Dissertation
Dissertation > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)

Construction and Identification of the Recombinant Lentiviral Vector Expressing p55PIK

Author CaoZhanJiang
Tutor CaoZhiXin
School Huazhong University of Science and Technology
Course General Surgery
Keywords Lentiviral p55PIK Proliferation
CLC R373
Type Master's thesis
Year 2011
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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 .

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