PTD4-apoptin Induced Apoptosis Of Hela Cells And Its Effects On Bak/bax And AIF Genes

Objective: Expression of the PTD4-apoptin fusion protein by the prokaryotic express ion vctor of the pGEX-6p-1-PTD4-apoptin,and get the soluble fusion protein of the PT D4-apoptin by isolation and purification,verify the PTD4-apoptin fusion protein promote activity of the apoptosis in Hela;to investigate the effect on PTD4-apoptin fusion propte in on pro-apoptotic genes bak/bax,cytochrome c,Caspase-3 and apoptosis inducing facto r(AIF),and the role of the AIF in apoptin induced apoptosis of tumor cells,the apopti n induced mechanism of apoptosis of tumor cells.Methods: Transforming prokaryotic expression vetor pGEX-6p-1-PTD4-apoptin to str ain of Ecoli.BL21(DE3),induced by IPTG,etraction and purification of the protein.After PTD4-apoptin fusion protein add the media of the Hela cells,observe the phenom enon of Hela apoptosis,verfy that the apoptosis of the fusion protein,and detection of t he inhibitory effect of fusion protein on Hela cells by CCK8 kit.Then,the changes of bak/bax expression,the content of cytochrome c and Caspase-3 in the cytoplasm,and th e content of AIF in mitochondria and cytoplasm in Hela cells were dectectd by RT-PC R and western-blot after PTD4-apoptin action.Inhibition of AIF release from mitochondr ia by cyclosporin A,detection of inhibitory effect of PTD4-apoptin on Hela cells and va lidation of AIF effect on PTD4-apoptin induced apoptosis of Hela cells.Result: The soluble PTD4-apoptin fusion protein,which can induce the apoptosis of Hela cells and significantly inhibit the growth of Hela cells,was successfully obtained.PTD4-apoptin fusion protein induce significantly increased expression level of mRNA an d protein of bak/bax in Hela cells.The contents of cytochrome c and Caspase-3 in cyto plasm were significantly,and the conten of AIF in cytoplasm significantly higher than t hat in the mitochondria.PTD4-apoptin fusion protein inhibited the growth of Hela cells by inhibiting the release of AIF from mitochondria to the cytoplasm.Concusion: PTD4-apoptin fusion protein could induce the apoptosis of Hela cell act ivity;PTD4-apoptin probably by increasing the expression of bak/bax,cause the content o f cytochrome c and Caspase-3 in cytoplasm increased,the change of mitochondrial mem brane permeability and AIF from mitochondria released into the cytoplasm,causing apop tosis of Hela cells.