HBV融合抗原真核表达载体pIRES-neo-HBAg的构建及表达

Construction and expression of eukaryotic expression plasmid pIRES-neo-HBAg carrying HBV fusion antigen gene

目的构建HBV融合抗原真核表达质粒pIRES-neo-HBAg,并验证其在293T细胞中的表达。方法以含有HBV融合抗原的质粒pVAX1-HBV为模板,PCR扩增该融合基因。PCR产物经纯化后,将其克隆至载体pMD18-T中,构建pMD18-T-HBV质粒,经酶切和测序鉴定后,将其定向克隆入真核表达载体pIRES-neo,获得真核表达质粒pIRES-neo-HBAg。将该重组表达质粒瞬时转染人293T细胞,采用Western blot法、流式细胞术和免疫荧光细胞化学技术验证HBV融合抗原的表达。结果成功构建了HBV融合抗原真核表达质粒pIRES-neo-HBAg,Western blot法、流式细胞术和免疫荧光细胞化学技术结果显示融合抗原能够在293T细胞中表达。结论成功构建了HBV融合抗原真核表达质粒pIRES-neo-HBAg。

Objective To construct a eukaryotic expression plasmid harboring HBV fusion antigen gene, and to express it in 293T cells. Methods The HBV fusion gene fragment was amplified by PCR from the plasmid pVAXI-HBV containing HBV fusion gene. After purified, the product was cloned into pMDI8-T vector. The recombinant plasmid was confirmed by endonuclease digestion and sequencing analysis, and then subcloned into eukaryotic expression vector plRES-neo. Then the recombinant expression plasmid plRES-neo-HBAg was transfered into 293T cells by LipofectamineTM 2000. The expression of HBV fusion antigen was identified by Western blotting, flow cytometry and immunofluorescence cytochemistry. Results The eukaryotic expression vector plRES-neo-HBAg was constructed successfully. The expression of fusion antigen could be detected in the plRES-neo-HBAg transfected 293T cells by Western blotting, immunofluorescence cytochemistry and flow cytome- try. Cendmion The eukaryotic expression plasmid plRES-neo-HBAg is successfully constructed and the fusion antigen is expressed in 293T cells.