产气荚膜梭菌EF-Tu基因的原核表达及其多克隆抗体的制备

Prokaryotic expression of Clostridium perfringens EF-Tu gene and preparation of the polyclonal antibody

通过PCR方法扩增产气荚膜梭菌ATCC13124株EF-Tu基因,利用原核表达载体pGEX-6P-1构建重组质粒pGEX-EF,经双酶切和测序鉴定正确后转化E.coli Rosseta,进行IPTG诱导表达,表达产物纯化后免疫大白兔制备多克隆抗体,应用间接ELISA、Western-blot和间接免疫荧光方法对所得抗体进行检测。结果显示,经1.0mmol/L IPTG诱导5h后蛋白表达量最高,并且主要以包涵体的形式存在,分子质量为69ku。间接ELISA方法检测的多抗血清效价为1∶262 144。Western-blot结果证实,重组蛋白与产气荚膜梭菌ATCC13124株制备的多克隆抗体发生特异性结合,具有良好的免疫原性。Western-blot和间接免疫荧光结果表明,多抗血清能分别与pGEX-EF重组蛋白、ATCC13124菌体裂解液、pcDNA3.1-EF-Tu瞬时转染后的BHK-21细胞及ATCC13124感染的小鼠肠组织中菌体发生特异性结合反应。上述结果表明,此多克隆抗体能够应用于临床诊断。

The EF-Tu gene of Clostridium perfringens ATCC13124 was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1 to construct a recombinant expression vector pGEX-EF.The pGEX-EF was transformed into E.coli Rosseta.The purified protein was used to inject rabbit for preparation of polyclonal antibody,which was verified by the indirect ELISA,Western-blot and indirect immunofluorescence.The optimal expression condition was induced by IPTG at 1.0 mmol/L for 5 hours.The recombinant protein was mainly in inclusion form with molecular weight of 69 ku.The ELISA titer of polyclonal antibody was approximate 1∶262 144.Western-blot analysis revealed that antiserum against Clostridium perfringens ATCC13124 could specifically bind to the recombinant expressed protein and bacterium lysate of Clostridium perfringens.Furthermore,indirect immunofluorescence showed that the polyclonal antibody could specifically recognize the expressed protein in BHK-21 cells and Clostridium perfringens in the bacterium smear and in intestine of bacterium infected mice.