空肠弯曲菌AhpC蛋白的克隆表达及抗原性分析

目的构建空肠弯曲菌烷基过氧化氢还原酶（ahpC）基因的原核表达系统,克隆表达AhpC蛋白,用Western blot以及ELISA方法检测表达蛋白的抗原性,为筛选空肠弯曲菌感染后特异性抗体检测试剂的制备奠定基础。方法用PCR方法从中国分离菌株ICDCCJ07001的染色体DNA中扩增出ahpC基因,将目的基因插入表达载体pGEX-4T-1后转化入大肠杆菌E.coliBL21（DE3）,IPTG诱导重组质粒pGEX-ahpC的表达。SDS-PAGE分析表达产物,采用GST标签亲和层析柱纯化表达蛋白。应用兔免疫血清、临床感染者血清以及健康无感染者血清,利用Western blot以及ELISA的方法分析AhpC蛋白的抗原性并初步评价其在ELISA检测方法中的应用。结果 PCR扩增及双酶切鉴定证实重组质粒构建成功。重组表达蛋白经飞行质谱鉴定为空肠弯曲菌AhpC蛋白,Western blot及ELISA检测结果显示AhpC具有特异抗原性。结论成功构建了ahpC重组表达载体pGEX-4T-1/ahpC,并在大肠杆菌中高效表达。空肠弯曲菌AhpC蛋白具有抗原性,可以作为空肠弯曲菌感染后血清抗体检测的特异抗原。

Cloning,expression,purification and antigenicity analysis of AhpC in Campylobacter jejuni

To clone and express the AhpC protein of Campylobacter jejuni in E.coli and characterize its antigenicity and specificity.The ahpc gene was amplified by PCR from C.jejeuni strain ICDCCJ07001,and cloned into pGEX-4T-1.The recombinant PGEX-4T-1-ahpC was transformed to E.coli BL21（DE3） for protein expression.The expression product was analyzed and identified by SDS-PAGE and MALDI TOF-TOF 4700 Proteomics Analyzer.The expressed protein was identified as Campylobacter jejuni AhpC protein by Proteomics Analyzer.Both Western blot and ELISA showed that AhpC had specific antigenicity for Campylobacter jejuni.The expression and purification of recombinant AhpC antigen with good antigenicity and specificity will facilitate the research and clinical application in the diagnosis of C.jejuni infection.