Immunologic Research

, Volume 61, Issue 3, pp 281–293 | Cite as

Construction and characterization of VL–VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins

  • Xianzhi He
  • Lei Zhang
  • Pengchong Liu
  • Li Liu
  • Hui Deng
  • Jinhai Huang


Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the VL–VH tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (VL) and heavy chain (VH) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic VH–VL tail-parallel co-expression vectors based on the “5′-VH-ivs-IRES-VL-3′” mode, the ivs-IRES fragment and VL genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and VH genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing VL–VH tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional VL–VH tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.


Staphylococcal enterotoxins Germ-line genes, variable region Genetically engineered recombinant antibodies Disulfide stable Self-assembly 



This work was supported by the National Natural Science Foundation of China (No. 31272540) and Tianjin science and technology support key project plan (13ZCZDNC08800) in China.

Conflict of interest

The author (s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplementary material

12026_2015_8623_MOESM1_ESM.doc (278 kb)
Supplementary material 1 (DOC 278 kb)


  1. 1.
    al-Daccak R, Mehindate K, Poubelle PE, Mourad W. Signalling via MHC class II molecules selectively induces IL-1 beta over IL-1 receptor antagonist gene expression. Biochem Biophys Res Commun. 1994;201:855–60.CrossRefPubMedGoogle Scholar
  2. 2.
    Assenmacher M, Lohning M, Scheffold A, Manz RA, Schmitz J, Radbruch A. Sequential production of IL-2, IFN-gamma and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes. Eur J Immunol. 1998;28:1534–43.CrossRefPubMedGoogle Scholar
  3. 3.
    Babaei A, Zarkesh-Esfahani SH, Gharagozloo M. Production of a recombinant anti-human CD4 single-chain variable-fragment antibody using phage display technology and its expression in Escherichia coli. J Microbiol Biotechnol. 2011;21:529–35.CrossRefPubMedGoogle Scholar
  4. 4.
    Baines JE, McGovern RM, Persing D, Gostout BS. Consensus-degenerate hybrid oligonucleotide primers (CODEHOP) for the detection of novel papillomaviruses and their application to esophageal and tonsillar carcinomas. J Virol Methods. 2005;123:81–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Baker MD, Papageorgiou AC, Titball RW, Miller J, White S, Lingard B, Lee JJ, Cavanagh D, Kehoe MA, Robinson JH, Acharya KR. Structural and functional role of threonine 112 in a superantigen Staphylococcus aureus enterotoxin B. J Biol Chem. 2002;277:2756–62.CrossRefPubMedGoogle Scholar
  6. 6.
    Betley MJ, Borst DW, Regassa LB. Staphylococcal enterotoxins, toxic shock syndrome toxin and streptococcal pyrogenic exotoxins: a comparative study of their molecular biology. Chem Immunol. 1992;55:1–35.CrossRefPubMedGoogle Scholar
  7. 7.
    Boyce R, Chilana P, Rose TM. iCODEHOP: a new interactive program for designing COnsensus-DEgenerate Hybrid Oligonucleotide Primers from multiply aligned protein sequences. Nucleic Acids Res. 2009;37:W222–8.CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Buzby JC, Roberts T. Economic costs and trade impacts of microbial foodborne illness. World Health Statistics Q (Rapport trimestriel de statistiques sanitaires mondiales). 1997;50:57–66.Google Scholar
  9. 9.
    Carlsson R, Sjogren HO. Kinetics of IL-2 and interferon-gamma production, expression of IL-2 receptors, and cell proliferation in human mononuclear cells exposed to staphylococcal enterotoxin A. Cell Immunol. 1985;96:175–83.CrossRefPubMedGoogle Scholar
  10. 10.
    Chen L, Altman A, Mier W, Lu H, Zhu R, Haberkorn U. 99mTc-pertechnetate uptake in hepatoma cells due to tissue-specific human sodium iodide symporter gene expression. Nucl Med Biol. 2006;33:575–80.CrossRefPubMedGoogle Scholar
  11. 11.
    Cheng M, Chan SY, Zhao Q, Chan EY, Au SW, Lee SS, Cheung WT. Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes. PLoS ONE. 2011;6:e27406.CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Comas-Riu J, Rius N. Flow cytometry applications in the food industry. J Ind Microbiol Biotechnol. 2009;36:999–1011.CrossRefPubMedGoogle Scholar
