摘要
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 gain recombinant endostatin in Pichia pastoris (GS115)and study its inhibitory effect to transplanted liver cancer.Methods:Constructed Pichia expression vector pGAPZ a A transformed GS115 cells.SDS PAGE was used to assay the expression of endostatin in recombinant GS115.After purified with Heparin,MTT was used to assay inhibitory effect of recombinant endostatin to ECV 304 cells.Furthermore,nude mice were injected with HepG2.2.15 cells,internal inhibitory activity of recombinant endostatin to tumor was also detected.Results:PCR?DNA sequencing result showed Pichia expression vector pGAPZ a A was successfully constructed.MTT assay showed the recombinant endostatin could inhibit the proliferation of ECV 304 cells in vitro.Internal assay showed the recombinant endostatin could inhibit the growth of transplanted liver cancer.Conclusion:Recombinant human endostatin protein is successfully expressed in Pichia pastoris.
出处
《中国现代普通外科进展》
CAS
2002年第3期145-148,共4页
Chinese Journal of Current Advances in General Surgery
基金
中美合作课题 (CU 990 9 0 1)
