摘要
目的构建能够表达HBx蛋白的绿色增强荧光蛋白-HBx(pEGFP-N1-HBx)真核表达质粒,并探讨HBx蛋白对乙型肝炎病毒(HBV)复制的影响。方法以HBV Dimer为模板,PCR法扩增HBx基因,PCR产物测序分析正确后克隆至真核表达载体pEGFP-N1,命名为pEGFP-N1-HBx。将该质粒转染肝胚瘤细胞株HepG2,以RT-PCR法及荧光显微镜观察其表达情况,再通过基因共转染等技术研究HBx蛋白对HBV复制的影响。结果酶切鉴定和序列分析证实构建质粒含HBx基因,RT-PCR法检测和荧光显微镜观察表明构建的pEGFP-N1-HBx质粒在HepG2细胞中能够表达;质粒pEGFP-N1-HBx与pHBV48-X0共转染后,HepG2细胞上清液HBV-DNA、HBsAg和HBeAg分泌显著增多。结论 HBx基因克隆及其真核表达载体pEGFP-N1-HBx构建成功;HBx蛋白能够促进HBV复制及抗原分泌。
Objective To construct and express the eukaryotic expression vector of HBV x gene and study its eifect on the HBV replication in hepatocellular carcinoma cells. Methods A DNA fragment of 465 bp was amplified from recombinant plasmid HBV Dimer and then cloned into an expression vector pEGFP-N1. After being confirmed by restriction enzyme digestion and sequencing methods, the recombinant plasmid was named as pEGFP-N1-HBx that was subsequently transfected into HepG2 cells using LipofectamineTM2000. Fluorescent microscopy observation and RT-PCR were used to analyse the expression of the HBx gene. Afterwards, the effect on the HBV replication of HBx protein was assessed using transient co-transfection assays. Results Both restriction enzyme digestion and sequen- cing assays showed that the recombinant vector pEGFP-N1-HBx was successfully constructed. Fluorescent microscopy observation, RT-PCR indicated that the HBx gene expressed efficiently in the HepG2 cells. Moreover, the extracellular HBV DNA and HBV antigen secreted markedly after transient co-transfection assays. Conclusion HBx gene eukaryotic expression vector is successfully constructed and the HBx gene expressed efficiently in the HepG2 cells. HBx could promote the replication of HBV in the HepG2 cells.
出处
《安徽医科大学学报》
CAS
北大核心
2013年第11期1297-1299,共3页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81171662)
安徽省教育厅基金(编号:KJ2012Z163)
安徽医科大学校级科研基金(编号:2011XKJ049)
