摘要
目的 构建PGEX 6P 3 CCT原核表达载体 ,获得大量纯化的LMP1羧基端蛋白。方法 通过RT PCR技术从B95 8细胞中扩增EBVLMP1CCTcDNA ,克隆至PGEM Teasy载体上并测序。利用引物上的BamHI与EcoRI酶切位点将CCT基因插入PGEX 6P 3,转化大肠杆菌BL2 1 ,限制性内切酶消化鉴定。IPTG诱导重组菌株表达 ,用SDS PAGE与Westernblot对表达产物进行鉴定 ,并用Sepharose 4B亲和层析柱对表达产物进行纯化 ,PreScissionProteas酶对融合蛋白进行解离。结果 扩增的LMP1CCT长度为 5 97bp ,测序结果与已知序列吻合。重组子经酶切鉴定 ,获得PGEX 6P 3 CCT原核表达菌株 ,Westernblot表明重组蛋白能被LMP1单克隆抗体特异结合。获得约5 1kDa的PGEX 6P 3 CCT融合蛋白 ,分离纯化出约 2 1kDa的LMP1CCT蛋白。结论 成功获得EBVLM1CCT蛋白 ,为研究LMP1致瘤机制 。
Objective To construct recombinant expression plasmid of PGEX 6P 3 CCT and obtain pure protein of EBV LMP1 CCT.Methods EBV LMP1 CCT cDNA amplified from B95 8 cell by RT PCR technique was cloned into pGEM T easy and sequenced. It was then subcloned into expression vector PGEX 6P 3 and transformed into E.coli BL21. The EBV LMP1 CCT was expressed as a fusion recombinant protein and detected by Western blot. The fushion protein was purified by chromatography using Glutathione Sepharose 4B and was digested by the PreScission Proteas. Results The size of the amplified LMP1 CCT cDNA was 597bp.The DNA sequence of the cloned cDNA was the same as the published sequence. The recombinant prokaryotic expression plasmid PGEX 6P 3 CCT was constructed and the recombinant fusion protein was expressed in E.coli BL21 and detected by the anti LMP1 monoclonal antibody using Western blot. The 51kDa pGEX LMP1 fusion protein and the cleaved 21kDa LMP1 CCT protein were purified. Conclusions The pure LMP1 CCT recombinant protein provided the basic material for studying the oncogenic role of LMP1 and the development of gene therapy for NPC.
出处
《热带医学杂志》
CAS
2003年第2期128-132,175,共6页
Journal of Tropical Medicine
基金
国家自然科学基金 (No .39970 2 92 )
广东省自然科学基金研究项目 (No.990 1 83)
卫生部科学研究基金 (No .98 2 346 )资助
