Antiviral Sensitization And Apoptosis Induction Of NDPK-A And Its Mutants

Objective:In order to study the activity of antiviral sensitization and inducing apoptosis of NDPK-A and its mutants and their difference, nm23-H1 and its mutators were amplified and recombinant vectors pEGFP-nm23-H1, pEGFP-nm23-H1 C4S, pEGFP-nm23-H1 CallS were prepared.Methods:Taking pBV220-nm23-H1, pBV220-nm23-H1 C4S, pBV220-nm23-H1 CallS as templates, the nm23-H1 and its mutators were amplified using corresponding primers respectively and inserted into the vector pEGFP after digested with Xho I and EcoR I. Western blot was used to identify whether NDPK-A and its mutants were expressed after the recombinant vectors were transfected into cells. Simultaneously, NDPK-A and its mutants were located in cells by DAPI dyeing and laser focusing. Subsequently, pEGFP-nm23-H1, pEGFP-nm23-H1 C4S and pEGFP were transfected into the vero cell, and the stable cell lines were screened with G418 to dectect the anti-virus sensitization activity of NDPK-A and its mutants by CPE and plaque methods. In addition, pEGFP-nm23-H1, pEGFP-nm23-H1 C4S, pEGFP-nm23-H1 C4S and pEGFP were transiently transfected into A549 cell, then the cells with green fluroence were sorted using flow cytometry and dyed with PI to dectect the apoptosis of A549 affected by NDPK-A and its mutants.Results:We had succeeded in constructing recombinated vectors, and the NDPK-A and its mutants were expressed and located in the endochylema. the stable cell lines were achieved, and the CPE and plaque methods both proved that NDPK-A and NDPK-A C4S showed the synergistic effect of anti-virus to the antiviral drug nucleoside analogue, especially the synergistic effect of NDPK-A C4S is better than that of NDPK-A. Flow cytometry showed that NDPK-A and its mutants could urge the apoptosis of cancer cell A549, and the apoptosis rate among NDPK-A and its mutants follow that NDPK-A C4S>NDPK-A CallS>NDPK-A.Conclusions:The construction of recombinated vectors has been prepared for the research of NDPK-A and its mutants in vitro. The expression of NDPK-A and its mutants in cell and location in the endochylema is the capital terms for the research of the activity in cell of NDPK-A and its mutants. Comparing with NDPK-A wild type, its mutants showed higher activity in the synergistic effect of anti-virus and inducing cancer cell into apoptosis, which not only establishs the foundation but also provides the better outlook for study on the correlation between structure and bioactivities, the mechanism and the gene therapy of NDPK-A.