摘要
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 the HBc gene and to observe their expressions in the human hepatoma cell line. Methods We used pUC19/3.0HBV as a template to amplify the HBc gene with the PCR method, pcDNA3.0 and pEGFP-N1 were introduced to construct the expression vectors of pcDNA-HBc-HA and pEGFP-HBc, respectively. PCR, restriction endonuclease digestion and DNA sequencing were used to select the positive plasmids. Then, BELT402 cells were separately transfected with pcDNA-HBc-HA, pEGFP-HBc and their empty control vectors by liposome. The HBc mRNA and protein levels were further determined by RT-PCR, Western blot and a fluorescent microscope. Results The recombinant plasmids containing HBc gene were successfully constructed. Single and double enzyme digestion showed that both the recombinant plasmids contained the inserted gene fragments with the anticipated molecular weight. RT-PCR showed that high level of HBc mRNA was detected in BELT402 cells transfected with pcDNA-HBc-HA and pEGFP-HBc expression vectors, while there was no HBc gene expression in control cells transfected with pcDNA3.0 or pEGFP-N1. The green fluorescence protein was expressed in the pEGFP- HBc group and the pEGFP-N1 group, and HBc-HA protein was detected in the pcDNA-HBc-HA group by Western blot. Conclusion Two eukaryotic expression vectors with the HBc gene were successfully constructed and both of them were effectively expressed in BELT402 cells.
出处
《山东大学学报（医学版）》
CAS
北大核心
2010年第2期67-71,共5页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金资助课题(30700973)
