Lv S, Ye M, Wang X, Li Z, Chen X, Dou X, Dai Y, Zeng F, Luo L, Wang C, Li K, Luo X, Yan J, Li X
Neoplasma. 2015;62(4):531-40. doi: 10.4149/neo_2015_064.
Tumor vascular targeting is one of the most promising strategies in tumor therapy. Here we used E.coli to express a recombinant SP5.2/tTF fusion protein, which, as a tumor vascular targeting agent, consists of SP5.2 (a peptide selectively binding and targeting VEGFR-1 on tumor endothelial cells) and truncated tissue factor (tTF)and aimed to explore its anti-tumor activities.The SP5.2/tTF expression construct was synthesized by polymerase chain reaction (PCR) and recombined into plasmid pET22b(+). The fusion gene was verified by restriction mapping and sequencing. SP5.2/tTF was expressed in E. coli and then purified on a nickel-affinity chromatography column. The purified product was detected by SDS-PAGE. The pro-coagulant activity and binding of SP5.2/tTF to human umbilical vein endothelial cells (HUVECs) were monitored by FX activation analysis and fluorescent scanning confocal microscopy, respectively. The effect of SP5.2/tTF on tumor growth was analyzed in BALB/c mice bearing sarcoma 180 (S180) tumor. The tissue localization of SP5.2/tTF and its effect on tumor vessel thrombosis were observed by in vivo fluorescence imaging and histological studies, respectively. The fusion gene was successfully cloned into pET22b(+). SP5.2/tTF was abundantly expressed in bacterial cells and efficiently purified by nickel-affinity chromatography. Functional studies showed that the protein retained both the coagulation activity of tTF and the binding capacity of SP5.2 to HUVECs. In tumor xenograft studies, SP5.2/tTF selectively targeted the tumor, induced thrombosis, and led to retardation and even regression of tumor growth (growth inhibition ratio = 70%, P< 0.05). The recombinant fusion protein SP5.2/tTF inhibited tumor growth by selectively inducing thrombosis in tumor blood vessels.
肿瘤血管靶向是肿瘤治疗中最具前景的策略之一。在此，我们利用大肠杆菌表达一种重组SP5.2/tTF融合蛋白，作为肿瘤血管靶向剂，它由SP5.2（一种选择性结合并靶向肿瘤内皮细胞上VEGFR-1的肽）和截短的组织因子（tTF）组成，旨在探索其抗肿瘤活性。通过聚合酶链反应（PCR）合成SP5.2/tTF表达构建体，并将其重组到质粒pET22b(+)中。通过限制性酶切图谱和测序验证融合基因。SP5.2/tTF在大肠杆菌中表达，然后在镍亲和色谱柱上进行纯化。通过SDS-PAGE检测纯化产物。分别通过FX激活分析和荧光扫描共聚焦显微镜监测SP5.2/tTF的促凝血活性及其与人脐静脉内皮细胞（HUVECs）的结合。在携带肉瘤180（S180）肿瘤的BALB/c小鼠中分析SP5.2/tTF对肿瘤生长的影响。分别通过体内荧光成像和组织学研究观察SP5.2/tTF的组织定位及其对肿瘤血管血栓形成的影响。融合基因成功克隆到pET22b(+)中。SP5.2/tTF在细菌细胞中大量表达，并通过镍亲和色谱有效纯化。功能研究表明，该蛋白保留了tTF的凝血活性以及SP5.2与HUVECs的结合能力。在肿瘤异种移植研究中，SP5.2/tTF选择性靶向肿瘤，诱导血栓形成，并导致肿瘤生长延缓甚至消退（生长抑制率 = 70%，P < 0.05）。重组融合蛋白SP5.2/tTF通过选择性诱导肿瘤血管血栓形成来抑制肿瘤生长。
