中文摘要：

目的：在p53基因缺失型细胞株H1299中转染p53基因突变子175H,构建H1299-175H细胞模型,并观察小发夹环RNA（shRNA）的基因沉默效果。方法：采用引物重叠PCR定点突变技术合成p53基因突变子175H,并利用基因重组技术构建出表达载体,以脂质体转染法建立细胞模型;合成针对p53-175H的shRNA并构建出反转录病毒表达载体Psuper-175HR,应用磷酸钙共沉淀法导入包装293T细胞,并收集假病毒颗粒感染H1299细胞。应用Western blotting法检测P53蛋白表达。结果：转染p53基因突变子175H的H1299-175H细胞模型P53蛋白表达阳性;H1299-175H细胞模型转染shRNA后,P53蛋白表达下调。结论：成功构建H1299-175H细胞模型;构建的Psuper-175HR基因沉默载体可以有效地抑制p53基因突变子175H的表达。

英文摘要：

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.