含人干细胞白血病基因的重组慢病毒表达载体的构建及鉴定

Construction and identification of recombinant lentiviral vectors containing human stem cell leukemia gene

目的构建含人干细胞白血病(SCL)基因的重组慢病毒表达载体(GV287-SCL),为糖尿病膀胱异常病症(DCP)的基因治疗奠定基础。方法采用PCR法将人SCL基因质粒内的遗传物质扩增,与慢性病毒穿透质粒结合,构建重组穿梭质粒GV287-EGFP/SCL,共转染293T细胞,通过多次感染、扩增和提纯,得到重组慢病毒GV287-SCL。以不同浓度重组慢病毒原液感染293T细胞,采用Western blotting法检测目的基因,荧光标记法计算病毒滴度。结果人SCL基因扩增产物大小为1 036 bp,与目的基因相关片段中SCL c DNA大小相同。阳性转化子扩展后均形成503 bp条带,其大小与目的片段人SCL c DNA相当。重组质粒GV287-EGFP/SCL阳性克隆的目的基因SCL成功插入到GV287-EGFP,基因排序和Gen Bank信息库里的人SCL基因mRNA基本相同。重组慢病毒滴度为5×108TU/m L。结论成功构建了GV287-SCL,病毒滴度为5×108TU/m L。本研究为继续进行SCL基因体内转染及开展DCP的基因治疗提供了基础。

Objective To construct the recombinant lentiviral vectors containing human stem cell leukemia ( SCL) gene (GV287-SCL), and to lay the foundation for gene therapy of diabetic cystopathy (DCP).Methods The genetic material from plasmid of SCL gene was amplified by PCR , and was connected to the shuttle plasmid to construct GV287-EGFP/SCL, then they were used to transfect 293T cells.The recombinant lentivirus GV287-SCL was obtained by multi-ple infections, amplification and purification.293T cells were transfected by different concentrations of recombinant lenti-virus.We used Western blotting to detect the target gene, and fluorescence labeling method to calculate the virus titer. Results The size of amplification product from human SCL gene was 1036 bp, which was the same as SCL-cDNA in the target gene associated fragment.After the extension of positive transformants, each formed 503 bp band, whose size was consistent with human SCL cDNA.The SCL target gene of recombinant plasmid GV287-EGFP/SCL positive clones were successfully inserted into the GV287-EGFP, the genetic sequence was the same as the human SCL gene mRNA in Gen-bank, and the titer of recombinant lentiviral was 5.0 ×108 TU/mL.Conclusions The recombinant lentiviral vector GV287-SCL is successfully constructed, and the titer is 5.0 ×108 TU/ml.This study provides a foundation for SCL gene transfection in vivo and gene therapy for DCP.