VEGF和Smad7双基因过表达慢病毒载体的构建

目的：构建过表达VEGF和Smad7双基因的慢病毒载体，为勃起功能障碍基因治疗的研究提供有效工具。方法：根据GenBank中基因信息，设计合成VEGF和Smad7引物，采用overlap PCR方法扩增目的基因片段，运用基因重组技术将其克隆至慢病毒表达载体Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin，经酶切、测序对重组质粒进行验证。将重组质粒和Helper1.0、pHelper2.0辅助质粒共转染293T细胞，包装双基因过表达慢病毒并测试其滴度。结果：经酶切和测序鉴定表明VEGF和Smad7双基因重组慢病毒载体构建成功，荧光法测定重组慢病毒滴度高（2E+8TU/mL)。VEGF和Smad7重组慢病毒能高效转染293T细胞，Western blot检测显示VEGF和Smad7蛋白在靶细胞中过表达。结论：成功构建了携带VEGF和Smad7基因并能正确表达的高滴度的重组慢病毒载体。

The construction of lentiviral vector with over-express of VEGF gene and Smad7 gene

objective: To construct a lentiviral vector expressing the VEGF and the Smad7 gene, which is applied to gene therapy for erectile dysfunction.Methods: Primers of VEGF and Smad7 were designed according to the gene information of GenBank;The target gene fragment was amplified by overlap PCR.VEGF and Smad7 were cloned into lentivirus expression vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin by using gene recombination technique.The recombinant plasmids were identified by restriction enzyme digestion and DNA sequencing.The 293T cells were transfected with constructed plasmids,packaing system pHelper1.0 and pHelper2.0 plasmids.Lentivirus was packaged and the titer was determined. Results: The recombinant lentiviral vector of VEGF and Smad7 was successfully constructed by restriction enzyme digestion and DNA sequencing.Recombinantlentiviruses measured by fluorometry in high titer were obtained (2E + 8TU / ml).The gene VEGF and smad7 could be efficiently transfected into 293T cells by recombinant lentivirus,the overexpression of VEGF and smad7 protein in target cells were confirmed by Western Blotting. Conclusion: The recombinant lentiviral vector of VEGF and Smad7 genes can be produced successfully in high titer with correct expression.