Construction Of Non-fusion Dual Target Tk-ires-tum5Recombinant Adeno-associated Virus And Study In Vitro

Background:Prostate cancer (prostate cancer, PCa) incidence of sustained growth and sustainedincrease in mortality is a very important public issue. The current method of treatment ofPCa include surgery, radiation therapy and endocrine therapy. However, these methodsrely on non-PCa therapeutic effect is not ideal for hormones. Gene therapy is likely a wayto cure cancer. In this study, tumor endostatin build a functional fragment (Tum5) andsuicide gene (TK) non-fusion recombinant adenovirus-associated virus (adeno-associatedvirus, AAV), sustained expression of AAV infection PCa cells secrete Tum5protein,prevents cancer internal neovascularization, dual target therapy PCa.Objective:1.Build pAAV-TK, pAAV-Tum5, pAAV-TK-IRES-Tum5, pAAV-Tum5-IRES-TKplasmid.2.The rAAV-TK, rAAV-Tum5, rAAV-TK-IRES-Tum5, rAAV-Tum5-IRES-TK virusparticles packaging, concentration, purification and identification.3.Recombinant adeno-associated virus rAAV-TK, rAAV-Tum5, rAAV-TK-IRES -Tum5, rAAV-Tum5-IRES-TK function in vitro experiments.Methods:1.Tum5and HSV-TK genes into pIRES-MCS various sites by gene recombinationtechnology,build pAAV-TK, pAAV-Tum5, pAAV-TK-IRES-Tum5, pAAV-Tum5-IRES-TK plasmid by digestion Identification of plasmid correct.2.Using AAV Helper free system transfect HEK293cells to packaging virus; adopt"chloroform-PEG/NaCl precipitation-chloroform extraction" separation, concentrationand purification of viruses.3.Culture HUVEC and PC3cells. by fluorescence microscopy, quantitative real-timePCR, Western blot detection of viral infection efficiency, cell growth was observed byinverted microscope, by MTT assay and flow cytometry apoptosis and cell cycle changes.Results:1.Successfully constructed pAAV-TK, pAAV-Tum5, pAAV-TK-IRES-Tum5, pAAV-Tum5-IRES-TK plasmid. Successfully been concentrated recombinant adeno-associatedvirus rAAV-Tum5, rAAV-TK, rAAV-Tum5-IRES-TK, rAAV-TK-IRES-Tum5.2.The rAAV-Tum5, rAAV-TK, rAAV-Tum5-IRES-TK, rAAV-TK-IRES-Tum5wereable to successfully infect PC3and HUVEC cells, and the ability to express the targetprotein, Tum5protein can inhibit the ability of HUVEC tube formation, promote HUVECapoptosis, TK gene transfected PC3cells whose apoptosis is much larger than the controlgroup.Conclusion:Constructed AAV-TK-IRES-Tum5recombinant adeno-associated virus can infectPC3and HUVEC cells and the ability to express the target protein. Tum5protein inhibitsthe ability of HUVEC tube formation and promote apoptosis, TK gene promotes apoptosisin PC3cells, lay the foundation for further in vivo experiments.