人干细胞白血病基因重组慢病毒载体的构建及其在Cajal样间质细胞中的表达

Construction of recombinant human stem cell leukemia gene lentiviral vector and its expression in Cajal-like interstitial cells

目的构建含人干细胞白血病(SCL)基因的重组慢病毒载体,并转染体外培养的Cajal样间质细胞(ICC),为进一步利用慢病毒表达载体行体内实验奠定基础。方法通过聚合酶链反应(PCR)将人SCL基质粒内的遗传物质扩增,与慢病毒载体质粒GV287-EGFP结合,构建重组质粒GV287-EGFP/SCL,通过Western blot检测及基因测序对阳性克隆进行鉴定,并测定病毒滴度。体外分离、培养及鉴定ICC,重组慢病毒载体以感染复数(MOI)值为0.5、1.0、5.0、10.0、50.0和100.0时,分别转染ICC,通过激光共聚焦显微镜观察,计算不同MOI值的转染效果,确定最佳MOI值。重组慢病毒载体以最佳MOI值感染ICC作为实验组,以空载体转染的ICC(空载体组)及未转染的ICC(空白组)作为对照,通过逆转录聚合酶链反应(RT-PCR)检测SCL基因在ICC中的表达。结果经Western blot检测及基因测序鉴定SCL重组慢病毒表达载体构建成功,并成功转染ICC,激光共聚焦显微镜下可观察到强绿色荧光。MOI为10时转染效果最佳,转染效率>85%;RT-PCR结果表明,实验组SCL基因的表达量高于空载体组和空白组。结论成功制备人SCL基因重组慢病毒载体,并能高效转染ICC,介导SCL基因在ICC中表达。

Objective To construct recombinant lentiviral vectors containing human stem cell leukemia （SCL） gene and to observe its ability in transfecting Cajal-like interstitial cells and mediating SCL gene expression. Methods The SCL gene was amplified from plasmid with SCL gene by PCR, connected to the shuttle plasmid GV287-EGFP to construct GV287-SCL, the obtained replication-defective recombinant lentiviral GV287-SCL was propagated in 293T cells according to the steps of Invitrogen Lipofectamine 2000. The recombinant lentiviral GV287-SCL was purified and titrated by dilution technique. The transfected Cajal-like interstitial cells were cultured in vitro, the distribution and efficiency of recombinant lentiviral mediated SCL were observed by the expression of green fluorescence protein （GFP） under the fluorescent microscope. RT-PCR were used to measure the expression of SCL mRNA in the transfected Cajal-like interstitial cells. Results Western blot and gene sequencing confirmed that the SCL gene was successfully inserted into the lentiviral vector, and SCL recombinant lentiviral vector was successfully transfected into Cajal-like interstitial cells. The adenovirus had a high transfection efficiency of up to 85% when MOI was 10. RT-PCR showed the expression of SCL mRNA in the Cajal interstitial cells of the experimental group was lower than that of the vector group and the blank group. Conclusions The recombinant lentiviral vector containing human SCL gene has been successfully constructed by homogenous recombination in bacteria, and it has a high transfection efficiency and can mediate expression of SCL gene in Cajal-like interstitial cells.