【摘要】：骨形態(tài)發(fā)生蛋白(Bone Morphogenesis Proteins,BMPs)是一類具有誘 骨活性的蛋白質(zhì)生長因子。骨形態(tài)發(fā)生蛋白.4(BMP4)是BMPs中誘導(dǎo) 成骨活性較強(qiáng)的一種。目前已有研究者嘗試在不同的表達(dá)系統(tǒng)中進(jìn)行重組 表達(dá),但是在表達(dá)產(chǎn)物中,無法獲得活性較高的二聚體形式的蛋白。BMP4 在胞內(nèi)是以前體的形式合成的,包括信號肽、前導(dǎo)肽和成熟肽三個部分。 研究表明,在目前發(fā)現(xiàn)的絕大部分的以含前導(dǎo)肽的前體形式合成的蛋白質(zhì) 在其前導(dǎo)肽不存在的情況下均無法正確折疊。因此認(rèn)為,Pro肽在某些蛋 白質(zhì)的折疊過程中扮演了分子內(nèi)分子伴侶(IMC)的角色。本研究希望在 畢赤酵母中將BMP4前導(dǎo)肽與成熟肽融合表達(dá),以此來探討B(tài)MP4前導(dǎo)肽 在輔助成熟肽的折疊以及二硫鍵的配對過程中是否起到一定的作用。主要 的研究工作和研究結(jié)果包括:(1)將hBMP4前導(dǎo)肽與成熟肽片段克隆到 分泌型表達(dá)載體質(zhì)粒pPIC9K多克隆位點(diǎn)上,構(gòu)建能表達(dá)hBMP4前導(dǎo)肽 與成熟肽的重組質(zhì)粒pPIC9K-hBMP4/pro-ori;(2)以這一表達(dá)載體為基礎(chǔ), 對hBMP4前導(dǎo)肽與成熟肽之間內(nèi)源性蛋白水解酶識別位點(diǎn)進(jìn)行定點(diǎn)突 變,將真核細(xì)胞中堿性氨基酸蛋白酶(furin)識別的位點(diǎn)(RXXR)改為酵 母細(xì)胞中絲氨酸蛋白酶(Kex2P)識別的位點(diǎn)Kex2,并以此構(gòu)建了重組質(zhì) 粒pPIC9K-hBMP4/pro-mut;(3)表達(dá)質(zhì)粒經(jīng)轉(zhuǎn)化酵母宿主菌株GSll5, PCR鑒定基因整合后,搖瓶培養(yǎng),甲醇誘導(dǎo)表達(dá)并用點(diǎn)雜交來快速篩選高 表達(dá)菌株。表達(dá)產(chǎn)物經(jīng)SDS-PAGE和Western blotting分析表明表達(dá)產(chǎn)物 具有良好的抗原性和特異性,內(nèi)源性蛋白水解酶識別位點(diǎn)的改造能夠使前 導(dǎo)肽有效切割,切割后BMP4成熟肽的分子量為22KD,與標(biāo)準(zhǔn)蛋白一致, 但不存在同二聚體形式的產(chǎn)物。
[Abstract]:Bone Morphogenesis proteins (BMPs) are a kind of protein growth factors with bone inductive activity. Bone morphogenetic protein 4 (BMP4) is one of BMPs with high osteogenic activity. So far, researchers have tried to recombine in different expression systems, but in the expressed products, The protein. BMP4 in the form of high activity dimer can not be synthesized in the intracellular form, which consists of three parts: signal peptide, precursor peptide and mature peptide. It has been found that most of the proteins synthesized in the form of precursors containing precursor peptides can not be folded correctly without the existence of the precursor peptides. Therefore, it is suggested that protopeptide plays the role of intramolecular chaperone (IMC) in the folding of some egg white matter. In this study, we hope to express BMP4 prepeptide and mature peptide in Pichia pastoris. In order to investigate whether BMP4 prepeptide plays a role in assisting the folding of mature peptides and the matching of disulfide bonds. The main research work and results are as follows: (1) cloning hBMP4 precursor peptide and mature peptide fragment into secretory expression Vector plasmid pPIC9K polyclonal site, The recombinant plasmid pPIC9K-hBMP4 / pro-orii was constructed to express hBMP4 prepeptide and mature peptide. (2) based on this expression vector, hBMP4 prepeptide was synthesized. The identification site of endogenous proteolytic enzyme was suddenly changed with mature peptide. The (furin) recognition site (RXXR) of alkaline amino acid protease in eukaryotic cells was replaced by Kex2 site recognized by serine protease (Kex2P) in yeast cells, and the recombinant plasmid was constructed. PPIC9K-hBMP4 / pro-mutt; (3) the expression plasmid was identified after gene integration by the transformed yeast host strain GSll5. In shaking flask culture, methanol induced expression and dot hybridization were used to quickly screen high expression strains. SDS-PAGE and Western blotting analysis showed that the expressed product had good antigenicity and specificity. The modification of the recognition site of endogenous proteolytic enzyme can make the preconductors cut effectively. The molecular weight of the BMP4 mature peptide was 22kD, which was consistent with the standard protein, but there was no dimer product.
【學(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q78;Q516

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