was washed twice with PBS and the INT 407 cell adhered bacterial cells, including the remaining extracellular bacteria were killed by treating for 2 hours with 2 ml/ well of gentamycin at a concentration of 100 micrograms/ml. On completion of gentamycin treatment the cell monolayer in each well was again washed thrice with PBS and the infected INT 407 cells were lysed by treating with 1% of Triton X-100 in PBS at 37°C for 10 minutes in a total volume of 400 ul/ well to release the intracellular bacterium. Released bacterium was subsequently enumerated by plating on selective XLT agar media. Sample data is given in Table 14.
Table 14
No. of INT 407 Internalized Salmonella Colonies (CFU) Test supernatant from Induced secretory Vector having Camelid Antibody fragment insert in plasmid Test Control supernatant from Induced secretory empty vector 2xYT growth Media control
4 TNTC TNTC
TNTC: Colonies too numerous to count

Example 9


Salmonella inhibition by modified L.reuteri



[0234] Approximately 1.25x108 CFU (Colony forming Units) of modified L.reuteri having surface expressed salmonella specific camelid heavy chain antibodies were mixed with approximately 1.25x108 CFU of salmonella challenge dose.

[0235] One of the two controls comprised of approximately 1.25 x108 CFU of the host parental strain of L.reuteri mixed with approximately 1.25x108 CFU of salmonella challenge dose and the second control comprised of only the same salmonella challenge dose mixed with blank cell culture media without any lactobacillus bacterium, all in a total volume of 2 ml each.

[0236] The test bacterial mixtures including the two controls were incubated at 37°C at 110RPM for two-hours. Subsequently, entire contents of 2 ml of each test mixture including the two controls were seeded onto at least 90% confluent INT 407 cell monolayer in each well of the six well culture plates and further incubated at 37°C for two-hours to allow bacterial invasion to occur.

[0237] Upon completion of incubation, INT 407 monolayer cells in each well was washed twice with 1XPBS and the INT 407 cell adhered bacterial cells, including the remaining extracellular bacteria were killed by treating for two-hours with 2 ml/ well of gentamycin (Abbott Healthcare Pvt. Ltd.) at a concentration of 100 µg/ml. On completion of gentamycin treatment, the cell monolayer in each well was washed thrice with 1XPBS and the infected INT 407 cells were lysed by treating with 1% Triton X-100 in PBS at 37°C for 10 minutes in a total volume of 400 µl/ well to release the intracellular bacterium. Released bacterium was subsequently enumerated by plating on selective XLT agar media. Results are summarized in the Table 15 (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 55),Table 16 (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 61), and Table 17(polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 63 or 69) below.
Table 15
Sr. NoSampleNo. of INT 407 Internalized Salmonella Colonies (CFU)Salmonella INT 407 Cell Invasion Reduction Percent
1 Modified L. reuteri construct With antibody Expressed in MuB 186 83.91 by the modified construct
2 Parental L. reuteri strain 1018 11.93 by the parental strain
3 2xYT growth media Control. 1156 -----
Table 16
Sr. NoSampleNo. of INT 407 Internalized Salmonella Colonies (CFU)Salmonella INT 407 Cell Invasion Reduction Percent
1 Modified L. reuteri construct Wit h antibody Expressed in CnBP 229 78.97 by the modified construct
2 Parental L. reuteri strain 1089 12.94 by the parental strain
3 2xYT growth media Control. 1251  
Table 17
Sr. NoSampleNo. of INT 407 Internalized Salmonella Colonies (CFU)Salmonella INT 407 Cell Invasion Reduction Percent
1 Modified L. reuteri construct With antibody Expressed in CnBP& MuB 25 98.01 by the modified construct
2 Parental L. reuteri strain 1093 13.32 by the parental strain
3 2xYT growth media Control. 1261 -----

Example 10


Co-culture assay of Salmonella and L. reuteri modified strain with VHH antibody insert in MuB



[0238] The antagonistic, aggregating and growth inhibitory effect of the modified Lactobacillus reuteri constructs in comparison with the parental strain 1LB7 L.reuteri strain on salmonella was observed on the basis of reduction in salmonella colony forming units (CFU), when grown (co-cultured) with the lactobacillus cultures. Growing cultures of salmonella and lactobacillus were cultured together with a fixed CFU of 1.5x104 salmonella and 5x106 lactobacillus, in equal volumes of PBS. Sampling was performed every 2 hours up to six hours and the samples were plated on salmonella selective XLT agar media, to enumerate the viable salmonella bacterium in the sample of the test mixtures. The growth inhibitory effect is compared with the untransformed parental host L.reuteri 1LB7 and media control. Table 18 (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 55) shows the results.
Table 18
Sampling IntervalModified Test Strain Having Surface Displayed Camelid Antibody.Untransformed Parental Host 1LB7 ControlBacterial Growth Media Control% Reduction Of Salmonella in Co-Culture With Parental Host Strain% Reduction Of Salmonella in Co-Culture With Modified Strain.
0hr 42 64 74 ---- ----
2hrs 45 83 92 9.78 51.08
4hrs 115 204 299 31.77 61.53
6hrs 256 600 700 14.28 63.42

Example 11


Co-culture assay of Salmonella and L.reuteri modified strain with VHH antibody insert in MuB and CnBP.



[0239] Table 19 depicts the results of the effect of a modified L.reuteri strain having surface displayed camelid antibody in MuB and CnBP (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 63 or 69).
Table 19
Sampling IntervalModified Test Strain Having Surface Displayed Camelid Antibody.Untransformed Parental Host 1LB7 ControlMedia Control% Reduction Of Salmonella in Co-Culture With Parental Host Strain% Reduction Of Salmonella in Co-Culture With Modified Strain.
0hr 31 33 30 ----- -----
2hrs 30 42 64 34.37 53.12
4hrs 40 93 132 29.54 69.69
6hrs 130 291 365 20.27 64.38
24hrs observation:-

Example 12


Co-culture assay of Salmonella and L. reuteri modified strain with VHH antibody insert in CnBP.



