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  本文關(guān)鍵詞：AAV9受體親和純化法初探　出處：《華僑大學》2016年碩士論文　論文類型：學位論文

  更多相關(guān)文章： AAV9 基因治療 親和純化 受體

【摘要】：研究背景腺相關(guān)病毒(Adeno-asscociated virus,AAV)是一種能夠介導目的基因長效表達的新型基因轉(zhuǎn)移載體。其中,9型腺相關(guān)病毒(AAV9)可靶向心肌細胞及神經(jīng)膠質(zhì)細胞,并可透過血腦屏障,在臨床應用中有較為理想的應用前景。由于研究中的廣泛使用,AAV9的大規(guī)模純化成為其亟待解決的問題。傳統(tǒng)純化方法費時費力且純化效率不高,存在安全隱患,針對于此,選擇一種特異性強、操作穩(wěn)定,并適用于AAV9大規(guī)模生產(chǎn)的純化方法至關(guān)重要。親和色譜法(Affinity chromatography,AC)是一種能將特定的病毒粒子、蛋白質(zhì)和DNA混合物分離的層析技術(shù),主要依靠病毒表面衣殼與色譜填料的生物活性配基或者配對受體之間的可逆性結(jié)合發(fā)揮作用,具有選擇性良好、分辨率高、病毒粒子與基質(zhì)能較好結(jié)合,并具有較高的回收率等特點,在眾多領(lǐng)域備受親睞。近年來,親和層析技術(shù)也被用于AAV純化領(lǐng)域,如抗體特異性親和法、受體特異性親和法。近期研究發(fā)現(xiàn)AAV9可以特異性結(jié)合末端為β-1,4半乳糖基的受體,為AAV9受體親和色譜純化提供了可能。研究目的通過篩選與AAV9特異性結(jié)合的物質(zhì),構(gòu)建一種AAV9受體特異性的親和純化法,為建立AAV9受體親和純化系統(tǒng)做前期準備。研究方法(1)通過查閱文獻資料篩選確定候選配體分子;(2)采用物理結(jié)合實驗、細胞抑制實驗及生物信息學篩選等方法篩選確定能與AAV9特異性結(jié)合的物質(zhì);(3)構(gòu)建配體親和純化柱,純病毒上樣,通過改變純化柱的平衡緩沖液、洗脫緩沖液及pH等條件優(yōu)化純化柱柱效;(4)純化病毒生產(chǎn)原液,使用銀染檢測純化病毒的純度、Western Blot檢測純化病毒的特異性及使用Real-time PCR檢測純化病毒的滴度。研究結(jié)果(1)通過查閱文獻資料篩選確定了8種候選配體分子,分別為: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)采用物理結(jié)合實驗、細胞抑制實驗及生物信息學篩選等方法確定能與AAV9特異性結(jié)合的物質(zhì)為純化柱配基,分別為D-半乳糖、乳糖酸、D-半乳糖醛;(3)同時建立了乳糖酸純化柱、D-半乳糖醛酸純化柱及D-半乳糖純化柱三種純化柱,并通過對純化條件如平衡緩沖液、洗脫緩沖液及pH等的考察,最終確定以10mM Tris-HCl,pH=8緩沖液中添加4m M CaCl2為平衡緩沖液,并以添加20mM NaCl為起始洗脫液;(4)以D-半乳糖為配基的親和純化柱純化病毒生產(chǎn)原液,按上述純化條件,檢測純化樣品,驗證了病毒的特異性與純度。研究結(jié)論成功構(gòu)建了以D-半乳糖、D-半乳糖醛酸、乳糖酸為配體的純化柱,為建立AAV9受體親和純化系統(tǒng)做前期準備;但純化柱與AAV9的親和力不高,純化效率較低,有待進一步優(yōu)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.

【學位授予單位】：華僑大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R450

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本文編號：1394709