HBeAg融合表达载体的构建及表达

Construction of fusion expression vector and expression of HBeAg and green fluorescent protein

目的构建HBeAg融合表达载体，并探讨其在HepG2细胞的表达。方法采用PCR的方法从质粒PHBV1.5中扩增乙型肝炎病毒前C／C基因，克隆到pEC3FP-C1的多克隆位点区EcoRⅠ和BamHⅠ位点，构建HBeAg融合表达载体，通过酶切、PCR及测序鉴定，并把质粒转染HepG2细胞，用荧光显微镜、RT-PCR、免疫组化、ELISA检测融合蛋白的表达。结果通过酶切、PCR及测序鉴定，成功构建了HBeAg融合表达载体，并且该载体可以在HepG2细胞胞浆中表达融合蛋白。结论成功构建了乙肝病毒HBeAg融合表达载体，为以后用RNAi进行HBeAg的抑制研究奠定基础。

Objective To construct a fusion expression vector and discuss the expression of HBeAg and green fluorescent protein in HepG2 cell, Methods Amplifying the prec/c gene from the vector PHBV1.5 by PCR, the product of PCR was cloned into the EcoR Ⅰ and BamH Ⅰ site of pEGFP-C1, the recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing, then it was transfected into HepG2 cells to ensure the expression of HBeAg and green fluorescent protein. Results The Results of the restriction enzyme digestion, PCR and sequencing confirmed the vector was constructed successfully, furthermore it can express HBeAg and green fluorescent protein in cytoplast. Conclusion The vector was constructed successfully. It lays foundation for using RNAi to suppress the expression of HBeAg.