森林脑炎病毒包膜糖蛋白胞外区的表达、纯化及免疫原性分析  

Expression, purification and immunogenicity analysis of the extracellular domain of tick-borne encephalitis virus envelope glycoprotein

作　　者：陈洋 张剑 陈华 薛潘 武福星 袁明城 袁若栋 董阳超 叶伟 叶传涛 雷迎峰 CHEN Yang;ZHANG Jian;CHEN Hua;XUE Pan;WU Fuxing;YUAN Mingcheng;YUAN Ruodong;DONG Yangchao;YE Wei;YE Chuantao;LEI Yingfeng(Department of Microbiology and Pathogen Biology,School of Basic Medical Sciences,Air Force Medical University,Xi'an 710032,China;Department of Obstetrics and Gynecology,Tangdu Hospital,Air Force Medical University,Xi'an 710038,China;Department of Infectious Diseases,Tangdu Hospital,Air Force Medical University,Xi'an 710038,China)

机构地区：[1]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032 [2]空军军医大学唐都医院妇产科,陕西西安710038 [3]空军军医大学唐都医院传染科,陕西西安710038

出　　处：《空军军医大学学报》2023年第6期536-540,共5页Journal of Air Force Medical University

基　　金：陕西省重点研发计划项目(2021ZDLSF01-05);空军军医大学军事医学珠峰工程人才计划项目(zfrclyf)。

摘　　要：目的构建森林脑炎病毒(TBEV)森张株包膜糖蛋白E胞外区的原核表达质粒,并在大肠埃希菌中诱导表达、纯化及初步分析其免疫原性。方法从GenBank下载TBEV森张株E蛋白胞外区基因序列(Eecto),密码子优化后交由公司合成,通过双酶切的方法克隆至pET28a载体,获得重组质粒pET28a-TBEV-Eecto。经过酶切和测序分析后,将正确的重组质粒转化BL21(DE3)感受态,用1 mmol/L异丙基-β-D-硫化半乳糖苷诱导表达后通过SDS-PAGE分析重组E蛋白胞外区的表达形式。纯化的包涵体用6 mol/L盐酸胍进行变性溶解,用含有非表面活性剂NDSB-201的缓冲液进行复性,之后超滤浓缩、镍柱亲和层析纯化及蛋白印迹鉴定。将获得的纯化TBEV-Eecto蛋白免疫BALB/c小鼠,利用免疫血清进行蛋白印迹、免疫荧光实验分析其特异性。结果重组质粒pET28a-TBEV-Eecto经酶切后的片段与预期大小一致,测序分析正确。重组蛋白TBEV-Eecto主要以包涵体形式表达,经过盐酸胍变性溶解、NDSB-201复性、超滤及镍柱亲和纯化后获得纯化蛋白,SDS-PAGE和Western blotting分析均显示其与预期大小一致。纯化的TBEV-Eecto蛋白免疫小鼠血清可特异性识别真核表达的TBEV-PrME蛋白,提示TBEV-Eecto具有良好的免疫原性。结论成功构建TBEV森张株E蛋白胞外区的原核表达质粒并在大肠杆菌中表达。纯化的TBEV-Eecto蛋白具有较好的免疫原性,为森林脑炎的血清学诊断方法和亚单位疫苗研制提供了基础。Objective To construct prokaryotic expression plasmid of the extracellular domain of envelope glycoprotein E of tick-borne encephalitis virus(TBEV)Senzhang strain,and induce expression,purification and preliminary analysis of its immunogenicity in Escherichia coli.Methods The gene sequence of extracellular domain of E protein of TBEV Senzhang strain was obtained from Genbank,then synthesized by the company after codon optimization,and cloned into pET28a vector by double endonuclease digestion to obtain the recombinant plasmid pET28a-TBEV-Eecto.After restriction endonuclease digestion and sequencing analysis,the correct recombinant plasmid was transformed into BL21(DE3),which was induced by 1 mmol/L isopropyl-β-D-thiogalactopyranoside.The expression form of recombinant E protein in extracellular domain was analyzed by SDS-PAGE.The purified inclusion bodies were denatured and dissolved with 6 mol/L guanidine hydrochloride,and then renatured with NDSB-201 buffer,which was concentrated via ultrafiltration,purified by nickel ion affinity column chromatography,and identified by Western blotting.TBEV-Eecto protein was immunized to BALB/c mice,and Western blotting and immunofluorescence assay were used to analyze the specificity of immune serum.Results The fragment size of recombinant plasmid pET28a-TBEV-Eecto was consistent with the expected size,and the sequencing analysis was correct.The recombinant protein TBEV-Eecto was mainly expressed in the form of inclusion bodies.The purified protein was obtained after guanidine denaturation,ultrafiltration and nickel ion column affinity chromatography.SDS-PAGE and Western blotting analysis showed that its size was consistent with the expected size.The purified TBEV-Eecto protein could specifically recognize the eukaryotic expression of TBEV-PrME protein in mouse serum,suggesting that TBEV-Eecto has good immunogenicity.Conclusion The prokaryotic expression plasmid of E protein in extracellular domain of TBEV Senzhang strain is successfully constructed and expressed in Escheric

关 键 词：森林脑炎病毒 包膜蛋白 胞外区 原核表达 免疫原性 

分 类 号：R373.31[医药卫生—病原生物学]