人源TRAIL胞外段基因的克隆、表达与功能的初步检测  

Cloning and function of extraeellular fragment of human TRAIL gene

作　　者：赵昆朋[1] 王玉刚[2] 陈居杲[1] 黎燕[2] 马远方[1] 沈倍奋[2] 

机构地区：[1]河南大学医学院细胞与分子免疫室,开封475001 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《中华微生物学和免疫学杂志》2007年第5期457-460,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金项目（No.30571697）

摘　　要：目的 获得人源TRAIL胞外段结构域在大肠杆菌中可溶性表达，并初步鉴定其功能。方法 RT-PCR法从人外周血单个核细胞中扩增TRAIL胞外段基因，克隆入pGEM-T-Easy载体，DNA序列测定鉴定正确性。为了便于纯化目的蛋白，将其克隆入原核表达载体pET-30a，并在其氨基端加上组氨酸标签。采用E．coliBL21（DE3）表达，Ni亲和柱分离纯化目的蛋白。SDS-PAGE和Westernblot鉴定原核表达产物。MTT法、PI染色法及瑞氏．姬姆萨染色法观察TRAILHis蛋白的生物学活性。结果 所表达目的蛋白相对分子质量（Mr）与预期蛋白Mc相一致，该蛋白分别可以与抗TRAIL多克隆抗体及抗组氨酸标签抗体反应，并可抑制人淋巴瘤细胞系Jurkat细胞的增殖和诱导Jurkat细胞凋亡。结论 获得了具有生物学活性的TRAIL蛋白，为对其生物学功能及肿瘤治疗的进一步研究奠定了基础。Objective To obtain recombinant soluble TRAIL protein with biological activity. Methods Human TRAIL extracellular gene was amplified from PBMC and cloned into pGEM-T-Easy vector for sequence analysis. The expression vector pET-30a/TRAIL was constructed by DNA recombinant method, and the recombinant protein was expressed in E. coli BL21（DE3）. The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE and Western blot. The proliferation inhibition activity of TRAIL-His was detected by MTT method. PI staining and Wright-Giemsa staining assay were used to detect cell apoptnsis. Results The target protein expressed in E. coli BL21（DE3） have the same Mr as that of expected and could be recognized by anti- TRAIL Poly-Ab and anti-His McAb. The protein could also inhibit the proliferation and induce apoptosis of Jurkat cells. Conclusion Recombinant human TRAIL protein with biological activity was obtained. This prospective research laid solid foundations for further research on its biological activity and application in anti-tumor therapy.

关 键 词：TRAIL 原核表达 免疫印迹 

分 类 号：R686[医药卫生—骨科学]