羊源A型产气荚膜梭菌α毒素基因截短表达及其间接ELISA方法的建立与应用

Truncated expression of alpha-toxin gene of sheep-derived Clostridium perfringens type A and establishment and application of indirect ELISA method

基于GenBank登录的产气荚膜梭菌α毒素的氨基酸序列进行抗原决定簇及抗原性的分析,确定优势抗原区(101 aa~334 aa)。设计合成1对引物,以A型产气荚膜梭菌的DNA为模板,应用PCR方法扩增出702 bp大小的基因片段,克隆至表达载体pET32a(+)中进行原核表达。经亲和层析纯化的蛋白,Western blot鉴定相对分子质量约为43 kDa。以纯化的截短α毒素蛋白为包被抗原建立了间接ELISA方法,最适条件:2 mg/L重组蛋白100μL/孔、4℃过夜包被,血清样本1∶50稀释、37℃孵育40 min,HRP标记二抗1∶2500稀释、37℃孵育40 min,底物37℃显色10 min后终止反应。使用建立的方法对226份临床羊血清进行检测,阳性率为71.68%。结果表明,该方法具有良好的特异性、敏感性和重复性,可用于羊群疫苗免疫后的抗体监测及流行病学调查,为试剂盒的开发奠定了基础。

Based on the amino acid sequence of Clostridium perfringens alpha toxin registered in GenBank,the antigenic determinant and antigenicity were analyzed to determine the dominant antigenic region(101 aa-334 aa).A pair of primers were designed and synthesized.Using the DNA of Clostridium perfringens type A as the template,a gene fragment of 702 bp in size was amplified by PCR method,and cloned into the expression vector pET32 a(+)for prokaryotic expression.The protein purified by affinity chromatography was identified by Western blot with a molecular weight of about 43 kDa.An indirect ELISA method was established with purified truncatedαprotein as the coating antigen.The optimal conditions were:2 mg/L recombinant protein,100μL/well,overnight coating at 4℃,serum samples diluted 1:50,and incubated at 37℃for 40 min,the HRP-labeled secondary antibody was diluted 1:2500,incubated at 37℃for 40 min,and the reaction was terminated after the substrate developed color at 37℃for 10 min.Using the established method,226 clinical sheep sera were tested,and the positive rate was 71.68%.The experimental results show that the method has good specificity,sensitivity and repeatability,and can be used for antibody monitoring and epidemiological investigation after sheep vaccine immunization,which lays a foundation for the development of the detection kit.