产气荚膜梭菌plc基因的原核表达及其多克隆抗体的制备  

Prokaryotic expression of Clostridium perfringens plc gene and preparation of the polyclonal antibody against the expressed protein

作　　者：郑晓星[1] 解真真[1] 任晓峰[1] 王衡[1] 王勇[1] 杨静红[1] 任玉东[2] 马德星[1] 李广兴[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]东北农业大学工程学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2012年第10期1048-1052,共5页Chinese Veterinary Science

基　　金：黑龙江省博士后基金项目(LBH-Z05025);黑龙江省自然科学基金项目(ZJN0702-01);国家自然科学基金项目(31172295);兽医生物技术国家重点实验室开放基金项目(SKLVBF201207)

摘　　要：采用PCR方法扩增编码产气荚膜梭菌α毒素去除信号肽的plc基因片段,利用原核表达载体pGEX-6P-1构建重组质粒pGEX-plc,经双酶切和测序鉴定正确后转化E.coli BL21(DE3),进行IPTG诱导表达,表达产物经切胶纯化后作为免疫原制备多克隆抗体,应用间接ELISA和Western-blot方法对所得抗体进行检测。结果显示,经1.0mmol/L IPTG诱导4～5h后重组蛋白表达量最高,并且主要以包涵体的形式存在,分子质量为66ku。用间接ELISA方法检测的多抗血清效价达到1∶65 536。Western-blot结果证实,重组蛋白与制备的多克隆抗体发生特异性结合,具有良好的反应原性,而且多抗血清能够检测产气荚膜梭菌分泌的天然α毒素蛋白。上述结果表明,此多克隆抗体能够应用于临床诊断。The plc gene encoding Clostridium perfringensα toxin excluding signal peptide was amplified by PCR and cloned into prokaryotic expression vector pGEX-6p-1 to construct a recombinant expression vector pGEX-plc.The pGEX-plc was transformed into E.coli BL21（DE3）.The purified expressed protein was injected into a rabbit and the polyclonal antibody was then prepared,which were identified by the indirect ELISA and Western-blot analysis.It was found that the highest level expression of the recombinant protein was induced by IPTG at 1.0 mmol/L from 4 to 5 h,and the expressed product was mainly in inclusion form with molecular weight of 66 ku as certified by SDS-PAGE.The ELISA titer of the polyclonal antibody was approximately 1∶65 536.Western-blot analysis revealed that the antiserum against pGEX-plc had a specific affinity for the expressed protein.Furthermore,naturally secreted C.perfringensα toxin could also be detected by the polyclonal antibody.

关 键 词：产气荚膜梭菌 plc基因 多克隆抗体 蛋白质印迹 

分 类 号：S852.616.3[农业科学—基础兽医学]