非洲猪瘟EP364R蛋白的原核表达及间接ELISA方法的建立

Expression of EP364R Protein of African Swine Fever Virus in Prokaryotic Cells and Development of an Indirect ELISA Detection Method

本研究根据非洲猪瘟病毒(ASFV)Georgia 2007/1毒株的EP364R基因序列(GenBank登录号:FR682468.1)合成EP364R基因序列,通过PCR技术扩增目的基因EP364R,并克隆到pET-32a(+)载体,成功构建了重组质粒pET-32a-EP364R,鉴定正确后将其转化至大肠杆菌BL21(DE3)感受态细胞中成功诱导表达,纯化后的重组蛋白利用SDS-PAGE、Western blot进行鉴定。以纯化的pET-32a-EP364R重组蛋白为诊断抗原,成功建立了检测ASFV的间接ELISA抗体检测方法。将建立的间接ELISA检测方法与法国ID-Vet非洲猪瘟抗体试剂盒分别对临床84份血清进行检测,总符合率达89.3%,表明该方法具有良好的特异性、敏感性和重复性。为ASFV的快速诊断、流行病学调查和血清学抗体检测提供了快速实用的检测手段。

The EP364R gene sequence of African Swine Fever Virus(ASFV)was synthesized according to the Georgia 2007/1 strain(GenBank accession number:FR682468.1)and amplified by PCR for insertion into pET-32a(+)vector.The resulting recombinant plasmid pET-32a-EP364R was verified and transformed into E.coli BL21(DE3)competent cells for protein expression.The purified recombinant protein was demonstrated in SDS-PAGE and Western blot.The purified recombinant protein was used as the coating antigen to develop an indirect ELISA method for detection of ASFV.The indirect ELISA method developed here was used to detect 84 clinical serum samples together with French ID-Vet African swine fever antibody kit.The agreement of these two methods reached to 89.3%,indicating that our indirect ELISA method had good specificity,sensitivity and repeatability.The availability of this new indirect ELISA method provided a rapid and practical tool for rapid diagnosis,epidemiological investigation and serological antibody detection of ASFV.