p53基因RNA干扰载体的构建及其在ST细胞系中的稳定表达  

Generation and characteristic analysis of a stable ST cell line with p53 gene expression knock-down using sh RNA-expressing plasmids

作　　者：沈海燕[1,2,3] 张春红[1,2,3] 刘志成[1,2,3] 孙俊颖[1,2,3] 卢宇 张建峰[1,2,3] 

机构地区：[1]广东省农业科学院动物卫生研究所,广东广州510640 [2]广东省兽医公共卫生公共实验室,广东广州510640 [3]广东省畜禽疫病防治研究重点实验室,广东广州510640 [4]国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《中国兽医科学》2016年第8期979-985,共7页Chinese Veterinary Science

基　　金：国家自然科学基金青年基金项目(31302101);国家国际科技合作专项(2014DFA31730);广东省科技计划项目(2014B070706011;2014A010107022;2015A020209081;2013B010102012);公益性行业(农业)科研专项(201303046)

摘　　要：利用质粒介导的RNA干扰技术,建立稳定沉默p53基因的ST细胞系。通过改造p EGFP-C1载体,构建了猪源U6启动子启动编码p53 sh RNA序列的载体,将其转染ST细胞,用G418筛选细胞,并分析获得细胞的生长特性。RT-PCR和Western-blot结果显示,质粒介导的sh RNA有效地沉默了p53基因的表达;流式细胞术和细胞生长特性分析结果显示,与对照组细胞相比,p53基因沉默组ST细胞处于S期的细胞数量增多,而且p53基因沉默组ST细胞生长较快。本研究结果证实质粒介导的sh RNA高效、稳定地沉默了ST细胞中p53基因的表达。The study was to establish a stable ST cell line with p53 gene silencing via shRNA-expressing plasmind mediated RNA interference（RNAi）.ThisRNAi cell line established the foundation for the further research on explore the significance of p53 inhibition in the virus infection, shRNA-expressing plasmid targeting the p53 gene and the negative shRNA-expressing plasmid were constructed and transfected into ST cells. The positive cell clones were selected using G418,and isolated using the method of partial digestion. The expression of p53 was detected by SYBR Green I PCR and western blot. The effect of the p53 gene silencing on the cell cycle and cell proliferation was analyzed by flow cytometry and CCK-8 method,respectively. The results of SYBR Green I PCR and Western-blot demonstrated that the expression of p53 was substantially down-regulated by RNAi. The flow cytometry and CCK-8 results indicared that p53 shRNA-expression plasmid arrested the cell cycle in S phase,and promoted the cell proliferation. This RNAi cell line will be a useful tool for studying the biological functions of p53.

关 键 词：P53基因 sh RNA载体 RNA干扰 细胞系 

分 类 号：S852.4[农业科学—基础兽医学]