EB病毒潜伏膜蛋白2A_(1-119)重组质粒的表达及鉴定  

Expression and identification of the gene of LMP2A_(1-119) of Epstein Barr virus

作　　者：张琴[1] 薛向阳[1] 夏克栋[1] 李文姝[1] 陈韶[1] 张丽芳[1] 

机构地区：[1]温州医学院微生物学和免疫学教研室分子病毒和免疫研究所,浙江温州325035

出　　处：《温州医学院学报》2010年第4期319-321,共3页Journal of Wenzhou Medical College

基　　金：国家自然科学基金资助项目(30671882)

摘　　要：目的:构建含EB病毒(Epstein-Barr virus,EBV)潜伏膜蛋白(LMP)2A N端1-119基因的重组质粒,探讨其在原核表达系统中的表达情况。方法:采用RT-PCR方法从B95-8细胞中获得LMP2A1-119外显子基因片段,并克隆到pGEX-4T-1原核表达载体,转化大肠杆菌BL21(DE3)后,经IPTG诱导表达融合蛋白,通过Ni-NTA离子交换柱层析纯化,进一步采用SDS-PAGE和Western blot鉴定。结果:pGEX/LMP2A1-119重组质粒构建成功,并在大肠杆菌中得到表达。SDS-PAGE结果表明表达产物相对分子质量约为50 kD,West-ern blot结果证实其为目的蛋白。结论:EBV LMP2A1-119蛋白可在pGEX-4T-1原核表达系统中表达,为进一步研究其免疫学特性打下了基础。Objective：To construct the recombinant plasmid pGEX/LMP2A1-119 and to probe express situation of the LMP2A1-119 of Epstein-Barr virus（EBV） in Escherichia coli（E.coli） in the prolaryotic expression.Methods：The exon gene fragment encoding 1-119 amino acid of LMP2A was amplified by RT-PCR and was cloned into the expression veteor of pGEX-4T-1 for forming the PGEX/LMP2A1-119 recominant with which is transformed E.coli BL21（DE3）.The PGEX/LMP2A1-119 protein was expressed in E.colia,nd then purified with Ni-NTA agarose column ion-exchange chromatography.The protein was analyzed with SDS-PAGE and identified with Western-blot.Results：The PGEX/LMP2A1-119 recombinant plasmid was successfully constructed and fusion protein could be expressed in prokaryotic expression system.SDS-PAGE analysis showed the relative molecular mass of this fusion protein as 50×103,and the specificity of this fusion protein was confirmed with Western-blot.Conclusion：The fusion protein of the PGEX/LMP2A1-119 can be successfully expressed in prokaryotic expression system.It lays the foundation for the detecting its immunity effectiveness.

关 键 词：疱疹病毒4型 人 潜伏膜蛋白2A PGEX-4T-1 原核表达 重组 遗传 

分 类 号：R3[医药卫生—基础医学]