[0240] Table 20 depicts the results of the effect of a modified L. reuteri strain having surface displayed camelid antibody in CnBP (polynucleotide encoding antibody having amino acid sequence as set forth in SEQ ID NO: 61).
Table 20
Sampling IntervalModified Test Strain Having Surface Displayed Camelid Antibody.Untransformed Parental Host 1LB7 ControlMedia Control% Reduction Of Salmonella in Co-Culture With Parental Host Strain% Reduction Of Salmonella in Co-Culture With Modified Strain.
0hr 41 35 32    
2hrs 31 51 61 16.31 49.18
4hrs 53 108 128 15.62 58.59
6hrs 129 242 322 24.84 59.93

Example 13


Usage of anti-salmonella VHH antibodies and fragments thereof in milk based food preparation



[0241] To test the usage of anti-salmonella VHH antibodies and fragments thereof as described in the present disclosure in various food preparation formulations, a formulation was made by blending the VHH antibody fragments, obtained from the culture supernatant of the induced secretory bacillus vector into 70 % of skimmed milk powder dissolved in ultrapure water. Induced secretory bacillus vector culture supernatant solution was used as neat and as 1:2 dilution in PBS. The antibody solution was added at the rate of 10% in a volume of 1.5 ml of the 70% skimmed milk solution. After addition of the antibody solution, the skimmed milk solution was vortexed at 500 rpm for 30 seconds five times. Negative control comprised of 10% induced culture supernatant of secretory bacillus vector without the antibody gene fragment insert.

[0242] Subsequently, salmonella bacterium at a challenge dose of 10,000 organisms in 10µl was added to the skimmed milk solution having the antibodies, as well as the control without antibodies. Representative samples from the skimmed milk test solutions with added antibodies in two concentrations, i.e. the neat solution and the 1:2 diluted solutions, as well as the control were drawn and plated on XLT4 agar media to enumerate the salmonella colony forming units. Table 21 shows the results of the assay in tabulated format.
Table 21
Sampling IntervalTest- Culture supernatant from Induced secretory Vector having Camelid Antibody fragment insert in plasmid.Test- Culture supernatant from Induced secretory Vector having Camelid Antibody fragment insert in plasmid , diluted 1:2 in PBSControl- Culture supernatant from Induced secretory without antibody gene insertControl- 2xYT growth media
0 hr. 68 68 58 49
2 hr. 118 350 945 1103
4 hr. 445 850 TNTC TNTC
TNTC: colonies too numerous to count


[0243] Figure 1 depicts the graphical representation of salmonella colony forming units at various time points. It can be inferred from Figure 1 that the number of salmonella colony forming units is significantly less in samples that have the supernatant from the induced secretory vector having camelid antibody than samples with no antibody. The number of colonies in cultures without antibody were too numerous to count (TNTC). As Figure 1 suggests, by 4 hours, the fold inhibition of salmonella colony forming units in culture that has supernatant from the induced secretory vector is at least more than four-fold. This data suggests that the antibody fragments are stable and retain their function when incorporated into a food preparation, and is able to substantially reduce the salmonella colony forming units.

Example 14


Usage of anti-salmonella VHH antibodies and fragments thereof in an egg based food preparation



[0244] A formulation of egg yolk was developed with the culture supernatants of induced secretory bacillus vector having camelid VHH genes. Egg yolk was diluted 1:2 in PBS solution comprising of 2% Tween 80. To this egg yolk solution, VHH antibody test solution was added at a rate of 20% and the mixture was vortexed at 1000 rpm for 30 seconds five times. The culture supernatant from the induced empty secretory bacillus vector was used as control. Representative samples were drawn from the test egg yolk formulation and the control at two hour intervals from the start till four hours and were immediately plated in XLT4 agar media to enumerate the salmonella colony forming units. Table 22 shows the results in tabulated format.
Table 22
Sampling IntervalTest-Culture supernatant from Induced secretory bacillus vector having Camelid aAntibody fragment gene insert in plasmidControl - Culture supernatant from Induced secretory bacillus vector without Camelid antibody gene insert in plasmidControl-2xYT growth Media control
0 hr. 363 373 362
2 hr. 620 1133 1456
4hr. 924 1960 2376


[0245] Figure 2 depicts the graphical representation of salmonella colony forming units at various time points. It can be inferred from Figure 2 that by four hours, the salmonella colony forming units is decreased by more than two-fold in case of the culture comprising supernatant from induced secretory vector having camelid antibody fragment insert in plasmid. This data suggests that the antibody fragments are stable and retain their function when incorporated into a food preparation, and is able to substantially reduce the salmonella colony forming units.

Example 15


Efficacy of heat-inactivated modified Lactobacillus reuteri on inhibition of salmonella growth



[0246] The antagonistic, aggregating and growth inhibitory effect of both the heat inactivated modified Lactobacillus construct and the parental Lactobacillus reuteri strain 1LB7 on Salmonella was observed on the basis of reduction in salmonella colony forming units (CFU) during co-culture.

[0247] Growing cultures of lactobacillus were inactivated by heating for 30 minutes at 85°C. Complete inactivation was checked by carrying out three blind passages of the inactivated cultures in MRS growth media. Growing cultures of Salmonella and the inactivated modified Lactobacillus, including the control host parental strain 1LB7 were mixed together at a rate of 1x104 CFU of salmonella bacterium with 1.5x106 CFU of lactobacillus bacterium. Representative culture samples starting from the 0 hour, were taken every 2 hours up to 6 hours, and then at 24 hours. The samples were plated on XLT4 agar media to enumerate the salmonella bacterium present in the samples. The antagonistic, aggregating and growth inhibitory effect against salmonella by the inactivated modified and transformed lactobacillus reuteri strain was compared with the inactivated untransformed parental host strain 1LB7.

