RT-PCR法克隆目的基因构建表达hVEGF165的慢病毒载体

bstract： Objective To clone target gene by RT-PCR method and construct lentivirus vectors expressing hVEGF165 gene. Methods RT-PCR technology was employed to clone hVEGF165 gene,and this gene was cloned to lentivirus vector pLVX-EF1α-IRES2-AcGFP1 to construct a lentiviral vector pLVX-EF1α-hVEGF165-IRES2-AcGFP1, Bacterial colonies PCR and sequencing analysis were used for identification.Then，Lenti-X 293T cells were transfected with main vector pLVX-EF1α-hVEGF165-IRES2-AcGFP1, packaging plasmid gag-pro、vpr-pol、Tet-Off?、tat-IRES-rev and coated plasmid env（VSV-G）. lentiviral vectors were packaged and thetiter was determined. Results The hVEGF165 gene fragment was cloned successfully,and the lentiviral vector plasmid pLVX-EF1α-hVEGF165-IRES2-AcGFP1 was identified existing hVEGF165 gene fragment by Bacterial colonies PCR, DNA sequencing analysis confirmed that hVEGF165 gene sequencing was exactly the same with that reported by Genbank. After transfection，a large number of Lenti-X 293T cells with Green fluorescence was observed by fluorescent microscopy. The concentration of the virus titer was 1×108TU／mL. Conclusion hVEGF165 gene expression lentiviral vectors Can be successfully constructed by RT-PCR method to clone target gene.