  13. 13.
    Dellabona P, Peccoud J, Kappler J, Marrack P, Benoist C, Mathis D. Superantigens interact with MHC class II molecules outside of the antigen groove. Cell. 1990;62:1115–21.CrossRefPubMedGoogle Scholar
  14. 14.
    Demarest SJ, Glaser SM. Antibody therapeutics, antibody engineering, and the merits of protein stability. Curr Opin Drug Discov Devel. 2008;11:675–87.PubMedGoogle Scholar
  15. 15.
    Druar C, Saini SS, Cossitt MA, Yu F, Qiu X, Geisbert TW, Jones S, Jahrling PB, Stewart DI, Wiersma EJ. Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus’ soluble glycoprotein. Immunogenetics. 2005;57:730–8.CrossRefPubMedGoogle Scholar
  16. 16.
    Du J, Zhang C, Fu J, Chen Z, Xiao Q. Preparation of a monoclonal antibody against polyhedrin of Ectropis obliqua nucleopolyhedrovirus. Chin J Biotechnol (Sheng wu gong cheng xue bao). 2012;28:76–85.Google Scholar
  17. 17.
    Evenson ML, Hinds MW, Bernstein RS, Bergdoll MS. Estimation of human dose of staphylococcal enterotoxin A from a large outbreak of staphylococcal food poisoning involving chocolate milk. Int J Food Microbiol. 1988;7:311–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Fraser JD. High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature. 1989;339:221–3.CrossRefPubMedGoogle Scholar
  19. 19.
    Froyen G, Hendrix D, Ronsse I, Fiten P, Martens E, Billiau A. Effect of VH and VL consensus sequence-specific primers on the binding and neutralizing potential of a single-chain FV directed towards HuIFN-gamma. Mol Immunol. 1995;32:515–21.CrossRefPubMedGoogle Scholar
  20. 20.
    Hense M, Domann E, Krusch S, Wachholz P, Dittmar KE, Rohde M, Wehland J, Chakraborty T, Weiss S. Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Cell Microbiol. 2001;3:599–609.CrossRefPubMedGoogle Scholar
  21. 21.
    Huang J, Liu Y, Liu Y, Sun Y, Zhuang S, Wang J, Zhang L. Distribution detecton, expression & purification of staphylococcal enterotoxin K SEK. Chinese J Cell Mol Immunol. 2010;26(3):220–2.Google Scholar
  22. 22.
    Kappler J, Kotzin B, Herron L, Gelfand EW, Bigler RD, Boylston A, Carrel S, Posnett DN, Choi Y, Marrack P. V beta-specific stimulation of human T cells by staphylococcal toxins. Science. 1989;244:811–3.CrossRefPubMedGoogle Scholar
  23. 23.
    Karsunke XY, Wang H, Weber E, McLean MD, Niessner R, Hall JC, Knopp D. Development of single-chain variable fragment (scFv) antibodies against hapten benzo[a]pyrene: a binding study. Anal Bioanal Chem. 2012;402:499–507.CrossRefPubMedGoogle Scholar
  24. 24.
    Kong T, Li XB, Liu GW, Xie GH, Wang Z, Zhang ZG, Zhang Y, Sun J, Tang J. Preparation of specific monoclonal antibodies against chelated copper ions. Biol Trace Elem Res. 2012;145:388–95.CrossRefPubMedGoogle Scholar
  25. 25.
    Le Loir Y, Baron F, Gautier M. Staphylococcus aureus and food poisoning. Genet Mol Res (GMR). 2003;2:63–76.Google Scholar
  26. 26.
    Li J, Wang Y, Wang Z, Dong Z. Effect of VL and VH consensus sequence-specific primers on the binding and expression of a mini-molecule antibody directed towards human gastric cancer. Chin Med Sci J/Chin Acad Med Sci (Chung-kuo i hsueh k’o hsueh tsa chih). 2000;15:133–9.Google Scholar
  27. 27.
    Lin YC, Anderson MJ, Kohler PL, Strandberg KL, Olson ME, et al. Proinflammatory exoprotein characterization of toxic shock syndrome Staphylococcus aureus. Biochemistry. 2011;50:7157–67.CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Masuko K, Okazaki S, Satoh M, Tanaka G, Ikeda T, Torii R, Ueda E, Nakano T, Danbayashi M, Tsuruoka T, Ohno Y, Yagi H, Yabe N, Yoshida H, Tahara T, Kataoka S, Oshino T, Shindo T, Niwa S, Ishimoto T, Baba H, Hashimoto Y, Saya H, Masuko T. Anti-tumor effect against human cancer xenografts by a fully human monoclonal antibody to a variant 8-epitope of CD44R1 expressed on cancer stem cells. PLoS ONE. 2012;7:e29728.CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    McCormick JK, Yarwood JM, Schlievert PM. Toxic shock syndrome and bacterial superantigens: an update. Annu Rev Microbiol. 2001;55:77–104.CrossRefPubMedGoogle Scholar