[0248] Table 23 shows the results in tabulated format
Table 23
Sampling IntervalTest- Inactivated Modified L. reuteri Strain having VHH antibody gene insert in MuBControl-Inactivated Untransformed L. reuteri Parental Host strain 1LB7
0hr 54 62
2hrs 35 120
4hrs 68 475
6hrs 78 752
24 hrs 121 Mat
Mat: complete bacterial growth on the plate with merged colonies


[0249] Figure 3 depicts the graphical representation of salmonella colony forming units when co-cultured with heat-inactivated modified Lactobacillus. It can be inferred from Figure 3 that even up to 24 hours, the inactivated modified Lactobacillus strain that displays on its surface the camelid VHH antibody is able to effectively inhibit the growth of Salmonella. This data suggests that the modified Lactobacillus is effective even when it is heat-inactivated and incapable of growth and self-replication.

Example 16


Anti-salmonella activity of camelid VHH antibodies against Salmonella serovars



[0250] Bacillus subtilis secretory vector with cloned camelid antibody gene fragments were induced with IPTG (1mM, 12 hours at 37°C on shaker at 180RPM) and the culture supernatant was collected by centrifugation and filtered through 0.45um filter. Similar treatment was given to the supernatant of the induced plasmid, devoid of camelid heavy chain antibody gene fragment insert and the 2xYT growth media in which the Bacillus cultures was grown and these were used as controls.

[0251] Test supernatant and the two controls were subsequently challenged with Salmonella serovars, having bacterium inoculums of approximately 10,000 cells in a total test volume of 2 ml and incubated at 37° C under 180 r.p.m shaking. Representative samples from the test and the two control reactions were drawn at 0, 2, 4, 6 and 24 hours of incubation and were plated on selective XLT4 agar media to enumerate the Salmonella colony forming units. Table 24-27 denote the results using different Salmonella serovars.
Table 24
Anti Salmonella Biological activity on Salmonella typhimurium
Sampling IntervalTest-Culture supernatant from Induced secretory bacillus vector having Camelid antibody gene fragment insert in plasmidControl- Culture supernatant from Induced secretory bacillus vector without Camelid antibody gene insert in plasmidControl- 2xYT growth Media
0 98 109 114
2 91 295 398
4 63 TNTC TNTC
6 48 TNTC TNTC
24 1 Mat Mat
TNTC : Colonies too numerous to count.
Mat: Complete Bacterial growth on the plate with merged colonies.
Table 25
Anti Salmonella Biological activity on Salmonella gallinarum
Sampling IntervalTest-Culture supernatant from Induced secretory bacillus vector having Camelid antibody gene fragment insert in plasmidControl- Culture supernatant from Induced secretory bacillus vector without Camelid antibody gene insert in plasmidControl- 2xYT growth Media
0hrs 85 89 93
2hrs 63 119 166
4hrs 48 146 190
6hrs 49 TNTC TNTC
24hrs 2 Mat Mat
TNTC : Colonies too numerous to count.
Mat: Complete Bacterial growth on the plate with merged colonies.
Table 26
Anti Salmonella Biological activity on Salmonella newport
Sampling IntervalTest-Culture supernatant from Induced secretory bacillus vector having Camelid antibody gene fragment insert in plasmidControl- Culture supernatant from Induced secretory bacillus vector without Camelid antibody gene insert in plasmidControl- 2xYT growth Media
0hrs 42 52 57
2hrs 20 100 149
4hrs 4 TNTC TNTC
6hrs 3 Mat Mat
24hrs 0 Mat Mat
TNTC : Colonies too numerous to count.
Mat: Complete Bacterial growth on the plate with merged colonies.
Table 27
Anti Salmonella Biological activity on Salmonella abony
Sampling IntervalTest-Culture supernatant from Induced secretory bacillus vector having Camelid antibody gene fragment insert in plasmidControl- Culture supernatant from Induced secretory bacillus vector without Camelid antibody gene insert in plasmidControl- 2xYT growth Media
0hrs 76 74 83
2hrs 43 100 324
4hrs 5 TNTC TNTC
6hrs 2 Mat Mat
24hrs 0 Mat Mat
TNTC : Colonies too numerous to count.
Mat: Complete Bacterial growth on the plate with merged colonies.


[0252] Figure 4 show that the supernatant containing the secreted camelid VHH antibodies effectively inhibits Salmonella typhimurium growth upto 24 hours.

[0253] Figure 5 show that the supernatant containing the secreted camelid VHH antibodies effectively inhibits Salmonella gallinarium growth upto 24 hours.

[0254] Figure 6 show that the supernatant containing the secreted camelid VHH antibodies effectively inhibits Salmonella newport growth upto 24 hours.

[0255] Figure 7 show that the supernatant containing the secreted camelid VHH antibodies effectively inhibits Salmonella abony growth upto 24 hours.

[0256] Overall, Figures4-7 collectively show that the camelid VHH antibody is effective against a wide range of Salmonella species members, and can be used as a pan inhibitor of Salmonella growth and infection.

SEQUENCE LISTING



[0257] 

