Ma Y, Jin X-B, Chu F-J, Bao D-Mei, Zhu J-Y
Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, People's Republic of China.
Appl Microbiol Biotechnol. 2014 Sep;98(18):7923-33. doi: 10.1007/s00253-014-5818-0. Epub 2014 Jun 8.
Recombinant human endostatin (rEndostatin or endostar) has been shown to inhibit endothelial cells proliferation, migration, and angiogenesis and exhibits a broad spectrum of activities against solid tumors. However, rEndostatin is easily degradable and evenly distributed to all tissues. Selectively delivering rEndostatin to the lesion site might be more potent. The circumsporozoite protein (CSP) coats the malarial sporozoite and targets the liver for infection; I-plus of N end of CSP could specifically bind to the liver. Based on this, we hypothesize the fusion protein with introducing the CSP I-plus sequence into rEndostatin (rES-CSP) of which not only targets the liver, but also inhibits endothelial cells proliferation, migration, and tube formation. Therefore, it selectively reduces angiogenesis of hepatocellular carcinoma (HCC) and improves the anti-HCC effect. In this study, we synthesized a novel rES-CSP fusion gene by SOE-PCR and expressed the fusion protein in Escherichia coli BL2l (DE3). The suitable conditions were optimized by an orthogonal test (L(25)(5)(4)). The yields were 12 mg/l culture medium following refolding and purification on nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography matrices. The purified rES-CSP is specifically targeted to the hepatocyte and inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner and showed potent antiangiogenic capability on HUVECs tube formation assay and chick embryo chorioallantoic membrane (CAM) assay. These results lay the foundation for the further study of its targeting and anti-HCC in vivo and provide a feasible and convenient approach to produce liver-targeting drugs for treatment of the liver diseases.
重组人内皮抑素（rEndostatin或恩度）已被证明可抑制内皮细胞增殖、迁移和血管生成，并对实体瘤表现出广泛的活性。然而，rEndostatin易降解且均匀分布于所有组织。将rEndostatin选择性地递送至病变部位可能更有效。环子孢子蛋白（CSP）覆盖疟原虫子孢子并靶向肝脏进行感染；CSP N端的I-plus可特异性结合肝脏。基于此，我们推测将CSP I-plus序列引入rEndostatin（rES-CSP）的融合蛋白不仅靶向肝脏，还能抑制内皮细胞增殖、迁移和管腔形成。因此，它可选择性降低肝细胞癌（HCC）的血管生成并提高抗HCC效果。在本研究中，我们通过SOE-PCR合成了一种新型rES-CSP融合基因，并在大肠杆菌BL2l（DE3）中表达了融合蛋白。通过正交试验（L(25)(5)(4)）优化了合适的条件。在镍-次氮基三乙酸（Ni-NTA）金属亲和色谱基质上进行复性和纯化后，产量为12 mg/l培养基。纯化的rES-CSP特异性靶向肝细胞，并以剂量依赖性方式抑制人脐静脉内皮细胞（HUVECs）的增殖和迁移，在HUVECs管腔形成试验和鸡胚绒毛尿囊膜（CAM）试验中显示出强大的抗血管生成能力。这些结果为其在体内的靶向性和抗HCC进一步研究奠定了基础，并为生产用于治疗肝脏疾病的肝靶向药物提供了一种可行且便捷的方法。
