人T-bet和HBV-L共表达质粒的构建及体外表达

Construction and expression in vitro of recombinant plasmid for co-expression of human T-bet and HBV-L

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 eukaryotic vector for co-expression of human transcription factor T-bet and hepatitis B virus surface antigen large protein（HBV-L）genes, and determine its expression in vitro. Methods Human T-bet and HBV-L genes were amplified by PCR using the c DNA of peripheral blood mononuclear cells（PBMCs） and plasmid cp DNA3.1-HBV-L as templates respectively, and cloned into eukaryotic expression plasmid p IRES. The constructed recombinant plasmid p IRES-T-bet-HBV-L was transfected to 293 T cells, and the expressions of target genes were detected by RT-PCR, ELISA and immunofluorescence assay（IFA）. Results Restriction analysis and sequencing proved that recombinant plasmid p IRES-T-bet-HBV-L was constructed correctly. RT-PCR showed that both the m RNAs of T-bet and HBV-L were expressed in 293 T cells transfected with recombinant plasmid p IRES-T-bet-HBV-L. However, both ELISA and IFA proved the expressions of T-bet and HBV-L proteins. Conclusion Eukaryotic expression plasmid p IRES-T-betHBV-L was constructed successfully and expressed in 293 T cells.