Toll样受体4小干扰RNA表达载体的构建及鉴定筛选  

Construction and identification of small interference RNA plasmid expression vectors of Toll like receptor 4

作　　者：耿红莲[1] 王爱华[1] 陆慧琦[1] 赵文静[1] 邓燕[1] 竺蓓[1] 仲人前[1] 

机构地区：[1]第二军医大学长征医院实验诊断科全军临床免疫中心,上海200003

出　　处：《中国实验诊断学》2010年第10期1509-1511,共3页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金(30700762)

摘　　要：目的构建并筛选到能有效抑制TLR4基因表达的小干扰RNA表达载体,用于研究TLR4基因功能。方法 RNA干扰采用体外构建小干扰RNA表达载体法,将构建好的表达载体转染单核细胞株,分别用流式细胞术和实时荧光定量RT-PCR法检测TLR4蛋白和mRNA表达变化,筛选抑制效率最高的小干扰RNA表达载体。结果成功构建了TLR4小干扰RNA表达载体,并经电泳及测序证实;编号为S8-1的载体转染入细胞后,TLR4阳性率为27.03%,显著低于对照组的35.12%;流式细胞术和实时荧光定量RT-PCR法检测均证实编号为S8-1小干扰RNA表达载体的抑制效率最高,可用于后续实验。结论本实验所构建的编号为S8-1小干扰RNA表达载体可有效抑制TLR4基因表达,小干扰RNA表达载体法能够抑制体外培养细胞株TLR4的表达,可用于研究TLR4基因功能。Objective To construct and identify the small interference RNA（siRNA） plasmid expression vectors of Toll like receptor 4（TLR4） that can inhibit the expression of TLR4 efficiency.Methods Cultured monocyte cell line U937 was transfection with the TLR4 siRNA plasmid expression vectors.And then the expression of TLR4 was measured by using flow cytometry and real-time RT-PCR.Results The siRNA plasmid expression vectors were constructed successfully.The positive rate and the mean mRNA copy number of TLR4 was down regulated significantly after the trsnfection of No.S8-1 siRNA plasmid expression vector.Conclusion No.S81 siRNA plasmid expression vector can inhibit the expression of TLR4 and can be used in the TLR4 gene function study.

关 键 词：TOLL样受体4 小干扰RNA 表达载体 转染 

分 类 号：R346[医药卫生—基础医学]