J4 ›› 2010, Vol. 36 ›› Issue (2): 220-224.

• 基础研究 • 上一篇    下一篇

利用RNA干扰技术沉默p53基因突变子175H的效果评价

易贺庆1,2|马淑梅1|刘扬1|孔德娟1|高琳1|赵广通1|焦本蒸1|刘晓冬1   

  1. 1.吉林大学公共卫生学院 卫生部放射生物学重点实验室,吉林 长春130021;2.吉林省长春市卫生监督所|吉林 长春 130021
  • 收稿日期:2009-10-14 出版日期:2010-03-28 发布日期:2010-03-28
  • 通讯作者: 刘晓冬 (Tel:0431-85619418,E-mail:liuxd2002@yahoo.com) E-mail:liuxd2002@yahoo.com
  • 作者简介:易贺庆(1980-)|男|吉林省农安县人|在读医学博士|主要从事辐射分子生物学研究。
  • 基金资助:

    国家自然科学基金资助课题(30500142,30770649)

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

YI He-Qing1,2, MA Chu-Mei1, LIU Yang1, KONG De-Juan1, GAO Lin1, ZHAO An-Tong1, JIAO Ben-Zheng1, LIU Xiao-Dong1   

  1. 1.Key Laboratory of |Radiobiology,Ministry of Health,School of Public Health,Jilin University,Changchun 130021,China;2.Institution of Public Health Inspection of Changchun, Changchun 130021,China
  • Received:2009-10-14 Online:2010-03-28 Published:2010-03-28

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。

关键词: 基因, p53;突变子;基因沉默

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

中图分类号: 

  • R342.2