<110> AbGenics
Ghosh, sambuddha

<120> EXPRESSION OF A SINGLE CHAIN ANTIBODY AGAINST SALMONELLA IN LACTOBACILLUS

<130> PD010352IN-SC

<160> 272

<170> PatentIn version 3.5

<210> 1
<211> 7
<212> PRT
<213> artificial sequence

<220>
<223> antibody A CDR1

<400> 1

<210> 2
<211> 7
<212> PRT
<213> artificial sequence

<220>
<223> antibody A CDR2

<400> 2

<210> 3
<211> 15
<212> PRT
<213> artificial sequence

<220>
<223> antibody A CDR3

<400> 3

<210> 4
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody B CDR1

<400> 4

<210> 5
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody B CDR2

<400> 5

<210> 6
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody C CDR1

<400> 6

<210> 7
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody C CDR2

<400> 7

<210> 8
<211> 9
<212> PRT
<213> artificial sequence

<220>
<223> antibody CDR3

<400> 8

<210> 9
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody D CDR1

<400> 9

<210> 10
<211> 6
<212> PRT
<213> artificial sequence

<220>
<223> antibody D CDR2

<400> 10

<210> 11
<211> 16
<212> PRT
<213> artificial sequence

<220>
<223> antibody D CDR3

<400> 11

<210> 12
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody E CDR1

<400> 12

<210> 13
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody H CDR1

<400> 13

<210> 14
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody H CDR2

<400> 14

<210> 15
<211> 18
<212> PRT
<213> artificial sequence

<220>
<223> antibody H CDR3

<400> 15

<210> 16
<211> 7
<212> PRT
<213> artificial sequence

<220>
<223> antibody I CDR2

<400> 16

<210> 17
<211> 6
<212> PRT
<213> artificial sequence

<220>
<223> antibody J CDR2

<400> 17

<210> 18
<211> 6
<212> PRT
<213> artificial sequence

<220>
<223> antibody K CDR2

<400> 18

<210> 19
<211> 9
<212> PRT
<213> artificial sequence

<220>
<223> antibody M CDR1

<400> 19

<210> 20
<211> 9
<212> PRT
<213> artificial sequence

<220>
<223> antibody M CDR2

<400> 20

<210> 21
<211> 14
<212> PRT
<213> artificial sequence

<220>
<223> antibody M CDR3

<400> 21

<210> 22
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody P CDR1

<400> 22

<210> 23
<211> 7
<212> PRT
<213> artificial sequence

<220>
<223> antibody M CDR2

<400> 23

<210> 24
<211> 18
<212> PRT
<213> artificial sequence

<220>
<223> antibody P CDR3

<400> 24

<210> 25
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody Q CDR1

<400> 25

<210> 26
<211> 8
<212> PRT
<213> artificial sequence

<220>
<223> antibody Q CDR2

<400> 26

<210> 27
<211> 17
<212> PRT
<213> artificial sequence

<220>
<223> antibody Q CDR3

<400> 27

<210> 28
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody A CDR1

<400> 28
ggtcatacgt attatggacc ttgt 24

<210> 29
<211> 20
<212> DNA
<213> artificial sequence

<220>
<223> antibody A CDR2

<400> 29
attagtccta gtggtgggag 20

<210> 30
<211> 45
<212> DNA
<213> artificial sequence

<220>
<223> antibody A CDR3

<400> 30
gattcagggg gactctgcag ccatcgtgag cgcgactatg acatt 45

<210> 31
<211> 23
<212> DNA
<213> artificial sequence

<220>
<223> antibody B CDR1

<400> 31
gggttccgaa tattatggtt ccc 23

<210> 32
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> antibody B CDR2

<400> 32
attgttccta ttggtgggag t 21

<210> 33
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody C CDR1

<400> 33
ggattcggaa tcggtagttt cccc 24

<210> 34
<211> 18
<212> DNA
<213> artificial sequence

<220>
<223> antibody C CDR2

<400> 34
attggtagtg attatacg   18

<210> 35
<211> 26
<212> DNA
<213> artificial sequence

<220>
<223> antibody C CDR3

<400> 35
gacgtacttg actaccaccc agattt 26

<210> 36
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody D CDR1

<400> 36
aaatggagct acacgtacta ttgt 24

<210> 37
<211> 17
<212> DNA
<213> artificial sequence

<220>
<223> antibody D CDR2

<400> 37
attgatagtg aaggcac 17

<210> 38
<211> 52
<212> DNA
<213> artificial sequence

<220>
<223> antibody D CDR3

<400> 38
caatggtggt tattgcctca gaccccgtca actcgccgcg gattatgagt at 52

<210> 39
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody E CDR1

<400> 39
ggattcggaa tcggtagttt cgcc 24

<210> 40
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody H CDR1

<400> 40
ggagattcca tcactaccta ccac 24

<210> 41
<211> 20
<212> DNA
<213> artificial sequence

<220>
<223> antibody H CDR2

<400> 41
ataaatgatg atgctaattc 20

<210> 42
<211> 52
<212> DNA
<213> artificial sequence

<220>
<223> antibody H CDR3

<400> 42
tttgaggtgc gtccctggga ccgactctgg tcatccttat tcgtataact ac 52

<210> 43
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> antibody I CDR2

<400> 43
attagtccta ttggtgggag t 21

<210> 44
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> antibody J CDR2

<400> 44
atgattccta ttggtgggag t 21

<210> 45
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> antibody K CDR2

<400> 45
attattccta ttggtgggag t 21

<210> 46
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody M CDR1

<400> 46
gaatacaccg ctattaccta ctgt 24

<210> 47
<211> 18
<212> DNA
<213> artificial sequence

<220>
<223> antibody M CDR2

<400> 47
atcaatcgcg gtggtggt 18

<210> 48
<211> 46
<212> DNA
<213> artificial sequence

<220>
<223> antibody M CDR3

<400> 48
gtacaaacag accggtgatt gtgggatctt ccaattcttt ggaaac 46

<210> 49
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody P CDR1

<400> 49
ggatcgaccg ccagtatgta ctgc 24

<210> 50
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> antibody P CDR2

<400> 50
attagtggag atgataaagg g 21

<210> 51
<211> 49
<212> DNA
<213> artificial sequence

<220>
<223> antibody P CDR3

<400> 51
cgcgagcgac aacaactggt gaacgtctac acgcccggac gtacgaatt 49

<210> 52
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> antibody Q CDR1

<400> 52
ggagataccc tcagtaccta ctgc 24

<210> 53
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> antibody Q CDR2

<400> 53
atttatcgtc ttagggatat g 21

<210> 54
<211> 48
<212> DNA
<213> artificial sequence

<220>
<223> antibody Q CDR3

<400> 54
tgtgtgcgac tattcggtac ttgtcagcta gtcgaagatt ttgaacta 48

<210> 55
<211> 126
<212> PRT
<213> artificial sequence

<220>
<223> antibody A

<400> 55



<210> 56
<211> 379
<212> DNA
<213> artificial sequence

<220>
<223> antibody A

<400> 56

<210> 57
<211> 125
<212> PRT
<213> artificial sequence

<220>
<223> antibody B

<400> 57



<210> 58
<211> 375
<212> DNA
<213> artificial sequence

<220>
<223> antibody B

<400> 58

<210> 59
<211> 119
