中文摘要：

目的：构建髓系细胞触发受体-1（TREM-1）的真核表达质粒。方法：利用RT-PCR从人外周血总RNA中扩增TREM-1基因的全长cDNA,插入克隆载体pUCm-T,BamHI和Pst I,酶切后将目的片段插入真核表达载体pEGFP-C3,构建含有TREM-1的重组质粒pEGFP-TREM-1,转染293细胞,用荧光显微镜观察、Western-blot鉴定蛋白表达。将pEGFP-TREM-1重组质粒转染THP-1细胞,100 ng/ml LPS刺激24 h后,半定量RT-PCR检测TNF-α和IL-1β基因的mRNA水平。结果：经酶切和基因测序证实,克隆的基因片段为TREM-1基因;经转染的293细胞在荧光显微镜下可见荧光,并能表达TREM-1蛋白;重组TREM-1蛋白具有生物学活性,能增强TNF-α和IL-1β基因的mRNA水平。结论：成功构建了能够表达TREM-1的真核表达质粒pEGFP-C3-TREM-1,为进一步研究该基因的功能奠定了实验基础。

英文摘要：

Objective： To construct a eukaryotic expression plasmid containing human triggering receptor expressed on myeloid cells-1（TREM-1） gene.Methods： The entire gene coding region of human TREM-1 was amplified from total RNA of human peripheral blood by means of RT-PCR.The fragment of TREM-1 was cloned to vector pUCm-T.After digestion by restriction endonuclease BamH I and Pst I,the fragment was subcloned into the eukaryotic expressing vector pEGFP-C3.This recombinant vector was transfected into 293 cells using liposome.The expression level of TREM-1 was determined by fluorescence microscope and Western blot assay.The recombinant TREM-1 vector was transfected into THP-1 cells.After stimulation with 100 ng/ml LPS for 24 h,the mRNA levels of TNF-α and IL-1β were measured using RT-PCR.Results： The expression vector was constructed,and the result of the DNA sequencing showed that the constructed plasmid containing the TREM-1 gene.Fluorescence microscope and Western blot analysis showed that TREM-1 protein was expressed in 293 cells successfully.After transfection into THP-1 cells,recombinant TREM-1 could upregulate the mRNA levels of TNF-α and IL-1β.Conclusion： Eukaryotic expression plasmid pEGFP-TREM-1 is successfully constructed and showed biological activity.