ASFV多表位融合基因MeP72基因的克隆、表达及其间接ELISA检测方法建立  

Expression of multi epitope fusion antigen gene MeP72 of ASFV in Pichia pastoris and establishment of an indirect ELISA assay

作　　者：郭晶 孟庆玲[1] 乔军[1] 李重阳[1] 伍晔晖 王立霞[1] 马帅 才学鹏[2] Jing Guo;Qingling Meng;Jun Qiao;Chongyang Li;Yehui Wu;Lixia Wang;Shuai Ma;Xuepeng Cai(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《生物技术》2018年第6期548-553,共6页Biotechnology

基　　金：国家“十三·五”重点研发计划项目子课题(“基于复合多表位融合抗原的非洲猪瘟病毒间接ELISA抗体检测试剂盒的研制”,No.2017YFD0502300);兵团中青年科技创新领军人才计划(“动物传染病诊断与防控技术”,No.2016BC001)

摘　　要：[目的]建立以重组多表位融合蛋白reMeP72为包被抗原的ASFV间接ELISA检测方法。[方法]预测分析ASFV结构蛋白P72的优势抗原表位,串连优势抗原表位并优化稀有密码子以合成多表位融合抗原基因MeP72。在毕赤酵母表达系统中诱导表达,分析重组融合蛋白的反应原性。用reMeP72做包被抗原,建立ASFV的ELISA间接检测方法。[结果]成功构建了402bp的MeP72基因片段。SDS-PAGE和WesternBlotting分析该蛋白在14kDa有条带,证明其反应原性良好。经ELISA反应条件优化,建立的ELISA方法与猪的其他病原的阳性血清不发生交叉反应,且可检测到稀释倍数为1∶512ASFV阳性血清,证明其特异性和敏感性良好。[结论]建立了抗原包被浓度为15μg/mL,一抗、二抗稀释倍数分别为1∶200、1∶5000倍的特异性和敏感性良好的reMeP72-ELISA检测方法。[Objective]The ASFV indirect ELISA detection method was established for recombinant multi epitope fusion protein reMeP72 as inclusion antigen.[Methods]The antigenic epitopes of ASFV structural protein P72 were predicted and analyzed.Then,the fusion antigen gene MeP72 was constructed according to the dominant antigen epitope and expressed in Pichia pastors expression system.ASFV indirect ELISA detection method was established that reMeP72 be used to coated antibody.[Results]MeP72 gene fragment for 402 bp was successfully constructed.It was showed has a band at 14 kDa by SDS-PAGE test and Western Blotting with ASFV positive serum,which proved the good reactivity of reMeP72.ELISA reaction conditions were optimized,and the established ELISA method did not cross react with other positive serums of pigs,and the positive serum of ASFV still could be detected after diluted of 1∶512 times,which prove the specificity and sensitivity of the method were good.[Conclusion]A specific and sensitive reMeP72-ELISA method was developed for the detection of antigen-coated antibodies with a concentration of 15 μg /mL,Dilution times of primary and secondary antibodies being 1∶200 and 1∶5 000,respectively.

关 键 词：非洲猪瘟病毒 MeP72 毕赤酵母表达 间接ELISA检测方法 

分 类 号：Q784[生物学—分子生物学]