牛传染性鼻气管炎病gB蛋白的截短表达及间接ELISA方法的建立

Development of an indirect ELISA for detection of antibodies against infectious bovine rhinotracheitis virus truncated glycoprotein B

为建立检测牛传染性鼻气管炎病毒（IBRV）的间接ELISA方法,本研究根据Gen Bank登录的IBRV全基因组序列,设计引物扩增糖蛋白B（g B）基因编码第190～524氨基酸残基（aa）的基因,构建重组质粒p ET-g B进行原核表达。SDS-PAGE结果显示目的蛋白在大肠杆菌中以包涵体形式表达,大小约为40 ku。Western blot鉴定表明目的蛋白具有良好的反应原性。以纯化的g B蛋白作为包被抗原建立了间接ELISA检测方法,该方法敏感性好,特异性强,与其他病原血清无交叉反应,其组内组间变异系数均低于11%。与IDEXX公司的g B-ELISA试剂盒进行对比,阳性符合率为84.28%,阴性符合率为85.71%。采用本研究建立的方法对黑龙江省10个农场的血清样品进行了IBR的流行性调查,阳性率最高为65.52%,最低为5.56%,平均阳性率为18.01%。本研究建立的ELISA方法为进出口检疫和该病防制工作提供一种可选方法。

In this study, a pair of primers were designed to amplify the gene fragment encoding the sequence of aa190-aa524 of glycoprotein B （gB） infectious bovine rhinotracheitis virus and the amplified product was inserted into pET-30a vector to construct recombinant plasmid of pET-gB and transformed into E.coli Rosetta. The expression results showed that the target protein was expressed mainly in form of inclusion body with a MW of 40 ku, which was recognized by IBRV positive serum. In addition, an indirect ELISA was developed with the truncate gB as a coating antigen for detecting antibody against IBRV. The assay was specific to detect antibody against IBRV, but no cross reactions with positive sera to other related diseases, and the coefficient of variation was less than 11%. Furthermore, the assay were compared with the commercial detection kit of IDEXX gB-ELISA and the results showed that the positive and negative coincidence rates were 84.28% and 85.71%, respectively. While, a total of 1,377 bovine sera from different regions of Heilongjiang were detected by this assay and the average positive rate was 18.01%. These results demonstrated that the ELISA method is potential to be applicated in the import and export quarantine.