不同结构形式的HBX蛋白对其在肝细胞癌HepG2中分布的影响  

The different structure forms of HBx protein influence their intracellular distribution in hepatocellular carcinoma HepG2 cells

作　　者：彭亮[1] 张瑞[2] 赵晶[2] 闵婕[1] 蒲长宇[1] 周后龙[1] 杨安钢[2] 冯英明[3] 

机构地区：[1]第四军医大学唐都医院肿瘤科,陕西西安710038 [2]第四军医大学基础医学院生物化学和分子生物学教研室,陕西西安710032 [3]第四军医大学研究生院,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2011年第6期611-614,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助(810300453087300630901771)

摘　　要：目的:本研究选用不同的真核表达载体,分别克隆并构建了包含HBX基因或其与GFP以不同方式融合表达的重组质粒,探究不同结构的HBX蛋白对其在细胞内定位的影响。方法:以本实验室构建好的pcDNA3.0-HBX质粒为摸板,采用PCR方法扩增Flag-HBX,克隆至pMD-18T载体中,测序正确后,分别亚克隆至不同载体,构建重组质粒pFlag-HBX-IRES2-EGFP,pEGFP-C3-Flag-HBX,pFlag-HBX-EGFP-N3,鉴定正确后瞬时转染肝癌HepG2细胞,通过间接免疫荧光染色显示HBX蛋白的细胞内定位和分布。结果:成功构建出三种Flag-HBX的真核重组质粒;不同重组质粒转染HepG2细胞后,间接免疫荧光显示与GFP不同融合形式的HBX蛋白在细胞内存在不同的分布特征。结论:为研究HBX在肝癌发生发展中的作用提供了有意义的实验依据,尤其是对体外细胞转染结果的解释提供了借鉴。AIM： Different eukaryotic expression vectors were selected in this research,and then we respectively cloned and constructed recombinant plasmids contained HBX gene or its different fusion forms with GFP.We are aimed to explore the influence of the HBX protein with different structure on its intracellular localization.METHODS： Flag-HBX gene was amplified from pcDNA3.0-HBX plasmid in our laboratory,cloned into pMD-18T vectors,sequenced.and then subcloned into different vectors.After right sequenced,respective recombinant plasmids of pFlag-HBX-IRES2-EGFP,pEGFP-C3-Flag-HBX and pFlag-HBX-EGFP-N3 were transiently transfected into hepatoma HepG-2 cells.The intra-cellular localizations and distributions of HBX protein were examined by indirect immunofluorescence.RESULTS： Three different Flag-HBX eukaryotic expression vectors were successfully constructed.After transfection of them into HepG2 cells respectively,indirect immunofluorescence demonstrated that HBX protein fused with GFP in different forms show distint intra-cellular distribution characteristics.CONCLUSION： We have provided significant experimental evidences for research of the role of HBX protein in the development of hepatocellular carcinoma,and especially the references for explanation of the outcomes in vitro transfection experiments.

关 键 词：HBX 肝癌HEPG2细胞 定位 间接免疫荧光 

分 类 号：R392.12[医药卫生—免疫学]