猪萨佩罗病毒VP1蛋白的原核表达及其间接ELISA方法的建立  

Prokaryotic expression of VP1 protein of porcine sapelovirus and establishment of indirect ELISA detection method

作　　者：李娜娜 陶洁[1] 李本强[1] 石迎[1] 程靖华 乔长涛[1] 刘惠莉 LI Na-na;TAO Jie;LI Ben-qiang;SHI Ying;CHENG Jing-hua;QIAO Chang-tao;LIU Hui-li(Institute of Animal Science and Veterinary Medicine,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China;Shanghai Normal University,Shanghai 200234,China)

机构地区：[1]上海市农业科学院畜牧兽医研究所,上海201106 [2]上海师范大学,上海200234

出　　处：《中国兽医科学》2021年第4期435-440,共6页Chinese Veterinary Science

基　　金：上海市科技兴农重点攻关项目[沪农科创字(2019)第3-2号];国家“十三五”重点研发项目(2018YFD0500102)。

摘　　要：为建立猪萨佩罗病毒(PSV)的快速检测方法,本试验以重组VP1蛋白为包被抗原建立了检测PSV Ig G的间接ELISA方法。RT-PCR从PSV分离株SHCM2019中扩增VP1基因,将其克隆到原核表达载体p ET28a,对重组质粒pET28a-VP1进行原核表达,经Western-blotting检测后,利用VP1重组蛋白建立检测猪血清中PSV Ig G抗体的间接ELISA方法。结果显示,表达的VP1蛋白分子质量约为40 ku,与预期大小相符,Western-blotting表明,重组蛋白与PSV抗体阳性血清具有良好反应原性。以VP1蛋白为抗原建立的ELISA方法与猪流行性腹泻病毒(PEDV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)及牛病毒性腹泻病毒(BVDV)的阳性血清均无交叉反应,特异性好;批内和批间的变异系数均小于10%,重复性好。利用建立的间接ELISA方法对95份猪临床血清样本进行检测,阳性率为44.21%。本研究建立的检测方法为PSV的血清学诊断提供了可靠手段。In order to establish a rapid detection method for porcine sapelovirus(PSV),the indirect ELISA method was established for detection of IgG antibody against PSV by using the recombinant VP1 protein as coating antigen.The VP1 segment amplified from the SHCM2019 isolate by RT-PCR,was cloned into vector pE T28 a to perform prokaryotic expression.The indirect ELISA method was established for detection of IgG antibody in porcine serum by using the recombinant VP1 protein detected Western-blotting.The results showed that the expressed VP1 protein was about 40 ku,which was consistent with the expected size.Western-blotting test indicated that the recombinant protein had good reactivity with the anti-PSV serum.The ELISA method had no cross reaction with PEDV,PRV,PCV2,CSFV and BVDV positive serums,which showed its strong specificity.The coefficient of variation of the intra-assay and inter-assay were less than 10%,with good repeatability.The established indirect ELISA method was used to detect 95 clinical porcine serums,and the positive rate was 44.21%.The detection method established in this study provides a reliable means for serological diagnosis of PSV.

关 键 词：猪萨佩罗病毒 VP1蛋白 原核表达 间接ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]