人类成熟型TGF-β_1真核表达载体的构建及表达

Construction of the huaman mature TGF-β_1 and ITS expression

目的:构建人类成熟型TGF-β1的真核表达载体,转染成纤维细胞进行表达,为其在基因治疗中的应用提供实验依据。方法:TRIzol法抽提人新鲜胎盘组织的总RNA,RT-PCR法扩增人成熟型TGF-β1基因,构建真核重组载体pIRES2-EGFP-hTGF-β1,并采用脂质体介导法转染成纤维细胞。结果:从人新鲜胎盘组织中成功提取得到含有hTGF-β1基因的总RNA。重组载体pIRES2-EGFP-hTGF-β1转染成纤维细胞后,经荧光显微镜观察可见绿色荧光。结论:成功构建了人成熟型TGF-β1基因的真核表达载体pIRES2-EGFP-hTGF-β1,转染成纤维细胞株后hTGF-β1获得成功表达。

Objective：To establish human mature TGF-β1 gene cloning and expression vector and through its expression.Eukaryotic expression was transfected into fibroblast cells and primarily studied,thus,we could provide experimental proves on gene therapy application of hTGF-β1.Methods：The cDNA fragment encoding human mature TGF-β1 was amplified by RT-PCR from the total RNA of healthy person＇s placenta by TRIzol agent.We constructed the eukaryotic vetor of hTGF-β1,that was pIRES2-EGFP-hTGF-β1.After being correctly identified,it was transfected into fibroblast cells with lipofectin reagent,and RT-PCR was used to test its mRNA in fibroblast cells after 48 h.Results：We successfully extracted the total RNA containing hTGF-β1 gene from human placenta tissue.After the recombinant vetors pIRES2-EGFP-hTGF-β1 was transfected into fibroblast cells,we saw the green fluorescent light.Conclusion：We successfully constructed the recombinant vetors pIRES2-EGFP-hTGF-β1.hTGF-β1 gene is expressed after the recombinant vetor pIRES2-EGFP-hTGF-β1 is transfected into fibroblast cells.