炎症信号受体TLR-4基因的克隆及真核表达

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pcDNA3 TLR4。用Dosper阳离子转染试剂包裹质粒 ,转染人胚肾 2 93细胞 (HEK 2 93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 2 72 6bp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 2 93细胞表达。本研究显示重组真核表达质粒 pcDNA3 TLR4在HEK 2 93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

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

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.