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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。
OBJECTIVE: To construct and express specific double-stranded single-chain antibodies specific for alpha-toxin (CPA) in order to prevent and treat related diseases such as enterotoxemia and gas gangrene caused by alpha-toxin of type A Clostridium perfringens. The biological activity of a preliminary study. Methods: The scFv gene of Clostridium perfringens type A toxin scFv was screened from the whole human phage antibody library and used as template to amplify two single-chain antibody fragments by PCR and introduced into the middle by primer design The recombinant plasmid was ligated to G4S or (G4S) 3 and subcloned into prokaryotic expression vector p ET-28a (+). The recombinant plasmid was transformed into E.coli BL21 and induced by IPTG. The recombinant protein was identified by purification and purified by Western blotting and indirect ELISA. Antigen binding activity; Biological activity of bivalent single-chain antibody was tested by inhibition of CPA-hydrolyzed lecithin activity and hemolytic activity by in vitro detection and in vivo mouse challenge protection test. Results: The results of double enzyme digestion and sequencing showed that the scFv 2-5 and sc (Fv) 2-15 double-stranded scFv were screened correctly and analyzed by 12% SDS-PAGE after induction Western blot and indirect ELISA analysis showed that the constructed bivalent single chain antibody had specific binding activity with the antigen CPA and the binding activity of the sc (Fv) 2-15 to the antigen Was significantly higher than that of sc (Fv) 2-5 and sc Fv, demonstrating further that sc (Fv) 2-15 neutralizes toxins more readily than sc (Fv) 2-5 and sc Fv in vitro and in vivo . Conclusion: The human full-length bivalent single chain antibody against Clostridium perfringens type A toxin A has been successfully prepared, which lays the foundation for further research on the diagnosis and treatment of various diseases caused by the toxin.