戊型肝炎病毒ORF2蛋白与新型融合标签Halo Tag的融合表达  

Fusion expression of hepatitis E virus ORF2 protein with Halo Tag fusion tag

作　　者：马天武[1] 黄芬[1] 井申荣[1] 曾韦锟[1] 禹文海[2] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500 [2]中国医学科学院北京协和医学院医学生物学研究所医学灵长类中心实验动物部,昆明650118

出　　处：《中国生物制品学杂志》2012年第12期1583-1586,共4页Chinese Journal of Biologicals

基　　金：云南省教育厅基础研究面上项目(2011Y382)

摘　　要：目的在大肠杆菌KRX中表达戊型肝炎病毒（Hepatitis E virus,HEV）ORF2与新型融合标签Halo Tag融合蛋白。方法采用RT-PCR法从4型HEV毒株中扩增ORF2基因部分片段（aa 382~620）,插入含新型融合标签Halo Tag的原核表达载体pFN18the中,构建重组表达质粒pFN18the-ORF2,转化大肠杆菌KRX,鼠李糖诱导表达Halo-ORF2融合蛋白,纯化后Western blot分析融合蛋白的反应原性。结果经RT-PCR扩增出717 bp的目的基因片段;重组表达质粒pFN18the-ORF2经双酶切和测序鉴定构建正确;表达的融合蛋白Halo-ORF2相对分子质量为57 900,表达量约占菌体总蛋白的15%,以包涵体形式表达,纯化后纯度达90%以上,可与HEV IgG阳性血清特异性结合。结论在大肠杆菌KRX中表达了Halo-ORF2融合蛋白,为HEV结构蛋白抗原表位的筛选及戊肝血清学诊断的研究奠定了基础。Objective To express hepatitis E virus（HEV）ORF2 protein with Halo Tag fusion tag in E.coli KRX strain.Methods Partial ORF2 gene（aa 382 ~ 620）was amplified from HEV type 4 by RT-PCR and inserted into prokaryotic expression vector pFN18the containing Halo Tag fusion tag.The constructed recombinant plasmid pFN18the-ORF2 was transformed to E.coli KRX and induced with rhamnose.The expressed Halo-ORF2 fusion protein was purified and analyzed for reatogenicity by Western blot.Results The target gene fragment at a length of 717 bp was amplified by RT-PCR.Restriction analysis and sequencing proved that recombinant plasmid pFN18the-ORF2 was constructed correctly.The expressed fusion protein,with a relative molecular mass of 57 900,contained about 15% of total somatic protein and reached a purity of more the 90% after purification,which showed specific binding to HEV IgG positive serum.Conclusion Recombinant Halo-ORF2 fusion protein was expressed in E.coli KRX,which laid a foundation of screening of antigenic epitope of structural protein of HEV and study on serological diagnosis of HE.

关 键 词：肝炎病毒 戊型 ORF2蛋白 HALOTAG 融合蛋白 原核细胞 基因表达 

分 类 号：R373.21[医药卫生—病原生物学] Q786[医药卫生—基础医学]