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目的:纯化毕赤酵母菌GS115株表达的分泌型人endostatin蛋白,并对其抑癌活性进行研究。方法:利用SepharoseG-25及Heparin亲和层析柱纯化重组蛋白,并以MTT法测定重组endostatin对人脐静脉血管内皮细胞(ECV～304)增殖的抑制活性;另以HepG2.2.15细胞注射裸鼠成瘤后,体内验证重组蛋白的抑瘤活性。结果:MTT结果显示纯化后的endostatin在体外可特异性抑制人脐静脉内皮细胞的增殖,最高抑制率为86.0%;动物实验证明其可明显抑制荷瘤动物肿瘤的生长。结论:经纯化后的重组endostatin具有较强抑癌活性,将为临床抗血管生成治疗实体瘤研究奠定基础。
OBJECTIVE: To purify secreted human endostatin protein expressed by Pichia pastoris GS115 strain and study its anti-tumor activity. METHODS: The recombinant protein was purified by Sepharose G-25 and Heparin affinity chromatography. The inhibitory activity of recombinant endostatin on the proliferation of human umbilical vein endothelial cells (ECV-304) was determined by MTT assay. In addition, HepG2.2.15 cells were injected with nude mice After tumorigenesis, the anti-tumor activity of the recombinant protein was verified in vivo. Results: The results of MTT showed that the purified endostatin could specifically inhibit the proliferation of human umbilical vein endothelial cells in vitro with the highest inhibition rate of 86.0%. Animal experiments show that it can significantly inhibit tumor growth in tumor-bearing animals. Conclusion: The purified recombinant endostatin has a strong anti-tumor activity, which will lay the foundation for clinical anti-angiogenesis therapy of solid tumors.