人骨形成蛋白-2成熟肽在巴斯德毕赤酵母中的分泌表达  

Secreted expression of mature peptide of human bone morphogenetic protein-2 in Pichia pastoris

作　　者：董文[1,2] 杨莉莉[3] 孙健[1] 刘增君[1] 

机构地区：[1]天津市红桥医院骨科 [2]天津医科大学附属第一中心医院骨科,天津300192 [3]天津医科大学肿瘤医院研究所免疫室天津市"肿瘤防治"重点实验室

出　　处：《海南医学》2008年第3期37-39,共3页Hainan Medical Journal

摘　　要：目的利用甲醇毕赤酵母表达系统,高效分泌表达人骨形成蛋白-2成熟肽(hBMP-2)。方法利用PCR方法扩增得到hBMP-2基因,构建其毕赤酵母真核表达载体pPICZaC/hBMP2,电转化巴斯德毕氏酵母X-33,于28℃进行甲醇诱导分泌表达,用PCR法、SDS-PAGE、WesternBlot等方法筛选获得高效表达rhBMP-2的工程菌株,并进行了表达产物的纯化和活性测定。结果表达产物分泌至培养上清中,rhBMP-2含量达100mg·L-1。SDS-PAGE初步验证了表达产物的分子量,经WesternBlot和ELISA检测到重组表达产物的特异性结合活性。结论构建了重组hBMP-2的基因工程菌,并在毕赤酵母中实现了高效分泌表达,为进一步研究其活性和功能奠定了基础。Objective To construct and express mature peptide of human bone morphogenetic protein-2 （hBMP-2） in Pichia pastoris. Methods The gene encoding of hBMP-2 was amplification by PCR and cloned into Pichia pastoris expression vector pPICZaC. The recombinant pPICZaC/hBMP2 was transformed into the Pichia pastoris X-33 strain via electroporation. Then the rhBMP-2 was expressed induced by methanol at 28℃. The high level expression was selected and assayed by the methods of PCR, SDS-PAGE and Western Blot. The rhBMP-2 was purified and the bioactivity of it was initially assayed. Results The rhBMP-2 was secreted into the supernatant and the concentration reached 100mg·L^-1. The molecular mass was initially identified by SDSPAGE. And the rhBMP-2 was further identified by Western Blot and ELISA with specific antibody binding activity. Conclusions The rhBMP-2 was successfully constructed and expressed in Pichia Pastoris. And this contributes to further study of its function and activity.

关 键 词：人骨形成蛋白-2成熟肽 毕赤酵母 表达 

分 类 号：R68[医药卫生—骨科学]