hBmp4在甲醇酵母中的分泌表达及hBmp4全序列优化对其表达的影响
发布时间：2018-12-18 12:03
【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OD_(600)=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。
[Abstract]:Bone morphogenetic protein (Bone Morphogenetic Proteins,BMPs) is a kind of protein growth factor which belongs to an extracellular glycosylated protein dimer with high homology at the end of C'. Bone morphogenetic egg white 4 (BMP4), also known as bone morphogenetic protein 2B, has high osteoinductive activity and promotes bone and cartilage. In addition to osteogenesis, BMP4 also has a wide range of biological activities. HBMP4 also plays an important role in the development of embryo, limb bud, heart, eye, kidney, nerve and so on. In this study, we try to establish the expression technique of ". HBmp4 mature fragment in Pichia pastoris (Pichiapastoris)." The main research work included: transforming plasmid pPIC9k-hBmp4/dm expressing mature hBmp4 fragment into yeast mother host strain. After PCR identification gene integration, shaking flask culture was carried out. Methanol induced expression and dot hybridization were used to screen high expression strains. Based on the expression vector, the secondary structure of mRNA was predicted according to the preference of yeast codon, the analysis of AT content and the combination of software RNAstructure (Version 4.0). The. Pichia pastoris in the mature segment of hBmp4 was mutated from low frequency codon to synonymous codon with high frequency to achieve high efficient expression. The results of SDS-PAGE and WESTERN-BLOT analysis showed that the molecular weight of rhBMP4 was 26kD, and there was some glycosylation on the original 13KD molecule, and there was no dimer product. WESTERN-BLOT confirmed that the expressed product had good antigenicity and specificity. The observation of different induction conditions showed that the medium of pH6.0 was maintained with 0.5% methanol. The biomass of OD_ (600) was about 10 before induction, and the expression reached the highest level at 4 days after induction. Using protein Bradford total egg white quantification, combined with EUSA to quantify the target protein rhBMP4:, the expression of specific protein in the supernatant reached 17.731 mg / L, Accounting for 22.115% of the total protein content; After the whole sequence of hBmp4 mature fragment was optimized, the expression amount of single copy strain was three times higher than that of unoptimized sequence of corresponding copy number.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78
[Abstract]:Bone morphogenetic protein (Bone Morphogenetic Proteins,BMPs) is a kind of protein growth factor which belongs to an extracellular glycosylated protein dimer with high homology at the end of C'. Bone morphogenetic egg white 4 (BMP4), also known as bone morphogenetic protein 2B, has high osteoinductive activity and promotes bone and cartilage. In addition to osteogenesis, BMP4 also has a wide range of biological activities. HBMP4 also plays an important role in the development of embryo, limb bud, heart, eye, kidney, nerve and so on. In this study, we try to establish the expression technique of ". HBmp4 mature fragment in Pichia pastoris (Pichiapastoris)." The main research work included: transforming plasmid pPIC9k-hBmp4/dm expressing mature hBmp4 fragment into yeast mother host strain. After PCR identification gene integration, shaking flask culture was carried out. Methanol induced expression and dot hybridization were used to screen high expression strains. Based on the expression vector, the secondary structure of mRNA was predicted according to the preference of yeast codon, the analysis of AT content and the combination of software RNAstructure (Version 4.0). The. Pichia pastoris in the mature segment of hBmp4 was mutated from low frequency codon to synonymous codon with high frequency to achieve high efficient expression. The results of SDS-PAGE and WESTERN-BLOT analysis showed that the molecular weight of rhBMP4 was 26kD, and there was some glycosylation on the original 13KD molecule, and there was no dimer product. WESTERN-BLOT confirmed that the expressed product had good antigenicity and specificity. The observation of different induction conditions showed that the medium of pH6.0 was maintained with 0.5% methanol. The biomass of OD_ (600) was about 10 before induction, and the expression reached the highest level at 4 days after induction. Using protein Bradford total egg white quantification, combined with EUSA to quantify the target protein rhBMP4:, the expression of specific protein in the supernatant reached 17.731 mg / L, Accounting for 22.115% of the total protein content; After the whole sequence of hBmp4 mature fragment was optimized, the expression amount of single copy strain was three times higher than that of unoptimized sequence of corresponding copy number.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78
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