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  • 學位論文

醣奈米粒子作為高親和性探針應用於目標蛋白質濃縮及鑑定

Glyconanoparticle as a High Affinity Probe for Target Protein Enrichment and Identification

指導教授 : 汪根欉
共同指導教授 : 林俊成(Cheng-Chung Lin)

摘要


醣類在細胞辨識、細菌感染或毒素的入侵扮演相當重要角色。而單一醣類與蛋白質間的作用力,往往並不強,自然界為提升蛋白質對醣類的作用力與辨識能力,利用多配位基與多受體的多價性效應(Multivalancy)來解決這項問題。 金奈米粒子由於其容易調控粒子大小、可修飾粒子表面、大的表面積/體積比與特殊的光學性質。因此金奈米粒子已被應用於與各種蛋白質、抗體、凝集素及小分子偶合,並有許多的應用。金奈米粒子由於其粒徑在數十奈米至數百奈米間,正好與蛋白質作用的細胞大小相近,因此金奈米粒子對醣類與蛋白質作用力的研究,可說是一相當優良的載體。 類志賀氏毒素由O157:H7等能致病細菌所分泌,其結構為AB5型式。類志賀氏毒素利用B5與細胞表面上Pk抗原結合後,將具酵素活性的A單元能進入細胞中毒殺細胞,進而造成嚴重疾病。因此B單元與細胞表面Pk抗原作用則此毒素作用的關鍵。 綠膿桿菌則由於容易生長,因此成為醫院院內感染的一株重要潛伏性細菌。其感染途徑則與其膜上二種凝集素有關,被稱為PA-IL及PA-IIL,分別辨識細胞表面半乳糖與岩藻糖。而其結構則為單體四聚物,在辨識細胞表面醣體後,進而可分泌多醣體的生物膜而保護細菌不受外來藥物作用。 本論文將利用醣包覆的金奈米粒子,合成不同長度鏈長的醣類分子裝配於4、13及20 nm的金奈米粒子上。並以化學呈色法來定量各大小金奈米粒子上醣體的數目,以分析醣體數目對多價性作用力的關係。經計算,短鏈Pk配位基平均在每一個4、13及20 nm的金奈米粒子上有60、826及1498個配位基,而在長鏈則有著113、1298、1970個配位基。 論文中還利用表面電漿共振光譜儀測量類志賀氏毒素與綠膿桿菌I型凝集素與晶片上Pk抗原類似物親和力。並經改變稀釋介質來減少醣包覆金奈米粒子對晶片表面非專一性吸附的行為,再以醣包覆金奈米粒子與類志賀氏毒素及綠膿桿菌I型凝集素進行BIAcore競爭性實驗,可發現20 nm長鏈Pk抗原類似物包覆的金奈米粒子,其對類志賀氏毒素的作用力,相對於Pk單體而言有著448萬倍的提升,而金奈米粒子上每一醣體的貢獻更是高達22萬倍的提升。而對於綠膿桿菌的多價性作用力,相對於半乳糖有著31萬倍的提升,這樣的多價性作用力的提升,是在過去多價性作用力當中罕見。 另外與陳玉如老師實驗室合作開發的NBAMS技術,除可將MALDI-TOF偵測綠膿桿菌I型凝集素的極限推至78 fmol,且可減少MALDI-TOF在分析生物樣品中常遇到鹽類問題,另外NBAMS更是成功應用於綠膿桿菌I型凝集素其鍵結活性區的快速鑑定,利用醣包覆金奈米粒子與凝集性作用時,可將其鍵結活性區保護不受蛋白質水解

並列摘要


In this thesis, six Pk encapsulated AuNPs (Pk-AuNPs) with the combination of three different sizes (4 nm, 13 nm and 20 nm) and two different lengths of linker were synthesized. The carbohydrate ligand amount on each AuNP was determined by anthrone method. For the Pk ligand with short linker, there are 60, 826 and 1498 ligands on each 4 nm, 13 nm and 20 nm AuNPs, respectively, while those for the long linker ligand are 113, 1298 and 1970, respectively. The multivalent interaction between Pk-AuNPs and B subunit of shiga-like toxin (Slt-B) were evaluated by surface plasma resonance (SPR) competition assays. The inhibition avidity results showed that the binding affinity was affected by nanoparticle size, linker length and the ligand density on nanoparticle surface. In comparison with the binding affinity of mono Pk ligand with Slt-B, the highest affinity increment was showed by the 20-Pk-l-AuNP (20 nm size and long tether) and its binding affinity was enhanced by 4.48x108 times. Pk-AuNP was also used as affinity probe to simultaneously enrich and isolate the target PA-IL at a femto-mole level from a protein mixture. The captured protein was directly identified by treating with protease and analyzing the hydrolytic peptide fragments. Furthermore, the binding-epitope-containing peptides were revealed by MS-MS analysis of remained peptides on AuNP after washing step. The high affinity AuNP probe was also used as affinity probe to purify recombinant Slt-B from E. Coli cell lysat. The purity of extracted protein is higher than 95% based on the MALDI-TOF and SDS-PAGE analyses, and the purification yield is 1.085 mg of Slt-B from 100 mL of cell culture. Combination with sliver enhancement, a Pk-AuNP based method for the detection of shiga-like toxin was developed. The monoclonal antibody for the Slt-A was immobilized on the glass slide. Thus, the toxin was captured by antibody and the carbohydrate-binding site (Slt-B) was exposed. The presence of toxin was then visualized by staining the chip with the high affinity Pk-AuNP followed by silver enhancement. Although the detection limit of current un-optimized Pk-AuNP based method is not as sensitive as ELISA, our method provides a relative cost effective, rapid and accurate platform.

參考文獻


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被引用紀錄


黃立德(2013)。建構功能化奈米粒子應用於生物分子分離偵測與複合材料的合成〔博士論文,國立清華大學〕。華藝線上圖書館。https://doi.org/10.6843%2fNTHU.2013.00020

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