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目的:构建含有转铁蛋白受体(TFR)和血管内皮生长因子(VEGF)的双启动子慢病毒载体,体外转染中华小型猪骨髓间充质细胞并检测其表达。方法:PCR法分别扩增TFR和VEGF基因,分别克隆入pLenti-GFP-Neo慢病毒载体的CMV启动子和SV40启动子后,构建出双启动子调控的慢病毒表达载体pLenti-TG-VEGF。利用Lipofectin2000试剂将pRsv-REV、pMDlg-pRRE、pMD2G共转染293T细胞进行慢病毒包装,72 h后收集病毒上清,感染中华小型猪骨髓间充质细胞,并通过Western blot法检测TFR和VEGF的表达情况。结果:成功构建了含有TFR和VEGF基因的双启动子慢病毒载体;包装好的慢病毒颗粒可成功感染中华小型猪骨髓间充质细胞;Western blot法检测TFR和VEGF高水平的表达。结论:成功构建了双启动子调控的慢病毒载体pLenti-TG-VEGF,并建立其慢病毒表达系统,从而为进一步探讨活体内观察移植细胞携带治疗基因的MRI基因成像应用的可行性奠定了基础。
OBJECTIVE: To construct a double promoter lentiviral vector containing transferrin receptor (TFR) and vascular endothelial growth factor (VEGF), and to transfect Chinese miniature pig bone marrow mesenchymal cells in vitro and detect their expression. Methods: The TFR and VEGF genes were amplified by PCR and cloned into the CMV promoter and SV40 promoter of pLenti-GFP-Neo lentiviral vector respectively to construct the lentiviral vector pLenti-TG-VEGF. 293T cells were co-transfected with pRsv-REV, pMDlg-pRRE and pMD2G by Lipofectin2000 reagent, and then lentivirally packaged. After 72 h, the virus supernatant was collected and infected with Chinese miniature pig bone marrow mesenchymal cells. TFR and VEGF The expression of the situation. Results: The double promoter lentiviral vector containing TFR and VEGF gene was successfully constructed. The packaged lentivirus particles could successfully infect Chinese miniature pig bone marrow mesenchymal cells. The high level expression of TFR and VEGF was detected by Western blot. CONCLUSION: The double promoter-mediated lentiviral vector pLenti-TG-VEGF was successfully constructed and its lentiviral expression system was established, which laid the foundation for the further study on the feasibility of using MRI gene imaging in transplanted cells to carry the therapeutic gene .