密码子优化提高狂犬病病毒CTN-1株核蛋白在大肠埃希菌中的表达  

Codon optimization improves the expression of nucleoprotein of rabies virus CTN-1 strain in E.coli

作　　者：任元雪 高鑫 刘茜[1,2] 卢学新 朱武洋[1,2] Ren Yuanxue;Gao Xin;Liu Qian;Lu Xuexin;Zhu Wuyang(Key Laboratory of Biosafety,National Health and Family Planning Commission,National Institute for Viral Disease Control and Prevention,China CDC,Beijing 102206,China;Center for Biosafety Mega-Science,Chinese Academy of Sciences,Wuhan 430071,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,卫健委生物安全重点实验室,北京102206 [2]中国科学院生物安全大科学研究中心,武汉430071

出　　处：《中华微生物学和免疫学杂志》2021年第5期333-337,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家重点研发计划(2019YFC1200701)。

摘　　要：目的:获取具有生物活性的狂犬病病毒核蛋白在大肠埃希菌表达系统中高效表达。方法:实验使用密码子优化技术重新编码狂犬病病毒CTN-1株核蛋白基因,人工合成全长基因并克隆至pET-43.1a表达载体,在大肠埃希菌BL21(DE3)株中诱导表达,并使用Western blot检测其反应原性。结果:诱导后SDS-PAGE分析显示在相对分子质量50×103处出现明显的表达条带,与预期蛋白质条带大小一致。其中在大肠埃希菌A600为0.5左右,37℃诱导5 h时表达产量最高(约为32.3%)。大量表达时核蛋白的表达形式主要为包涵体,后经Ni 2+螯合层析纯化,获得纯度为95%以上的目的蛋白。纯化产物经Western blot鉴定,与疫苗免疫小鼠血清呈阳性反应,表明大肠埃希菌表达CTN-1株核蛋白具有生物活性。结论:本实验成功建立了CTN-1株核蛋白在大肠埃希菌表达系统的高效表达方法,并获得了高纯度目的蛋白,为进一步的临床诊断和新型疫苗制备提供基础资料。Objective To obtain the high-efficiency expression of the biological active rabies virus nucleoprotein in the prokaryotic expression system.Methods This experiment uses codon optimization technology to re-encode the nucleoprotein gene of rabies virus CTN-1 strain,artificially synthesize the full-length gene and clone it into pET-43.1a prokaryotic expression vector,induced expression in BL21(DE3)strain of Escherichia coli(E.coli),and used Western blot to detect its reactogenicity.Results The results showed that after induction,SDS-PAGE electrophoresis analysis showed that an obvious expression band appeared at a molecular weight of 50×103,which was consistent with the expected protein band size.Among them,the E.coli concentration A600 is about 0.5,and the expression yield is the highest(about 32.3%)when induced at 37℃for 5 h.Nucleoprotein expression product is mainly inclusion body when it is expressed in large quantities.After purification by Ni2+chelating chromatography,the purity of the target protein can reach over 95%.The purified product was identified by Western blot and positively reacted with the sera of mice immunized with rabies vaccine,indicating that the prokaryotic expression of the CTN-1 strain nucleoprotein has biological activity.Conclusions This experiment successfully established a high-efficiency expression method for the nucleoprotein of the CTN-1 strain in the prokaryotic expression system,and obtained high-purity target protein,which provides a basis for further clinical diagnosis and preparation of new vaccines.

关 键 词：狂犬病病毒 CTN-1株 核蛋白 原核表达 密码子优化 

分 类 号：R373.9[医药卫生—病原生物学]