人骨形成蛋白-7成熟肽基因在巴斯德毕氏酵母中的分泌表达  

Secreted Expression of Mature Peptide Gene of Human Bone Morphogenic Protein-7 in Pichia pastoris

作　　者：刘蕾[1] 王慧[2] 颜光涛[1] 史晶[2] 包士中[2] 荫俊[2] 

机构地区：[1]中国人民解放军总医院基础所生化室,北京100853 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2006年第6期862-864,共3页Letters in Biotechnology

摘　　要：目的:利用甲醇酵母表达系统,构建重组人骨形成蛋白-7(rhBMP-7)成熟肽的基因工程菌,并实现其分泌表达。方法:采用PCR方法,依据GenBank数据库中hBMP-7成熟肽序列(AccessionNo.NM_001719)设计并合成一对引物,从已构建的含hBMP-7的克隆载体中扩增获得人骨形成蛋白-7成熟肽基因片段,并以pPIC9K为表达载体构建重组表达质粒,电转化巴斯德毕氏酵母SMD1168,于30℃进行甲醇诱导分泌表达,经筛选获得rhBMP-7成熟肽表达株,并进行了表达产物的活性测定。结果:表达产物存在于培养上清中,约占分泌蛋白总量的8%。经Western印迹分析可以检测到重组表达产物,ELISA检测其具有特异性结合活性。结论:构建了重组人骨形成蛋白-7成熟肽的基因工程菌并在甲醇酵母中实现了分泌表达,为进一步的活性及功能研究奠定了基础。Objective： To construct and express mature peptide of human bone morphogenic protein-7 （hBMP-7） in Pichia pastoris. Methods： A pair of primers based on mature peptide of hBMP-7 gene sequence were designed according to C.enBank （Accession No.NM_001719）. The DNA sequence encoding the mature peptide of hBMP-7 was amplified by PCR and cloned into P.pastoris expression vector pPIC9K. The recombinant pPIC9K-hBMP7 was linearized and electroporated into P.pastoris SMD1168 strain. Then expression was induced by methanol at 30℃ in secreted form. The expressing strain was selected and assayed. Results： Cloned gene was expressed in soluble form by secreting into culture medium. Recombinant protein was of 8% total secreted protein and easily identified by Western blot and ELISA with specific antibody binding activity. Conclusion： The successful cloning and expression of rhBMP-7 mature peptide in P.pastoris are conductive to further study of its function.

关 键 词：人骨形成蛋白 成熟肽 巴斯德毕氏酵母 分泌表达 

分 类 号：Q785[生物学—分子生物学] Q786