炎症信号受体TLR-4基因的克隆及真核表达

首都医科大学学报 ›› 2004, Vol. 25 ›› Issue (1): 40-43.

炎症信号受体TLR-4基因的克隆及真核表达

梁峰¹, 胡大一¹, 王伯瑶², 陈槐卿²

1. 首都医科大学附属北京同仁医院心脏中心;2. 四川大学华西医学中心

收稿日期:2003-05-21 修回日期:1900-01-01 出版日期:2004-01-15 发布日期:2004-01-15

Cloning and Expression of Inflammatory Signal TLR-4 Gene in HEK293 Cell

Liang Feng¹, Hu Dayi¹, Wang Boyao², Chen Huaiqing²

1. Cardiac Center, Beijing Tongren Hospital, Capital University of Medical Science;2. West China Medical Center, Sichuan university

Received:2003-05-21 Revised:1900-01-01 Online:2004-01-15 Published:2004-01-15

摘要/Abstract

摘要： 为探讨TLR-4基因在真核细胞的表达情况,用RT-PCR技术从人脐静脉血管内皮细胞总RNA扩增TLR-4全密码子cDNA序列。限制性内切酶HindⅢ和BamHⅠ双酶切真核表达质粒pcDNA3和TLR-4cDNA片段,构建重组质粒pcDNA3TLR-4。用Dosper阳离子转染试剂包裹质粒,转染人胚肾293细胞(HEK293细胞)。用免疫荧光细胞化学染色及流式细胞术分析TLR-4基因表达。结果:从人脐静脉血管内皮细胞扩增出2726bp的TLR-4cDNA片段。经PCR、酶切及序列测定分析证实质粒pcDNA3内插入了TLR-4cDNA片段,免疫荧光细胞化学染色和流式细胞术分析均检测到TLR-4基因在人胚肾293细胞表达。本研究显示重组真核表达质粒pcDNA3-TLR-4在HEK293细胞表达TLR-4蛋白,有利于进一步研究其相应配体和信号传导通路.

关键词: Toll样受体-4, 克隆, 真核表达

Abstract: This study was to examine TLR-4 gene expression in HEK 293 cells. Total RNA was isolated from human umbilical vein endothelial cells(HUVECs) by using trizol re agent. The sequences coding for TLR-4 were amplified and inserted into eukaryot ic expression vector pcDNA3 at HindⅢ and BamHΙsites. The pcDNA3-TLR-4 was tran sfected into HEK 293 cells by using Dosper liposomal transfectional reagent. The expression of TLR-4 was determined on the HEK 293 cells transfected with pcDNA 3 -TLR-4 by fluorescence immunocytochemisty and flow cytometry. 2 726 bp TLR-4 cD NA fragment was amplified from HUVECs by RT-PCR. The recombinant plasmid pcDNA3 -TLR-4 was confirmed by PCR, enzyme digestion and sequencing. Both immunocytoflu orescent staining and flow cytometric analysis showed TLR-4 expression in the H EK 293 cells that was transfected with pcDNA3-TLR-4. This study shows that when tran s fected with pcDNA3-TLR-4, the HEK 293 cells express TLR-4, in order to furthr d efine its ligands and signal pathway.

Key words: TLR-4, clone, eukaryotic expression

中图分类号:

R503

梁峰;胡大一;王伯瑶;陈槐卿. 炎症信号受体TLR-4基因的克隆及真核表达[J]. 首都医科大学学报, 2004, 25(1): 40-43.

Liang Feng;Hu Dayi;Wang Boyao;Chen Huaiqing. Cloning and Expression of Inflammatory Signal TLR-4 Gene in HEK293 Cell[J]. Journal of Capital Medical University, 2004, 25(1): 40-43.

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