论文部分内容阅读
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 the prokaryotic expression vector LMP1-CTAR2 and purify the CTLA2 protein of LMP1. Methods: EBVLMP1 CTAR2 cDNA was amplified by RT-PCR from B95-8 cells and cloned into PGEM-T vector for sequencing. Subcloning technique was used to construct PGEX-6P-3-CTAR2 recombinant expression vector. The expressed product was identified by SDS-PAGE and western blot, and the expressed product was isolated and purified by Sepharose 4B affinity chromatography. Results: A 225 bp fragment was amplified by PCR. The sequencing result was consistent with the known LMP1 CTAR2 sequence. The prokaryotic expression vector pGEX-6P-3-CTAR2 was successfully constructed. Western blot analysis showed that the expressed product could specifically bind to anti-LMP1 monoclonal antibody. PGEX-6P-3-CTAR2 fusion protein of Mr about 37000 was successfully isolated and LMP1 CTAR2 protein of Mr about 15000 was purified. Conclusion: The EBV LMP1 CTAR2 protein was successfully obtained and could be used to study the mechanism of phage screen and CTAR2 tumorigenesis.