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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。
OBJECTIVE: In this study, different eukaryotic expression vectors were selected and cloned and constructed recombinant plasmid containing HBX gene or its fusion with GFP in different ways to explore the effect of different structures of HBX protein on intracellular localization. METHODS: Flag-HBX was amplified by PCR from pcDNA3.0-HBX plasmid constructed in our laboratory and cloned into pMD-18T vector. After correct sequencing, the vector was subcloned into different vectors to construct recombinant plasmid pFlag-HBX-IRES2-EGFP, pEGFP-C3-Flag-HBX and pFlag-HBX-EGFP-N3 were transiently transfected into HepG2 cells. Indirect immunofluorescence staining showed the intracellular localization and distribution of HBX protein. Results: Three kinds of Flag-HBX eukaryotic recombinant plasmids were constructed successfully. After transfection with different recombinant plasmids, indirect immunofluorescence showed that there were different distribution characteristics of HBX proteins with different fusion forms in HepG2 cells. Conclusion: This study provides a meaningful experimental basis for studying the role of HBX in the development and progression of hepatocellular carcinoma, especially for the explanation of transfection results in vitro.