人骨形态发生蛋白-7成熟肽的表达及其生物学活性的初步研究

发布时间：2018-03-05 11:41

本文选题：人骨形态发生蛋白-7　切入点：毕氏酵母　出处：《中国人民解放军军医进修学院》2007年硕士论文　论文类型：学位论文

【摘要】： 骨形态发生蛋白(Bone morphogenetic proteins，BMPs)是一种多功能蛋白，是转化生长因子(transforming growth factor-beta，TGF-β)超家族成员之一，为目前发现的一类具有异位成骨能力的细胞因子，它能刺激间充质细胞向成骨细胞分化。人骨形态发生蛋白-7(hBMP-7)又称为成骨蛋白-1(Osteogenic Protein-1，OP-1)，由于其具有较强的骨诱导能力而受到人们广泛关注。它与Ⅰ型胶原蛋白的缓释体移入骨干缺损部位，会导致有功能的新骨形成，显示出应用于骨与关节及软骨缺损修复的前景。而且同时对神经系统、造血系统的分化发育也有调控作用。 hBMP-7基因编码431个氨基酸，由N端的信号肽、中间区域的前肽和C端的成熟肽三部分组成，翻译后的前体需要进一步的加工修饰，包括切掉信号肽和前肽，其成骨活性主要表现在C端的成熟肽区。hBMP-7成熟肽含有139个氨基酸，单体分子量约15.7kD，带有3个糖基化位点及7个半胱氨酸残基。天然hBMP-7存在同源或异源二聚体形式，二聚体的hBMP-7是其生物学活性的主要形式。制备hBMP-7成熟肽主要的研究工作包括以下三个方面： 1．采用pMAL-p2x表达载体，在原核系统中获得了hBMP7-MBP融合蛋白的可溶性高效表达，并通过亲和纯化得到纯度约为80％的融合蛋白，利用Factor Xa蛋白酶对融合蛋白酶切后，得到目的蛋白hBMP-7成熟肽，以单体形式存在。 2．鉴于毕氏酵母(Pichia pastoris)表达系统能对重组蛋白进行翻译后必要的剪接、正确的折叠和修饰，糖基化形式更接近于高等真核生物，因此选用此表达系统，将hBMP-7成熟肽基因克隆入表达载体pPIC9K，用SalⅠ酶将重组质粒线性化，电转化入Pichia pastoris SMD1168细胞，G418抗性筛选出阳性整合子，，确定Mut~+表型进行甲醇快速诱导，并对其培养和诱导条件进行一定的探索和优化。经过整合子的筛选和表达条件的优化，最终筛选到目的蛋白表达量占上清蛋白8％的分泌表达株，表达量为0.6mg／L，酶联免疫检测及蛋白印迹结果表明，表达的重组蛋白与抗hBMP-7抗体具有特异结合活性，提示重组表达蛋白具有天然蛋白的分子构象。 3．为了提高目的蛋白的表达量，依据毕氏酵母的密码子偏好性，对hBMP-7成熟肽基因进行基因修饰，最终确定以含三个精氨酸突变位点的酵母菌作为表达株进行扩大培养，经Ni-NTA柱纯化得到纯度为80％的蛋白样品，基因修饰后的hBMP-7成熟肽在毕氏酵母中的表达水平提高了约4倍，产量为2.5mg／L。所得hBMP-7成熟肽在还原状态下以分子量约18kD的单体形式存在，在非还原状态下以二聚体形式存在，分子量约为36kD左右，重组蛋白经PNGase F糖苷酶酶切分析证明其在毕氏酵母中发生了N糖基化。细胞学实验证实制备的hBMP-7成熟肽具有一定的生物学活性，能够促进成纤维细胞向成骨细胞转化。综上所述，本研究利用毕氏酵母系统成功分泌表达了hBMP-7成熟肽，并能形成有生物学活性的二聚体形式，为今后hBMP-7的功能研究奠定了基础。
[Abstract]:Bone morphogenetic protein (Bone morphogenetic, proteins, BMPs) is a multifunctional protein, transforming growth factor (transforming growth factor-beta, TGF- beta) superfamily, is a type of the current found with cytokines of ectopic osteogenesis, it can stimulate mesenchymal cells to differentiate into osteoblasts. Human bone morphogenetic protein -7 (hBMP-7) also known as osteogenic protein -1 (Osteogenic Protein-1 OP-1), because of its strong ability of bone induction have received widespread attention. It and type I collagen release into the backbone of the defect site, will lead to new bone formation function, applied to the show repair of bone and joint cartilage defects and prospects. And at the same time on the nervous system, the differentiation and development of hematopoietic system also has a regulatory role.
The hBMP-7 gene encoding a 431 amino acid signal peptide N, by the end of the propeptide and the C end of the middle region of the mature peptide is composed of three parts, the precursor need further modification after translation, including cut off the signal peptide and peptide, its osteogenic activity is mainly manifested in the mature peptide of C end.HBMP-7 mature peptide monomer containing 139 amino acids, molecular weight of about 15.7kD, with 3 glycosylation sites and 7 cysteine residues. There are two natural hBMP-7 homologous or heterologous dimers form dimers with two hBMP-7 is the main form of its biological activity. The preparation work of hBMP-7 mature peptide mainly includes the following three aspects:
1. the pMAL-p2x expression vector, the soluble hBMP7-MBP fusion protein was highly expressed in prokaryotic system, and obtained by affinity purification of about 80% purity of fusion protein, fusion protein digested by Factor Xa protease, the target protein hBMP-7 mature peptide, a monomer.
2. in Pichia pastoris (Pichia pastoris) expression system can be translated to the recombinant protein necessary for splicing, correct folding and modification, glycosylated form closer to higher eukaryotes, so the use of this expression system, the hBMP-7 mature peptide gene was cloned into the expression vector pPIC9K. The recombinant plasmid Sal 1 enzyme linear electric pastoris was transformed into Pichia SMD1168 cells, G418 resistance screening positive for integron, determine Mut~+ phenotype of methanol induced rapid and carries on the exploration and optimization of culture and induction conditions. After optimizing the expression conditions and screening of integrons, the final screening to the target protein accounted for 8% of the supernatant protein the expression and secretion of strains, the expression level of 0.6mg / L, ELISA and Western blot results showed that the expression of recombinant protein with anti hBMP-7 antibody has a specific binding activity, suggesting that the recombinant protein A molecular conformation that has natural proteins.
3. in order to improve the expression level of target protein, according to codon preference of Pichia pastoris, gene modification of hBMP-7 mature peptide gene, determined by yeast containing three arginine mutation strains as were purified by Ni-NTA column chromatography, the purity was 80% protein samples, the expression level of gene modification after the hBMP-7 mature peptide in Pichia pastoris was improved about 4 times, the yield for the monomer form of 2.5mg / L. from hBMP-7 mature peptide in the reduced state with molecular weight of about 18kD, the reduced state to two dimer form of non molecular weight is about 36kD, the recombinant protein was PNGase F glycosidase enzyme analysis confirms the glycosylation of N in Pichia pastoris. Cytological experiments confirmed that the prepared hBMP-7 mature peptide with certain biological activity, can promote fibroblast cells to osteoblast like cells.
To sum up, this study successfully secreted hBMP-7 mature peptide by Pichia pastoris system, and formed a two mer form with biological activity, which laid the foundation for further research of hBMP-7 function.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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