狂犬病病毒N蛋白主要抗原区的表达及SPA-ELISA抗体检测方法的建立

Development of SPA-ELISA for detection of antibodies against rabies virus based on expression of main antigenic determinant of Nucleoprotein

摘要:

为有效监测免疫狂犬病疫苗后的抗体水平，以狂犬病病毒N蛋白的主要抗原表位所在区域N1蛋白为包被抗原，建立了SPA-ELISA抗体检测方法。用RT-PCR方法分别扩增出狂犬病病毒Flury LEP株N基因及其N1区域 (1 000~1 353 bp)，将二者分别克隆至原核表达载体pGEX-6P-1中，并将重组的表达载体分别转化至大肠杆菌BL21(DE3) 中，经1 mmol/L IPTG诱导后进行免疫印迹分析。结果表明，重组的RV N和RV N1均以包涵体形式表达。与表达全长RV N相比，RV N1的表达量显著高于前者，且该重组蛋白同样具有良好的反应原性。用纯化的重组RV N1作为诊断抗原建立了检测犬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是2 mg/L，血清的最佳稀释度为1∶100，Protein A-HRP的稀释度为1∶4 000。用建立的ELISA方法对102份临床血清样品进行检测，与商品化试剂盒相比，二者的符合率为94.1％。试验结果表明基于主要抗原表位所在区域N1蛋白的SPA-ELISA方法可有效用于犬RV抗体水平的检测，为检测RV免疫抗体的ELISA试剂盒的研制奠定了基础。

Abstract:

To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000?1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-β-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.