猪链球菌自溶素的原核表达及其抗体间接ELISA检测方法的建立

Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody

【目的】建立快速、准确检测猪链球菌自溶素(atl)抗体的间接ELISA方法。【方法】根据GenBank登录的猪链球菌atl基因序列设计1对引物,采用PCR方法从猪源链球菌中获得atl基因序列;构建pET-32a-atl和pGEX-4T-1-atl重组质粒,并将重组质粒转化大肠杆菌BL21感受态细胞中进行诱导表达;以表达纯化后的atl-His融合蛋白为免疫原,免疫新西兰大白兔,制备多克隆抗体;以兔多克隆抗体为一抗,用Western blotting方法检测atl-GST融合蛋白的反应原性;以纯化后的atl-GST融合蛋白为包被抗原,猪链球菌感染阳性血清作为标准血清,建立猪链球菌自溶素抗体的间接ELISA检测方法。利用本试验建立的ELISA方法与商用猪链球菌2型ELISA抗体检测试剂盒同时对184份血清样品进行检测并用本试验建立的方法进行临床样品检测。【结果】从猪链球菌中成功扩增出atl基因;构建的重组质粒,经诱导表达和纯化,获得大小为40 ku的atl-His和48 ku的atl-GST融合蛋白;经Western blotting验证,兔抗atl-His抗血清与atl-GST融合蛋白具有良好的反应原性;间接ELISA结果显示,该检测方法的阴阳临界值为0.318,阳性血清稀释至1∶1 280检测仍为阳性;批内批间变异系数均<10%,表明该方法具有良好的重复性及敏感性。本试验所建立的ELISA方法检测出96份阳性样品,商用猪链球菌2型ELISA抗体检测试剂盒检测出66份阳性样品,符合率为71.7%。应用建立的方法检测458份不同饲养阶段的猪血清样品,其中检出277份阳性样品,阳性率为60.4%。【结论】本试验成功建立了检测猪链球菌自溶素抗体的间接ELISA方法并进行了初步应用,为猪链球菌病血清流行病学调查奠定了基础。

【Objective】 This study was aimed to establish a rapid and accurate indirect ELISA method for detecting Streptococcus suis autolysin(atl) antibody.【Method】 A pair of primers was designed according to the Streptococcus suis atl gene sequence registered in GenBank, and the atl gene sequence was obtained from Streptococcus suis by PCR.The recombinant plasmids pET-32 a-atl and pGEX-4 T-1-atl were constructed, and then were transferred into BL21 competent cells for induction expression.The purified atl-His fusion protein was used as immunogen to immunize New Zealand White rabbits to prepare polyclonal antibody.The reactivity of atl-GST fusion protein was detected by Western blotting, using the purified atl-GST fusion protein as the coating antigen and the positive serum of Streptococcus suis infection as the standard serum, an indirect ELISA method for the detection of Streptococcus suis autolysin antibody was established.184 serum samples were detected by the ELISA method established in this test and the commercial Streptococcus suis type 2 ELISA antibody detection kit at the same time, and the clinical samples were detected by the method established in this test.【Result】 atl gene was successfully amplified from Streptococcus suis.The constructed recombinant plasmids was induced, expressed and purified to obtain 40 ku atl-His and 48 ku atl-GST fusion proteins.Western blotting result showed that rabbit anti atl-His antiserum had good reactivity with atl-GST fusion protein.The results of indirect ELISA showed that the critical value of negative serum and positive serum was 0.318,and the positive serum was still positive when diluted to 1∶1280.The intra-batch and inter-batch coefficients of variation were both less than 10%,indicating that the method had good reproducibility and sensitivity. 96 positive samples were detected by the ELISA method and 66 positive samples were detected by the commercial Streptococcus suis type 2 ELISA antibody test kit, with a compliance rate of 71.7%.A total of 458 pig serum samples from different feeding stages of some pig farms in Jiangxi were detected by the established method, of which 277 positive samples were detected, and the positive rate was 60.4%.【Conclusion】 The indirect ELISA method for detecting Streptococcus suis autolysin antibody infection was successfully established and preliminarily applied, which laid a foundation for the seroepidemiological investigation of Streptococcus suis.