猪传染性胃肠炎病毒S基因B、C位点的克隆与表达及间接ELISA方法的建立  

Cloning and Expression of the SBC Gene of Transmissible gastroenteritis virus and Development of an Indirect TGEV SBC-ELISA

作　　者：尹杰[1,2] 杨恒[2] 曹三杰[2] 黄小波[2] 文心田[2] 李洁萍 余正强 周军 

机构地区：[1]四川省遂宁市畜牧食品局,四川遂宁629000 [2]四川农业大学动物医学院动物传染病与基因芯片实验室/动物疫病与人类健康四川省重点实验室,四川雅安625014 [3]四川省遂宁市船山区畜牧食品局,四川遂宁629000

出　　处：《中国兽医杂志》2015年第1期7-10,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(31072144,30901084);国家公益性行业(农业)科研专项(201203056)

摘　　要：针对猪传染性胃肠炎病毒(TGEV)S基因B、C抗原位点编码基因片段(SBC,下同)设计合成1对引物,用PCR方法扩增SBC基因,将该基因片段定向插入到原核表达载体p ET-32a(+)中,构建原核表达载体p ET-32a-SBC。阳性质粒转化宿主菌BL21(DE3),在IPTG的诱导下表达大小约38 k D的蛋白,重组蛋白以包涵体的形式存在。Western blot分析表明,表达产物具有良好的免疫学活性。以纯化的表达产物作为诊断抗原建立了检测TGEV抗体的间接ELISA(TGEV SBC-ELISA)方法。表明建立的间接ELISA方法特异性和重复性良好,敏感性比血清中和试验高,可用于TGEV的检疫和流行病学调查。The SBC gene of TGEV was amplified with PCR method. The PCR product was cloned into pET-32a （＋）to get a prokaryotic recombinant plasmid pET-32a-SBC. The target gene was successfully expressed in the cell BL21 （DE3） After induction by 1.0 mol/L IPTG, a recombinant protein about 38 kD in size were expressed Western blot analysis showed that the recombinant protein could react with TGEV positive serum. An indirect ELISA （TGEV SBC-ELISA）was developed using purified recombinant SBC from the inclusion bodies as described previously, which had excellent specificity to TGEV. These results suggested that the developed ELISA had good stability and specificity. Compared with serum neutralization test, the indirect ELISA was more sensitive. So this assay can be used as a tool for diagnosis and quarantine of TGEV.

关 键 词：猪传染性胃肠炎病毒 克隆与表达 诊断 

分 类 号：S858.28[农业科学—临床兽医学]