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.
作者
曲哲会
张喜文
齐志颖
李卓燕
李盼盼
吕硕硕
张雯
赵聘
焦凤超
赵云焕
黄立
王丹娜
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)
出处
《黑龙江畜牧兽医》
CAS
北大核心
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)。
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
duck Tembusu virus
E protein
prokaryotic expression system
reactogenicity
solubility analysis
作者简介
曲哲会(1980-),男,副教授,博士,研究方向为动物病毒致病与免疫机制,quzhehuil980@sina.cn;通信作者:王丹娜(1980-),女,高级兽医师,硕士,研究方向为兽用生物制品研发,wangdanna00@163.com.
