猪繁殖与呼吸综合征病毒重组N蛋白间接ELISA检测方法的建立

摘要

The constructed recombinant plasmids (pGEX-6P-N) were expressed in E. coli BL21 and offering optimum condition for inducement. The expression protein was purified through Glutathione Sepharose 4B affinity adsorption columm The purified recombinant protein were used as coating antigen to determine the optimal coating conditions (such as coating antigen quantity,the dilution of serum sampie,the working concentration of HRP labeled goat antibody pig IgG, substrate response time and so on)and relative standard of measurement, an indirect ELISA method for detecting PRRSV antibodies were established , by use of which 360 serum samples were detected by the method and market ELISA kit (IDEXX) respcctively. Compared with the market ELISA kit,the repeatability between the same and different batch were 1. 52％ and 6. 17％ respectively, and the coincident rate between the two methods reached 92. 5％. The results showed that the developed indirect ELISA method had the advantages such as high repeatability and strong specificity and the method can be used to detect the serum antibodies of PRRSV.%在最佳条件下,通过对重组质粒pGEX-6P-N进行诱导表达,并利用Glutathione Sepharose 4B亲和吸附柱对表达产物(重组N蛋白)进行纯化.用重组N蛋白作为包被抗原,通过对相关条件进行优化(如抗原包被量,血清稀释度,酶标二抗最佳工作浓度,底物作用时间等),确定判定标准,最终初步建立了检测PRRSV抗体的间接ELISA方法.用建立的间接ELISA方法检测360份血清样品,并与IDEXX公司研制的ELISA试剂盒检测的结果相比较,批内重复性试验变异系数均值为1.52%,批间变异系数为6.17%,符合率达92.5%,试验表明,建立的间接ELISA检测方法具有良好的重复性和特异性,可用于PRRSV血清抗体的检测.