聚乙二醇限进亲和介质的制备及应用研究

发布时间：2018-03-16 11:14

本文选题：聚乙二醇　切入点：亲和介质　出处：《广西大学》2016年硕士论文　论文类型：学位论文

【摘要】：近年来,固定化金属离子亲和层析(IMAC)技术因其分离快速、分离条件温和,成本低等优势而得到广泛应用。但是在一些复杂的混合体系中,由于蛋白质大分子会与目标小分子竞争亲和位点,降低了亲和介质对活性小分子的分离纯化效果而使其应用范围受到了限制。为了减弱实际体系中大分子蛋白质的干扰,近年来发展起来的限进性介质具有一定的优势,其主要的特点是在吸附介质表面修饰一层半渗透屏障形成体积排阻效应,并结合IMAC的亲和力选择性富集目标活性分子。因此,根据上述思路,研究制备了一种具有高载量、高选择性限进性亲和吸附介质。详细的实验结果如下：(1)采用乳化交联法制备磁性壳聚糖微球(M-CS)作为亲和介质的载体,微球平均粒径为25μm。使用环氧氯丙烷对载体进行活化,当溶胀时间为4h、活化温度为50℃、活化时间为6h、环氧氯丙烷体积分数50%、NaOH浓度为0.4M时,环氧基密度为260μmol·g-1。通过亚氨基乙二酸(IDA)作为间隔臂螯合亲和铜离子(Cu2+),制备M-CS@Cu2+。选择牛血清蛋白(BSA)为大分子蛋白模型,考察了离子强度、pH值、吸附时间、配基密度(Cu2+)对吸附效果的影响,当缓冲液离子浓度为0.02M、pH=7、吸附时间为60min,配基密度为140μmol·g-1时,对BSA (1mg·mL-1)的吸附量达到117.6mg·g-1。(2)用聚乙二醇mPEG1900对亲和介质进行表面修饰,当以三氟化硼为催化剂,催化剂用量(摩尔比)1：0.08、反应时间6h,反应温度50℃时,mPEG1900接枝率达26.46%,并通过红外光谱(FT-IR)、扫描电子显微镜(SEM)、差式量热分析仪(DSC)及热失重(TG)对其进行了表征。研究了接枝率对阻挡BSA吸附效果的影响,限进亲和介质对BSA的吸附量随着mPEG1900的接枝率升高而降低。当介质配基密度为140μmol·g-1,表面mPEG1900接枝率为26.46%时,介质对BSA的阻拒率达到50.68%。(3)为了进一步探索限进亲和介质的应用效果,以Val-Ser-Leu-Pr o-Glu-Try (VW-6)为小分子肽模型,BSA为大分子蛋白模型分子,建立了模拟混合体系以及酪蛋白酶解体系,比较了mPEG1900修饰前后亲和介质在混合体系以及酪蛋白酶解体系中对小分子的吸附选择性以及对大分子的阻拒效果。结果表明限进亲和介质对VW-6的吸附效果有提高,对大分子BSA的吸附具有一定的阻挡效果。说明限进性金属离子亲和介质能够有效的富集小分子多肽,减少大分子蛋白的非特异性吸附,为提高活性小分子的分离效率提供了一种可行的方法。
[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.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O658.1

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