牛源无乳链球菌sip蛋白抗原优势区的原核表达及其间接ELISA检测方法的建立

Prokaryotic expression of sip gene of bovine Streptococcus agalactiae and establishment of indirect ELISA

为测定牛源无乳链球菌表面免疫相关蛋白(sip)的抗原性,本实验通过建立间接ELISA方法,对该蛋白的抗原性进行分析及鉴定。根据无乳链球菌sip基因序列,设计一对引物,以临床分离菌株基因组DNA为模板,PCR扩增sip抗原表位优势区编码的基因序列,并将其与原核表达载体pET-30a(+)连接,构建重组质粒pET-sip,转化宿主菌BL21中,经IPTG诱导,获得重组蛋白,western blot分析表明,重组蛋白具有良好的抗原性。以纯化的表达产物作为包被抗原,建立检测无乳链球菌抗体的间接ELISA方法,确定抗原最佳包被浓度为3.125μg/mL,血清最佳稀释度为1∶160,酶标二抗最适稀释度为1∶4 000。应用该方法对无乳链球菌、化脓性链球菌、大肠杆菌和金黄色葡萄球菌兔阳性血清进行检测,结果表明,该方法具有良好的特异性和灵敏度。

To express the sip gene of bovine Streptococcus agalactiae and establish indirect ELISA assay for antibody detection, the antigenic epitope sequence of sip gene was amplified by PCR from clinical isolated strains using a pair of primers derived from the published sequence. The sip gene sequence was cloned into prokaryotic expression vector pET-30a （＋） for expression in bacteria BL21 and its immunogenicity was confirmed by western blot analysis. An indirect ELISA assay was established using purified expression product as coating antigen for detection of S. agalactiae antibody again. Preliminary application in detecting rabbit anti-S, agalactiae serum, S.pyogenes serum, E. coli serum and S. aureus serum showed that this method had good specificity and sensitivity.