禽呼肠孤病毒σC重组蛋白的表达纯化与免疫原性分析  

Expression,purification and immunogenicity evaluation of the σC gene of avian reovirus

作　　者：王孟月 吴芃 程艺 田辉 张素玲 王彦伟 李向东 逄文强 田克恭 WANG Meng-yue;WU Peng;CHENG Yi;TIAN Hui;ZHANG Su-ling;WANG Yan-wei;LI Xiang-dong;PANG Wen-qiang;TIAN Ke-gong(National Research Center For Veterinary Medicine,Luoyang,Henan 471000,China)

机构地区：[1]国家兽用药品工程技术研究中心,河南洛阳471000

出　　处：《中国兽医学报》2020年第9期1720-1725,共6页Chinese Journal of Veterinary Science

基　　金：郑洛新国家自主创新示范区创新引领型产业集群专项资助项目(181200211700);洛阳市河洛英才计划资助项目(田克恭主持)。

摘　　要：对禽呼肠孤病毒(avian reovirus,ARV)σC基因进行大肠杆菌密码子优化,并将优化后的σC基因及其截短片段(122/156/192~326位氨基酸)克隆至pET-28a(+)载体,分别构建pET-28a(+)-σC,pET-28a(+)-σC-122,pET-28a(+)-σC-156和pET-28a(+)-σC-192重组载体。将测序鉴定正确的阳性质粒分别转化E.Coli BL21(DE3)感受态细胞,IPTG诱导后产物经Western blot分析检测,显示σC及其截短蛋白σC-122、σC-156和σC-192在大肠杆菌中获得正确表达,相对分子质量分别为37000,25000,22000和18000,均能与ARV阳性血清发生特异性反应。本研究通过温度调控双水相萃取以及无机盐多步蛋白盐析沉淀前处理工艺,去除大肠杆菌表达重组蛋白中99%的内毒素。纯化后的σC及其截短蛋白,免疫21日龄SPF鸡,并于免后28 d进行抗体检测,结果显示截短蛋白免疫组血清抗体阳性比例仅为4/10,但重组蛋白σC免后抗体阳性比例可达到8/10,与全病毒类似,可作为ARV亚单位疫苗开发的候选蛋白。In this study,avian reovirusσC gene was optimized for Escherichia coli expression.The optimizedσC gene and its truncated fragments(122/156/192-326 amino acids)were cloned into the pET-28 a(+)vector,and these recombinant pET-28 a(+)-σC,pET-28 a(+)-σC-122,pET-28 a(+)-σC-156 and pET-28 a(+)-σC-192 expressing vectors were constructed.The recombinant plasmids were identified by sequencing analysis,and the recombinant fusion proteins were expressed in E.Coli BL21(DE3).Western blot results showed that the recombinant proteins had specific reaction against ARV positive serum,and the relative molecular quality ofσC,σC-122,σC-156 andσC-192 are 37000,25000,22000 and 18000,respectively.The temperature controlled double water phase extraction and multistep protein salting out precipitation method were used to remove of endotoxin from the protein solutions.The purified recombinant proteins were injected into 21-day-old SPF chickens,and the ARV-specific antibody titers were determined at 28 d after immunization.The results indicated that only 4/10 chickens in the truncated proteins vaccinated groups showed seroconversion.But the recombinant proteinσC induced a somewhat similar seroconversion(8/10)with the positive control group.Therefore,the recombinantσC is a candidate protein for a subunit vaccine to prevent ARV in chickens.

关 键 词：禽呼肠孤病毒 σC基因 大肠杆菌 双水相萃取 盐析沉淀 

分 类 号：S852.65[农业科学—基础兽医学]