利用RNA干扰技术沉默p53基因突变子175H的效果评价

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。

Evaluation of effect of RNAi on silence of p53 mutant 175H

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-175...