鼠源Fcε-Fcγ融合蛋白的瞬时基因表达、大量制备及其对小鼠的抗过敏作用

发布时间：2018-01-16 23:01

本文关键词：鼠源Fcε-Fcγ融合蛋白的瞬时基因表达、大量制备及其对小鼠的抗过敏作用　出处：《华东理工大学》2011年博士论文　论文类型：学位论文

【摘要】：过敏性疾病普遍存在于人们的日常生活中,随着全球范围内的空气和环境的恶化,发病率呈逐年上升的趋势。哮喘是一种最典型的过敏性疾病,也是在工业化国家最常见的慢性疾病,被世界卫生组织列为疾病中的四大顽症之一。哮喘的治疗是世界公认的医学难题,人们一直在寻找安全有效的哮喘治疗药物,除了基于传统药物的改良和优化外,更多地把希望寄托在新的治疗靶点和生物药物的开发上。目前处于临床研究中的治疗哮喘的生物药物有十多种,另有更多种临床前研究中的候选药物。阻断IgE-多价抗原复合体与肥大细胞和嗜碱性粒细胞表面的受体FcεRI的结合可以从源头上阻止过敏反应的发生,是治疗过敏疾病的主要方向。抗过敏融合蛋白(AAFP)由人Fcε和Fcγ构成,可以交联肥大细胞和嗜碱粒细胞表面的介导过敏反应的Fcε受体Ⅰ(FcεRI)和抑制性Fcγ受体Ⅱb (FcγRⅡb),抑制肥大细胞和嗜碱粒细胞的活化及过敏炎性介质的释放,阻断过敏的发生。受体的特异性决定AAFP只作用于人的细胞,这就给AAFP的功能和作用机理的研究带来材料来源的限制。鉴于此,本文设计了鼠源的AAFP (mAAFP),利用瞬时基因表达(Transient gene expression, TGE)技术快速大量制备目的蛋白,并采用Protein A亲和层析纯化mAAFP。通过小鼠的被动皮肤过敏反应实验,研究了mAAFP抗过敏药物的药效,为评估AAFP可以作为治疗人类过敏性疾病药物的前期开发研究奠定了基础。瞬时基因表达技术可以在短时间内获得一定量的目的蛋白而被广泛研究和应用。但这种技术的应用效果常因蛋白而异。因此,本实验需要针对mAAFP建立一个快速高产的瞬时基因表达体系。TGE过程复杂,许多因素影响其转染效率和目的蛋白的产率,如宿主细胞、表达载体、转染试剂和转染过程等等。目前,大部分动物细胞表达的蛋白药物都是在CHO细胞中生产的,采用CHO细胞作为TGE的宿主细胞,可以确保前期研发样品与最后临床应用产品在分子结构上的一致性(如糖基化等)。本实验首次成功使用两种无血清培养基CDAGT-CHO和PF-CHO进行了瞬时基因表达。前者的特点是可以提供高转染效率,后者的特点足促进蛋白的表达。针对mAAFP的特性,我们在6孔板内优化了其瞬时转染体系。首先对比了两种不同商业化载体在转染效率上的差别,选择了pID作为后续实验的表达载体。之后,从转染试剂、转染时DNA/PEI复合物的共沉淀、转染时DNA与PEI的用量、转染时的细胞密度等多方面对其转染过程进行了优化。结果表明,利用PEI介导载体pID-EG瞬时转染表达mAAFP的最优条件是：DNA (4μg/106 cells)与PEI (DNA:PEI=1:2.5)在20%培养体积的DMEM/F12培养基中共沉淀5 min形成DNA/PEI复合物,加入细胞密度为2×106 cells/ml的CHO-S细胞培养体系中进行转染,6 h后补加等体积的PF-CHO培养基完成转染并进行后期培养。利用优化的瞬时转染条件,在1.3 L生物反应器中瞬时表达了mAAFP, (?)且蛋白表达量只有2 mg。为了进一步提高蛋白表达量,采用葡萄糖限制性流加及降低温度控制策略对转染后的培养过程进行了调控,提供足够的营养物质给转染后的细胞,以延长反应器中细胞的生长和存活率维持时间。结果显示,采用降低温度流加培养工艺对转染后的培养过程进行控制,与原来的分批培养相比,培养时间延长了一倍,蛋白总产量提高了14倍,达到30 mg。这一结果表明,对瞬时转染后的培养过程进行调控可以显著提高蛋白的表达。将此过程进一步成功放大到5L生物反应器中,最终蛋白浓度达到了25 mg/L。利用rProtein A Sepharose Fast Flow介质纯化mAAFP,通过优化蛋白的洗脱条件,收获的mAAFP纯度达到98%,收率达到85%以上。通过SDS-PAGE、Western blot对纯化得到的mAAFP进行了验证。被动皮肤过敏反应(PCA)实验可以用于检测抗原或者评价抗过敏药物的效果。在本研究中,PCA结果成功揭示了mAAFP可以抑制IgE介导的I型过敏反应,其抑制效率可以达到96%。在6.13mg/kg的mAAFP剂量下,其抑制效果在体内可以持续24天甚至更久。这一现象说明,mAAFP一旦注射入体内迅速与肥大细胞或嗜碱性粒细胞表而的受体结合后,其在体内不易被清除,其半衰期长于其结构中的任一免疫球蛋白。这一结果显示了AAFP蛋白具有治疗过敏性疾病的潜力,并为其深层次的分子机理研究以及AAFP在人体内的药效研究奠定了基础。
[Abstract]:Allergic diseases are common in people's daily life, with the deterioration of global air and environment, the incidence rate is increasing year by year. Asthma is one of the most typical allergic diseases, is the most common chronic disease in industrialized countries, the WHO is listed as one of the four major chronic diseases. The treatment of asthma is recognized worldwide medical problem, people have been searching for a safe and effective drug for the treatment of asthma, in addition to improving and optimizing the traditional medicine based on more put their hopes in the development target and biological treatment. The new biological drugs currently in clinical research in the treatment of asthma more than 10 otherwise, more in preclinical studies of candidate drugs. Blocking IgE- binding multivalent antigen complex with mast cells and basophilic cell surface receptor Fc epsilon RI from the source to stop The occurrence of anaphylaxis is the main direction for the treatment of allergic diseases.
Anti allergy fusion protein (AAFP) composed of Fc and Fc epsilon gamma, the cross-linking of mast cells and basophils surface mediated allergic reaction Fc epsilon receptor (Fc - RI) and inhibitory Fc receptor gamma (Fc gamma B R II B), inhibition of mast cell and basophil cell activation and allergic inflammatory mediators release, blocking allergies. The receptor specificity AAFP only effects on human cells, which bring source material restrictions on research to the function and mechanism of AAFP. In view of this, this paper designed a murine AAFP (mAAFP), transient gene expression using (Transient gene expression, TGE) technology and rapid preparation of target protein, and purified by using Protein A mAAFP. passive cutaneous anaphylaxis in mice affinity chromatography, pharmacodynamic study of anti allergy drug mAAFP, for the evaluation of AAFP can be used as a pre treatment of human allergic disease development The research laid the foundation.
