西尼罗病毒E蛋白的原核表达及其多克隆抗体的制备  

Prokaryotic expression and polyclonal antibody preparation of West Nile virus E protein

作　　者：张颖[1,2] 曹增国 李恩涛 李国华 闫飞虎[2] 莫若 李武健[2,3] 迟航 王化磊 冯娜 高玉伟 杨松涛 赵永坤 夏咸柱 ZHANG Ying;CAO Zeng-guo;LI En-tao;LI Guo-hua;YAN Fei-hu;MO Ruo;LI Wu-jian;CHI Hang;WANG Hua-lei;FENG Na;GAO Yu-wei;YANG Song-tao;ZHAO Yong-kun;XIA Xian-zhu(College of Wildlife and Protected Area,Northeast Forestry University,Harbin 150040,China;Key Laboratory of Jilin Province for Zoonosis Prevention and Control/Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Changchun 130122,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China;College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]东北林业大学野生动物与自然保护地学院,黑龙江哈尔滨150040 [2]军事科学院,军事医学研究院军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [3]吉林大学动物医学学院,吉林长春130062 [4]华南农业大学兽医学院,广东广州510642 [5]石河子大学动物科技学院,新疆石河子832003 [6]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国兽医科学》2020年第5期570-575,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0501804)。

摘　　要：利用PCR技术扩增西尼罗病毒(WNV)E蛋白基因并将其插入原核表达载体pET-30a(+),构建重组质粒pET-30a(+)-WNV-E,并转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导目的蛋白表达,对诱导条件进行优化,利用His镍柱亲和层析法纯化目的蛋白,纯化后的蛋白经SDS-PAGE与Westernblot鉴定正确后,免疫BALB/c小鼠制备多克隆抗体并进行间接免疫荧光鉴定。结果显示:WNV E蛋白在大肠杆菌中以包涵体形式被成功表达,蛋白最佳诱导条件为37℃,0.8 mmol/L IPTG诱导5 h,经间接免疫荧光鉴定多克隆抗体能特异性识别WNV E蛋白。本研究成功表达并纯化了WNV E蛋白,证明该蛋白具有良好的免疫原性,为WNV抗原、抗体检测方法的建立和新型疫苗的研发及相关研究奠定了基础。The gene encoding West Nile virus(WNV)E protein was amplified by PCR and inserted into the prokaryotic expression vector pET-30a(+),constructing the recombinant plasmid pET-30a(+)-WNV.The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3)and induced with IPTG.Meanwhile,the induction condition was optimized.The expressed recombinant E protein was purified by His nickel column affinity chromatography and identified by SDS-PAGE and Western-blot.Then,the purified E protein was used as immunogen for BALB/c mice by intramuscular injection route after mixed with Freund adjuvant.The prepared polyclonal antibody was detected for specificity by indirect immunofluorescence assay(IFA).The results showed that the E protein was expressed in a form of inclusion body in E.coli.The optimal induction of the protein was induced at 37℃and 0.8 mmol/L IPTG for 5 h.In conclusion,the WNV E protein was successfully expressed and purified.This protein has good immunogenicity,which lays the foundation for the establishment of detection methods of WNV antigen and antibody,the development of new vaccines and related research.

关 键 词：西尼罗病毒 原核表达 E蛋白 多克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]