<212> PRT
<213> artificial sequence

<220>
<223> antibody C

<400> 59



<210> 60
<211> 359
<212> DNA
<213> artificial sequence

<220>
<223> antibody C

<400> 60

<210> 61
<211> 125
<212> PRT
<213> artificial sequence

<220>
<223> antibody D

<400> 61



<210> 62
<211> 381
<212> DNA
<213> artificial sequence

<220>
<223> antibody D

<400> 62

<210> 63
<211> 119
<212> PRT
<213> artificial sequence

<220>
<223> antibody E

<400> 63



<210> 64
<211> 361
<212> DNA
<213> artificial sequence

<220>
<223> antibody E

<400> 64

<210> 65
<211> 119
<212> PRT
<213> artificial sequence

<220>
<223> antibody F

<400> 65



<210> 66
<211> 358
<212> DNA
<213> artificial sequence

<220>
<223> antibody F

<400> 66

<210> 67
<211> 119
<212> PRT
<213> artificial sequence

<220>
<223> antibody G

<400> 67

Val Thr Val cys Ser Gly Arg 115

<210> 68
<211> 361
<212> DNA
<213> artificial sequence

<220>
<223> antibody G

<400> 68

<210> 69
<211> 128
<212> PRT
<213> artificial sequence

<220>
<223> antibody H

<400> 69



<210> 70
<211> 386
<212> DNA
<213> artificial sequence

<220>
<223> antibody H

<400> 70

<210> 71
<211> 125
<212> PRT
<213> artificial sequence

<220>
<223> antibody I

<400> 71

<210> 72
<211> 374
<212> DNA
<213> artificial sequence

<220>
<223> antibody I

<400> 72

<210> 73
<211> 125
<212> PRT
<213> artificial sequence

<220>
<223> antibody J

<400> 73

<210> 74
<211> 374
<212> DNA
<213> artificial sequence

<220>
<223> antibody J

<400> 74

<210> 75
<211> 124
<212> PRT
<213> artificial sequence

<220>
<223> antibody K

<400> 75

<210> 76
<211> 377
<212> DNA
<213> artificial sequence

<220>
<223> antibody K

<400> 76

<210> 77
<211> 124
<212> PRT
<213> artificial sequence

<220>
<223> antibody L

<400> 77

<210> 78
<211> 373
<212> DNA
<213> artificial sequence

<220>
<223> antibody L

<400> 78

<210> 79
<211> 127
<212> PRT
<213> artificial sequence

<220>
<223> antibody M

<400> 79

<210> 80
<211> 379
<212> DNA
<213> artificial sequence

<220>
<223> antibody M

<400> 80

<210> 81
<211> 128
<212> PRT
<213> artificial sequence

<220>
<223> antibody N

<400> 81

<210> 82
<211> 386
<212> DNA
<213> artificial sequence

<220>
<223> antibody N

<210> 83
<211> 129
<212> PRT
<213> artificial sequence

<220>
<223> antibody O

<400> 83

<210> 84
<211> 386
<212> DNA
<213> artificial sequence

<220>
<223> antibody O

<400> 84

<210> 85
<211> 130
<212> PRT
<213> artificial sequence

<220>
<223> antibody P

<400> 85

<210> 86
<211> 390
<212> DNA
<213> artificial sequence

<220>
<223> antibody P

<400> 86

<210> 87
<211> 128
<212> PRT
<213> artificial sequence

<220>
<223> antibody Q

<400> 87

<210> 88
<211> 382
<212> DNA
<213> artificial sequence

<220>
<223> antibody Q

<400> 88

<210> 89
<211> 129
<212> PRT
<213> artificial sequence

<220>
<223> antibody R

<400> 89

<210> 90
<211> 382
<212> DNA
<213> artificial sequence

<220>
<223> antibody R

<400> 90

<210> 91
<211> 129
<212> PRT
<213> artificial sequence

<220>
<223> antibody S

<400> 91

<210> 92
<211> 383
<212> DNA
<213> artificial sequence

<220>
<223> antibody S

<400> 92

<210> 93
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody A

<400> 93

Asp Gly Phe Lys
145

<210> 94
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody A

<400> 94

<210> 95
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody B

<400> 95



<210> 96
<211> 435
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody B

<400> 96

<210> 97
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody C

<400> 97



<210> 98
<211> 419
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody C

<400> 98

<210> 99
<211> 147
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody D

<400> 99



<210> 100
<211> 441
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody D

<400> 100

<210> 101
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody E

<400> 101



<210> 102
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site antibody E

<400> 102

<210> 103
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody F

<400> 103



<210> 104
<211> 418
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody F

<400> 104

<210> 105
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody G

<400> 105



<210> 106
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody G

<400> 106

<210> 107
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody H

<400> 107

<210> 108
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody H

<400> 108

<210> 109
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody I

<400> 109

<210> 110
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody I

<400> 110



<210> 111
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody J

<400> 111

<210> 112
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody J

<400> 112



<210> 113
<211> 144
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody K

<400> 113

<210> 114
<211> 437
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody K

<400> 114

<210> 115
<211> 144
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site antibody L

<400> 115

<210> 116
<211> 433
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody L

<400> 116

<210> 117
<211> 147
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody M

<400> 117



<210> 118
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody M

<400> 118

<210> 119
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody N

<400> 119



<210> 120
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody N

<400> 120

<210> 121
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody O

<400> 121



<210> 122
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody O

<400> 122

<210> 123
<211> 150
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody P

<400> 123



<210> 124
<211> 450
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody P

<400> 124

<210> 125
<211> 150
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody Q

<400> 125



<210> 126
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody Q

<400> 126

<210> 127
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody R

<400> 127

<210> 128
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody R

<400> 128

<210> 129
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody S

<400> 129

<210> 130
<211> 443
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 1 antibody S

