Construction And Expression Of Optimized Novel Human Immunodeficiency Virus Type-1 Ientiviral Vector Containing Vascular Endothelial Growth Factor 164 And Enhanced Green Fluorescent Protein

Vascular endothelial growth factor (VEGF) has been believed to be a specific mitogen for endothelial cells subserving angiogenesis and permeability in development and after injury. Recent studies have depicted the localization of VEGF and its receptors on neurons and astrocytes and it has been shown to induce neuritic growth and to provide neuroprotection particularly after ischemia or spinal cord injuries. Therefore, it becomes a major candidate factor in gene therapy for ischemic stroke, while how to choose the vectors is the key problem.Objective:Construct four plasmids system of HIV-1 lentiviral vector containing VEGF164 and enhanced green fluorescent protein (EGFP). Then they co-transfect 293T cells, in order to construct optimized novel HIV-1 lentiviral vector containing VEGF164 and EGFP.Methods:①Amplified with RT-PCR, the section about 573bp was obtained. This section was cloned into carrier pMD-18T so that we got reconstructive pMD18T-VEGF164.②The plasmids,pHR'CMV-EGFP,pIRES2-EGFP,were digested by restriction enzymes (Bam HI, Bst XI), then they were joined to construct the reconstructive pHR'CMV-IRES2-EGFP.③The plasmids, pHR'CMV-IRES2-EGFP, pMD18T-VEGF164, were digested by restriction enzyme Bam HI, then they were joined to construct the reconstructive plasmid.④The recombinant was identified by digestion and confirmed by sequening.⑤The four plasmid system, the reconstructive plasmid(pHR'CMV-VEGF164-IRES2-EGFP), pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G, was co-transfected into 293T cells. Collecting the supernatant of virus, we obtained lentiviral particles by high-speed centrifugation. Then observe fluorescence of the cells by fluorescence microscope and detect the expression of target gene by RT-PCR.Result:①The RT-PCR products were observed and photographed under ultraviolet lam, we saw three bright bands, they were 645bp(VEGF188),573bp (VEGF164),441bp (VEGF121). We obtained rat VEGF164 by recycling the target fragments.②The vector plasmid(pHR'CMV-VEGF164-IRES2-EGFP) was identified by restriction enzyme digestion analysis(Bam HI). The electrophoretogram displayed 9.8Kb and 573bp DNA fragments that were produced. Then it was identified by restriction enzyme digestion analysis (BstⅪ, XhoⅠ) again. The electrophoretogram displayed 9.7Kb and 720bpDNA fragments.③The gene segment was confirmed by sequencing. Contrasted with Genebank, finding that there was a base mutation on VEGF164 sequence, but it would not influence the expression of VEGF protein.④The four plasmid system, pHR'CMV-VEGF164-IRES2-EGFP, pCMV-VSV-G, pCMVA8.9, pRSV-Rev, was co-transfected into 293T cells.24 hours later, green fluorescence was observed by fluorescence microscopy, and it was evident at 48 hours. Seventy percent of the cells in each field of view were EGFP-positive cells. The lentiviral particles infected the 293T cells again, green fluorescence was observed by fluorescence microscopy, We extracted total RNA from 293T after infected, then RT-PCR, we found that the target gene expressed in the infected group, while had not express in the control.Conclusion:①We extracted total RNA from Wistar rats blood, then got the VEGF164 gene by RT-PCR, and the sequencing proved successful.②pHR'CMV-VEGF164-IRES2-EGFP was constructed by connecting reaction, and identified by restriction enzyme digestion analysis.③This research constructed four plasmids system of HIV-1 lentiviral vector containg VEGF164 and EGFP, they were co-transfected into 293T cells. Then collected the supernatant of virus and gained the optimized novel HIV-1 lentiviral vector containing VEGF164 and EGFP.