Archives of Virology

, Volume 164, Issue 4, pp 983–994 | Cite as

Construction and characterization of porcine single-chain fragment variable antibodies that neutralize transmissible gastroenteritis virus in vitro

  • Fanqing Zhang
  • Yuxue Chen
  • Liang Yang
  • Jianguo ZhuEmail author
Original Article


Transmissible gastroenteritis virus (TGEV) infection causes severe diarrhea in piglets and imposes a significant economic burden on pig farms. Single-chain fragment variable (scFv) antibodies effectively inhibit virus infection and could be a potential therapeutic reagent for preventing disease. In this study, a recombinant scFv antibody phage display library was constructed from peripheral blood lymphocytes of piglets infected with TGEV. The library was screened with four rounds of biopanning using purified TGEV antigen, and scFv antibodies that bound to TGEV were obtained. The scFv gene was subcloned into the pET-28a(+), and the constituted plasmid was introduced into Escherichia coli BL21 (DE3) for protein expression. All three scFv clones identified had neutralizing activity against TGEV. An immunofluorescence assay and western blot analysis demonstrated that two scFv antibodies reacted with the spike protein of TGEV. These results indicate that scFv antibodies provide protection against viral infection in vitro and may be a therapeutic candidate for both prevention and treatment of TGEV infection in swine.



This work was financially supported by the Key Project of Science and Technology for Agriculture of Shanghai (Hu Nong Ke Gong Zi (2015) no. 1-8).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The animal experiment was performed in accordance with the recommendations in the Guidelines for the Use of Laboratory Animals provided by the Science and Technology Commission of Shanghai Municipality (STCSM). The protocol was approved by the ethics committee of the Shanghai JiaoTong University, School of Agriculture and Biology.


