死亡受体5胞外区域的重组、表达及活性鉴定

目的: 构建死亡受体5(death receptor 5,DR5)胞外区域(eDR5)的表达载体,表达纯化重组蛋白并鉴定其生物特性.方法: 通过重叠 PCR 获得 DR5 胞外段编码序列,构建 pET-22b( )/DR5 表达载体,转化大肠杆菌 BL21(DE3),IPTG 诱导表达,Ni2 柱亲和纯化,SDS-PAGE、直接 ELISA 鉴定纯化产物的纯度和特异性,用 MTT 法检测eDR5 蛋白阻断DR5 单克隆抗体 FMU1.5 和 TRAIL 诱导人胶质瘤细胞株 U343(高表达DR5)、U373(低表达DR5 )细胞凋亡的作用.结果: 获得了 DR5 胞外段编码序列,目的蛋白在上清及包涵体中都有表达, 表达量占菌体总蛋白的 30% 以上,纯化的重组蛋白纯度达 95% 以上,蛋白产量达 9 mg/ml.ELISA 结果表明所纯化蛋白为eDR5.eDR5 蛋白可部分阻断 FMU1.5 和 TRAIL 诱导人胶质瘤细胞株 U343 细胞凋亡的作用,其阻断率与 DR5 表达相关.结论: 死亡受体 5 胞外段基因的成功重组、表达及纯化,为进一步的功能研究奠定了基础.

Construction and expression of extracellular domain of human death receptor 5 and detection of its biological activity

Objective: To construct the expressing vector of the extracellular domain of death receptor 5 (DR5), express it E.coli, identify the purified DR5 protein, and study its biological activity. Methods: The extracellular domain of DR5(eDR5) was assembled by overlapping PCR. The expression vector pET-22b(+)/ DR5 was constructed and transformed into E.coli BL21(DE3). The expression of eDR5 protein was induced by IPTG and purified by Ni 2+ -affinity chromatographic column. The purity and specificities were detected by SDS-PAGE and ELISA, respectively. The blocking effects of purified eDR5 on FMU1.5-induced apoptosis of U343, U373 cells were observed. Results: The extracellular domain of DR5 was obtained by overlapping PCR. The eDR5 protein was expressed in both supernatants and inclusion bodies with a yield more than 30% of total bacterial proteins. The purity of eDR5 was more than 95% and the yield reached 9 mg/ml. The result of ELISA showed the purified protein was eDR5. Purified eDR5 partially blocked the apoptosis of U343 cells induced by FMU1.5 and TRAIL. Conclusion: The successful construction, expression, and purification of the extracellular domain of DR5 protein lays a foundation for further study of DR5 function.