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目的构建2种包含乙肝病毒核心蛋白(HBc)基因的真核表达载体,检测其在人肝癌细胞系BEL7402中的表达。方法以质粒pUC19/3.0HBV作为模板,PCR扩增HBc基因,分别以pcDNA3.0和pEGFP-N1为母本,构建含HBc基因的真核表达载体pcDNA-HBc-HA及pEGFP-HBc。通过单、双酶切、PCR及DNA测序鉴定其正确性。利用脂质体将其分别转染入人肝癌细胞系BEL7402,并采用RT-PCR、Western blot及荧光显微镜观察的方法,分别检测其mRNA、蛋白质水平的表达。结果获得与预期分子量大小一致的DNA片段,转染2种质粒的BEL7402细胞均可高水平表达HBc mRNA,而空载体转染组则无表达,转染pEGFP-HBc转染组及pEGFP-N1转染组,均有较强的绿色荧光表达,pcDNA-HBc-HA转染组细胞可检测到HBc-HA融合蛋白的表达。结论成功构建2种携载HBc基因的真核表达载体,并可在人肝癌细胞系BEL7402中有效表达。
Objective To construct two eukaryotic expression vectors containing hepatitis B virus core protein (HBc) gene and detect their expression in human hepatocellular carcinoma cell line BEL7402. Methods HBc gene was amplified by PCR using plasmid pUC19 / 3.0HBV as template. The eukaryotic expression vectors pcDNA-HBc-HA and pEGFP-HBc containing HBc gene were constructed with pcDNA3.0 and pEGFP-N1 as the female parent respectively. The correctness was confirmed by single and double enzyme digestion, PCR and DNA sequencing. The recombinant plasmid was transfected into human hepatocellular carcinoma cell line BEL7402 by liposome respectively. The mRNA and protein levels were detected by RT-PCR, Western blot and fluorescent microscopy. Results The DNA fragment with the expected molecular weight was obtained. BEL7402 cells transfected with the two plasmids could express HBc mRNA at high level, but no expression in the transfected group and transfected with pEGFP-HBc and pEGFP-N1 The results showed that the expression of HBc-HA fusion protein was detected in cells transfected with pcDNA-HBc-HA. Conclusion Two eukaryotic expression vectors carrying HBc gene were successfully constructed and expressed in human hepatocellular carcinoma cell line BEL7402.