蜱传脑炎病毒非结构蛋白1的原核表达、纯化及应用  

Prokaryotic expression,purification and application of non⁃structural protein 1 of tick⁃borne encephalitis virus

作　　者：张婷婷 王伟 陈子杨 李景良 张颖 孙宏亮 常军亮 ZHANG Ting-ting;WANG Wei;CHEN Zi-yang;LI Jing-liang;ZHANG Ying;SUN Hong-liang;CHANG Jun-liang(Changchun Institute of Biological Products,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,吉林长春130012 [2]黑龙江红十字(森工总)医院急诊科,黑龙江哈尔滨150040

出　　处：《中国生物制品学杂志》2022年第11期1351-1356,共6页Chinese Journal of Biologicals

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

摘　　要：目的原核表达、纯化蜱传脑炎病毒(tick-borne encephalitis virus,TBEV)非结构蛋白1(non-structural protein 1,NS1),并评价其在蜱传脑炎(tick-borne encephalitis,TBE)临床诊断中的应用价值。方法根据GenBank登录的TBEV“森张”株NS1基因序列设计引物,PCR扩增TBEV NS1基因,克隆至原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-4T-1-NS1,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经GST bestarose 4FF亲和层析纯化后,Western blot检测其反应原性;以重组NS1蛋白为固相抗原,采用间接ELISA法检测临床血清。结果重组表达质粒pGEX-4T-1-NS1经双酶切及测序鉴定证明构建正确;表达的重组蛋白相对分子质量约65000,表达量为22.1%,主要以包涵体形式存在;纯化蛋白纯度达93%以上,与TBEV全病毒感染小鼠血清具有良好的反应原性。采用NS1作为包被抗原的间接ELISA法检测临床血清,特异性为95%,敏感度为100%。结论成功在大肠埃希菌中表达并纯化了TBEV NS1蛋白;作为病毒特异性抗原,经间接ELISA法验证,该蛋白在临床疾病的血清学诊断上具有一定的应用价值。Objective To express the non-structural protein 1(NS1)of tick-borne encephalitis virus(TBEV)in prokaryotic cells,purify the expressed product and evaluate its application value in the clinical diagnosis of tick-borne encephalitis(TBE).Methods Primers were designed according to the NS1 protein gene sequence of TBEV Sen-Zhang strain registered in GenBank,with which NS1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1.The constructed recombinant plasmid pGEX-4T-1-NS1 was transformed to E.coli BL21(DE3)and induced with IPTG.The expressed recombinant protein was purified by GST bestarose 4FF affinity chromatography,and analyzed for reactogenicity by Western blot.Clinical serum samples were determined by indirect ELISA using recombinant NS1 protein as a solid phase antigen.Results Restriction analysis and sequencing proved that recombinant plasmid pGEX-4T-1-NS1 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 65000,contained about 22.1%of total somatic protein,mainly existed in a form of inclusion body.The purified recombinant protein reached a purity of more than 93%,and showed good reactogenicity with the mouse serum infected with whole TBEV.The specificity of indirect ELISA method developed using NS1 as coating antigen was 95%,while the sensitivity was 100%,in determination of clinical serum samples.Conclusion TBEV NS1 was successfully expressed in E.coli and purified.As a virus-specific antigen,the protein showed a certain application value in the serological diagnosis of clinical disease as proved by indirect ELISA.

关 键 词：蜱传脑炎病毒 流行性乙型脑炎病毒 非结构蛋白1 原核表达 酶联免疫吸附测定 

分 类 号：R373.31[医药卫生—病原生物学] Q786[医药卫生—基础医学]