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目的:克隆TNF相关的诱导凋亡配体胞膜外段基因（the extracellular regionof the human TRAIL cDNA,TPAILex）,构建表达载体并在大肠杆菌DH5α中高效表达有生物学活性的TRAIL蛋白胞膜外段。方法:抗CD3McAb刺激正常人外周血淋巴细胞,用RT-PCR、巢式-PCR方法从活化的淋巴细胞中获得人TRAIL基因及其胞膜外段oDNA,并克隆到pGEM-T Easy载体中,DNA测序鉴定。将TRAIL的胞膜外段基因插入表达载体pGEX-2T,构建重组表达质粒pGEX/TRAILex,用IPTC诱导表达TRAIL蛋白的胞膜外段,GST-Agarose4B层析柱纯化 GST-TRAILex融合蛋白,Western-blot鉴定纯化产物。结果:抗CD3 McAb能诱导正常人外周血单个核细胞TRAIL的高表达,成功地克隆了TRAIL胞膜外段基因,构建了重组表达载体pGEX/TRAILex,IPTG诱导后得到高效表达的TRAIL蛋白胞膜外段,经12％SDS-PAGE分析,可观察到一分子量和理论值相符（约54kD）的诱导表达带。Kodak软件分析表面GST-TRAIL胞膜外段融合蛋白的表达量占菌体蛋白总量的28％,进一步分析证实TRAIL蛋白的胞膜外段主要以可溶性的形式表达,用GST-Agarose4B层析柱纯化超声破菌后的上清,每升细菌培养液可得到9.8mg纯度的95％以上的GST-Tex。Western-blot结果证实54kD的表达带可与鼠抗TRAIL McAb起特异性反应。结?
OBJECTIVE: To clone the TNF-related extracellular region of the human TRAIL cDNA (TPAILex), construct an expression vector and express the biologically active TRAIL protein extracellular membrane segment in E. coli DH5α . Methods: Human peripheral blood mononuclear cells were stimulated with anti-CD3McAb. The human TRAIL gene and its extracellular domain oDNA were obtained from activated lymphocytes by RT-PCR and nested-PCR, and cloned into pGEM-T Easy vector. DNA sequencing identification. The extracellular membrane fragment of TRAIL was inserted into the expression vector pGEX-2T to construct the recombinant expression plasmid pGEX / TRAILex. The outer membrane of TRAIL protein was induced by IPTC, the GST-TRAILex fusion protein was purified by GST-Agarose 4B chromatography, Western- blot identification of purified product. RESULTS: Anti-CD3 McAb could induce the high expression of TRAIL in normal human peripheral blood mononuclear cells. The outer segment of TRAIL gene was successfully cloned and the recombinant expression vector pGEX / TRAILex was constructed. The highly expressed TRAIL protein membrane was induced by IPTG The outer segment, induced by a 12% SDS-PAGE analysis, showed a molecular weight and theoretical value consistent (about 54kD) induced expression zone. Kodak software analysis of surface GST-TRAIL outer membrane fusion protein expression accounted for 28% of the total bacterial protein, further analysis confirmed that the extracellular membrane of TRAIL protein is mainly expressed in soluble form, with GST-Agarose4B column Purified sonicated supernatant, bacterial culture broth per liter can be obtained 9.8mg purity of 95% or more of GST-Tex. Western-blot results confirmed that the expression of 54kD band with the mouse anti-TRAIL McAb from the specific reaction. Knot?