【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。更多还原

【Abstract】 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.更多还原