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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
To investigate the expression of TLR4 gene in eukaryotic cells, full-length TLR4 cDNA sequence was amplified by RT-PCR from total RNA of human umbilical vein endothelial cells. The restriction endonucleases Hind Ⅲ and BamH Ⅰ double digestion of eukaryotic expression plasmid pcDNA3 and TLR 4 cDNA fragments to construct recombinant plasmid pcDNA3 TLR4. Plasmid was wrapped with Dosper cationic transfection reagent and transfected into human embryonic kidney 293 cells (HEK2 93 cells). TLR4 gene expression was analyzed by immunofluorescence cytochemistry and flow cytometry. Results: The 7272 bp TLR4 cDNA fragment was amplified from human umbilical vein endothelial cells. The TLR4 gene fragment was inserted into the pcDNA3 plasmid by PCR, restriction analysis and sequencing analysis. The expression of TLR4 gene in human embryonic kidney 293 cells was detected by immunofluorescence staining and flow cytometry. This study showed that recombinant eukaryotic expression plasmid pcDNA3 TLR4 expressed TLR4 protein in HEK2 93 cells, which is conducive to further study of its corresponding ligand and signal transduction pathway