抗A型产气荚膜梭菌α毒素全人源双价单链抗体的构建、表达及其活性的初步研究

doi:10.13523/j.cb.20170403

中国生物工程杂志

2017, Vol. 37

Issue (4): 18-25 DOI: 10.13523/j.cb.20170403

研究报告

抗A型产气荚膜梭菌α毒素全人源双价单链抗体的构建、表达及其活性的初步研究

王冬冬, 张国利, 岳玉环, 吴广谋, 田园, 刘雨玲, 吉元刚, 王金鹏, 李建, 潘荣荣, 马洪圆

军事医学科学院军事兽医研究所长春 130122

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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摘要： 目的：为防治A型产气荚膜梭菌α毒素引起的肠毒血症及气性坏疽等相关疾病，构建并高效表达中和α毒素（CPA）的特异性双价单链抗体，并对其生物学活性进行初步研究。方法：以全人源噬菌体抗体库中筛选得到的抗A型产气荚膜梭菌α毒素单链抗体scFv基因为模板，PCR的方法扩增两条单链抗体片段并通过引物设计引入中间连接肽G4S或（G4S）3，亚克隆至原核表达载体pET-28a（+），转化E.coli BL21，IPTG诱导表达、鉴定及表达产物的层析纯化；Western blot和间接ELISA方法检测与抗原的免疫结合活性；通过体外检测抗体抑制CPA水解卵磷脂的活性和溶血活性以及体内小鼠攻毒保护试验初步研究双价单链抗体的生物学活性。结果：双酶切鉴定及基因测序结果表明构建的双价单链抗体sc（Fv）2-5和sc（Fv）2-15均正确，诱导表达后经12% SDS-PAGE分析，两者均以包涵体形式表达且蛋白分子量符合理论值大小，Western blot和间接ELISA分析结果显示，构建的双价单链抗体与抗原CPA具有特异结合活性，且sc（Fv）2-15与抗原的结合活性明显高于sc（Fv）2-5和scFv，体外与体内生物活性试验结果进一步证明，sc（Fv）2-15中和毒素的能力较sc（Fv）2-5和scFv具有明显优势。结论：成功制备了抗A型产气荚膜梭菌α毒素的全人源双价单链抗体，为进一步研究该毒素引发的各类疾病的诊断和治疗奠定了基础。

关键词： 双价单链抗体; 生物活性检测; A型产气荚膜梭菌α毒素; 表达与纯化

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.

Key words: Bivalent single chain antibody Expression and purification The alpha-toxin of Clostridium perfringens type A Biological activity detection

收稿日期: 2016-11-20 出版日期: 2017-04-25

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通讯作者: 张国利 E-mail: Zhangguoli2001@126.com

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引用本文:

王冬冬, 张国利, 岳玉环, 吴广谋, 田园, 刘雨玲, 吉元刚, 王金鹏, 李建, 潘荣荣, 马洪圆. 抗A型产气荚膜梭菌α毒素全人源双价单链抗体的构建、表达及其活性的初步研究[J]. 中国生物工程杂志, 2017, 37(4): 18-25.

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.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170403 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I4/18

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