摘要
依据F种肠道病毒(enterovirus species F,EV-F)SD-S67毒株的基因组序列设计合成引物,RT-PCR扩增出该毒株的VP1基因,并将其克隆到原核表达载体pGEX-4T-1的BamHⅠ/EcoRⅠ位点,表达出VP1重组蛋白。以纯化VP1重组蛋白为免疫原,制备兔源多克隆抗体,并以制备的抗体建立了检测F种肠道病毒的双抗体夹心ELISA方法,同时对山东和河南省部分地区牛群感染EV-F进行了初步的流行病学调查。结果显示,捕获抗体的最佳包被量为400 ng/孔,HRP酶标抗体的最佳稀释倍数为1∶400;确定的方法判断标准为样品的D_(490)>0.217时判定为阳性。建立的ELISA方法可以特异性检测出EV-F病毒抗原,而EV-E和BVDV病毒抗原检测结果均为阴性,表明方法具有较好的特异性。以建立的ELISA方法检测不同稀释倍数的阳性样品,结果稀释1000倍后的样品检测结果仍为阳性,表明建立的方法具有较高的敏感性。应用建立的方法检测部分样品,组内变异系数为1.05%~9.24%,组间变异系数为1.39%~6.78%,说明该方法具有良好的重复性。同时部分样品的ELISA和PCR方法的检测结果显示,两者的符合率为100%。以该方法对山东、河南两地牛群样品进行检测,结果显示牛群的EV-F的感染率分别为12%和8%。结果表明,建立的F种肠道病毒双抗体夹心ELISA方法具有较高的特异性、敏感性,且快速简便,可用于F种肠道病毒感染的诊断与流行病学调查。
According to the genomic sequence of the SD-S67 strain(EV-F),primers were designed,synthesized,and used to amplify the VP1 gene using RT-PCR.The amplified fragment was cloned into BamHⅠ/EcoRⅠsites of prokaryotic expression vector pGEX-4 T-1-VP1.Recombinant VP1 protein was expressed,purified and used to prepare the VP1 polyclonal antibody by administering the rabbit with the purified VP1 recombinant protein emulsified by Freund’s complete adjuvant as immunogen.A double antibody sandwich ELISA method was established to detect virus antigen of EV-F with the prepared antibody.The results showed that the coating amount of capture antibody was 400 ng/well,and the dilution of antibody labeled with horseradish peroxidase(HRP)was 1∶400.The criteria was determined and the sample was tested as positive when its D_(490)>0.217.Specificity assay showed that only antigen of enterovirus species F could be detected,while antigen of BVDV and BEV were negative.Sensitivity assay showed that the positive fecal samples were still detected as positive even they were diluted 1000 times.Meanwhile the intra-group coefficient of variation was 1.05%-9.24%,and the inter-group coefficient of variation was 1.39%-6.78%,indicating that the method has good repeatability.Furthermore,comparison detection of 20 samples by ELISA and RT-PCR demonstrated that two methods share 100%coincidence rate,indicating that the established ELISA method had a high sensitivity.Primary investigation on EV-F infection among cattle herds from Shandong and Henan provinces was performed using the established ELISA,the results showed that the EV-F infection rate in cattle was 12%and 8%,respectively.In summary,the above results showed that the established double antibody sandwich ELISA has high specificity and sensitivity,it is rapid and simple and can be used for the diagnosis and epidemiological investigation on EV-F infection.
作者
王浴光
常晓冉
胡俊英
刘叙
蔡梦露
章凡
胡卉琪
王新平
WANG Yuguang;CHANG Xiaoran;HU Junying;LIU Xu;CAI Menglu;ZHANG Fan;HU Huiqi;WANG Xinping(College of Veterinary Medicine,Jilin University,Changchun 130062,China;Ministry of Education Key Laboratory for Zoonosis Research,Jilin University,Changchun 130062,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2022年第1期89-94,共6页
Chinese Journal of Veterinary Science
基金
国家“十三五”规划重点研发计划资助项目(2016YFD0500904,2017YFD0500104)。
