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

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.