Transient gene expression can be obtained in a short period of time a certain amount of target protein is widely studied and applied. But the application of this technique for protein varies. Therefore, the need for mAAFP to establish a rapid high transient gene expression system of.TGE process is complicated, many factors affect the transfection efficiency and protein the yield, such as host cell, expression vector, transfection reagent and the transfection process and so on. At present, most of the animal cell expression of protein drugs are produced in CHO cells, using CHO cells as TGE host cells, can ensure the consistency of the early development of sample and finally clinical application of the molecular structure of the product (such as sugar etc.).
This is the first successful use of two kinds of serum free medium CDAGT-CHO and PF-CHO were transient gene expression. The characteristic is that it can provide high transfection efficiency, foot characteristics of the latter promotes protein expression. Aiming at the characteristics of the mAAFP, we optimized the transfection system in 6 hole plate. The comparison of the difference between the two different commercial carriers in the transfection efficiency, choose the pID as the expression vector of the follow-up experiments. Then, from the transfection reagent, transfection of DNA/PEI complex co precipitation, the amount of DNA and PEI transfection, transfected cell density of many aspects such as optimization of the transfection process. The results show that the missile carrier transient transfection of pID-EG expression of mAAFP's optimal conditions is the use of PEI: DNA (4 g/106 cells) and PEI (DNA:PEI=1:2.5) in the 20% volume of culture medium of DMEM/F12 co precipitation in 5 min formation of the DNA/PEI complex, adding cell density The transfection was carried out in the CHO-S cell culture system of 2 x 106 cells/ml. After 6 h, the transfection was supplemented with equal volume PF-CHO medium and later culture was carried out.
Based on Instantaneous Optimization of transfection conditions, in 1.3 L bioreactor transiently expressed mAAFP, (?) and the expression of only 2 mg. in order to further improve the amount of protein expression by glucose restriction and flow and reduce the temperature control strategy on the training process after transfection of regulation, to provide sufficient nutrients for transfection after the cell, to extend the celligen growth and survival duration. The results showed that the lower temperature fed batch culture process to control the process training after transfection, compared with the original batch cultivation, cultivation time doubled, total protein yield increased 14 times, reaching 30 mg. results show that the process of training after transient transfection control can significantly improve the expression of the protein. This process will be further enlarged successfully in 5L bioreactor, the final protein concentration reached 25 mg/L. by rP Rotein A Sepharose Fast Flow medium purifies mAAFP. By optimizing the elution condition of protein, the purity of harvested mAAFP reaches 98%, and the yield is above 85%. Through SDS-PAGE, Western blot, the purified mAAFP is verified.
Passive cutaneous anaphylaxis (PCA) test can be used for the detection of antigen or anti allergy drug evaluation results. In this study, I type PCA allergic reaction results revealed that mAAFP can inhibit IgE mediated, the inhibition efficiency can reach 96%. at the dose of mAAFP 6.13mg/kg, its inhibitory effect in vivo can last for 24 days even more. This phenomenon shows that mAAFP once injected into the body quickly and mast cells or basophilic cell surface receptor binding, the clearance in the body is not easy, the half-life is longer than any immunoglobulin in its structure. The results showed that the AAFP protein has the potential for the treatment of allergic diseases, and laid the foundation for the efficacy of the deep research on the molecular mechanism of AAFP in the body.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

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