<400> 130



<210> 131
<211> 146
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody A

<400> 131

<210> 132
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody A

<400> 132

<210> 133
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody B

<400> 133

<210> 134
<211> 435
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody B

<400> 134

<210> 135
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody C

<400> 135



<210> 136
<211> 419
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody C

<400> 136

<210> 137
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody D

<400> 137



<210> 138
<211> 441
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody D

<400> 138

<210> 139
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody E

<400> 139



<210> 140
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody E

<400> 140

<210> 141
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody F

<400> 141



<210> 142
<211> 418
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody F

<400> 142

<210> 143
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody G

<400> 143



<210> 144
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody G

<400> 144

<210> 145
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody H

<400> 145



<210> 146
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody H

<400> 146

<210> 147
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody I

<400> 147



<210> 148
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody I

<400> 148

<210> 149
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody J

<400> 149

<210> 150
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody J

<400> 150

<210> 151
<211> 144
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody K

<400> 151

<210> 152
<211> 437
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody K

<400> 152



<210> 153
<211> 144
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody L

<400> 153

<210> 154
<211> 433
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody L

<400> 154



<210> 155
<211> 147
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody M

<400> 155

<210> 156
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody M

<400> 156

<210> 157
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody N

<400> 157

<210> 158
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody N

<400> 158

<210> 159
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody O

<400> 159



<210> 160
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody O

<400> 160

<210> 161
<211> 150
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody P

<400> 161



<210> 162
<211> 450
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody P

<400> 162

<210> 163
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody Q

<400> 163



<210> 164
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody Q

<400> 164

<210> 165
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody R

<400> 165



<210> 166
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody R

<400> 166

<210> 167
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody S

<400> 167

<210> 168
<211> 443
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 2 antibody S

<400> 168

<210> 169
<211> 146
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody A

<400> 169

<210> 170
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody A

<400> 170



<210> 171
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody B

<400> 171

<210> 172
<211> 435
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody B

<400> 172



<210> 173
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody C

<400> 173

<210> 174
<211> 419
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody C

<400> 174

<210> 175
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody D

<400> 175

<210> 176
<211> 441
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody D

<400> 176

<210> 177
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody E

<400> 177

<210> 178
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody E

<400> 178

<210> 179
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody F

<400> 179



<210> 180
<211> 418
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody F

<400> 180

<210> 181
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody G

<400> 181



<210> 182
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody G

<400> 182

<210> 183
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody H

<400> 183



<210> 184
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody H

<400> 184

<210> 185
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody I

<400> 185



<210> 186
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody I

<400> 186

<210> 187
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody J

<400> 187



<210> 188
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody J

<400> 188

<210> 189
<211> 143
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody K

<400> 189

<210> 190
<211> 437
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody K

<400> 190

<210> 191
<211> 143
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody L

<400> 191

<210> 192
<211> 433
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody L

<400> 192

<210> 193
<211> 146
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody M

<400> 193

<210> 194
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody M

<400> 194



<210> 195
<211> 147
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody N

<400> 195

<210> 196
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody N

<400> 196



<210> 197
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody O

<400> 197

<210> 198
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody O

<400> 198

<210> 199
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody P

<400> 199



<210> 200
<211> 450
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody P

<400> 200

<210> 201
<211> 147
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody Q

<400> 201



<210> 202
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody Q

<400> 202

<210> 203
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody R

<400> 203



<210> 204
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody R

<400> 204

<210> 205
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody S

<400> 205



<210> 206
<211> 443
<212> DNA
<213> artificial sequence

<220>
<223> mub insertion site 3 antibody S

<400> 206

<210> 207
<211> 146
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody A

<400> 207



<210> 208
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody A

<400> 208

<210> 209
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody B

<400> 209

<210> 210
<211> 435
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody B

<400> 210

<210> 211
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody C

<400> 211

<210> 212
<211> 419
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody C

<400> 212



<210> 213
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody D

<400> 213

<210> 214
<211> 441
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody D

<400> 214



<210> 215
<211> 138
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody E

<400> 215

<210> 216
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody E

<400> 216



<210> 217
<211> 139
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody F

<400> 217

<210> 218
<211> 418
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody F

<400> 218

<210> 219
<211> 138
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody G

<400> 219

<210> 220
<211> 421
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody G

<400> 220

<210> 221
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody H

<400> 221



<210> 222
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody H

<400> 222

<210> 223
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody I

<400> 223



<210> 224
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody I

<400> 224

<210> 225
<211> 145
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody J

<400> 225



<210> 226
<211> 434
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody J

<400> 226

<210> 227
<211> 144
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody K

<400> 227



<210> 228
<211> 437
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody K

<400> 228

<210> 229
<211> 144
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody L

<400> 229



<210> 230
<211> 433
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody L

<400> 230

<210> 231
<211> 147
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody M

<400> 231



<210> 232
<211> 439
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody M

<400> 232

<210> 233
<211> 148
<212> PRT
<213> artificial sequence

<220>

<210> 234
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody N

<400> 234

<210> 235
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody O

<400> 235

<210> 236
<211> 446
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody O

<400> 236



<210> 237
<211> 150
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody P

<400> 237

<210> 238
<211> 450
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody P

<400> 238

<210> 239
<211> 148
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody Q

<400> 239

<210> 240
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody Q

<400> 240

<210> 241
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody R

<400> 241



<210> 242
<211> 442
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody R

<400> 242

<210> 243
<211> 149
<212> PRT
<213> artificial sequence

<220>
<223> cnbp insertion site antibody S

<400> 243



<210> 244
<211> 443
<212> DNA
<213> artificial sequence

<220>
<223> cnbp insertion site antibody S

<400> 244

<210> 245
<211> 20
<212> DNA
<213> artificial sequence

<220>
<223> lactobacillus identification forward

<400> 245
agagtttgat cctggctcag   20

<210> 246
<211> 20
<212> DNA
<213> artificial sequence

<220>
<223> lactobacillus identification reverse

<400> 246
ccgtcaattc ctttgagttt   20

<210> 247
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> 1 reuteri identification forward

