CHO细胞表达牛病毒性腹泻病毒E2-E^(rns)融合蛋白及免疫原性分析  

Bovine viral diarrhea virus E2-E^(rns)fusion protein expressed in CHO cells and its immunogenicity analysis

作　　者：李亚军 郝荣增 茹毅 龚真莉[1] 刘艳红[1] 郑海学[1] LI Ya-jun;HAO Rong-zeng;RU Yi;GONG Zhen-li;LIU Yan-hong;ZHENG Hai-xue(State Key Laboratory for Animal Disease Control and Prevention/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2023年第10期1207-1215,共9页Chinese Veterinary Science

基　　金：中国农业科学院兰州兽医研究所所级基本科研业务费(110231160042036,1610312021013,1610312022009);甘肃省自然科学基金项目(22JR5RA030);“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目(2022SDZG02)。

摘　　要：本研究旨在利用CHO细胞表达系统制备牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)E2-E^(rns)融合蛋白,并分析其免疫原性。以BVDV-1 NADL株基因序列为基础,构建融合表达BVDV E2和E^(rns)蛋白的真核重组表达质粒pcDNA3.1-BVDV-E2-E^(rns),转染CHO细胞,进行上清分泌表达;离心收集细胞培养上清进行亲和层析纯化,通过SDS-PAGE电泳和Western-blot鉴定纯化蛋白的免疫反应性;使用纯化后的融合蛋白免疫新西兰大白兔,通过细胞免疫荧光(IFA)试验和间接ELISA试验鉴定E2-E^(rns)融合蛋白免疫家兔血清的抗体反应性和抗体效价。结果,通过CHO细胞表达系统,成功制备了纯化的BVDV E2-E^(rns)融合蛋白,Western-blot鉴定结果显示,His标签单克隆抗体和BVDV阳性血清均能够与纯化的融合蛋白发生特异性免疫反应。间接ELISA和IFA试验结果显示,一免后第7天血清抗体呈阳性,并持续至免疫后第28天,血清抗体效价水平可达1∶512000,且该血清抗体可以与MDBK细胞中感染的BVDV发生特异性免疫反应。上述结果表明,本研究成功利用CHO细胞表达系统制备并纯化了BVDV E2-E^(rns)融合蛋白,并通过体内和体外试验证明该融合蛋白具有良好的免疫原性,为BVD的诊断方法及其新型亚单位疫苗研制奠定了基础。This study was to produce E2-E^(rns)fusion protein of bovine viral diarrhea virus(BVDV)by using suspension CHO cells expression system and to analyze its immunogenicity.In this study,the recombinant eukaryotic expression plasmid pc DNA3.1-BVDV-E2-E^(rns)was constructed based on the gene sequence of BVDV-1 NADL strain.The E2-E^(rns)fusion protein was secreted and expressed in cells supernatant after transfect the recombinant expression plasmid pc DNA3.1-BVDV-E2-E^(rns)into CHO cells.The supernatant of cell culture was collected by centrifugation and purified by affinity chromatography.The immunoreactivity of the purified protein was determined by SDS-PAGE electrophoresis and Western-blot.The purified fusion protein was used to immunize New Zealand white rabbits.The antibody reactivity and antibody titer of rabbit serum immunized with E2-E^(rns)fusion protein were determined by immunofluorescence(IFA)and indirect ELISA assay.In result,the purified BVDV E2-E^(rns)fusion protein was successfully prepared by suspension CHO cell expression system.Western-blot identification results showed that both His labeled monoclonal antibody and BVDV positive serum could react with the purified fusion protein.The results of indirect ELISA and IFA showed that serum antibodies could be detected in the 7th day after prime immunization,and the antibody level was maintained at a high titer until the 28th day after immunization.The antibody titer was 1∶512000,and the serum antibody could have a specific immune reaction with the BVDV in infected MDBK cells.In conclusion,the purified BVDV E2-E^(rns)fusion protein was successfully prepared and purified by the suspended CHO cell expression system,and the recombinant protein have a good immunogenicity was texted,which provided a research data for the development of BVD diagnosis and novel subunit vaccine.

关 键 词：牛病毒行腹泻病毒 E2-E^(rns)融合蛋白 真核表达 CHO培养 免疫原性 

分 类 号：S852.653[农业科学—基础兽医学]