Construction Of Recombinant Adenoviruses Carrying Melittin Gene And Study On Their Tumour Inhibition In Vitro

OBThCTIVE: To consmiCt recombinant adenoviruses caping melittin gene, underthe control of CMV (cytomegalovirus ) promoter or AFP(Q -fetoProtein) transcriPtionreguatory element (rAFP), and observe their tUrnor idriition in viho, as well as theireffeCtS on theorigenicity ofhePatOcarcinoma cells.METHODS: Melittin gene was synthesized chendcally, with itS rare codonsconverted into human optical ones, its trdrislation initiation region oPtimized with Kozakconservative sequence, and was driven by CMV promoter or rAFP. Anerwars gene ofinterest was recombined into adenovira backbone plasmd through a new sbolifiedbacterial homologous recombinan system, then the recombinam adenoviral plasnddswere linearized with Pac I and transfected 293 cells mediated by lipofectin to generatethe desired recombinan adenoviruses. After the resutan viruses containing melittin geneinfected the hepatocarcinoma cells, transcriPion of meldri gene was verified by methodof RT-PCR, While proliferation inhibition of Which observed by Mri assay and 3H-TdRincorporation. In order to observe effect of transgene on tUmorigenicity ofhepatocarcinoma cells, AFP was measuremented by ELISA double antibody sandwichedmethod, and eXPression of CD54 by flow cytomecy ComPared with CMV promoter wealso observed Whether transgene, under the control of rAFP could sPecifically inhibitproliferation of AFPTositive cells.RESULTS: Recombinan adenoviral vectors containing melittin gene wersuccessfully contricted, and it was me1ittin gene that could be transcripted proved byRT-PCR. At 48th hour after infection with adenovirus, indicated by MTT assay and 3H-TdR incorporation, changes of cell proliferation were mainly caused by virus but melittin gene. This period were characterized by proliferation of hepatocarcinoma cells inhibited and stimulated respectively by low or high titers of viruses. At 72th hour, effect of viruses per se disappeared, meanwhile inhibition of melittin gene on cell proliferation appeared, with positive correlation to logarithmic titers of viruses, suggesting which was caused by the expression of melittin gene. About effect of transgene on tumorigenicity of hepatocarcinoma cells, melittin gene showed decreasing expression of AFP and CD54. Comparing between rAFP and CMV promoter indicated that transgene, under the control of rAFP, could indeed specifically inhibit proliferation of AFP-positive cells.CONCLUSIONS: After transducted into cells, melittin gene could be transcripted and translated, furthermore, its effect was enough to inhibit tumour cell proliferation, and change their biological behavior, indicating it is possible that melittin play antitumour role by transgene.