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| Construction, Expression and Preliminary Study on Activity of Human Bivalent Single Chain Antibody Against the Alpha-toxin of Clostridium perfringens Type A |
| WANG Dong-dong, ZHANG Guo-li, YUE Yu-huan, WU Guang-mou, TIAN Yuan, LIU Yu-ling, JI Yuan-gang, WANG Jing-peng, LI Jian, PAN Rong-rong, MA Hong-yuan |
| Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China |
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Abstract To prevent from and treat for enterotoxemia and gas gangrene and other diseases caused by the alpha -toxin of Clostridium perfringens type A(CPA), the specific bivalent scFv against CPA was constructed and expressed, together with preliminary study on its biological activity. Methods With scFv gene screened from Human phage antibody library against CPA as a template, to introduce connecting peptide G4S or (G4S)3 by primer design and amplify two single chain antibody fragment by PCR, then clone into the prokaryotic expression vector pET-28a (+). Recombinant plasmid was transformed and expressed in Ecoli BL21 (DE3) by IPTG induction.The expressed protein were purified by column chromatography. Indirect enzyme-linked immunosorbent assay (ELISA) was used to detect antigen binding activity. To prove the neutralizing potential of the double single chain antibody, alpha-toxin was preincubated with the double single chain and subsequently tested for its lecithinase activity in an egg yolk diffusion turbidity (EYDT) assay, its hemolytic activity in a hemolysis test, and its lethal effect on mice after intravenously administration. Results:Double enzyme digestion and gene sequencing results showed that the sc (Fv) 2-5 and sc(Fv) 2-15 were constructed and expressed correctly. The analysis conducted by 12% SDS-PAGE showed that proteins was expressed in a high level as inclusion bodies and the molecular weight consistent with theoretical value. Indirect ELISA and Western blot detection showed that the sc (Fv) 2-5 and sc(Fv) 2-15 have a strong immune binding activity and that the latter were much better. The activity test in vivo and in vitro indicated that the neutralizing ability of sc(Fv) 2-15 was higher than that of sc (Fv) 2-5 and scFv. Conclusion:Human bivalent single chain antibody against the Alpha-toxin of Clostridium perfringens Type A was successfully prepared, which lay a foundation for further study on the diagnosis and treatment of various diseases caused by this toxin.
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Received: 20 November 2016
Published: 25 April 2017
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Cite this article:
WANG Dong-dong, ZHANG Guo-li, YUE Yu-huan, WU Guang-mou, TIAN Yuan, LIU Yu-ling, JI Yuan-gang, WANG Jing-peng, LI Jian, PAN Rong-rong, MA Hong-yuan. Construction, Expression and Preliminary Study on Activity of Human Bivalent Single Chain Antibody Against the Alpha-toxin of Clostridium perfringens Type A. China Biotechnology, 2017, 37(4): 18-25.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170403 OR https://manu60.magtech.com.cn/biotech/Y2017/V37/I4/18
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[1] Shreya D, Uppalapati S R, Kingston J J, et al. Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model. Molecμlar Immunology, 2015, 65(1):51-57.
[2] 许崇波,马从林,朱平,等.抗A型产气荚膜梭菌α毒素单克隆抗体杂交瘤细胞系的建立与中和效应.卫生研究,1998,27(1):464-466. Xu C B,Ma C L, Zhu P, et al. Establishment of hybridoma cell lines secreting monoclonal antibodies against Alpha-toxin of Clostridium perfringens type A and the netrulization effect of the produced McAbs. Journal of Hygiene Research, 1998, 27(1):464-466.
[3] 严丹丹,杨福辉,方瑾.抗人大肠癌双价单链抗体基因的构建及表达. 世界华人消化杂志, 2006, 14(24):2395-2400. Yan D D,Yang F H, Fang J. Construction and expression of bivalent single-chain antibody against human colorectal carcinoma. World Chinese Journal of Digestology, 2006, 14(24):2395-2400.
[4] Zhao Y, Kang L, Gao S, et al. Expression and purification of functional Clostridium perfringens, alpha and epsilon toxins in Escherichia coli. Protein Expression & Purification, 2011, 77(2):207-213.
[5] Garcia J P, Beingesser J, Bohorov O, et al. Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana. Toxicon, 2014, 88(2):93-98.
[6] Yamashita M, Katakura Y, Shirahata S. Recent advances in the generation of human monoclonal antibody. Cytotechnology, 2007, 55(2):55-60.
[7] Huang B C, Davern S, Kennel S J. Mono and bivalent binding of a scFv and covalent diabody to murine laminin-1 using radioiodinated proteins and SPR measurements:effects on tissue retention in vivo. Journal of Immunological Methods, 2006, 313(1-2):149-160.
[8] Weatherill E E, Cain K L, Heywood S P, et al. Towards a universal disulphide stabilised single chain Fv format:importance of interchain disulphide bond location and vL-vH, orientation. Protein Engineering, Design and Selection, 2012, 25(7):321-329.
[9] 马媛媛,何健民,康永杰.外源性蛋白在大肠杆菌中可溶性表达的策略综述. 世界科技研究与发展,2015,37(5):627-630. Ma Y Y, He J M, Kang Y J. Reviews on strategies of enhance heterologous protein soluble expression in Escherichia coli. World Sci-Tech R&D, 2015, 37(5):627-630.
[10] Zhou L, Zhao Z, Li B, et al. TrxA mediating fusion expression of antimicrobial peptide CM4 from multiple joined genes in Escherichia coli. Protein Expression & Purification, 2009, 64(2):225-230.
[11] Zhang J, Lv X, Xu R, et al. Soluble expression, rapid purification, and characterization of human interleukin-24(IL-24) using a MBP-SUMO dual fusion system in Escherichia coli. Applied Microbiology and Biotechnology, 2015, 99(16):6705-6713. |
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