空肠弯曲菌PEB1A蛋白的原核表达及间接ELISA方法的建立

Prokaryotic Expression of PEB1A Protein of Campylobacter jejuni and Establishment of an Indirect ELISA

为了建立检测血清中空肠弯曲菌(Campylobacter jejuni)抗体的间接ELISA方法,试验通过PCR扩增并克隆空肠弯曲菌PEB1A基因,构建原核表达载体pET32a-PEB1A,将该表达载体转化大肠杆菌BL21(DE3)感受态细胞,诱导表达获得约47ku的可溶性目的蛋白,通过Western blotting证明所表达的重组蛋白具有良好的生物学活性。以纯化后的重组蛋白作为包被抗原,建立空肠弯曲菌抗体间接ELISA方法,对其特异性、敏感性、重复性进行检测。结果表明,本试验建立的空肠弯曲菌抗体间接ELISA检测方法临界值为0.3424,本方法仅与空肠弯曲菌阳性血清发生特异性反应,与其他抗血清无交叉反应,具有较强的特异性,批内、批间的重复性试验变异系数均<5%,具有较好的重复性和稳定性。该方法的建立可应用于血清中空肠弯曲菌抗体的快速检测,为进一步防制空肠弯曲菌腹泻提供依据。

In order to develop an indirect ELISA method to detect Campylobacter jejuni antibody,PEB1A gene of Campylobacter jejuni was cloned and amplified by PCR,the prokaryotic expression vector pET32a-PEBIA was constructed,and then transferred into the expression strain E.coli BL21(DE3),and obtained about47ku of soluble protein.Western blotting result showed that the expressed recombinant protein had good biological activity,an indirect ELISA method for detecting antibody against Campylobacter jejuni was developed using expressed PEB1A protein as coating antigen,and detected its specificity,sensitivity,repeatability,respectively.The results showed that the established method for detection of Campylobacter jejuni antibody critical value was0.3424.This method only specifically reacts with Campylobacter jejuni positive sera,and had no cross-reactivity with other antiserum and strong specificity.In addition,the coefficients of variations in both inter-and intra-assay were less than5%indicating that it had good repeatability and stability.The establishment of this method could be applied to the rapid detection of Campylobacter jejuni in serum,and provided basis for further prevention and control of Campylobacter jejuni diarrhea.