狂犬病病毒基质蛋白的原核表达及其间接ELISA方法的建立

摘要

目的 以原核表达的狂犬病病毒M蛋白作为检测抗原,建立间接ELISA方法用来检测狂犬病病毒抗体.方法 为表达狂犬病病毒(RV)基质蛋白(M),采用RT-PCR方法从狂犬病病毒Flury-LEP株中扩增RV基质蛋白基因,双酶切后定向克隆至原核表达载体pET-32a(+),构建重组质粒pET-M.将重组质粒pET-M转化至宿主菌E.coli Rosetta中进行表达.SDS-PAGE和Western blot分析确定蛋白表达量和特异性.用纯化的重组M蛋白作为包被抗原建立检测犬RV抗体的间接ELISA方法,通过优化反应条件,确定抗原最佳包被量、血清的最佳稀释度、Protein A-HRP的最佳稀释度.结果 经PCR、双酶切及测序鉴定,重组质粒pET-M构建成功,将重组质粒进行转化后诱导表达,经SDS-PAGE电泳分析得到高效表达的M蛋白,重组蛋白可被RV阳性血清特异性识别,表明基质蛋白具有良好的反应原性.用表达的狂犬病病毒M蛋白建立了检测RV抗体的间接ELISA方法,用该间接ELISA方法与以狂犬病病毒作为诊断抗原的商品化ELISA试剂盒分别对93份临床血清样品进行检测,结果两者的符合率为89.2%.结论 用原核表达的重组M蛋白作为包被抗原建立的间接ELISA方法可用做检测犬RV抗体水平的参考.%To evaluate the effectiveness of rabies vaccination, we developed the indirect-KLISA method to detect the antibodies against rabies virus (RV) using the prokaryotic expression of matrix protein as antigen. M gene of RV LEP-Flury strain was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The product was purified and cloned into the prokaryotic expression vector pET-32a (+ ) to obtain the cloning expressed plasmid pET-M, double digestion, and sequence analysis were carried afterwards. The recombinant plasmid was identified by PCR, double enzyme digestion and sequence analysis. They were transformed into Rosetta competent cells for expression. SDS-PAGE was performed to analyze the M protein expression production. Results showed that M gene was highly expressed in E. coli, and the molecular weight of the protein was 42 ku. Western-blotting analysis showed that the recombinant protein was recognized specifically by positive serum of RV. Based on the purified RV M protein, an indirect ELISA method for the detection of the RV antibodies was established. The optimal concentration of RV M coating the ELISA plate was 6μg/mL. The optimal concentration of serum samples and SPA-HRP was 1 : 200 and 1 : 2 000 respectively. Compared with commercially available ELISA kit, the coincidence rate of indirect ELISA was 89. 2%. Our results show that the developed indirect ELISA based on the RV M is useful for the detection of the RV antibody in clinical samples.