论文部分内容阅读
目的构建人转录因子T-bet基因与乙肝病毒表面抗原大蛋白全长基因(hepatitis B virus surface antigen large protein,HBV-L)的共表达载体,并检测其在体外的表达。方法利用PCR及基因重组技术,以人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)的cDNA为模板,扩增获得人T-bet全长基因,以含有HBV-L的质粒pcDNA3.1-HBV-L为模板,扩增获得HBV-L基因,将上述2个基因克隆至真核表达质粒p IRES中,获得共表达质粒p IRES-T-bet-HBV-L。将该质粒转染293T细胞,经RT-PCR、ELISA和细胞免疫荧光试验检测其在真核细胞中的表达。结果限制性酶谱分析及测序证实重组质粒p IRES-T-bet-HBV-L构建成功;该质粒在体外转染293T细胞后,可表达T-bet与HBV-L两者的mRNA;细胞免疫荧光试验和ELISA检测结果证实,该质粒转染293T细胞后可表达T-bet和HBV-L蛋白。结论成功构建了共表达质粒pIRES-T-bet-HBV-L,该质粒能在293T细胞中表达。
Objective To construct a co-expression vector of human transcription factor T-bet gene and hepatitis B virus surface antigen large protein (HBV-L) and test its expression in vitro. Methods The full-length human T-bet gene was amplified by PCR and gene recombination technique using the cDNA of human peripheral blood mononuclear cells (PBMC) as a template. The recombinant plasmid pcDNA3.1- HBV-L was used as a template to amplify the HBV-L gene. The above two genes were cloned into the eukaryotic expression plasmid pIRES to obtain the co-expression plasmid pIRES-T-bet-HBV-L. The plasmid was transfected into 293T cells and its expression in eukaryotic cells was detected by RT-PCR, ELISA and cell immunofluorescence assay. Results Restriction endonuclease analysis and DNA sequencing confirmed that the recombinant plasmid pES-T-bet-HBV-L was constructed successfully. The plasmid transfected 293T cells could express mRNA of T-bet and HBV-L. Fluorescence and ELISA tests confirmed that the plasmid transfected 293T cells can express T-bet and HBV-L protein. Conclusion The co-expression plasmid pIRES-T-bet-HBV-L was successfully constructed and expressed in 293T cells.