乙肝病毒X基因真核表达载体pEGFP-X的构建及其表达

Construction and expression of eukaryotic expression vector for hepatitis B virus X gene containing EGFP

目的:构建pEGFP-X真核表达载体,观察其在肝癌细胞株中的表达。方法:从质粒pcDNA3.1(+)-X酶切获HBV X基因片段,克隆至pEGFP-N1;用酶切、PCR和测序鉴定pEGFP-X载体;用脂质体法,将重组载体(pEGFP-X)和空载体(pEGFP-N1)瞬时转染肝癌细胞细胞株BEL-7402;分别用RT-PCR、荧光显微镜鉴定HBV X和EGFP基因表达。结果:鉴定证实pEGFP-X载体构建成功;该质粒瞬时转染的BEL-7402有HBV X基因表达,并发绿色荧光。结论:构建的pEGFP-X真核载体能在BEL-7402中表达,绿色荧光蛋白示踪利于表达HBV X基因稳定细胞株的筛选,并为探讨HBV X基因在HCC中的致瘤机理提供实验模型奠定基础。

Objective ： To construct eukaryotic expression vector for hepatitis B virus X（HBV X） gene with enhanced green fluorescence protein （ EGFP ） , and confirm its expression in hepatocellular carcinoma （ HCC ） cell fine.Method： HBV X gene was cloned from pcDNA3. 1 （＋）-X by enzyme digestion and inserted to pEGFP-N1,The rector was confirmed by enzyme digestion ,PCR assay and sequencing.Then pEGFP-X was transfected to HCC cell line Bel-7402.After transient transfection, expressions of HBV X and EGFP gene were detected by RT-PCR and fluorescence microscope respectively. Results： The results showed that the recombinant plasmid could express HBV X gene efficiently in Bel-7402, which showed green fluorescence. Conclution： The pEGFP-X was constructed and the fused HBV X-EGFP gene was expressed in Bel-7402 successfully.These facilitate the study of the effect of HBV X protein on the development of HCC.