【摘要】：骨形態(tài)發(fā)生蛋白(Bone Morphogenetic Proteins,BMPs)是一類蛋白質(zhì)生長因 子,屬于細(xì)胞外的糖基化的蛋白二聚體,C’末端具有較高同源性。骨形態(tài)發(fā)生蛋 白4(BMP4)又稱骨形態(tài)發(fā)生蛋白2B,具有高效骨誘導(dǎo)活性,可促進(jìn)骨、軟骨,牙 本質(zhì)等硬組織的形成及缺損的修復(fù)。BMP4除了誘導(dǎo)成骨作用之外,還具有廣泛的 生物學(xué)活性。研究證實,hBMP4對胚胎、肢芽以及心、眼、腎、神經(jīng)等組織器官 的發(fā)育也有重要的調(diào)節(jié)作用。 本研究嘗試建立".hBmp4成熟片段在畢赤酵母(Pichiapastoris)中的表達(dá)技術(shù)。 主要研究工作包括:將表達(dá)hBmp4成熟片段的載體質(zhì)粒pPIC9k-hBmp4/dm轉(zhuǎn)化酵 母宿主菌株,PCR鑒定基因整合后,搖瓶培養(yǎng),甲醇誘導(dǎo)表達(dá)并用點雜交來快速 篩選高表達(dá)菌株;以這一表達(dá)載體為基礎(chǔ),依據(jù)酵母密碼子使用偏好性,AT含量 分析,結(jié)合軟件RNAstructure(Version 4.0)預(yù)測mRNA二級結(jié)構(gòu),將hBmp4成熟片 段內(nèi).Pichia pastoris使用低頻密碼子突變?yōu)槭褂妙l率較高的同義密碼予以期實現(xiàn)高 效表達(dá)。 表達(dá)產(chǎn)物經(jīng)SDS-PAGE和WESTERN-BLOT分析表明rhBMP4的分子量為 26KD,在原有13KD的分子上帶有一定程度的糖基化,不存在同二聚體形式的產(chǎn) 物;WESTERN-BLOT證實表達(dá)產(chǎn)物具有良好的抗原性和特異性:對不同誘導(dǎo)條件 的觀察表明:以pH6.0的培養(yǎng)基,保持以終濃度為0.5%的甲醇進(jìn)行誘導(dǎo),調(diào)整誘導(dǎo) 前菌體生物量在OD_(600)=10左右,誘導(dǎo)4d表達(dá)量達(dá)到最高;利用蛋白質(zhì)Bradford總蛋 白定量,結(jié)合EUSA對目的蛋白rhBMP4:進(jìn)行定量,表達(dá)上清中特異性蛋白的表達(dá) 量達(dá)到17.731mg/L,占總蛋白含量的22.115%;hBmp4成熟片段全序列優(yōu)化后,單 拷貝菌株的表達(dá)量與對應(yīng)拷貝數(shù)的未優(yōu)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.
【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

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