PRRSV E蛋白的表达及检测抗体胶体金试纸的研制  

Expression of the E protein of porcine reproductive and respiratory syndrome virus and development of colloidal gold strip for the detection of antibodies against PRRSV

作　　者：杨欢[1] 吴天成[1] 刘禹[1] 郑鑫[1] 崔焕忠[1] 

机构地区：[1]吉林农业大学动物科技学院,长春130118

出　　处：《黑龙江畜牧兽医》2014年第5期19-21,26,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：科技部农业科技成果转化资金项目(2012GB2B1001 06);吉林省世行贷款农产品质量安全项目(2011-Y28);吉林农业大学启动基金项目(201221);吉林省科技发展计划项目(20110224);吉林省留学人员科技创新创业项目(2011-15)

摘　　要：为了建立快速检测猪繁殖与呼吸综合征病毒(PRRSV)抗体的方法,试验采用RT-PCR扩增ORF5基因,克隆至原核表达载体pET-28a上,诱导重组质粒转化菌表达,SDS-PAGE分析目的蛋白表达情况,Western-blot检测表达蛋白免疫活性,亲和层析纯化表达蛋白。用SPA标记胶体金,分别以纯化E融合蛋白和羊抗猪抗体为检测线和质控线制备胶体金试纸,用所研制的试纸检测猪血清样品40份,并与IDEXX ELISA试剂盒的检测结果进行比较。结果表明:成功表达E融合蛋白,表达蛋白具有良好的反应原性,纯度为90%;制备的胶体金试纸与IDEXX ELISA试剂盒检测符合率为92%。说明该种方法可检测PRRSV抗体。To establish a method of rapid detection of antibodies against porcine reproductive and respiratory syndrome virus （PRRSV）, the ORF5 gene was amplified by RT - PCR and cloned into prokaryotic expression vector pET28a, and then the recombinant plasmid was trans- formed into Escheriehia coil BI21 （ DE3 ） and the expression was induced by IPTG. SDS - PAGE and Western - blot were used to analyze the ex- pression of the target protein and detect the immune activity of the expressed protein, respectively, and the expressed protein was purified by af- finity chromatography. The colloidal gold strip for detecting antibodies against PRRSV was developed using Staphylococcal protein A （SPA） la- beled with colloidal gold, taking the purified E fusion protein and goat anti -pig antibodies as test line and control line, respectively. 40 pig ser- um samples were tested by colloidal gold strip, and the detection results were compared with the results using IDEXX ELISA kit. The result showed that the E fusion protein was successfully expressed, and the expressed protein had good reactogenicity, with the purity of 90%. The co- incidence rate between the colloidal gold strip and IDEXX ELISA kit for the detection was 92%. The results indicate that the method with colloi- dal gold strip can be used for the detection of antibodies against PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒(PRRSV) 糖蛋白E 表达 胶体金试纸 抗体 

分 类 号：Q786[生物学—分子生物学]