EB病毒LMP1-CTAR2结构域的原核表达及蛋白质纯化  

Prokaryotic expression and purification of EBV LMP1-CTAR2

作　　者：余琳[1,2] 张雪雁[3] 李孜[3] 

机构地区：[1]广州医学院医学检验系,广州510182 [2]广州医学院第一附属医院检验科,广州510120 [3]广州医学院病原生物学与免疫学教研室,广州510182

出　　处：《中国卫生检验杂志》2010年第7期1583-1585,共3页Chinese Journal of Health Laboratory Technology

基　　金：广东省自然科学基金研究项目(No.990183);广州医学院基金项目(03-K-54)

摘　　要：目的:构建LMP1-CTAR2原核表达载体,纯化LMP1羧基端活化区2(CTAR2)蛋白。方法:通过RT-PCR技术从B95-8细胞中扩增EBVLMP1 CTAR2 cDNA,克隆至PGEM-T载体后测序。运用亚克隆技术构建PGEX-6P-3-CTAR2重组表达载体。用SDS-PAGE与western blot对表达产物进行鉴定,利用Sepharose 4B亲和层析柱对表达产物进行分离和纯化。结果:PCR扩增出225 bp的基因片段,测序结果与已知的LMP1 CTAR2序列吻合。成功构建PGEX-6P-3-CTAR2原核表达载体,western blot分析表明表达产物能与抗LMP1单克隆抗体特异结合。成功分离出Mr约37000的PGEX-6P-3-CTAR2融合蛋白,纯化出Mr约15000的LMP1 CTAR2蛋白。结论:成功获得EBV LMP1 CTAR2蛋白,可用于噬菌体筛库和CTAR2致瘤机制研究。Objective：To construct prokaryotic expression vector of CTAR2,then to purify the CTAR2 protein.Methods：Amplified from B95-8 cell by RT-PCR,EBV LMP1 CTAR2 cDNA is cloned into pGEM-T carrier,sequenced and then subcloned into the expression vector（PGEX-6P-3）,whose products are identified with Western blot and SDS-PAGE.The CTAR2 protein is separated and purified by Glutathione Sepharose 4B and PreScission Proteas respectively.Results：The amplified 225 bp DNA sequence is the same as the published sequence of CTAR2.The prokaryotic expression vector（PGEX-6P-3-CTAR2） is constructed and its products can be combined with the monoclonal antibody of LMP1 in Western blot.The 37KD fusion protein of PGEX-6P-3-CTAR2 is separated and the 15KD pure protein of CTAR2 is purified.Conclusion：The CTAR2 protein is obtained,which provides the basis for further research on carrying out phage display peptide library and oncogenic mechanism of CTAR2.

关 键 词：EPSTEIN-BARR病毒 羧基端活化区2 原核表达 蛋白质纯化 

分 类 号：R-33[医药卫生]