Construction and Identification of the Recombinant Lentiviral Vector Expressing p55PIK
|School||Huazhong University of Science and Technology|
|Keywords||Lentiviral p55PIK Proliferation|
Objective: To construct expression of p55PIK lentiviral vector Lenti p55PIK and observe the impact the high expression p55PIK tumor cell proliferation . The the steps methods: get the people p55PIK full coding region fragment was amplified using PCR method by restriction enzyme digestion , ligation, transformation and plasmid extraction recombinant lentiviral expression plasmid p55PIK-pCDF, digestion and gene sequencing . The liposomal transfection Law 293FT cells transfected pCDF- p55PIK pFIV pVSVG, packaging can produce green fluorescent lentiviral Lenti - p55PIK . Tumor cells will be of lentiviral Lenti-p55PIK infection , the expression p55PIK determined by Western blot , the expression of cell-associated antigen Ki67 proliferation by flow cytometry , observed of p55PIK cell proliferation . Results : PCR product was subjected to agarose gel electrophoresis to obtain about 1400bp bands, size and p55PIK coding gene sequence of the same size . Plasmid by digestion of recombinant p55PIK-pCDF preliminary identification , proof of the p55PIK coding gene sequences inserted into the pCDF plasmid . Constructed plasmid by restriction enzyme digestion the preliminary identification evacuation sequencing , results in full compliance with the p55PIK coding gene sequences . The the pCDF-p55PIK, pFIV with pVSVG transfected 293FT cells packaging , packaging cell efficiency can be observed under a fluorescence microscope , cracking cells resulting virus Lenti - p55PIK . Infected tumor cells displayed green fluorescence in the cells were observed under a fluorescence microscope , Lenti-p55PIK Western the blot assay p55PIK of expression was significantly higher than the empty virus group and blank control group ( P lt; 0.01 ) , flow Ki67 expression , proliferation and cell-associated antigen - cell technology detects results show the Lenti-p55PIK group was significantly higher than the empty virus group and blank control group , (P lt; 0.05). Conclusion: We successfully constructed people p55PIK lentiviral expression vector Lenti - p55PIK high in tumor cells the expression people p55PIK protein , but also observed p55PIK promote the proliferation of cell-associated antigen Ki67 expression .