本文選題：干擾素　切入點(diǎn)：融合蛋白　出處：《中國(guó)科學(xué)技術(shù)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：基因重組干擾素如IFN-α(IFN-α包括IFNα-2a,IFNα-2b,IFNα-1b等)及其修飾體(如多價(jià)干擾素)已經(jīng)作為抗病毒和免疫調(diào)節(jié)類藥物,被廣泛用于臨床,治療多種病毒性疾病和抗腫瘤等。所謂天然的干擾素α(IFN-α)是指細(xì)胞經(jīng)過誘導(dǎo)而表達(dá)產(chǎn)生的細(xì)胞因子,通過信號(hào)傳導(dǎo)繼而發(fā)揮抗病毒、抗細(xì)胞增殖和調(diào)節(jié)免疫應(yīng)答作用,然而,作為一個(gè)小分子蛋白,IFN-α在血漿中清除的速度比較快,其半衰期僅為數(shù)十分鐘,導(dǎo)致在臨床治療中需要重復(fù)多次用藥才能達(dá)到治療效果。這樣不僅給患者增加痛苦和經(jīng)濟(jì)負(fù)擔(dān),還會(huì)出現(xiàn)藥物的峰谷現(xiàn)象,難以達(dá)到治療效果并容易產(chǎn)生副作用。因此,迫切需要探索一系列增長(zhǎng)IFN在體內(nèi)的半衰期的方法。在本研究中,為了延長(zhǎng)IFN-α在血漿中的半衰期,我們利用免疫球蛋白具有在血液循環(huán)中長(zhǎng)效半衰期的特性,構(gòu)建了編碼人源IFNα-2b和免疫球蛋白Fc片段的基因重組融合蛋白。同時(shí),對(duì)Fc片段中可誘導(dǎo)細(xì)胞殺傷活性(包括ADCC和CDC活性)的個(gè)別位點(diǎn)進(jìn)行了改造,以降低免疫球蛋白Fc片段可能導(dǎo)致的副作用。構(gòu)建的融合蛋白既能保持干擾素的活性并帶有天然的N端,又具有抗體的優(yōu)越性,其Fc片段的CH2,CH3區(qū)域可以用于ProteinA的親和層析,為蛋白純化提供了方便;而且保留的抗體鉸鏈link部分使兩個(gè)有效分子之間的功能互不影響。為了進(jìn)一步延長(zhǎng)融合蛋白在血漿中的半衰期,我們通過定點(diǎn)突變的方式對(duì)IgG的Fc片段進(jìn)行改造,使Fc與FcRn結(jié)合能力更強(qiáng)。另外,本研究中還采用了新型的甲醇誘導(dǎo)型酵母表達(dá)系統(tǒng),達(dá)到高效和低成本的效果。研究中對(duì)表達(dá)融合蛋白畢赤酵母的高密度發(fā)酵培養(yǎng)以及產(chǎn)物純化工藝進(jìn)行初步探索。經(jīng)過純化的融合蛋白,其純度可以達(dá)到90%以上,對(duì)于后期的蛋白活性檢測(cè)和臨床試驗(yàn)研究提供材料,為將來的藥物開發(fā)設(shè)計(jì)和應(yīng)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.

【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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相關(guān)期刊論文 前2條

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本文編號(hào)：1573525