小鼠Toll样受体9基因的克隆、表达及其活性的初步鉴定  

Cloning and expression of toll-like receptor 9 of murine and identification of its biological activity

作　　者：彭丽娜[1,2] 邱亚峰[1] 繆剑华 李向东[1] 王晓杜[1] 袁经权[3] 王寿昆[2] 李力[3] 马志永[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]福建农林大学动物科学学院,福建福州350002 [3]广西壮族自治区药用植物园,广西南宁530023

出　　处：《福建农林大学学报（自然科学版）》2010年第2期154-158,共5页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：广西自然科学基金资助项目(08320152)

摘　　要：从小鼠巨噬细胞中提取细胞总RNA,采用RT-PCR技术扩增小鼠Toll样受体9(mTLR9)cDNA,构建真核表达质粒p3XFLAG-CMV-7.1-mTLR9,转染293T细胞,利用Western Blotting检测蛋白表达,并用双荧光素酶报告基因系统检测mTLR9对其介导的信号通路下游转录因子NF-κB转录活性影响.结果表明,本试验成功克隆到小鼠TLR9cDNA,构建的重组体转染细胞后表达的蛋白分子质量与预计相符,并能激活下游转录因子NF-κB的转录活性.Toll like receptor 9 is an important member of the toll like receptors family.In order to clone and express the murine TLR9 and identify its biological activity,the total RNA was extracted from mouse macrophage,TlR9 full length cDNA was amplified by RT-PCR,and cloned into p3XFLAG-CMV-7.1 plasmid.Recombined plasmid was transfected and expressed in 293T cell.The expressed protein was assayed by Western Blotting.NF-κB is a downstream transcription factor of signal pathway mediated by TLR9,its luciferase activity was measured by dual luciferase reporter system.The result of Western Blotting and luciferase analysis revealed that the murine TLR9 cDNA was cloned successfully,and the molecular weight of recombinant TLR9 was almost the same as predicted value.The transcription activity of NF-κB was activated by TLR9.This study provided a insight for constructing a stable transfected HEK293T cell line model which can express murine TLR9 gene,and for the futher study of molecular mechanisms of TLR9-mediated signaling pathway and biological function.

关 键 词：小鼠Toll样受体9 基因表达 NF-ΚB 转录活性 

分 类 号：Q786[生物学—分子生物学]