组织因子途径抑制因子/绿色荧光蛋白双顺反子真核表达载体构建及其在NIH 3T3细胞中表达

Construction of the bicistronic expression vector containing tissue factor pathway inhibitor and green fluorescent protein gene

目的构建含人组织因子途径抑制因子(TFPI)和绿色荧光蛋白(GFP)基因的双顺反子真核表达载体,并验证其在NIH 3T3细胞中的表达,为血管再狭窄的防治提供一个具有示踪和治疗双重作用的有效载体。方法以含有全长cDNA的pIRES-TFPI为模板,多聚酶链反应(PCR)扩增TFPI全长cDNA,经酶切后插入到pIRES2-AcGFP1-Nuc载体中,构建成双顺反子真核表达载体,重组质粒经酶切图谱分析、PCR扩增及测序鉴定后命名为pIRES2-AcGFP1-Nuc-TFPI。将其转染NIH3T3细胞,采用荧光显微镜观察GFP在细胞中的表达,以RT-PCR检测TFPI在细胞内的表达。结果经酶切图谱分析、PCR扩增及DNA测序证实双顺反子真核表达载体构建正确;荧光显微镜可观察到细胞内GFP的表达;RT-PCR证实经TFPI基因转染的细胞内TFPI mRNA表达增高。结论成功构建了包括TFPI和GFP的真核双表达载体,并使其在NIH3T3细胞中顺利表达。

Objective To construct an eukaryotic expression vector which is able to simultaneously express human tissue factor pathway inhibitor（TFPI） and green fluorescent protein（GFP） in eukaryotic cells,and provide a useful tool for investigating the feasibility of application of TFPI gene in the prevention of restenosis.Methods The full length cDNA encoding TFPI was amplified by polymerase chain reaction（PCR） with pIRES-TFPI containing the full length cDNA of human TFPI gene as a template,and subsequently inserted into pIRES2-AcGFP1-Nuc plasmid after digestion with the corresponding restriction endonuleases.The recombinant plasmid pIRES2-AcGFP1-Nuc-TFPI was confirmed by restriction endonuclease mapping,PCR amplification and DNA sequencing.NIH 3T3 cells were transfected with pIRES2-AcGFP1-Nuc-TFPI or pIRES2-AcGFP1-Nuc to investigate the expressions of TFPI and GFP in eukaryotic cells.Fluorescence microscopy was employed to observe the expression of GFP,while RT-PCR was utilized to analyze the expression of TFPI gene in NIH 3T3 cells.Results The expression vector pIRES2-AcGFP1-Nuc-TFPI was successfully constructed and resulted in simultaneously cellular expression of both TFPI and GFP when transfected into NIH 3T3 cells.Fluorescence microscopy confirmed the GFP expression in the cells and RT-PCR revealed that the expression of TFPI mRNA was increased in TFPI gene transfected cells.Conclusion It is demonstrated that bicistronic expression vector containing TFPI and GFP genes was successfully constructed,which could simultaneously express TFPI and GFP in NIH 3T3 cells.