  1. 1.
    Masters PS (2006) The molecular biology of coronaviruses. Adv Virus Res 66:193–292. CrossRefPubMedGoogle Scholar
  2. 2.
    Jones T, Pritchard G, Paton D (1997) Transmissible gastroenteritis of pigs. Vet Rec 141:427–428CrossRefPubMedGoogle Scholar
  3. 3.
    Schwegmann-Wessels C, Herrler G (2006) Transmissible gastroenteritis virus infection: a vanishing specter. Dtsch Tierarztl Wochenschr 113:157–159PubMedGoogle Scholar
  4. 4.
    Spaan W, Cavanagh D, Horzinek MC (1988) Coronaviruses: structure and genome expression. J Gen Virol 69(Pt 12):2939–2952. CrossRefPubMedGoogle Scholar
  5. 5.
    Laude H, Van Reeth K, Pensaert M (1993) Porcine respiratory coronavirus: molecular features and virus-host interactions. Vet Res 24:125–150PubMedGoogle Scholar
  6. 6.
    Krempl C, Schultze B, Laude H, Herrler G (1997) Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus. J Virol 71:3285–3287PubMedPubMedCentralGoogle Scholar
  7. 7.
    Delmas B, Gelfi J, L’Haridon R, Vogel LK, Sjostrom H, Noren O, Laude H (1992) Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357:417–420. CrossRefPubMedGoogle Scholar
  8. 8.
    Delmas B, Rasschaert D, Godet M, Gelfi J, Laude H (1990) Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein S. J Gen Virol 71(Pt 6):1313–1323. CrossRefPubMedGoogle Scholar
  9. 9.
    Reguera J, Ordono D, Santiago C, Enjuanes L, Casasnovas JM (2011) Antigenic modules in the N-terminal S1 region of the transmissible gastroenteritis virus spike protein. J Gen Virol 92:1117–1126CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Enjuanes L, Sune C, Gebauer F, Smerdou C, Camacho A, Anton IM, Gonzalez S, Talamillo A, Mendez A, Ballesteros ML et al (1992) Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus. Vet Microbiol 33:249–262CrossRefPubMedGoogle Scholar
  11. 11.
    Meng F, Ren Y, Suo S, Sun X, Li X, Li P, Yang W, Li G, Li L, Schwegmann-Wessels C, Herrler G, Ren X (2013) Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing spike genes from porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. PLoS One 8:e57468CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gerdts V, Zakhartchouk A (2017) Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses. Vet Microbiol 206:45–51. CrossRefPubMedGoogle Scholar
  13. 13.
    Langel SN, Paim FC, Lager KM, Vlasova AN, Saif LJ (2016) Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV): historical and current concepts. Virus Res 226:93–107. CrossRefPubMedGoogle Scholar
  14. 14.
    Bohl EH, Gupta RK, Olquin MV, Saif LJ (1972) Antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus. Infect Immun 6:289–301PubMedPubMedCentralGoogle Scholar
  15. 15.
    Wesley RD, Woods RD (2001) Partial passive protection with two monoclonal antibodies and frequency of feeding of hyperimmune anti-transmissible gastroenteritis virus (TGEV) serum for protection of three-day-old piglets from a TGEV challenge infection. J Vet Diagn Investig 13:290–296. CrossRefGoogle Scholar
  16. 16.
    Bestagno M, Sola I, Dallegno E, Sabella P, Poggianella M, Plana-Duran J, Enjuanes L, Burrone OR (2007) Recombinant dimeric small immunoproteins neutralize transmissible gastroenteritis virus infectivity efficiently in vitro and confer passive immunity in vivo. J Gen Virol 88:187–195. CrossRefPubMedGoogle Scholar
  17. 17.
    Unkauf T, Miethe S, Fuhner V, Schirrmann T, Frenzel A, Hust M (2016) Generation of recombinant antibodies against toxins and viruses by phage display for diagnostics and therapy. Adv Exp Med Biol 917:55–76. CrossRefPubMedGoogle Scholar
  18. 18.
    Bustamante-Cordova L, Melgoza-Gonzalez EA, Hernandez J (2018) Recombinant antibodies in veterinary medicine: an update. Front Vet Sci 5:175CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Golchin M, Aitken R (2008) Isolation by phage display of recombinant antibodies able to block adherence of Escherichia coli mediated by the K99 colonisation factor. Vet Immunol Immunopathol 121:321–331. CrossRefPubMedGoogle Scholar
  20. 20.
    Chai Z, Fu F, Jiang F, Tian H, Wang Z, Zheng N, Zhang X, Wang X, Li X (2014) Development of a neutralizing mouse-pig chimeric antibody with therapeutic potential against Haemophilus parasuis in Pichia pastoris. FEMS Microbiol Lett 354:85–91. CrossRefPubMedGoogle Scholar
  21. 21.
    Corti D, Voss J, Gamblin SJ, Codoni G, Macagno A, Jarrossay D, Vachieri SG, Pinna D, Minola A, Vanzetta F, Silacci C, Fernandez-Rodriguez BM, Agatic G, Bianchi S, Giacchetto-Sasselli I, Calder L, Sallusto F, Collins P, Haire LF, Temperton N, Langedijk JP, Skehel JJ, Lanzavecchia A (2011) A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333:850–856. CrossRefPubMedGoogle Scholar
  22. 22.
    Weisser NE, Hall JC (2009) Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol Adv 27:502–520. CrossRefPubMedGoogle Scholar
  23. 23.
    Schofield DJ, Pope AR, Clementel V, Buckell J, Chapple S, Clarke KF, Conquer JS, Crofts AM, Crowther SR, Dyson MR, Flack G, Griffin GJ, Hooks Y, Howat WJ, Kolb-Kokocinski A, Kunze S, Martin CD, Maslen GL, Mitchell JN, O’Sullivan M, Perera RL, Roake W, Shadbolt SP, Vincent KJ, Warford A, Wilson WE, Xie J, Young JL, McCafferty J (2007) Application of phage display to high throughput antibody generation and characterization. Genome Biol 8:R254CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Yokota T, Milenic DE, Whitlow M, Schlom J (1992) Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 52:3402–3408PubMedGoogle Scholar
  25. 25.
    Better M, Chang CP, Robinson RR, Horwitz AH (1988) Escherichia coli secretion of an active chimeric antibody fragment. Science 240:1041–1043CrossRefPubMedGoogle Scholar
  26. 26.
    Liu H, Wang Y, Duan H, Zhang A, Liang C, Gao J, Zhang C, Huang B, Li Q, Li N, Xiao S, Zhou EM (2015) An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication. Vet Microbiol 181:252–260. CrossRefPubMedGoogle Scholar
