目的：采用原核表达系统表达人Kininogen D560148TRAIL114281融合蛋白，并对其生物学活性进行研究。方法：PCR技术扩增Kininogen D560148和TRAIL114281的编码序列，分别构建原核表达载体pMALKininogen D560148（pMALKD5）、pMALTRAIL114281（pMALTRAIL）和pMALKininogen D560148 TRAIL114281（pMALKT），重组质粒分别转化大肠杆菌BL21，IPTG诱导表达融合蛋白MBPKD5、MBPTRAIL和MBPKT，并经亲和层析纯化。MTT法检测细胞的增殖，管状形成实验检测内皮细胞血管形成，流式细胞仪和电镜检测细胞凋亡。结果：成功构建原核表达载体pMALKD5、pMALTRAIL和pMALKT，并获得纯化的融合蛋白MBPKD5、MBPTRAIL和MBPKT。融合蛋白MBPKT与MBPKD5、MBPTRAIL相比可显著抑制内皮细胞ECV304和胰腺癌细胞SW1990的增殖、明显抑制ECV304细胞体外管腔的形成，同时，MBPKT剂量依赖性地诱导SW1990细胞凋亡。结论：融合蛋白Kininogen D560148 TRAIL114281既能诱导肿瘤细胞凋亡又能抑制血管生成，为进一步开发靶向性抗肿瘤药物奠定了基础

Objective:To express Kininogen D560148TRAIL114281 fusion protein using prokaryotic system and observe its biological functions.Methods: The Kininogen D560148 gene and TNFrelated apoptosisinducing ligand (TRAIL114281 ) gene were amplified by PCR and were cloned into pMAL expression vector to construct recombinant pMALKininogen D560148 (pMALKD5), pMALTRAIL114281 (pMALTRAIL) and pMALKininogen D560148TRAIL114281 (pMALKT) plasmids, respectively. The plasmids were transformed into E. coli BL21 and were efficiently expressed after IPTG induction. The purified MBPKD5, MBPTRAIL and MBPKT proteins were obtained by amylose resin affinity purification column. The proliferation of cells was measured by MTT; tube formation of endothelial cell was detected by tube formation assay; and the apoptosis of cells were observed by electron microscopic and FCM.Results: Prokaryotic expression vectors pMALKD5, pMALTRAIL and pMALKT and their purified fusion proteins MBPKD5, MBPTRAIL and MBPKT were successfully obtained. MBPKT significantly inhibited the proliferation of ECV304 endothelial cells, SW1990 pancreatic cancer cells and the tube formation of ECV304 cells compared with those of MBPKD5 and MBPTRAIL. Meanwhile, MBPKT dosedependently induced the apoptosis of SW1990 cells.Conclusion: Kininogen D560148TRAIL114281 fusion protein can inhibit the proliferation of tumor cells and angiogenesis of endothelial cells, which lays a foundation for further research on tumortargeting drugs.

上海市现代生物与新药产业发展基金（No. 024319115）