本文選題：聚乙二醇　切入點(diǎn)：親和介質(zhì)　出處：《廣西大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來,固定化金屬離子親和層析(IMAC)技術(shù)因其分離快速、分離條件溫和,成本低等優(yōu)勢(shì)而得到廣泛應(yīng)用。但是在一些復(fù)雜的混合體系中,由于蛋白質(zhì)大分子會(huì)與目標(biāo)小分子競(jìng)爭(zhēng)親和位點(diǎn),降低了親和介質(zhì)對(duì)活性小分子的分離純化效果而使其應(yīng)用范圍受到了限制。為了減弱實(shí)際體系中大分子蛋白質(zhì)的干擾,近年來發(fā)展起來的限進(jìn)性介質(zhì)具有一定的優(yōu)勢(shì),其主要的特點(diǎn)是在吸附介質(zhì)表面修飾一層半滲透屏障形成體積排阻效應(yīng),并結(jié)合IMAC的親和力選擇性富集目標(biāo)活性分子。因此,根據(jù)上述思路,研究制備了一種具有高載量、高選擇性限進(jìn)性親和吸附介質(zhì)。詳細(xì)的實(shí)驗(yàn)結(jié)果如下：(1)采用乳化交聯(lián)法制備磁性殼聚糖微球(M-CS)作為親和介質(zhì)的載體,微球平均粒徑為25μm。使用環(huán)氧氯丙烷對(duì)載體進(jìn)行活化,當(dāng)溶脹時(shí)間為4h、活化溫度為50℃、活化時(shí)間為6h、環(huán)氧氯丙烷體積分?jǐn)?shù)50%、NaOH濃度為0.4M時(shí),環(huán)氧基密度為260μmol·g-1。通過亞氨基乙二酸(IDA)作為間隔臂螯合親和銅離子(Cu2+),制備M-CS@Cu2+。選擇牛血清蛋白(BSA)為大分子蛋白模型,考察了離子強(qiáng)度、pH值、吸附時(shí)間、配基密度(Cu2+)對(duì)吸附效果的影響,當(dāng)緩沖液離子濃度為0.02M、pH=7、吸附時(shí)間為60min,配基密度為140μmol·g-1時(shí),對(duì)BSA (1mg·mL-1)的吸附量達(dá)到117.6mg·g-1。(2)用聚乙二醇mPEG1900對(duì)親和介質(zhì)進(jìn)行表面修飾,當(dāng)以三氟化硼為催化劑,催化劑用量(摩爾比)1：0.08、反應(yīng)時(shí)間6h,反應(yīng)溫度50℃時(shí),mPEG1900接枝率達(dá)26.46%,并通過紅外光譜(FT-IR)、掃描電子顯微鏡(SEM)、差式量熱分析儀(DSC)及熱失重(TG)對(duì)其進(jìn)行了表征。研究了接枝率對(duì)阻擋BSA吸附效果的影響,限進(jìn)親和介質(zhì)對(duì)BSA的吸附量隨著mPEG1900的接枝率升高而降低。當(dāng)介質(zhì)配基密度為140μmol·g-1,表面mPEG1900接枝率為26.46%時(shí),介質(zhì)對(duì)BSA的阻拒率達(dá)到50.68%。(3)為了進(jìn)一步探索限進(jìn)親和介質(zhì)的應(yīng)用效果,以Val-Ser-Leu-Pr o-Glu-Try (VW-6)為小分子肽模型,BSA為大分子蛋白模型分子,建立了模擬混合體系以及酪蛋白酶解體系,比較了mPEG1900修飾前后親和介質(zhì)在混合體系以及酪蛋白酶解體系中對(duì)小分子的吸附選擇性以及對(duì)大分子的阻拒效果。結(jié)果表明限進(jìn)親和介質(zhì)對(duì)VW-6的吸附效果有提高,對(duì)大分子BSA的吸附具有一定的阻擋效果。說明限進(jìn)性金屬離子親和介質(zhì)能夠有效的富集小分子多肽,減少大分子蛋白的非特異性吸附,為提高活性小分子的分離效率提供了一種可行的方法。
[Abstract]:In recent years, immobilized metal ion affinity chromatography (IMAC) technology because of its quick separation, mild separation condition, advantages of low cost and widely used. But in some complex hybrid systems, because the protein will compete with the goal of small molecule binding sites, low affinity purification effect of medium separation of active small molecules the application range is limited. In order to reduce the interference of large molecular proteins in the actual system, developed in recent years in limited medium has certain advantages, its main characteristic is adsorbed on the medium surface modification layer of semi permeable barrier formation volume exclusion effect, combined with the selective enrichment of IMAC affinity the activity of target molecules. Therefore, according to the above ideas, study on Preparation of a high load, high selectivity limit into the affinity adsorption medium. The detailed experimental results are as follows: (1) the milk Preparation of magnetic chitosan microspheres and chemical crosslinking method (M-CS) as a carrier of affinity medium, the average diameter of microspheres was 25 M. activated by epichlorohydrin on the carrier, when the swelling time was 4h, activation temperature is 50 DEG C, the activation time was 6h, the volume fraction of 50% epichlorohydrin, NaOH at a concentration of 0.4M the epoxy density is 260 mol - g-1. (IDA) by sub Aminoadipate as spacer chelating copper ion (Cu2+), the preparation of M-CS@Cu2+. bovine serum protein (BSA) as a model protein, the effects of ionic strength, pH value, adsorption time, ligand density (Cu2+) on the adsorption effect when the buffer concentration is 0.02M, pH=7, adsorption time 60min, ligand density of 140 mol - g-1, BSA (1mg - mL-1) the adsorption capacity reached 117.6mg / g-1. (2) surface modification of affinity medium with polyethylene glycol mPEG1900 with three boron fluoride as catalyst. Catalytic Agent 1:0.08 (molar ratio), reaction time 6h, reaction temperature is 50 DEG C, the grafting ratio of mPEG1900 was 26.46%, and by infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), differential thermal analysis (DSC) and thermogravimetric analysis (TG) were used to characterize the effects of the grafting rate. To stop the BSA adsorption effect, adsorption affinity of restricted access media for BSA decreased with the grafting ratio of mPEG1900 increased. When the medium ligand density of 140 mol - g-1, the surface grafting ratio of mPEG1900 is 26.46%, BSA medium on the block rate of 50.68%. (3) in order to further explore the application effect of affinity medium, with Val-Ser-Leu-Pr o-Glu-Try (VW-6) is a small molecular peptide model, BSA protein model molecules, established the simulation system and the mixed casein solution system, compared the mPEG1900 modified affinity medium in the mixed system and casein solution system in a small Selective adsorption of molecules and macromolecules to block the effect. The results show that the limit in affinity medium adsorption on VW-6 was increased, adsorption of BSA molecules has a barrier effect. That limit in metal ion affinity enrichment medium to small peptides effectively, reduce the nonspecific adsorption of protein molecules that provides a feasible method for improving the separation efficiency of active small molecules.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O658.1

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本文編號(hào)：1619668