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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 recombinant lentiviral vector (GV287-SCL) containing human SCL gene and lay the foundation for the gene therapy of diabetic bladder abnormalities (DCP). Methods The genetic material of human SCL gene was amplified by PCR and combined with the chimeric virus-penetrating plasmid. The recombinant shuttle plasmid GV287-EGFP / SCL was constructed and transfected into 293T cells. After multiple infection, amplification and purification, Get recombinant lentivirus GV287-SCL. 293T cells were infected with different concentrations of recombinant lentivirus stock solution. The target gene was detected by Western blotting and the virus titer was calculated by fluorescent labeling. Results The size of human SCL gene amplification product was 1 036 bp, which was the same as the size of SCL c DNA in the relevant gene fragment. The positive transformants formed a 503 bp band after expansion, which was comparable in size to the human SCL c DNA of the target fragment. The target gene SCL of the recombinant plasmid GV287-EGFP / SCL positive clones was successfully inserted into GV287-EGFP, and the human SCL gene mRNA was basically the same in the gene sequencing and Gen Bank database. Recombinant lentivirus titer of 5 × 108TU / m L. Conclusion GV287-SCL was successfully constructed and the virus titer was 5 × 108TU / m L. This study provided the basis for further in vivo transfection of SCL gene and gene therapy of DCP.