LMP-1基因慢病毒载体构建及其在大鼠骨髓间充质干细胞的表达

目的：构建人LMP-1重组慢病毒载体，体外转染大鼠骨髓间充质干细胞，检测LMP-1基因在大鼠骨髓间充质干细胞的表达。方法：利用PCR法从cDNA文库中钓取LMP-1基因，将其与经AgeI酶切线性化的慢病毒载体pGC-FU-EGFP相连接，转化感受态大肠杆菌，筛选出阳性克隆pGC-FU-LMP-1-EGFP，基因测序对其鉴定。经293T细胞包装后，收集富舍病毒颗粒LV-LMP-1-EGFP的细胞上清，浓缩并标定滴度，RT-PCR检测并鉴定。以最佳MOI值体外转染大鼠骨髓间充质干细胞，荧光显微镜观察转染是否成功，流式细胞仪检测转染效率，RT-PCR和Westernblot检测转染细胞LMP-1基因的表达。结果：①基因测序及RT-PCR检测证实携带人LMP-1基因的慢病毒载体构建成功，包装后获得滴度为2×10^8 TU／ml的LV-LMP-1-EGFP。②以MOI=100转染大鼠骨髓间充质干细胞，荧光显微镜下可见大量绿色荧光蛋白表达，转染效率可达93．5％，经RT-PCR与Westernblot检测，被转染细胞内有LMP-1基因表达。结论：成功构建携带人LMP-1基因的慢病毒载体，可高效转染大鼠骨髓间充质干细胞，被转染细胞可高效表达LMP-1基因。

Construction of lentivirus vector containing human LIM mineralization protein-1(LMP-1)and its expression in rat bone mesenchymal stem cells

Objective ：To construct a recombiant lentivirus vector of human LMP-1 and detect the expression of LMP-1 in infected rat bone mesenchymal stem cells. Methods ：LMP-1 gene from the cDNA library were extracted by Polymerase Chain Reaction （PCR）. The LMP-1 genes were connected into lentiviral vectors pGC-FU-EGFP which was linearized by Age I enzyme to produce recombiant ｜entivirus vector called as pGC-FU-LMP-1-EGFP ,then packaged by 293T cells. The virus supernant congtaining LV-LMP-1-EGFP was harvested,concentrated and titrated. The rat BMSCs were transfected with re- combiant lentivirus LV-LMP-1-EGFP at the most appropriate MOI. The mRNA and protein expression of LMP-1 were detected by RT-PCR and Western blot. Results： ①LV-LMP-1-EGFP was recombined successfully and the titer reached 2×10^8 TU/ml. ②The efficiency of infection was 93.5% ,which was get after LV-LMP-1-EGFP infected rat BMSCs at the most appropriate MOI=100. The expression of LMP-1 gene was confirmed by RT-PCR and Western blot. Conclusion：Lentivirus vector con- taining human LMP-1 gene is constructed successfully,which can transfected efficiently into rat BMSCs, and the infected rat BMSCs can effectively express LMP-1.