蜱传脑炎病毒衣壳蛋白的可溶性表达、纯化及初步应用  

Soluble expression, purification and preliminary application of capsid protein of tick-borne encephalitis virus

作　　者：聂倩文 张婷婷 常东英 刘君 陈子杨 常军亮 NIE Qian-wen;ZHANG Ting-ting;CHANG Dong-ying;LIU Jun;CHEN Zi-yang;CHANG Jun-liang(Biotechnology Laboratory,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China;不详)

机构地区：[1]长春生物制品研究所有限责任公司生物技术研究室,吉林长春13001 [2]长春理工大学生命科学技术学院,吉林长春130022

出　　处：《微生物学免疫学进展》2022年第6期1-6,共6页Progress In Microbiology and Immunology

基　　金：吉林省省级医药健康产业发展专项基金项目(20170311010YY)。

摘　　要：目的构建蜱传脑炎病毒(tick-borne encephalitis virus,TBEV)衣壳蛋白(capsid protein,C)原核表达质粒,并以其表达、纯化的C蛋白作为抗原检测临床血清,评价其在蜱传脑炎(tick-borne encephalitis,TBE)临床血清学诊断中的应用。方法登录Genbank对TBEV“森张”株C蛋白基因序列设计扩增引物,以PCR扩增C蛋白目的基因并克隆至原核表达载体pET-32a(+)后,构建重组表达质粒pET-32a-C。再将其转化至大肠埃希菌BL21(DE3)感受态细胞,采用IPTG诱导剂诱导表达,表达产物经Ni-NTA亲和层析纯化后,Western blot检测其反应原性;以制备的C蛋白作为包被抗原,用间接ELISA分别检测20份TBEV IgG抗体阳性临床血清、10份流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)阳性临床血清及20份阴性血清,并计算检测的特异性和敏感度。结果重组表达质粒pET-32a-C序列测定正确;经12%SDS-PAGE电泳分析,重组C蛋白主要为可溶性蛋白表达;纯化后蛋白纯度>90%,经Western blot鉴定可与TBEV全病毒血清抗体特异性结合。间接ELISA定性检测临床TBE血清,其特异性与敏感度分别为96.7%和95.0%。结论成功构建了TBEV衣壳蛋白重组表达质粒pET-32a-C并在大肠埃希菌中实现诱导表达;表达的重组C蛋白可与TBEV阳性血清特异性结合,在临床疾病的血清学诊断上具有一定的应用价值。Objective To construct a prokaryotic expression plasmid containing capsid protein C gene of tick-borne encephalitis virus(TBEV)and express the gene in E.coli,and to evaluate the application of purified C protein used as antigens in clinical serological diagnosis of tick-borne encephalitis(TBE).Methods The primers were designed to amplify the C protein gene of TBEV"Senzhang"strain registered in GenBank.The target gene of C protein was amplified by PCR and cloned into the prokaryotic expression vector pET-32a(+),and the recombinant expression plasmid pET-32a-C was constructed.Then it was transformed into Escherichia coli(E.coli)BL21(DE3)competent cells and induced by IPTG inducer.The expression products were purified by Ni-NTA affinity chromatography and its reactivity was detected by Western blot.Using purified C protein as coating antigen,10 clinical serum samples positive for TBEV,10 clinical serum samples positive for JEV and 20 serum samples negative for JEV were detected by indirect ELISA,and the specificity and sensitivity of the ELISA were calculated.Results The sequence of the recombinant expression plasmid pET-32a-C was correct;the recombinant C protein was mainly expressed as soluble protein detected by 12%SDS-PAGE;the purity of the target protein was more than 90%,and the protein could bind specifically to the serum antibodies against TBEV by Western blot analysis.The specificity and sensitivity of the indirect ELISA for detection of clinical TBE serum were 96.7%and 95.0%,respectively.Conclusion The recombinant expression plasmid pET-32a-C containing TBEV capsid protein gene was successfully constructed and expressed in E.coli The recombinant C protein can specifically bind to TBEV-antibody-positive serum.It has potential applications in the serological diagnosis of clinical diseases.

关 键 词：蜱传脑炎病毒 乙型脑炎病毒 衣壳蛋白 原核表达 纯化 酶联免疫吸附试验 

分 类 号：R392-33[医药卫生—免疫学]