猪繁殖与呼吸综合征病毒重组N蛋白原核表达及间接ELISA方法的建立

Prokaryotic Expression of Recombinant PRRSV N Protein and Establishment of Indirect ELISA Method

为制备猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)N蛋白多克隆抗体,建立检测PRRSV N蛋白抗体的间接ELISA方法,通过PCR扩增N蛋白基因,将N蛋白基因克隆至原核表达载体pET-32a(+),构建重组质粒pET-32a-N。将重组质粒转化至BL21(DE3)感受态细胞中,通过IPTG诱导N蛋白表达,将纯化后的重组N蛋白免疫小鼠,获得N蛋白多克隆抗体。将纯化后的N蛋白作为抗原,建立检测PRRSV N蛋白抗体的间接ELISA方法,并评估该方法的特异性、重复性和准确性。结果表明,表达的重组N蛋白呈可溶性,制备的N蛋白多克隆抗体ELISA效价能达到1∶2048000,IFA和Western blot结果表明制备的鼠多克隆抗体能特异性识别PRRSV N蛋白。建立的间接ELISA方法特异性良好,重复性试验变异系数均小于10%,且与同类型商品化试剂盒结果比较,符合率达到91.10%,可为PRRSV N蛋白的免疫学检测和结构功能的研究提供参考。

The study was aimed to prepare polyclonal antibody against PRRSV N protein and develop indirect ELISA to detect antibody against PRRSV N protein.The gene encoding PRRSV N protein was amplified and cloned into pET-32a(+) prokaryotic expression vector,resulting in the pET-32a-N recombinant plasmid.The recombinant plasmid was deliveried into BL21(DE3) cells.The expression of N protein was induced by IPTG and then purified.The polyclonal antibodies were obtained by immunizing mice with recombinant N protein.The indirect ELISA method for detecting antibodies to PRRSV N protein was developed and the specificity,reproducibility and accuracy of the method were also evaluated by using purified N protein as antigen.The results showed that the expressed recombinant N protein was soluble and the polyclonal antibody potency of 1∶2 048 000 was measured by indirect ELISA.Furthermore,both IFA and Western blot results showed that the prepared murine polyclonal antibody could specifically recognize PRRSV N protein.The indirect ELISA method based on the expressed N protein showed good specificity,the coefficient of variation of the reproducibility test was less than 10%,and the compliance rate with the results of the same type of commercial kits reached 91.10%.The results of the study provide an experimental basis for the immunological detection of PRRSV N proteins and further studies of their structure and function.