干扰素α和人免疫球蛋白Fc片段融合蛋白的构建与表达

发布时间：2018-03-06 05:26

本文选题：干扰素　切入点：融合蛋白　出处：《中国科学技术大学》2011年硕士论文　论文类型：学位论文

【摘要】：基因重组干扰素如IFN-α(IFN-α包括IFNα-2a,IFNα-2b,IFNα-1b等)及其修饰体(如多价干扰素)已经作为抗病毒和免疫调节类药物,被广泛用于临床,治疗多种病毒性疾病和抗肿瘤等。所谓天然的干扰素α(IFN-α)是指细胞经过诱导而表达产生的细胞因子,通过信号传导继而发挥抗病毒、抗细胞增殖和调节免疫应答作用,然而,作为一个小分子蛋白,IFN-α在血浆中清除的速度比较快,其半衰期仅为数十分钟,导致在临床治疗中需要重复多次用药才能达到治疗效果。这样不仅给患者增加痛苦和经济负担,还会出现药物的峰谷现象,难以达到治疗效果并容易产生副作用。因此,迫切需要探索一系列增长IFN在体内的半衰期的方法。在本研究中,为了延长IFN-α在血浆中的半衰期,我们利用免疫球蛋白具有在血液循环中长效半衰期的特性,构建了编码人源IFNα-2b和免疫球蛋白Fc片段的基因重组融合蛋白。同时,对Fc片段中可诱导细胞杀伤活性(包括ADCC和CDC活性)的个别位点进行了改造,以降低免疫球蛋白Fc片段可能导致的副作用。构建的融合蛋白既能保持干扰素的活性并带有天然的N端,又具有抗体的优越性,其Fc片段的CH2,CH3区域可以用于ProteinA的亲和层析,为蛋白纯化提供了方便;而且保留的抗体铰链link部分使两个有效分子之间的功能互不影响。为了进一步延长融合蛋白在血浆中的半衰期,我们通过定点突变的方式对IgG的Fc片段进行改造,使Fc与FcRn结合能力更强。另外,本研究中还采用了新型的甲醇诱导型酵母表达系统,达到高效和低成本的效果。研究中对表达融合蛋白毕赤酵母的高密度发酵培养以及产物纯化工艺进行初步探索。经过纯化的融合蛋白,其纯度可以达到90%以上,对于后期的蛋白活性检测和临床试验研究提供材料,为将来的药物开发设计和应用提供良好的前景。
[Abstract]:Recombinant interferon alpha (IFN- alpha IFN- alpha IFN alpha -2a including IFN, -2b, IFN alpha -1b) and its modification (such as multivalent as antiviral and interferon) have immunomodulatory drugs, is widely used in clinical treatment of various viral diseases and cancer. The so-called natural interferon alpha (IFN- alpha) refers to the cells after induction and expression of cytokines produced by signal transduction, then play antiviral, anti cell proliferation and regulation of immune response, however, as a small molecule protein, IFN- alpha in plasma clearance faster, its half-life is only tens of minutes, resulting in clinical treatment repeated treatment to achieve the therapeutic effect. It not only for patients with increased pain and economic burden, there will be a peak phenomenon of drug, to achieve the treatment effect and is easy to produce side effects. Therefore, the urgent need to explore a series of growth IFN Methods the in vivo half-life. In this study, in order to extend the IFN- alpha in plasma half-life, we use the properties of immunoglobulin has long half-life in blood circulation, construct the recombinant gene encoding human IFN alpha -2b and immunoglobulin Fc fragment fusion protein. At the same time, to induce cell killing the activity of Fc fragments (including ADCC and CDC activity) the individual loci were made, in order to reduce the side effects of immunoglobulin Fc fragments may result. This fusion protein can maintain the activity of interferon and N with a natural end, also has the superiority of antibodies, the Fc fragments of CH2, CH3 region for ProteinA affinity chromatography, protein purification and antibody provides a convenient; hinge part link retained between the two effective molecular functions do not affect each other. In order to further extend the fusion protein in the plasma half-life, We through the point mutation of IgG fragment of Fc transformation, the Fc and FcRn binding ability. In addition, this study also uses a new type of methanol inducible yeast expression system, achieve high efficiency and low cost. The effect on the expression of high density fermentation of fusion protein in Pichia pastoris and purification the process was preliminarily explored. After purified fusion protein, its purity can reach more than 90%, to provide materials testing late protein activity and clinical trials, provide good prospects for drug design and application in the future.

【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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