【作者】 蔡燕惠；

【导师】 张彦定；

【作者基本信息】 福建师范大学 ， 发育生物学， 2005， 硕士

【摘要】 骨形态发生蛋白（Bone Morphogenetic Proteins,BMPs）是一类蛋白质生长因子,属于细胞外的糖基化的蛋白二聚体,C’末端具有较高同源性。骨形态发生蛋白4（BMP4）又称骨形态发生蛋白2B,具有高效骨诱导活性,可促进骨、软骨,牙本质等硬组织的形成及缺损的修复。BMP4除了诱导成骨作用之外,还具有广泛的生物学活性。研究证实,hBMP4对胚胎、肢芽以及心、眼、肾、神经等组织器官的发育也有重要的调节作用。 本研究尝试建立".hBmp4成熟片段在毕赤酵母（Pichiapastoris）中的表达技术。主要研究工作包括:将表达hBmp4成熟片段的载体质粒pPIC9k-hBmp4/dm转化酵母宿主菌株,PCR鉴定基因整合后,摇瓶培养,甲醇诱导表达并用点杂交来快速筛选高表达菌株;以这一表达载体为基础,依据酵母密码子使用偏好性,AT含量分析,结合软件RNAstructure（Version 4.0）预测mRNA二级结构,将hBmp4成熟片段内.Pichia pastoris使用低频密码子突变为使用频率较高的同义密码予以期实现高效表达。 表达产物经SDS-PAGE和WESTERN-BLOT分析表明rhBMP4的分子量为26KD,在原有13KD的分子上带有一定程度的糖基化,不存在同二聚体形式的产物;WESTERN-BLOT证实表达产物具有良好的抗原性和特异性:对不同诱导条件的观察表明:以pH6.0的培养基,保持以终浓度为0.5%的甲醇进行诱导,调整诱导前菌体生物量在OD₆₀₀=10左右,诱导4d表达量达到最高;利用蛋白质Bradford总蛋白定量,结合EUSA对目的蛋白rhBMP4:进行定量,表达上清中特异性蛋白的表达量达到17.731mg/L,占总蛋白含量的22.115%;hBmp4成熟片段全序列优化后,单拷贝菌株的表达量与对应拷贝数的未优化序列相比提高了3倍。更多还原

【Abstract】 Bone morphogenetic proteins （ BMPs ） are dimeric secreted glycoproteins, each members shares sequence similarity in their C’terminal regions. BMPs have diverse biological activities and play critical roles in the migration, proliferation, and differentian of mesenchymal cells during embryogenesis and in the repair and regeneration of tissues during postfetal life. In our research, the recombinant human bone morphogenetic proteins 4 （ rhBMP4 ） was sussessfully expressed in the Pichia pastoris. Furthermore, investigations for rhBMP4 expression, small scale fermentations were carried out.The cDNA mature domain encoding hBMP4 was cloned into the expression vector, pPIC9K. And the reconstructed Pichia pastoris expression vector was introduced into Pichia pastoris strain for expression. For higher expression level, codons in the cDNA mature domain encoding hBMP4 were optimized according to the favorite for Pichia pastoris. Analysis of culture medium revealed that the molecular weight of rhBMP4 is about 26KD by SDS-PAGE and WESTERN-BLOT; the yield is about 17.731mg/L and accounts for 22.115% of the total proteins in the supernatants. Expression with favorite codons for Pichia pastoris increased about 3-fold in proportion to that with the original codons. The optimal time to harvest is 96h post induction with 0.5% of methanol, with the culture medium buffered at pH6.0 and the optimal OD₆00 at about 10 before induction.更多还原