携带IRES的hBDNF绿色荧光蛋白表达载体的构建和鉴定

Construct and identification of the pIRES2-enchanced green flurescent protein expression vector carrying hBDNF gene

目的构建表达人脑源性神经生长因子(hBDNF)基因的真核细胞表达载体,并且观察重组质粒在真核细胞中的表达情况。方法采用PCR方法从健康人基因组中扩增出该基因,将hBDNF的PCR产物用XhoⅠ和SalⅠ双酶切后克隆到用XhoⅠ和SalⅠ双酶切的带有EGFP和内部核糖体转入位点(IRES)的真核细胞表达载体pIRES2-EGFP中。对重组质粒进行双酶切和质粒测序鉴定后,采用阳离子脂质体Lipofectamine 2000将重组的质粒转染至真核细胞中,用RT-PCR和荧光显微镜检测基因在细胞中表达的情况。结果双酶切和质粒测序结果证明hBDNF已正确地克隆到真核表达载体pIRES2-EGFP中,质粒能在细胞中正常表达。结论成功构建了EGFP/hBDNF表达载体,为应用hBDNF治疗中枢神经系统疾病奠定坚实的基础。

Objective To construct the pIRES2-enchanced green flurescent protein expression vector carrying hBDNF gene and detect the expression of recombinant plasmid in 293-T cells. Method To amplify hBDNF gene from normal human genomics by PCR,insert the products of hBDNF PCR products restriction digested with Sal Ⅰ and Xho Ⅰ into eucaryotic expression vector pIRES2-EGFP with EGFP gene and internal ribosomes entry site（IRES）. To indetify recombinant plasmid by enzyme digestion and sequence analysis. To transfect recombinant plasmid into 293-T cells with Lipofectamine 2000 and detect the expression of recombinant plasmid by RT-PCR and fluorescence microscope. Results Results of enzyme digestion and sequence analysis of recombinant plasmid demonstrated that hBNDF gene was correctly inserted into eucaryotic expression vector pIRES2-EGFP. After tranfected into 293-T cells,recombinant plasmid could be expressed successfully in cells. Conclusion EGFP/hBDNF plasmid has been constructed successfully,thus providing an important and convenient tool to treat central nervous system diseases with hBDNF.