利用同源重组构建大鼠甘油二酯激酶γ(DGKγ)慢病毒过表达载体  

Construction of rat diacylglycerol kinase gamma lentivirus overexpression vector by homologous recombination

作　　者：李蕾[1] 谢建山 杜家政 师亮[1] 崔慧林[1] Li Lei;Xie Jianshan;Du Jiazheng;Shi Liang;Cui Huilin(Department of Histology and Embryology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China)

机构地区：[1]山西医科大学组织胚胎学教研室

出　　处：《中国组织工程研究》2020年第2期260-264,共5页Chinese Journal of Tissue Engineering Research

基　　金：山西省优势重点学科项目(2011-2014),项目参与人:崔慧林;山西医科大学基础医学科技培植基金计划(201410),项目负责人:崔慧林;山西医科大学科技创新基金项目(01200719),项目负责人:崔慧林~~

摘　　要：背景:慢病毒载体作为外源性转基因载体已被广泛应用,但是大鼠甘油二酯激酶γ(diacylglycerol kinaseγ,DGKγ)基因慢病毒载体未见报道。目的:用同源重组的方法构建大鼠DGKγ慢病毒过表达载体。方法:提取成年SD大鼠脑组织总RNA,以反转录得到的cDNA作为模板,通过PCR反应分段扩增大鼠DGKγ基因CDS区5'端1 029 bp和3'端1 362 bp,用同源重组技术将这2个片段与线性化载体进行定向连接,构建CMV-rat DGKγ-GFP慢病毒载体并进行PCR扩增及测序鉴定。经293T细胞包装后产生慢病毒,收集慢病毒感染293T细胞,荧光显微镜下观察细胞中GFP的表达并应用实时荧光定量PCR和Western blotting法检测细胞中DGKγmRNA和蛋白的表达。结果与结论:CMV-rat DGKγ-GFP慢病毒载体经PCR扩增和测序鉴定构建成功;经CMV-rat DGKγ-GFP慢病毒感染后的293T细胞,荧光显微镜下呈GFP阳性,实时荧光定量PCR显示DGKγmRNA的表达较空载体组显著升高(P<0.01),Western blotting显示DGKγ蛋白表达较空载体组极显著升高(P<0.001)。提示:成功构建了大鼠DGKγ慢病毒过表达载体,DGKγ在293T细胞有效高表达。BACKGROUND:Lentiviral vectors have been widely used as exogenous transgenic vectors.However,a recombinant lentiviral vector containing rat diacylglycerol kinaseγ(DGKγ)gene has not been reported.OBJECTIVE:To construct lentiviral overexpression vector of rat DGKγby homologous recombination.METHODS:Total RNA was extracted from the brain tissue of adult Sprague-Dawley rats,and the cDNA obtained by PCR was used as a template to amplify the 5'-end 1 029 bp and the 3'-end 1 362 bp of the rat DGKγgene CDS.Then,the two homologous recombination fragments were ligated into the plasmid vector.The positive clones were confirmed by PCR and DNA sequencing.The CMV-rat DGKγ-GFP lentiviral vector and the lentiviral packaging system were co-transfected into 293T cells for virus packaging and lentivirus was collected to infect 293T cells.The expression of GFP in infected 293T cells was observed under fluorescence microscope.Real-time PCR and western blot assay were used to detect the relative expression of DGKγmRNA in infected 293T cells.RESULTS AND CONCLUSION:The results of PCR simplification and sequencing indicated that the CMV-rat DGKγ-GFP lentiviral vector was successfully constructed.In 293T cells infected with CMV-rat DGKγ-GFP lentivirus,the expression of GFP was observed under fluorescence microscope and the DGKγmRNA expression was increased significantly than that of the vector group by real-time PCR(P<0.01).Western blot assay results showed that the DGKγprotein expression of the selected GFP-positive 293T cells was increased very significantly(P<0.001).To conclude,the rat DGKγlentiviral overexpression vector has been successfully constructed and maintains high expression in 293T cells.

关 键 词：甘油二酯激酶γ DGKγ 同源重组 慢病毒载体 过表达 

分 类 号：R496[医药卫生—康复医学] R394.3[医药卫生—临床医学]