不同包被抗原检测PRRS抗体间接ELISA方法的建立

Establishment of the indirect ELISA method for detecting PRRS antibody using different antigens

为了建立更加敏感、特异、准确、有效地检测抗猪繁殖与呼吸综合征病毒（PRRSV）抗体的间接ELISA方法，本研究利用RT—PCR方法分别扩增出PRRSVVR2332毒株的GP4和N蛋白完整编码基因，将2种基因片段分别和共同插入原核表达载体pET-32a中，成功构建重组质粒pET32a—GP4、pET-32a-N和pET-32a—GP4-N。将3种重组质粒分别转化至大肠杆菌BL21（DE3）中诱导表达出GP4蛋白、N蛋白和GP4N融合蛋白，并对表达出的蛋白进行纯化。将3种已纯化的重组蛋白分别作为间接ELISA的包被抗原，通过不断优化反应条件，最终建立了检测抗PRRSV抗体的间接ELISA方法。分别使用该3种方法检测从河南省多个地区收集的200份猪血清样品中的抗PRRSV抗体，并与商品化试剂盒进行比较，结果显示，GP4蛋白做抗原时检测阳性符合率为82．7％，N蛋白做抗原时检测阳性符合率为87．2％，GP4-N融合蛋白做抗原时检测阳性符合率为85．5％；因此，N蛋白是间接ELISA方法检测抗PRRSV抗体的优势包被抗原。

To establish more sensitive, specific, accurate and efficient indirect ELISA method for de tecting anti PRRSV antibody, complete coding genes of GP4 and N protein of PRRSV VR2332 strain were amplified using RT PCR method and the two gene fragments were inserted respectively or co inserted into the prokaryotic expression vector pET-32a（＋） to yield recombinant plasmids pET-32a-GP4, pET-32a-N and pET-32a GP4-N. The three recombinant plasmids were respectively used to transform the Escherichia coli BL21/DE3 cells, and induced to express the GP4, N protein and GP4-N fusion protein. The three recombinant proteins were purified and used as the coating antigens for the indirect ELISA. Finally,three indirect ELISA methods were established for detecting anti-PRRSV antibody, three indirect ELISA methods and commercialized kit were used respectively to detect the anti-PRRSV antibody of 200 pig serum samples collected from different re gions of Henan province. The results indicted that the coincidence rates of GP4, N protein and GP4 N fusion protein were 82.7%,87.2% and 85.5%,compared with the commercialized kit, respectively. So,the N protein was the best coating antigen of indirect ELISA method for detecting anti-PRRSV antibody.