<400> 247
gccgcctaag gtgggacaga t   21

<210> 248
<211> 20
<212> DNA
<213> artificial sequence

<220>
<223> 1 reuteri identification reverse

<400> 248
aacactcaag gattgtctga   20

<210> 249
<211> 32
<212> DNA
<213> artificial sequence

<220>
<223> MuB partial gene forward

<400> 249
gctctagaac ggttcactac aagtacgcag at   32

<210> 250
<211> 34
<212> DNA
<213> artificial sequence

<220>
<223> MuB partial gene reverse

<400> 250
cgagctctgc gaaaaaagac aaaaaggctc aacc   34

<210> 251
<211> 27
<212> DNA
<213> artificial sequence

<220>
<223> CnBP complete forward

<400> 251
gctctagaac tcataatatg gtctttg   27

<210> 252
<211> 28
<212> DNA
<213> artificial sequence

<220>
<223> CnBP complete reverse

<400> 252
cgagctcata atttaacatt aatttgtg   28

<210> 253
<211> 22
<212> DNA
<213> artificial sequence

<220>
<223> VHH large insert forward

<400> 253
gtcctggatc ccttctacaa gg   22

<210> 254
<211> 23
<212> DNA
<213> artificial sequence

<220>
<223> VHH large insert reverse

<400> 254
gggacgtcct gttgaactgt tcc   23

<210> 255
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> MuB inverse forward

<400> 255
gacgacttca tttggatcct tggt   24

<210> 256
<211> 37
<212> DNA
<213> artificial sequence

<220>
<223> MuB inverse reverse

<400> 256
acgcgtcgac catatggaag tcgtcaagga actcgaa   37

<210> 257
<211> 38
<212> DNA
<213> artificial sequence

<220>
<223> VHH insert for MuB forward

<400> 257
cgggatccag atgtgcagct gcaggagtct ggaggagg   38

<210> 258
<211> 43
<212> DNA
<213> artificial sequence

<220>
<223> VHH insert for MuB reverse

<400> 258
ggaattccat atgagtgcgg ccgctggaga cggtgacctg ggt   43

<210> 259
<211> 24
<212> DNA
<213> artificial sequence

<220>
<223> L reuteri MuB gene without restriction sites

<400> 259
acggttcact acaagtacgc agat   24

<210> 260
<211> 27
<212> DNA
<213> artificial sequence

<220>
<223> 1 reuteri MuB gene without restriction sites

<400> 260
tgcgaaaaaa gacaaaaagg ctcaacc   27

<210> 261
<211> 31
<212> DNA
<213> artificial sequence

<220>
<223> CnBP inverse forward

<400> 261
ggaattccat atgtctgaaa agtacttcgg t   31

<210> 262
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> CnBP inverse reverse

<400> 262
tagcttcttg acggatccat c   21

<210> 263
<211> 37
<212> DNA
<213> artificial sequence

<220>
<223> VHH insert for CnBP forward

<400> 263
cgggatccga tgtgcagctg caggagtctg gaggagg   37

<210> 264
<211> 43
<212> DNA
<213> artificial sequence

<220>
<223> VHH insert for CnBP reverse

<400> 264
ggaattccat atgagtgcgg ccgctggaga cggtgacctg ggt   43

<210> 265
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> CnBP phos oligos forward

<400> 265
actctagata tggtctttgc g   21

<210> 266
<211> 21
<212> DNA
<213> artificial sequence

<220>
<223> CnBP phospho oligos reverse

<400> 266
ataatttaac actagtttgt g   21

<210> 267
<211> 27
<212> DNA
<213> artificial sequence

<220>
<223> salmonella FimH forward

<400> 267
ggaattccat atgaaaatat actcagc   27

<210> 268
<211> 30
<212> DNA
<213> artificial sequence

<220>
<223> salmonella FimH reverse

<400> 268
ccgctcgagt tactaatcat aatcgactcg   30

<210> 269
<211> 29
<212> DNA
<213> artificial sequence

<220>
<223> salmonella OmPD forward

<400> 269
ggaattccat atgaaactta agttagtgg   29

<210> 270
<211> 32
<212> DNA
<213> artificial sequence

<220>
<223> salmonella OmPD reverse

<400> 270
ccgctcgagt tactagaact ggtagttcag ac   32

<210> 271
<211> 696
<212> DNA
<213> artificial sequence

<220>
<223> L.reuteri FimH seq

<400> 271

<210> 272
<211> 1089
<212> DNA
<213> artificial sequence

<220>
<223> L.reuteri OmPD seq

<400> 272






Claims

1. A recombinant Lactobacillus spp. expressing on the surface one or more chimeric proteins, wherein the chimeric protein comprises:

a. at least one single chain antibody against Salmonella surface protein, OmpD, having amino acid sequence as set forth in SEQ ID NO:61; and

b. at least one surface protein that is expressed on the surface of the recombinant Lactobacillus spp.,

wherein the surface protein expressed in the recombinant Lactobacillus spp. is MuB or CnBP.
 
2. The recombinant Lactobacillus spp. as claimed in claim 1, wherein the chimeric protein is encoded within the Lactobacillus spp. genome.
 
3. The recombinant Lactobacillus spp. as claimed in claim 1, wherein the single chain antibody is encoded by a polynucleotide consisting of the nucleic acid sequence of SEQ ID NO: 62.
 
4. The recombinant Lactobacillus spp. as claimed in claim 1, wherein the Lactobacillus spp. is Lactobacillus reuteri.
 
5. A food product comprising a recombinant Lactobacillus spp. as claimed in claim 1.
 
6. The food product as claimed in claim 5, further consisting of a carrier selected from the group consisting of a lubricant, a surfactant, solvent, emulsifier, wetting agent, animal feed, dye or oral solution.
 
