利用U2-OS细胞表达rhBMP-7的研究

发布时间：2018-05-22 10:33

本文选题：癌细胞 + U2-OS　；参考：《济南大学》2017年硕士论文

【摘要】：人骨形态发生蛋白-7(Human bone morphogenetic protein-7,hBMP-7),又名成骨蛋白-1(Osteogenic Protein,OP-1),属于转化生长因子-β(Transforming growth factor-β,TGF-β)超家族。hBMP-7能够诱导动物或人间充质干细胞分化为骨、软骨、韧带、肌腱和神经组织,在临床骨科领域中具有广阔的应用前景,可用于促进脊柱融合和治疗骨折、骨缺损、骨不连、股骨头缺血性坏死等。但是,在基因工程表达该蛋白的过程中遇到了一些难以克服的难题:原核表达系统由于缺乏翻译后加工、修饰机制,使表达的蛋白复性困难;毕赤酵母的糖基化形式与人类存在差异亦不利于该蛋白的表达;CHO细胞表达的重组人骨形态发生蛋白-7(rhBMP-7)虽有活性,但是表达量很低,尚不具备产业化条件,因此,国内外生产此蛋白的公司极少,价格昂贵。所以,能否克服如上困难是rhBMP-7能否真正实现产业化的关键。目的:人源癌细胞具有完整的翻译后加工机制、可无限增殖和体外易培养的特点,能否让其“弃恶从善”用于表达具有临床应用价值、结构复杂的蛋白质是一个具有挑战性的课题。本研究拟利用人源癌细胞U2-OS进行rhBMP-7蛋白的表达,以探讨人骨肉瘤U2-OS细胞作为表达系统表达rhBMP-7的可行性,本研究有可能为复杂结构蛋白质的高效表达提供一种新的解决方案。方法:(1)rhBMP-7在U2-OS细胞中的表达:构建真核表达质粒pcDNA3.1-rhBMP-7,利用Lipofectamine2000将真核表达质粒转染至U2-OS细胞中,采用实时荧光定量PCR法检测rhBMP-7基因在mRNA水平上的表达;Western Blot法检测rhBMP-7的表达。(2)分泌性rhBMP-7的表达:分析三种信号肽(hBMP-7自身信号肽B7、人基质金属蛋白酶-9信号肽M9、人纤溶酶原信号肽SP)对U2-OS细胞分泌表达rhBMP-7的影响。构建真核表达质粒pcDNA3.1-rhB7-h BMP7、pcDNA3.1-rhSP-hBMP7、pcDNA3.1-rhM9-hBMP7。利用Lipofectamine2000将真核表达质粒转染至U2-OS细胞中,采用实时荧光定量PCR法检测rhBMP-7基因在mRNA水平上的表达,Western Blot法检测U2-OS细胞内与其培养基中分泌表达的rhBMP-7。在Western Blot检测分泌表达的rhBMP-7的基础上进一步利用ELISA检测U2-OS细胞内与细胞外分泌表达的rhBMP-7。(3)rhBMP-7的纯化与分析:选用亲和层析法纯化细胞分泌表达的rhBMP-7蛋白;通过糖苷内切酶酶切样品rhBMP-7,对其进行糖基化分析;纯化的rhBMP-7作用于NIH3T3细胞,通过检测NIH3T3细胞的ALP活性,分析rhBMP-7的生物活性。结果:(1)Western Blot结果显示rhBMP-7蛋白分子量为65-75KDa,实验组细胞中rhBMP-7表达量高于阴性对照组。结果表明rhBMP-7可在U2-OS细胞中表达。(2)研究分泌性rhBMP-7的表达中,通过Western Blot与ELISA检测U2-OS细胞内与细胞外分泌表达的rhBMP-7,结果表明U2-OS细胞内与细胞外均有表达rhBMP-7。ELISA结果显示细胞外表达的rh BMP-7蛋白量高于细胞内表达的rhBMP-7,说明rhBMP-7实现了分泌表达,而且hBMP-7自身信号肽引导的U2-OS细胞分泌表达的rhBMP-7蛋白量较高。(3)经镍柱纯化得到rhBMP-7蛋白,通过BCA法测得rhBMP-7蛋白浓度为28.3mg/L,在缓冲液中可完全溶解。rhBMP-7经SDS-PAGE电泳显示条带单一,纯度较好。(4)通过糖苷内切酶(Endo H)酶切rhBMP-7对其进行糖基化分析。未经Endo H酶切的rhBMP-7单体rhBMP-7蛋白分子量为34-40KDa,二聚体rhBMP-7蛋白分子量为65-75KDa。经Endo H酶切的rhBMP-7单体蛋白分子量为17-20KDa,二聚体rh BMP-7蛋白分子量为34-40KDa。根据Endo H酶切rhBMP-7的结果得出:U2-OS细胞对表达rhBMP-7蛋白进行了糖基化修饰。(5)rhBMP-7对NIH3T3细胞碱性磷酸酶(ALP)的影响结果显示:含rhBMP-7培养基使NIH3T3细胞的ALP活性明显增强,而且呈现出明显的量、效关系,表明U2-OS细胞表达的rhBMP-7具有生物活性。结论:(1)本实验结果表明,可以利用U2-OS细胞表达rhBMP-7,而且信号肽会影响细胞对表达蛋白的分泌效率;hBMP-7自身信号肽引导U2-OS细胞分泌表达rhBMP-7的蛋白量较高。(2)U2-OS细胞对表达rhBMP-7蛋白进行了糖基化修饰,蛋白可溶。生物活性检测结果表明U2-OS细胞表达的rhBMP-7具有生物活性。(3)本实验的结果表明:U2-OS细胞可作为表达系统进行复杂结构蛋白质的制备,此为今后大规模表达和制备结构复杂的细胞因子、人源抗体、疫苗等奠定了实验基础。
[Abstract]:Human bone morphogenetic protein -7 (Human bone morphogenetic protein-7, hBMP-7), also known as osteogenic protein -1 (Osteogenic Protein, OP-1), belongs to transforming growth factor beta (Transforming growth beta, beta) superfamily can induce animal or human mesenchymal stem cells to differentiate into bone, cartilage, ligaments, tendons and nerve tissue, in clinical practice. There are broad applications in the field of Department of orthopedics, which can be used to promote spinal fusion and the treatment of fractures, bone defects, bone nonunion and avascular necrosis of the femoral head. However, there are some difficult problems to be overcome in the process of gene engineering expression of this protein: the prokaryotic expression system is due to the lack of post-translational processing, modification mechanism, and expression of protein. The glycosylation form of Pichia pastoris and human existence are not good for the expression of the protein. Although the recombinant human bone morphogenetic protein -7 (rhBMP-7) expressed by CHO cells is active, the expression is very low and there is no industrial condition. Therefore, there are few companies producing this protein at home and abroad, and the price is expensive. Difficulty is the key to the real industrialization of rhBMP-7. Objective: human cancer cells have a complete post-translational processing mechanism, which can grow infinitely and easily in vitro. It is a challenging task to make its "abandoned evil and good" to express the value of clinical application. The expression of rhBMP-7 protein in human cancer cell U2-OS is used to explore the feasibility of human osteosarcoma U2-OS cells as expression system for expression of rhBMP-7. This study may provide a new solution for the efficient expression of complex structural proteins. Method: (1) the expression of rhBMP-7 in U2-OS cells: Construction of eukaryotic expression plasmid pcDNA3.1-rhBMP- 7, the eukaryotic expression plasmid was transfected into U2-OS cells by Lipofectamine2000, and the expression of rhBMP-7 gene at mRNA level was detected by real-time fluorescence quantitative PCR; Western Blot method was used to detect the expression of rhBMP-7. (2) the expression of secretory rhBMP-7: analysis of three signal peptides (hBMP-7 itself signal peptide B7, human matrix metalloproteinase -9 signal peptide, human The effect of plasminogen signal peptide SP on the expression of rhBMP-7 in U2-OS cells. Construction of eukaryotic expression plasmid pcDNA3.1-rhB7-h BMP7, pcDNA3.1-rhSP-hBMP7, pcDNA3.1-rhM9-hBMP7. using Lipofectamine2000 to transfect eukaryotic expression plasmid into U2-OS cells, and detect the expression of rhBMP-7 gene on mRNA level by real-time fluorescent quantitative PCR. Ern Blot assay was used to detect the expression of rhBMP-7. secreted in U2-OS cells and the expression of rhBMP-7 in Western Blot by Western Blot to further purify and analyze rhBMP-7. (3) rhBMP-7 in U2-OS cells and exocrine expression by ELISA: affinity chromatography was used to purify the secretory expression of the rhBMP-7 protein; The glycoside endonuclease was cut to the sample rhBMP-7 to carry out the glycosylation analysis. The purified rhBMP-7 acted on the NIH3T3 cells and analyzed the bioactivity of rhBMP-7 by detecting the ALP activity of NIH3T3 cells. Results: (1) the Western Blot results showed that the rhBMP-7 protein molecular weight was 65-75KDa, and the rhBMP-7 expression in the tested group was higher than that of the negative control group. The results showed that rhBMP-7 could be expressed in U2-OS cells. (2) to study the expression of secretory rhBMP-7, the expression of rhBMP-7 in U2-OS cells and cell exocrine in U2-OS cells was detected by Western Blot and ELISA. The results showed that the RH BMP-7 protein in the U2-OS cell and outside cells expressed the RH BMP-7 protein amount of the cell appearance of the cells higher than that of the intracellular expression. 7, the expression of rhBMP-7 was expressed, and the expression of rhBMP-7 protein secreted by U2-OS cells guided by hBMP-7 self signal peptide was higher. (3) the rhBMP-7 protein was purified by the nickel column, the concentration of rhBMP-7 protein was 28.3mg/L by the BCA method, and the.RhBMP-7 was completely dissolved in the buffer solution, and the purity of the band was single and the purity was better. (4) The glycosylation analysis was carried out by the glucoside endonuclease (Endo H) enzyme cut rhBMP-7. The molecular weight of rhBMP-7 monomer rhBMP-7 protein was 34-40KDa without Endo H enzyme cut, and the molecular weight of the two polymer rhBMP-7 protein was 65-75KDa. through the Endo H enzyme. The results of cutting rhBMP-7 showed that the expression of rhBMP-7 protein was glycosylated by U2-OS cells. (5) the effect of rhBMP-7 on the alkaline phosphatase (ALP) of NIH3T3 cells showed that the ALP activity of NIH3T3 cells was obviously enhanced by the rhBMP-7 culture medium, and showed a significant amount and effect relationship, indicating that rhBMP-7 of the U2-OS cell expression was bioactive. Conclusions: (1) the experimental results show that the expression of rhBMP-7 can be expressed by U2-OS cells, and the signal peptide can affect the secretion efficiency of the expressed protein, and the hBMP-7 self signal peptide leads U2-OS cells to secrete a higher protein expression of rhBMP-7. (2) U2-OS cells have glycosylated, soluble protein and bioactivity detection for the expression of rhBMP-7 protein. The results showed that the rhBMP-7 expressed by U2-OS cells had biological activity. (3) the results of this experiment showed that U2-OS cells could be used as an expression system to prepare complex structural proteins, which laid the experimental basis for large-scale expression and preparation of complex cytokine, human antibody and vaccine in the future.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

【参考文献】