作　　者： ; ; ; ; ;

机构地区： 天津市内分泌研究所

出　　处： 《中国生物工程杂志》 2005年第7期61-65,共5页

摘　　要： 以重组质粒pUC-BMP2为模板PCR扩增人BMP-2成熟肽编码序列,将该序列克隆入pGEM-T载体进行DNA序列分析后亚克隆入分泌型毕赤酵母表达载体pPIC9K中。重组质粒oPIC9K-BMP2经BelⅡ酶切后回收线性化片段,聚乙二醇法转染毕赤酵母茵株GS115。PCR筛选整合有人BMP-2基因的酵母细胞重组子,以甲醇进行诱导表达,于酵母细胞培养基中可检测到rhBMP-2。体外培养条件下,所得rhBMP-2可增加2T3小鼠成骨细胞内碱性磷酸酶活性;体内实验.rhBMP-2冻干粉可于小鼠骨四头肌肌袋中诱导软骨细胞群产生。 A DNA coding sequence for the mature peptide of human BMP-2 was amplified by PCR and cloned into plasmid pGEM-T. After being verified by DNA sequence analysis, the target gene was subcloned into the shuttle plasmid pPIC9K of Pichia pastoris expression system, under the control of promoter AOX1 (alcohol oxidase 1). Recombinant plasmid pPlC9K-BMP-2 was linearized by BglⅡdigestion and then transformed into Pichia pastoris strain GS115.The transformant was selected by PCR and introduced to methanol induced expression. Firstly, Pichia pastoris GS115 was cultured in BMGY medium, 24h afterwards, the medium was changed into BMMY containing methanol of a final concentration of 0.5% V/V. 6days after culture, supernatant of the medium was collected and analyzed by SDS-PAGE. In supernatant a 24kDa protein was detected, which decreased to 16kDa after deglycosylation and was approved as rhBMP-2 by Western blot.2T3 mouse osteoblast cell line was cultured and rhBMP-2 form Pichia pastoris of different concentration was added into the medium, with rhBMP-2 purchased from R&D company used as control. As a result, these two kinds of rhBMP-2 had almost equal bio-ability of increasing the activity of ALP in this cultured 2T3 cell. Further in vivo study showed, embedded rhBMP-2 could induced myoblast differentiate into chondrocyte.

关 键 词： 巴斯德毕赤酵母 骨形态发生蛋白 序列分析 碱性磷酸酶活性 酵母表达载体 体外培养条件 重组质粒 扩增 聚乙二醇法 细胞培养基 编码序列 酵母菌株 酵母细胞 诱导表达 体内实验 成熟肽 分泌型 亚克隆 载体

领　　域： [生物学] [生物学]