  30. 30.
    Mead PS, Slutsker L, Griffin PM, Tauxe RV. Food-related illness and death in the united states reply to dr. hedberg. Emerg Infect Dis. 1999;5:841–2.CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    Mizuguchi H, Xu Z, Ishii-Watabe A, Uchida E, Hayakawa T. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Therapy J Am Soc Gene Therapy. 2000;1:376–82.CrossRefGoogle Scholar
  32. 32.
    Nehlsen K, Herrmann S, Zauers J, Hauser H, Wirth D. Toxin-antitoxin based transgene expression in mammalian cells. Nucleic Acids Res. 2010;38(5):e32.CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Ortega E, Abriouel H, Lucas R, Gálvez A. Multiple roles of Staphylococcus aureus enterotoxins: pathogenicity, superantigenic activity, and correlation to antibiotic resistance. Toxins. 2010;2:2117–31.CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Pinchuk IV, Beswick EJ, Reyes VE. Staphylococcal enterotoxins. Toxins. 2010;2:2177–97.CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Sasaki T, Terano Y, Shibata T, Kawamoto H, Kuzuguchi T, Kohyama E, Watanabe T, Ohyama T, Gemba M. Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies. Microbiol Immunol. 2005;49:589–97.CrossRefPubMedGoogle Scholar
  36. 36.
    Scherrer D, Corti S, Muehlherr JE, Zweifel C, Stephan R. Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep. Vet Microbiol. 2004;101:101–7.CrossRefPubMedGoogle Scholar
  37. 37.
    Schlievert PM. Role of superantigens in human disease. J Infect Dis. 1993;167:997–1002.CrossRefPubMedGoogle Scholar
  38. 38.
    Scholl P, Diez A, Mourad W, Parsonnet J, Geha RS, Chatila T. Toxic shock syndrome toxin 1 binds to major histocompatibility complex class II molecules. Proc Natl Acad Sci USA. 1989;86:4210–4.CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Shen J, Vil MD, Jimenez X, Iacolina M, Zhang H, Zhu Z. Single variable domain-IgG fusion. A novel recombinant approach to Fc domain-containing bispecific antibodies. J Biol Chem. 2006;281:10706–14.CrossRefPubMedGoogle Scholar
  40. 40.
    Singh PK, Agrawal R, Kamboj DV, Gupta G, Boopathi M, Goel AK, Singh L. Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B. Appl Environ Microbiol. 2010;76:8184–91.CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Spaulding AR, Salgado-Pabon W, Kohler PL, Horswill AR, Leung DY, Schlievert PM. Staphylococcal and streptococcal superantigen exotoxins. Clin Microbiol Rev. 2013;26:422–47.CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Stoneley M, Willis AE. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression. Oncogene. 2004;23:3200–7.CrossRefPubMedGoogle Scholar
  43. 43.
    Thomas DY, Jarraud S, Lemercier B, Cozon G, Echasserieau K, Etienne J, Gougeon ML, Lina G, Vandenesch F. Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster. Infect Immun. 2006;74:4724–34.CrossRefPubMedCentralPubMedGoogle Scholar
  44. 44.
    Urushibata Y, Itoh K, Ohshima M, Seto Y. Generation of Fab fragment-like molecular recognition proteins against staphylococcal enterotoxin B by phage display technology. Clin Vaccine Immunol (CVI). 2010;17:1708–17.CrossRefGoogle Scholar
  45. 45.
    Vagner S, Galy B, Pyronnet S. Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites. EMBO Rep. 2001;2:893–8.CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    Vu BG, Gourronc FA, Bernlohr DA, Schlievert PM, Klingelhutz AJ. Staphylococcal superantigens stimulate immortalized human adipocytes to produce chemokines. PLoS ONE. 2013;8(10):e77988.CrossRefPubMedCentralPubMedGoogle Scholar
  47. 47.
    Wood AC, Todd I, Cockayne A, Arbuthnott JP. Staphylococcal enterotoxins and the immune system. FEMS Microbiol Immunol. 1991;3:121–33.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Xianzhi He
    • 1
    • 2
  • Lei Zhang
    • 1
  • Pengchong Liu
    • 1
  • Li Liu
    • 1
  • Hui Deng
    • 1
  • Jinhai Huang
    • 1
  1. 1.School of Life ScienceTianjin UniversityTianjinChina
  2. 2.School of Chemical engineering & TechnologyTianjin UniversityTianjinChina

Personalised recommendations