人内皮抑素在毕赤酵母中的组成型表达及其活性检测  

Constitutive Expression of Human Endostatin in Pichia Pastoris and Bioactivity Assay

作　　者：梁丽敏[1] 李校坤[1] 苏志坚[1] 黄亚东[1] 吴晓萍[1] 郑青[1] 

机构地区：[1]暨南大学药学院生物制药教研室,广东广州510630

出　　处：《药物生物技术》2005年第5期294-297,307,共5页Pharmaceutical Biotechnology

基　　金：国家973项目子课题(No.G1999054204)

摘　　要：为获得大量具有生物学活性的重组人内皮抑素(Endostatin),在毕赤酵母中进行组成型表达的研究。将由毕赤酵母偏爱密码子组成的Endostatin cDNA插入组成型载体pGAPZαA中,构建表达载体pGAPZαA-ENDO,并转化到毕赤酵母X-33中。重组菌株在甘油醛-3-磷酸脱氢酶(GAP)启动子的调控作用下,进行组成型表达Endostatin蛋白。重组人Endostatin产量达到102mg/L。Western blot显示:表达蛋白能与兔抗Endostatin的多克隆抗体特异性结合。纯化后的重组Endostatin具有抑制人血管内皮细胞系ECV-304细胞增殖的活性。利用毕赤酵母组成型表达系统能获得大量具有生物活性的Endostatin蛋白。The aim is to produce large amount of human Endostatin with biological activity for further research. A high-yield expression of Endostatin in yeast Pichia pastoris was studied here. The synthetic Endostatin cDNA was inserted into a yeast constitutive vector, pGAPZαA. Under the control of the promoter GAP（glyceraldehydes 3-phosphate dehydrogenase） Endostatin was expressed constitutively. The expression level of Endostatin was 102 mg/L. This protein could be specifically immunoprecipitated by rabbit polyclonal antibodies against human Endostatin. After affinity purification, the Endostatin protein could restrain the proliferartion of endothelium cells in vitro. Human Endostatin with biological activity was successfully expressed by Pichia Pastris constitutive expression system.

关 键 词：重组人内皮抑素 毕赤酵母 组成型表达 

分 类 号：Q78[生物学—分子生物学]