TFR和VEGF基因双启动子调控的慢病毒载体构建及其表达研究  

Construction and expression of dual promoter lentiviral vector containing TFR and VEGF genes

作　　者：魏梦绮[1] 沈琦[2] 罗颖[3] 宦怡[1] 刘莹[1] 杨勇[1] 张劲松[1] 路凡[2] 郑敏文[1] 

机构地区：[1]第四军医大学西京医院放射科,陕西西安710032 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032 [3]第四军医大学基础部病理生理学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2011年第12期1319-1321,1324,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金面上项目(30370821)

摘　　要：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。AIM： To construct a dual promoter lentivira vector containing TFR and VEGF gene, and detect the expression of TFR and VEGF genes in MSCs of Chinese miniswine. METHODS： The TFR and VEGF gene were amplified by polymerase chain reaction and cloned into pLentiGFP-Neo after the CMV and SV40 promoter respectively, to generate the lentivira vector pLenti-TG-VEGF. The fourplasmids lentiviral vector system（pRsv-REV, pMDIg-pRRE, pMD2G and pLenti-TG-VEGF）were cotransfected into human embryonic kidney 293T cells with Lipofectin 2000 reagent. The packaged virus was harvested 72 h later. MSCs were infected by lentivirus carrying TFR and VEGF genes. Western blotting was used to detect the expression of TFR and VEGF genes in the viral infected MSCs. RESULTS： Restriction endonuclease digestion analysis and DNA sequencing demonstrated that the dual promoter lentivirus vector containing TFR and VEGF genes were constructed successfully. MSCs could be successfully infected by the recombinant ientivirus. Expression of TFR and VEGF protein could be detected in the infected MSCs by Western blotting. CONCLUSION： The dual promoter lentiviral vector containing TFR and VEGF genes are constructed successfully which provides basis for future research on Cardiac molecular imaging of cell transplantation.

关 键 词：慢病毒载体 转铁蛋白受体 血管内皮生长因子 骨髓间充质干细胞 

分 类 号：R392.12[医药卫生—免疫学]