IGFBP7基因慢病毒载体的构建及在K562细胞中的表达

Construction of Venus Vector Carrying IGFBP7 Gene and Its Expression in K562 Cells

本研究构建胰岛素样生长因子结合蛋白7(IGFBP7)基因的慢病毒载体,为研究该基因在白血病中的作用提供基础。采用限制性内切酶酶切获得目的基因,基因重组构建慢病毒载体质粒venus-IGFBP7,用293T细胞包装慢病毒颗粒,感染K562细胞,并采用多种方法鉴定。结果表明,所获IGFBP7基因经测序与GenBank比对序列一致,慢病毒载体质粒venus-IGFBP7经BamHⅠ酶切鉴定片段大小正确,荧光显微镜及流式细胞术检测到绿色荧光蛋白在293T及K562细胞中表达,RT-PCR和Western blot检测到IGFBP7 mRNA和蛋白在K562细胞表达。结论:成功构建带有IGFBP7基因的慢病毒载体,为研究IGFBP7基因在K562细胞中的作用奠定了基础。

Abstract The aim of this study was to construct venus vector carrying the gene encoding insulin-like growth factor binding protein 7 （ IGFBP7）, which provides an effective platform for exploring the function of this gene in leukemia. After digestion by restriction endonuclease, the IGFBP7 gene was recombined with the transfer plasmid. The venus particles were packaged using 293T cells to transinfect K562 cells, and identification was performed by means of flow cytometry, RT-PCR and Western blot. The results showed that the sequence of cloned IGFBP7 gene was the same as that in GenBank. The size of product restricted by BamI-I I was same as the predicted one. GFP expression was observed in 293T and K562 cells with the fluorescent microscopy and flow cytomety. The expression level of mRNA and protein of IGFBP7 was confirmed by RT-PCR and Western blotting in K562 cells. It is concluded that venus vector carrying IGFBP7 ~ene has been successfuUv constructed and orovides basis for exolorin~ function of 1GFBP7 in K562 cells.