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目的 :在毕赤酵母菌 (GS115 )中表达有活性的重组人分泌型血管内皮抑素蛋白 ,探讨其对鼠移植性肝癌的抑制作用。方法 :成功构建的分泌型酵母表达载体 ,经电转化法转入酵母菌中 ,SDS PAGE电泳筛选阳性重组子 ,经Heparin亲和层析柱进行初步纯化。MTT法测定重组人血管内皮抑素对脐静脉血管内皮细胞 (ECV 30 4 )增殖的抑制作用 ;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 :构建的酵母表达载体经PCR、测序鉴定完全正确。SDS PAGE电泳证实重组菌较空白菌多一明显条带 ,Heparin亲和层析后 ,1MNaCl洗脱的蛋白峰在体外可特异性抑制血管内皮细胞的增殖 ,体内可明显抑制鼠移植性肝癌的生长 ,并诱导肿瘤细胞凋亡。结论 :在毕赤酵母菌中成功表达了有活性的重组血管内皮抑素蛋白 ,对肿瘤治疗具有很好的开发前景。
Objective : To express active recombinant human secretory endostatin protein in Pichia pastoris (GS115) and to investigate its inhibitory effect on rat transplanted liver cancer. Methods: The secretory yeast expression vector successfully constructed was transformed into yeast by electroporation. The positive recombinants were screened by SDS PAGE electrophoresis and purified by Heparin affinity chromatography. MTT assay was used to determine the inhibitory effect of recombinant human endostatin on the proliferation of umbilical vein endothelial cells (ECV 30 4 ). After HepG 2 .15 cells were injected into nude mice, the inhibitory activity of the recombinant protein was verified in vivo. Results : The constructed yeast expression vector was completely verified by PCR and sequencing. SDS PAGE electrophoresis confirmed that the recombinant strain was more than that of the blank strain. After Heparin affinity chromatography, the protein peak eluted by 1M NaCl could specifically inhibit the proliferation of vascular endothelial cells in vitro, and the growth of rat transplanted liver cancer could be significantly inhibited in vivo. , and induce apoptosis of tumor cells. Conclusion: The active recombinant endostatin protein was successfully expressed in Pichia pastoris, which has a good prospect for the development of tumor therapy.