Construction And Expression Of Optimized Novel Human Immunodeficiency Virus Type-1 (HIV-1) Lentiviral Vector Containing Green Fluorescent Protein

Drug treatment and surgery operation are common therapeutic methods of cerebral ischemia. However, the limited curative effect of drug, the expensive cost of operation, the indication and the contraindication, all make these two ways not satisfied. With the development of scientific methods. Gene therapy is focused on, and vector is the key point. HIV-1 lentiviral vector can infect unseparated cells, can transfer longer fragment, can make objective gene have a long stabilized expression, and can not cause immune reaction of the host easily. It is an ideal gene vector, especially in the gene therapy of cerebral ischemia. HIV-1 lentiviral vector is constructed by reconstituting the genome of wide type HIV-1 lentivirus. Now lentiviral genome is seperated into three plasmids: packaging plasmid, envelope plasmid, vetor plasmid. By cotransfection of three plasmids, lentiviral particles are produced by 293T cells. This kind of lentiviral particle can only infect target cell or target tissue once, and dose not have the power of replication. In packaging plasmid, under the promoter of CMV, the gene sequences express all viral proteins except envelope protein and the assistant protein Vpu. In envelope plasmid, VSV-G gene took the place of env gene, then the lentiviral particle with VSV-G envelope can be produced at last. This kind of lentiviral particle can infect more kinds of cells or tissues, and they are more stable. Gene sequences in the three plasmids have less homology with each other. So probability of producing RCV decreased during coinfecton. In vector plasmid, there are objective gene or reporter gene, Rev response element (associates with RNA transport to cytoplasm from cell nucleu), cis-acting elements (needed by packaging,reverse transcription and integration). In this research, we constructed four plasmids system, in order to increse biosafety of HIV-1 lentiviral vector, wide type HIV-1 genome was segregated into four plasmids: vector plasmid, gag-pol plasmid, rev plasmid and envelope plasmid. Moreover, in vector plasmid, there was a deletion in U3 of the 3'tip. It made virus particle have character of self-inactiviating, and made target gene express under special promotor.Objective: Construct four plasmids system of HIV-1 lentiviral vector. Then they cotransfect 293T cells, in order to construct optimized novel HIV-1 lentiviral vector containing green fluorsecent protein and with VSV-G pseudo-capsule.Methods: Wide type HIV-1 genome was segregated into four plasmids: vector plasmid, gag-pol plasmid, rev plamid and envelope plasmid. These four kinds of plasmid were cleaved respectively by corresponding restriction enzyme, in order to confirm that they can release corresponding size gene fragments by electrophoretogram. Results told that four plasmids system was constructed successfully. Isolate plasmids according to endofree plasmid maxi kit. Four plasmids system cotransfected 293T cells, then assembled the cell supernatant. We can obtain lentiviral particles by high speed centrifugation, and determine the virus titre. Observe fluorescence of the cells by fluorescence microscope.Result:①Vector plasmid (pHR-CMV-EGFP) was cleaved by restriction enzyme (BamHI,XhoI), electrophoretogram displayed that 700bp and 8.6Kb fragments were released. gag-pol plasmid (pCMV△8.9) was cleaved by restriction enzyme (Spel, SalI), electrophoretogram displayed that 1.428Kb, 3.6Kb and 5.07Kb fragments were released. Rev plasmid (pRSV-Rev) was cleaved by restriction enzyme (BglII, XhoI), electrophoretogram displayed that 3.6Kb and 600bp fragments were released. Envelope plasmid (pCMV-VSV-G) was cleaved by restriction enzyme (XhoI), electrophoretogram displayed that 1.6Kb and 5.3Kb fragments were released. According to every plasmid's structure chart, four plasmids system were constructed successfully.②Four plasmids system cotransfected 293T cells, 48 hours later, green fluorescence was observed by fluorescence microscope, intensity of fluorescence was higher when it was 72 hours. Lentiviral particles tranfected 293T cell once again to detemined the viral titre, after 72 hours, calculated EGFP positive cells under fluorescence microscope, there were 80% cells which were EGFP positive in every visual field, the viral titre was 4×108TU/ml.Conclusion:①Construct four plasmids system of optimized novel HIV-1 lentiviral vector successfully by segregated wide type HIV-1 genome into four plasmids: vector plasmid, gag-pol plasmid, rev plamid and envelope plasmid. These plasmid were identified by cleavage and electrophoresis.②Four plasmids system cotransfected 293T produce cells, green fluorescence was observed by fluorescence microscope after 48 hours. The viral titre was 4×108TU/ml after centrifugalization.This research constructed optimized novel HIV-1 lentiviral vector containg green fluorescent protein and with VSV-G pseudo-capsule. The lentiviral particle titre can be 4×108TU/ml after centrifugalization. Our reserch can be as the base work of gene therapy in cerebral vascular diseases.