Abstract:

Abstract:Objective To establish the cell model expressing p53-175H and detect the gene silence effect of shRNA in H1299 (p53-/-) cell line. Methods PCR site-directed mutagenesis was used to gain p53-175H cDNA sequence,gene recombinant technique was used to construct the p53-175H expression vector and the vector was transferred into H1299（p53-/-）cells by liposome in vitro.The shRNA sequence targeting p53-175H was synthesized and inserted into retroviral vector Psuper to construct retro-Psuper-175HR.The retro-Psuper-175HR was transfected into packaging cell 293T using calcium phosphate co-precipitation and the viral soup containing sham-viral particle was used to infect H1299 cells directly.Western blotting was used to detect the protein expression of P53.Results The expression of P53 protein in the p53-175H cell model was positive.After transfection in H1299-175H cell model,the P53 protein expression was decreased.Conclusion The H1299-175H cell model is established successfully.Psuper-175HR gene silence vector can effectively suppress the expression of P53 mutant 175H in H1299-175H cells.

Key words: gene,p53;muton;gene silencing