AAV9受体亲和纯化法初探

发布时间：2018-01-07 23:27

本文关键词：AAV9受体亲和纯化法初探　出处：《华侨大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景腺相关病毒(Adeno-asscociated virus,AAV)是一种能够介导目的基因长效表达的新型基因转移载体。其中,9型腺相关病毒(AAV9)可靶向心肌细胞及神经胶质细胞,并可透过血脑屏障,在临床应用中有较为理想的应用前景。由于研究中的广泛使用,AAV9的大规模纯化成为其亟待解决的问题。传统纯化方法费时费力且纯化效率不高,存在安全隐患,针对于此,选择一种特异性强、操作稳定,并适用于AAV9大规模生产的纯化方法至关重要。亲和色谱法(Affinity chromatography,AC)是一种能将特定的病毒粒子、蛋白质和DNA混合物分离的层析技术,主要依靠病毒表面衣壳与色谱填料的生物活性配基或者配对受体之间的可逆性结合发挥作用,具有选择性良好、分辨率高、病毒粒子与基质能较好结合,并具有较高的回收率等特点,在众多领域备受亲睐。近年来,亲和层析技术也被用于AAV纯化领域,如抗体特异性亲和法、受体特异性亲和法。近期研究发现AAV9可以特异性结合末端为β-1,4半乳糖基的受体,为AAV9受体亲和色谱纯化提供了可能。研究目的通过筛选与AAV9特异性结合的物质,构建一种AAV9受体特异性的亲和纯化法,为建立AAV9受体亲和纯化系统做前期准备。研究方法(1)通过查阅文献资料筛选确定候选配体分子;(2)采用物理结合实验、细胞抑制实验及生物信息学筛选等方法筛选确定能与AAV9特异性结合的物质;(3)构建配体亲和纯化柱,纯病毒上样,通过改变纯化柱的平衡缓冲液、洗脱缓冲液及pH等条件优化纯化柱柱效;(4)纯化病毒生产原液,使用银染检测纯化病毒的纯度、Western Blot检测纯化病毒的特异性及使用Real-time PCR检测纯化病毒的滴度。研究结果(1)通过查阅文献资料筛选确定了8种候选配体分子,分别为:D-氨基半乳糖、乳糖酸、N-乙酰-D-半乳糖胺、肌醇半乳糖苷、D-半乳糖、D-半乳糖醛酸、低聚半乳糖、N-乙酰-D-乳糖胺;(2)采用物理结合实验、细胞抑制实验及生物信息学筛选等方法确定能与AAV9特异性结合的物质为纯化柱配基,分别为D-半乳糖、乳糖酸、D-半乳糖醛;(3)同时建立了乳糖酸纯化柱、D-半乳糖醛酸纯化柱及D-半乳糖纯化柱三种纯化柱,并通过对纯化条件如平衡缓冲液、洗脱缓冲液及pH等的考察,最终确定以10mM Tris-HCl,pH=8缓冲液中添加4m M CaCl2为平衡缓冲液,并以添加20mM NaCl为起始洗脱液;(4)以D-半乳糖为配基的亲和纯化柱纯化病毒生产原液,按上述纯化条件,检测纯化样品,验证了病毒的特异性与纯度。研究结论成功构建了以D-半乳糖、D-半乳糖醛酸、乳糖酸为配体的纯化柱,为建立AAV9受体亲和纯化系统做前期准备;但纯化柱与AAV9的亲和力不高,纯化效率较低,有待进一步优化。
[Abstract]:The research background of adeno-associated virus (Adeno-asscociated virus AAV) is a novel gene mediated gene transfer vector long-term expression. Among them, 9 type adeno-associated virus (AAV9) can be targeted myocardial cells and glial cells, and can penetrate the blood brain barrier has ideal application prospect in clinical application. Due to the widespread use of AAV9, the large-scale purification become a serious problem. The traditional purification method is time-consuming and the purification efficiency is not high, there are security risks, according to this, select a specific, stable operation, purification methods are very important and applicable to AAV9 mass production (Affinity affinity chromatography. Chromatography, AC) is a kind of specific virus particles, chromatography separation of protein and DNA mixture, rely mainly on the surface of the virus capsid and packing the bioactive ligand or pairing Reversible binding between receptors play a role, with good selectivity, high resolution, virus particles and the matrix have a good combination, and has characteristics of high recovery rate, is very popular in many fields. In recent years, affinity chromatography was also used for AAV purification, such as antibody affinity, receptor affinity method. Recent studies found that AAV9 could bind to -1,4 terminal beta galactosyl receptors, provides the possibility for AAV9 receptor affinity chromatography. The purpose of the study through a combination of screening and AAV9 specific substances, affinity purified method to construct a AAV9 receptor specificity, for the establishment of AAV9 receptor affinity purification system to make early preparations. Research methods (1) through the literature material screening to identify candidate ligand molecules; (2) the combination of physical experiment, cell inhibition test and bioinformatics screening methods such as screening to determine and AAV9. Specific binding material; (3) construct ligand affinity purification column, pure virus sample, purified by changing the column equilibration buffer, elution buffer and pH optimized purification column efficiency; (4) the production of purified virus solution, using silver staining detection of purity of purified virus, Western Blot virus titer detection and purification the specific use of Real-time and PCR detection for virus purification. Results (1) through the literature material screening identified 8 candidate ligands, respectively: D- galactosamine, lactobionic acid, N- acetyl -D- galactosamine, inositol half lactoside, D- galactose, D- galacturonic acid, Galacto sugar, N- acetyl -D- galactosamine; (2) the combination of physical experiment, cell inhibition test and bioinformatics screening method to determine the AAV9 binding and the material for the purification column ligand, respectively D- galactose, lactose acid, D- galactose aldehyde; (3) also established Lactose acid purification column, purified D- galacturonic acid purification column and D- galactose column three purification column, and the purification conditions such as buffer and elution of buffer and pH, finally confirmed the 10mM Tris-HCl pH=8 M CaCl2 add 4m buffer to balance the buffer, and to add 20mM NaCl as the starting eluent; (4) to D- galactose affinity purification column purified virus production liquid ligand, the purification conditions, detection of purified samples, verified the specificity and purity of the virus. The conclusion of the study is successfully constructed with D- galactose, D- galacturonic acid, lactobionic acid purification column in order to establish the AAV9 receptor ligand, affinity purification system to make early preparations; but the purification column and AAV9 affinity purification is not high, low efficiency, need to be further optimized.

【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R450

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