携带双报告基因真核表达载体pHSV1-TK-IRES2-EGFP在小鼠骨髓间充质干细胞内的表达

背景：单纯疱疹病毒胸腺嘧啶核苷激酶（herpes simplex virus 1-thymidine kinase,HSV1-TK）与绿色荧光蛋白（EGFP）双报告基因共表达载体构建及其在骨髓间充质干细胞内的表达研究报道较少。目的：构建pHSV1-TK-IRES2-EGFP真核表达载体,并检测其在体外培养的小鼠骨髓间充质干细胞内的表达。方法：应用聚合酶链式反应从质粒pHSV106中扩增出HSV1-TK基因后与pMD18-T载体连接,构成重组质粒pHSV1-TK/18T。重组质粒以限制性内切酶BglⅡ和SalⅠ进行双酶切,将其插入同样双酶切处理的pIRES2-EGFP载体内;并将新构成的重组质粒pHSV1-TK-IRES2-EGFP以脂质体法转染小鼠骨髓间充质干细胞。结果与结论：酶切鉴定结果表明,扩增的HSV1-TK基因序列正确,大小为1130bp;重组质粒载体内的HSV1-TK序列与GeneBank报告的序列完全一致;骨髓间充质干细胞转染后在荧光显微镜下可以观察到有特异性绿色荧光出现,HSV1-TK的mRNA有表达。实验成功构建了pHSV1-TK-IRES2-EGFP真核表达载体,在体外培养的小鼠骨髓间充质干细胞内能有效的表达。

Construction and expression of pHSV1-TK- IRES2-EGFP eukaryotic vector in mouse bone marrow mesenchymal stem cells

BACKGROUND： Up to date, thestudies regarding the construction of herpes simplex virus 1-thymidine kinase （HSV1-TK） and enhanced green fluorescent protein （EGFP） reporter gene co-expression vector and its expression in mouse bone marrow mesenchymal stem cells are few. OBJECTIVE： To construct a pHSV1-TK- IRES2-EGFP eukaryotic vector, and to observe the expression of HSV1-TK in mouse bone marrow mesenchymal stem cells. METHODS： The HSV1-TK cDNA fragments were obtained by polymerase chain reaction （PCR） from pHSV106 and Bgl Ⅱ, Sal Ⅰ two restriction sites were added, after T vector cloning, the recombinant plasmid pHSV1-TK/18T was digested by two restrictive endonucleases, and then HSV1-TK cDNA was collected and recombined with eukaryotic vector pIRES2-EGFP by using gene recombination technique. The recombinant plasmid pHSV1-TK- IRES2-EGFP was transfected into mouse bone marrow mesenchymal stem cells in vitro with lipofectamine transfection reagent. RESULTS AND CONCLUSION： A length of 1 130 bp with Bgl Ⅱ and Sal Ⅰ target gene sequences was obtained by PCR. And the pHSV1-TK- IRES2-EGFP expression plasmid was constructed successfully. The TK/GFP green fluorescent protein was detected after transfection. At the same time, the expression of HSV1-TK mRNA was determined. The eukaryotic vector pHSV1-TK- IRES2-EGFP was successfully constructed, and effectively expressed HSV1-TK mRNA in mouse bone marrow mesenchymal stem cells in vitro.