人骨形态发生蛋白6(hBMP6)在毕赤酵母中的分泌表达  

Expression and Secretion of Human Bone Morphogetic Protein-6 in Pichia pastoris

作　　者：米东[1] 杨爽[1] 胡芬[1] 张杰[1] 乔玉欢[1] 杜君[1] 朱天慧[1] 

机构地区：[1]南开大学医学院分子遗传实验室,天津300071

出　　处：《南开大学学报（自然科学版）》2010年第1期24-27,共4页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金(30700471);国家自然科学基金(30640029)

摘　　要：以重组质粒pcDNA6A-BMP6为模板PCR扩增hBMP6成熟肽cDNA编码序列,将该序列克隆到酵母分泌表达表达载体pPIC9K中.重组质粒pPIC9K-hBMP6经SacI酶切线性化,电击转化毕赤酵母GS115,PCR法筛选阳性转化子,利用梯度浓度G418筛选到七株高拷贝整合的阳性转化子。用甲醇进行诱导表达,SDSPAGE及Western-blot检测结果表明hBMP6成熟肽以分子量约为34和18 kD两种不同糖基化修饰的单体形式存在,具有良好的免疫原性.A coding sequence of mature human BMP6 was amplified by PCR and cloned into plasmid pPIC9K.Recombinant plasmid was linearized by SacI,then transformed into Pichia pastoris.The transformant was screened by PCR,and induced expression by methanol,seven strains haboring multi-copy of codon-optimized hBMP6 expression cassette were screened.The results of SDS- PAGE and Western-blot indicate that 34 and 18 kD proteins were produced in supernatant as monomers which have character of immunogenity.

关 键 词：毕赤酵母 BMP6 PPIC9K 分泌表达 

分 类 号：Q786[生物学—分子生物学]