7. A chimeric protein comprising a single chain antibody against Salmonella surface protein OmpD as set forth in SEQ ID NO: 61, and:

- a surface protein MuB, said chimeric protein is having at least a contiguous amino acid sequence as set forth in SEQ ID NO: 99, SEQ ID NO: 137, and SEQ ID NO: 175; or

- a surface protein CnBP, said chimeric protein is having a contiguous amino acid sequence as set forth in SEQ ID NO: 213.


 
8. A chimeric protein according to claim 7, wherein said chimeric protein is encoded by at least a contiguous nucleotide sequence as set forth in SEQ ID NO: 100, SEQ ID NO: 138, SEQ ID NO: 176 or wherein said chimeric protein is encoded by a contiguous nucleotide sequence as set forth in SEQ ID NO: 214.
 


Ansprüche

1. Rekombinanter Lactobacillus spp., exprimierend auf seiner Oberfläche einen oder mehr chimäre Proteine, wobei die chimären Proteine umfassen:

a. mindestens einen einkettigen Antikörper gegen ein Salmonellen-Oberflächenprotein OmpD, aufweisend eine wie in SEQ ID NO:61 dargelegte Aminosäuresequenz und

b. mindestens ein auf der Oberfläche des rekombinanten Lactobacillus spp. exprimiertes Oberflächenprotein,
wobei das in dem rekombinanten Lactobacillus spp. exprimierte Oberflächenprotein MuB oder CnBP ist.


 
2. Rekombinanter Lactobacillus spp. nach Anspruch 1, wobei das chimäre Protein im Genom des Lactobacillus spp. kodiert ist.
 
3. Rekombinanter Lactobacillus spp. nach Anspruch 1, wobei der einkettige Antikörper durch ein aus der Nukleinsäuresequenz von SEQ ID NO: 62 bestehendes Polynukleotid kodiert ist.
 
4. Rekombinanter Lactobacillus spp. nach Anspruch 1, wobei der Lactobacillus spp. Lactobacillus reuteri ist.
 
5. Lebensmittelprodukt umfassend einen rekombinanten Lactobacillus spp. nach Anspruch 1.
 
6. Lebensmittelprodukt nach Anspruch 5, ferner bestehend aus einem Trägerstoff ausgewählt aus der Gruppe bestehend aus einem Schmiermittel, einem Tensid, Lösungsmittel, Emulgator, Netzmittel, Tierfutter, Farbstoff oder einer Lösung zum Einnehmen.
 
7. Chimäres Protein umfassend einen einkettigen Antikörper gegen ein Salmonellen-Oberflächenprotein OmpD wie in SEQ ID NO: 61 dargelegt und:

- ein Oberflächenprotein MuB, wobei das chimäre Protein mindestens eine zusammenhängende Aminosäuresequenz wie in SEQ ID NO: 99, SEQ ID NO: 137 und SEQ ID NO: 175 dargelegt aufweist; oder

- ein Oberflächenprotein CnBP, wobei das chimäre Protein mindestens eine zusammenhängende Aminosäuresequenz wie in SEQ ID NO: 213 dargelegt aufweist.


 
8. Chimäres Protein nach Anspruch 7, wobei das chimäre Protein durch mindestens eine zusammenhängende Nukleotidsequenz wie in SEQ ID NO: 100, SEQ ID NO: 138, SEQ ID NO: 176 dargelegt kodiert ist oder wobei das chimäre Protein durch eine zusammenhängende Nukleotidsequenz wie in SEQ ID NO: 214 dargelegt kodiert ist.
 


Revendications

1. Un Lactobacillus spp. recombinant exprimant sur la surface une ou plusieurs protéines chimériques, dans laquelle la protéine chimérique comprend :

a. au moins un anticorps à chaîne unique dirigé contre la protéine de surface de Salmonella, OmpD, possédant la séquence d'acides aminés telle que décrite dans SEQ ID NO: 61 ; et

b. au moins une protéine de surface qui est exprimée sur la surface du Lactobacillus spp. recombinant,

dans laquelle la protéine de surface exprimée dans le Lactobacillus spp. recombinant est MuB ou CnBP.
 
2. Le Lactobacillus spp. recombinant selon la revendication 1, dans laquelle la protéine chimérique est codée au sein du génome du Lactobacillusspp..
 
3. Le Lactobacillus spp. recombinant selon la revendication 1, dans laquelle l'anticorps à chaîne unique est codé par un polynucléotide consistant en la séquence d'acide nucléique SEQ ID NO: 62.
 
4. Le Lactobacillus spp. recombinant selon la revendication 1, dans laquelle le Lactobacillus spp. est un Lactobacillus reuteri.
 
5. Un produit alimentaire comprenant un Lactobacillus spp. recombinant selon la revendication 1.
 
6. Le produit alimentaire selon la revendication 5, consistant en outre en un véhicule sélectionné dans le groupe consistant en un lubrifiant, un tensioactif, un solvant, un émulsifiant, un agent mouillant, de la nourriture pour animaux, un colorant ou une solution orale.
 
7. Une protéine chimérique comprenant un anticorps à chaîne unique dirigé contre la protéine de surface de Salmonella, OmpD, telle que décrite dans SEQ ID NO: 61, et ;

- une protéine de surface MuB, ladite protéine chimérique possédant au moins une séquence d'acides aminés contigus telle que décrite dans SEQ ID NO: 99, SEQ ID NO: 137, et SEQ ID NO: 175 ; ou

- une protéine de surface CnBP, ladite protéine chimérique possédant une séquence d'acides aminés contigus telle que décrite dans SEQ ID NO: 213.


 
8. Une protéine chimérique selon la revendication 7, dans laquelle ladite protéine chimérique est codée par au moins une séquence de nucléotides contigus telle que décrite dans SEQ ID NO: 100, SEQ ID NO: 138, SEQ ID NO: 176 ou dans laquelle ladite protéine chimérique est codée par une séquence de nucléotides contigus telle que décrite dans SEQ ID NO: 214.
 




Drawing

















Cited references

REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description