HCMV-IE1基因siRNA真核载体的构建及其效率研究  

Construction of a eukaryotic expression vector harboring the small interfering RNA targeting HCMV-IE1 gene and its gene silencing efficiency

作　　者：谢妮[1,2] 韩艳萍[1,2] 吴瑾滨[1,2] 袁建辉 

机构地区：[1]深圳大学医学院附属第一医院 [2]深圳市第二人民医院,广东深圳518035 [3]深圳市疾病与预防控制中心,广东深圳518020

出　　处：《南方医科大学学报》2010年第9期2077-2079,2083,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30500599);深圳市科技计划项目(201002054)~~

摘　　要：目的采用RNA干扰技术研究在体外对人巨细胞病毒即刻早期Ⅰ基因(HCMV-IE1)表达的沉默作用。方法根据HCMV-IE1基因表达框,设计siRNA干扰序列,重组至带U6启动子真核表达载体,构建针对HCMV-IE1基因的siRNA真核表达载体(pHCMV-IE1i);经测序鉴定后,脂质体法转染HEL细胞HCMVAD169株,应用荧光显微技术、流式细胞术和RT-PCR法检测分析pHCMV-IE1i对HCMV-IE1基因的表达干扰效果。结果测序结果显示针对HCMV-IE1基因的pHCMV-IE1i构建成功;荧光显微结果表明pHCMV-IE1i转入HEL细胞后可以特异抑制细胞中HCMV-IE1基因的表达;流式细胞术结果显示对照细胞组HCMV-IE1基因表达阳性率达60%以上,干扰细胞组HCMV-IE1基因表达阳性率仅达10%左右(P<0.05);RT-PCR检测结果发现pHCMV-IE1i能显著抑制HCMV-IE1基因的表达(P<0.05)。结论采用RNAi技术能有效抑制HCMV-IE1基因在细胞内的表达,对探讨其在防治HCMV感染疾病中的作用与意义提供了科学的实验依据。Objective To investigate the effect of RNA interference targeting human cytomegalovirus immediate early gene 1（HCMV-IE1） on the gene expression in vitro.Methods According to the sequence of HCMV-IE1 gene,the small interfering RNA（siRNA） sequences were designed and introduced into the eukaryotic expression vector containing the U6 promoter.After verification by sequence analysis,the recombinant eukaryotic plasmid（pHCMV-IE1i） was transfected into HEL HCMVAD169 cells.The effectiveness of HCMV-IE1 gene silencing was investigated by fluorescence microscopy,flow cytometry and RT-PCR.Results Sequence analysis confirmed successful construction of the recombinant eukaryotic expression plasmid pHCMV-IE1i.The expression of HCMV-IE1 was effectively suppressed by pHCMV-IE1i transfection in HEL cells as shown by fluorescence microscopy,flow cytometry（P〈0.05） and RT-PCR（P〈0.05）.Conclusion The expression of HCMV-IE1 can be effectively suppressed by RNA interference technique in vitro,which provides experimental data for prevention and treatment of HCMV infection.

关 键 词：人巨细胞病毒即刻早期I基因 基因表达 RNA干扰 真核载体 

分 类 号：R346[医药卫生—基础医学]