鸭坦布苏病毒E蛋白的原核表达与反应原性分析  

Prokaryotic expression and reactogenicity analysis of E protein of duck Tembusu virus

作　　者：曲哲会[1] 张喜文 齐志颖 李卓燕 李盼盼 吕硕硕 张雯 赵聘[1,2] 焦凤超 赵云焕[1,3] 黄立 王丹娜[4] QU Zhehui;ZHANG Xiwen;QI Zhiying;LI Zhuoyan;LI Panpan;LYU Shuoshuo;ZHANG Wen;ZHAO Pin;JIAO Fengchao;ZHAO Yunhuan;HUANG Li;WANG Danna(College of Animal Science and Veterinary Medicine,Xinyang Agriculture and Forestry University,Xinyang 464000,China;Henan Province Engineering Technology Research Center for Waterfowl Resources Development and Utilization and Epidemic Disease Prevention and Control,Xinyang 464000,China;Xinyang City Key Laboratory of Animal Husbandry and Environmental Control,Xinyang 464100,China;Beijing Biomedical Science and Technology Center,Zhaofenghua Biotechnology(Nanjing)Co.,Ltd.,Beijing102600,China)

机构地区：[1]信阳农林学院动物科技学院,河南信阳464000 [2]河南省水禽资源开发利用与疫病防控工程技术研究中心,河南信阳464000 [3]信阳市畜禽养殖与环境控制重点实验室,河南信阳464100 [4]兆丰华生物科技(南京)有限公司北京生物医药科技中心,北京102600

出　　处：《黑龙江畜牧兽医》2022年第23期67-71,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：河南省科技攻关项目(222102110188);信阳农林学院高水平科研孵化器建设项目(FCL202004);信阳农林学院青年科研基金项目(20200113);信阳农林学院科技创新团队建设项目(XNKJTD-014);信阳市创新应用专项(20200016)。

摘　　要：为了利用原核表达系统表达鸭坦布苏病毒(duck Tembusu virus, DTMUV)E蛋白,试验利用RT-PCR方法扩增DTMUV E基因,经核苷酸序列测定后,与pET-30a(+)载体连接,然后转化至大肠杆菌DH5α感受态细胞中,构建重组表达载体pET-DTMUV-E,再转化至大肠杆菌Rosetta(DE3)感受态细胞中,经IPTG诱导表达DTMUV E蛋白,通过SDS-PAGE分析表达产物,Western-blot方法鉴定反应原性,分别从诱导时间和IPTG浓度方面对表达条件进行优化,并进行表达产物的可溶性分析。结果表明:克隆得到大小约为1 503 bp的DTMUV E基因,并成功与pET-30a(+)载体连接,获得分子质量约65 ku的融合蛋白,可以与兔抗6×His多克隆抗体和鸡抗DTMUV多克隆抗体发生特异性免疫反应,终浓度为4 mmol/L的IPTG诱导4 h为最佳表达条件,重组蛋白DTMUV E主要以包涵体形式表达。说明利用原核表达系统表达的以包涵体形式存在的DTMUV E蛋白,具有较好的反应原性。In order to use a prokaryotic expression system to express the E protein of duck Tembusu virus(DTMUV), in this experiment, the DTMUV E gene was amplified by RT-PCR, and was ligated into the pET-30 a(+) vector after nucleotide sequencing, which was transformed into E. coli DH5α competent cells to construct the recombinant expression vector pET-DTMUV-E. pET-DTMUV-E was transformed into E. coli Rosetta(DE3) competent cells, which was induced by IPTG to express DTMUV E protein. The expression products and reactogenicity were identified by SDS-PAGE and Western-blot, and the expression conditions were optimized in terms of induction time and IPTG concentration, respectively, and the solubility of the expression products was analyzed. The results showed that the DTMUV E gene of about 1 503 bp was cloned and successfully ligated into the pET-30 a(+) vector. A fusion protein with a molecular weight of about 65 ku was obtained, which could specifically immunoreact with rabbit anti-His polyclonal antibody and chicken anti-DTMUV polyclonal antibody. The final concentration of 4 mmol/L IPTG induced for 4 h was the optimal expression condition, and it was expressed mainly in the form of inclusion bodies. The results indicated that the DTMUV E protein expressed in the inclusion body form using the prokaryotic expression system had good reactogenicity.

关 键 词：鸭坦布苏病毒 E蛋白 原核表达系统 反应原性 可溶性分析 

分 类 号：S852.6511[农业科学—基础兽医学]