Construction And Identification Of The Recombinant Lentiviral Vector Expressing P55PIK

Background and Objective: The objective of this researech is to construct a recombinant lentiviral vector expressing p55PIK, and we observe the effect of up-regulating p55PIK expression on cell proliferration.Methods: A pair of special primer were designed. The taget gene was amplified by PCR. After being digested by restriction endonucleases AscⅠa nd EcoRⅠ,the PCR product was connected to the plasmid pCDF. We amplified this recombinant plasmid in the DH5αcompetent cells. DNA sequencing conformed that the fragment inserted in the plasmid was the target gene.Then three plasmids of the lentiviral vector system were transformed to 293FT cells via liposome, so lentivirus was successfully packaged in 3 days.Green fluorescence could also be observed .We lysated those cells and got Lenti-p55PIK. After infecting the cancer cells in vitro, the expression of p55PIK was detected by Western blot,and expression of Ki67 was detected by flow cytometry.Results: After PCR product separated in the agrose electrophoresis,we could see a nearly 1400bp-length fragment,which was about the length of target gene.We slected the correct clone through endonucleases digesting and sent it to DNA sequencing. It was conformed that the fragment inserted in the plasmid pCDF accorded target gene exactly.In the progress of lentivirus packaging in the 293FT cells , we examined the efficiency by observing green fluorescence.When Lenti-p55PIK infected the cancer cells in vitro, green fluorescence was also observed.Compared with the blank lentivirus control and the blank control,Lenti-p55PIK could promote expression of p55PIK ( P<0.01 ) and Ki67(P<0.05),which is a cellular marker for proliferation.Conclusion: Lentivirus expressing p55PIK was successfully constructed. Lenti-p55PIK could promote expression of p55PIK and Ki67 in vitro.