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目的 :人的肿瘤坏死因子相关凋亡诱导配体 (TNF relatedapoptosisinducingligand ,TRAIL)基因经扩增后 ,克隆在大肠杆菌中表达并重新折叠并获得有活性的TRAIL蛋白。方法 :用PCR的方法从人胎盘cDNA文库中扩增TRAIL基因 ,经序列分析鉴定 ,再克隆到原核表达载体 pET11a ,经E .coliBL2 1(DE3)表达 ,电泳鉴定后 ,柱层析纯化 ,重新折叠 ,流式细胞术等方法检测其促凋亡活性。结果 :得到了TRAIL基因 ,并构建了可在大肠杆菌中表达的载体 pET11 TRAIL1(含TRAILcDNA序列 418 933)及可在真核细胞中表达的载体pLNCX TRAIL2 (含TRAILcDNA序列 88 933)。取前者在E .coliBL2 1(DE3)中表达 ,经纯化 ,重新折叠得到了复性的TRAIL蛋白。检测其对人白血病细胞株Jurkat有诱导凋亡的作用。结论 :从人胎盘cDNA文库中扩增TRAIL基因 ,在大肠杆菌中表达、重新折叠、纯化后 ,经检测得到了有活性的TRAIL蛋白
Objective : Human TNF-related apoptosis-inducing ligand (TRAIL) gene was amplified, cloned and expressed in E. coli and refolded to obtain active TRAIL protein. METHODS: TRAIL gene was amplified from human placental cDNA library by PCR, identified by sequence analysis, and then cloned into prokaryotic expression vector pET11a, expressed by E.coli BL21 (DE3), identified by electrophoresis, and purified by column chromatography. Folding, flow cytometry and other methods to detect its pro-apoptotic activity. Results: The TRAIL gene was obtained and the vector pET11 TRAIL1 (containing TRAIL cDNA sequence 418 933) and the vector pLNCX TRAIL2 (containing TRAIL cDNA sequence 88 933) expressed in eukaryotic cells were constructed. The former was expressed in E. coli BL21 (DE3), purified and refolded to obtain renatured TRAIL protein. The effect of apoptosis on the human leukemia cell line Jurkat was examined. CONCLUSION: TRAIL gene was amplified from human placental cDNA library, expressed, refolded and purified in E. coli. After detection, active TRAIL protein was detected.