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

发布时间：2018-06-23 19:09

本文选题：A型产气荚膜梭菌α毒素 + 双价单链抗体　；参考：《中国生物工程杂志》2017年04期

【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sc Fv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p ET-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sc Fv,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sc Fv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。
[Abstract]:Objective: in order to prevent and treat enterotoxemia and gas gangrene caused by Clostridium perfringens 伪 toxin type A, the bivalent single-chain antibody of neutralizing 伪 toxin (CPA) was constructed and expressed, and its biological activity was studied. Methods: two single-chain antibody fragments were amplified by PCR from the full-human phage antibody library against Clostridium perfringens 伪 -toxin single chain antibody sc Fv, and the intermediate ligand peptide was introduced by primer design. G4S or (G4S) 3 was subcloned into prokaryotic expression vector pET-28a (), and transformed into E. coli 21 and induced by IPTG. Western blot and indirect Elisa were used to detect the immunobinding activity of the expressed product. The biological activity of bivalent single chain antibody (bivalent single chain antibody) was preliminarily studied by in vitro detection of antibody inhibiting the activity of CPA hydrolyzed lecithin and lysotic activity as well as the protection test of mice against virus attack in vivo. Results: the bivalent single-chain antibodies sc (FV) 2-5 and sc (FV) 2-15 were confirmed by double enzyme digestion and gene sequencing. The expression of sc (FV) 2-5 and sc (FV) 2-15 were analyzed by 12SDS-PAGE. The results of Western blot and indirect Elisa analysis showed that the constructed bivalent single-chain antibody had specific binding activity to antigen CPA. The binding activity of sc (FV) 2-15 to antigen was significantly higher than that of sc (FV) 2-5 and sc Fv. The results of bioactivity tests in vitro and in vivo further proved that the ability of Sc (FV) 2-15 neutralizing toxin was superior to that of SC (FV) 2-5 and sc Fv. Conclusion: the full-human bivalent single-chain antibody against Clostridium perfringens 伪 toxin A was successfully prepared, which laid a foundation for the further study of the diagnosis and treatment of various diseases caused by the toxin.
【作者单位】：军事医学科学院军事兽医研究所;
【分类号】：R392

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