肿瘤坏死因子相关凋亡诱导配体的克隆、表达及重新折叠

The cloning, expression and refolding of TNF-related apoptosis inducing ligand

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 。

Objective: To clone human TRAIL(TNF related apoptosis inducing ligand) gene into E.coli expression vector to get active TRAIL protein after refolding and purification. Methods: The human TRAIL gene was amplified from the human placenta cDNA library, and cloned into prokaryotic expression vector pET11a after being sequenced. The plasmid was transformed into E.coli BL21 (DE3), followed by expression, refolding and purification. The protein activity was measured by flow cytometry. Results: The TRAIL 1 from 418 933 of the cDNA (the coding region of TRAIL was 111 281) and TRAIL 2 from 88 933 of the cDNA (coding for full length TRAIL) were obtained. The prokaryotic and eukaryotic expression vectors were pET11 TRAIL1 and pLNCX TRAIL2 respectively. The former was transformed and expressed in E.coli BL21 (DE3), then the refolded and purified protein was obtained. The apoptosis of human leukemia cell line Jurkat had been successfully induced by this TRAIL protein. Conclusion: The human TRAIL gene was amplified from the human placenta cDNA library, and after expression in E.coli , refolding and purification, the active TRAIL protein was obtained.