  27. 27.
    Pyo HM, Kim IJ, Kim SH, Kim HS, Cho SD, Cho IS, Hyun BH (2009) Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus. Vaccine 27:2030–2036. CrossRefPubMedGoogle Scholar
  28. 28.
    Harmsen MM, Fijten HP, Engel B, Dekker A, Eble PL (2009) Passive immunization with llama single-domain antibody fragments reduces foot-and-mouth disease transmission between pigs. Vaccine 27:1904–1911. CrossRefPubMedGoogle Scholar
  29. 29.
    Krempl C, Herrler G (2001) Sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins. J Virol 75:844–849CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Wang M, Zhang Y, Li B, Zhu J (2015) Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus. Res Vet Sci 100:109–114. CrossRefPubMedGoogle Scholar
  31. 31.
    Lee CM, Iorno N, Sierro F, Christ D (2007) Selection of human antibody fragments by phage display. Nat Protoc 2:3001–3008. CrossRefPubMedGoogle Scholar
  32. 32.
    Wang M, Zhang Y, Zhu J (2016) Anti-Staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice. Appl Microbiol Biotechnol 100:2153–2162. CrossRefPubMedGoogle Scholar
  33. 33.
    Hofmann M, Wyler R (1989) Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (PEDV). Vet Microbiol 20:131–142CrossRefPubMedGoogle Scholar
  34. 34.
    Lee DH, Jeon YS, Park CK, Kim S, Lee DS, Lee C (2015) Immunoprophylactic effect of chicken egg yolk antibody (IgY) against a recombinant S1 domain of the porcine epidemic diarrhea virus spike protein in piglets. Arch Virol 160:2197–2207. CrossRefPubMedGoogle Scholar
  35. 35.
    Garaicoechea L, Olichon A, Marcoppido G, Wigdorovitz A, Mozgovoj M, Saif L, Surrey T, Parreno V (2008) Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice. J Virol 82:9753–9764CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315–1317CrossRefPubMedGoogle Scholar
  37. 37.
    McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554. CrossRefPubMedGoogle Scholar
  38. 38.
    Wu CC, Lin EH, Lee YC, Tai CJ, Kuo TH, Wang HE, Luo TY, Fu YK, Chen HJ, Sun MD, Wu CH, Wu CW, Leu SJ, Deng WP (2010) Identification of a new peptide for fibrosarcoma tumor targeting and imaging in vivo. J Biomed Biotechnol 2010:167045CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Foy BD, Killeen GF, Frohn RH, Impoinvil D, Williams A, Beier JC (2002) Characterization of a unique human single-chain antibody isolated by phage-display selection on membrane-bound mosquito midgut antigens. J Immunol Methods 261:73–83CrossRefPubMedGoogle Scholar
  40. 40.
    Li F, Aitken R (2004) Cloning of porcine scFv antibodies by phage display and expression in Escherichia coli. Vet Immunol Immunopathol 97:39–51CrossRefPubMedGoogle Scholar
  41. 41.
    Haidaris CG, Malone J, Sherrill LA, Bliss JM, Gaspari AA, Insel RA, Sullivan MA (2001) Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens. J Immunol Methods 257:185–202CrossRefPubMedGoogle Scholar
  42. 42.
    Pansri P, Jaruseranee N, Rangnoi K, Kristensen P, Yamabhai M (2009) A compact phage display human scFv library for selection of antibodies to a wide variety of antigens. BMC Biotechnol 9:6CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Yang J, Yoshida R, Kariya Y, Zhang X, Hashiguchi S, Nakashima T, Suda Y, Takada A, Ito Y, Sugimura K (2010) Characterization of human single-chain antibodies against highly pathogenic avian influenza H5N1 viruses: mimotope and neutralizing activity. J Biochem 148:507–515. CrossRefPubMedGoogle Scholar
  44. 44.
    Khan L, Kumar R, Thiruvengadam R, Parray HA, Makhdoomi MA, Kumar S, Aggarwal H, Mohata M, Hussain AW, Das R, Varadarajan R, Bhattacharya J, Vajpayee M, Murugavel KG, Solomon S, Sinha S, Luthra K (2017) Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library. Sci Rep 7:45163CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Guirakhoo F, Catalan J, Monath T, Weltzin R (1996) Cloning, expression and functional activities of a single chain antibody fragment directed to fusion protein of respiratory syncytial virus. Immunotechnology 2:219–228CrossRefPubMedGoogle Scholar
  46. 46.
    Muller BH, Lafay F, Demangel C, Perrin P, Tordo N, Flamand A, Lafaye P, Guesdon JL (1997) Phage-displayed and soluble mouse scFv fragments neutralize rabies virus. J Virol Methods 67:221–233CrossRefPubMedGoogle Scholar
  47. 47.
    Lin Y, Li B, Ye J, Wang M, Wang J, Zhang Y, Zhu J (2015) Neutralization analysis of a chicken single-chain variable fragment derived from an immune antibody library against infectious bronchitis virus. Viral Immunol 28:397–404. CrossRefPubMedGoogle Scholar
  48. 48.
    Jacobs L, van der Zeijst BA, Horzinek MC (1986) Characterization and translation of transmissible gastroenteritis virus mRNAs. J Virol 57:1010–1015PubMedPubMedCentralGoogle Scholar
  49. 49.
    Sestak K, Meister RK, Hayes JR, Kim L, Lewis PA, Myers G, Saif LJ (1999) Active immunity and T-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins. Vet Immunol Immunopathol 70:203–221CrossRefPubMedGoogle Scholar
  50. 50.
    Zhang D, Huang X, Zhang X, Cao S, Wen X, Wen Y, Wu R, Liang E (2016) Construction of an oral vaccine for transmissible gastroenteritis virus based on the TGEV N gene expressed in an attenuated Salmonella typhimurium vector. J Virol Methods 227:6–13. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Fanqing Zhang
    • 1
  • Yuxue Chen
    • 1
    • 2
  • Liang Yang
    • 1
    • 2
  • Jianguo Zhu
    • 1
    • 3
    Email author
  1. 1.Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and BiologyShanghai JiaoTong UniversityShanghaiPeople’s Republic of China
  2. 2.Shanghai Frontan Animal Health Co., Ltd.ShanghaiPeople’s Republic of China
  3. 3.School of Agriculture and Biology, Shanghai Key Lab of Veterinary BiologyShanghai JiaoTong universityShanghaiPeople’s Republic of China

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