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標題: | 抗體徵招類共聚物的合成及其在標記細菌上的應用 Synthesis of Antibody-Recruiting Copolymers and their Applications in Targeting Bacteria |
作者: | Kuo-Lun Chien 簡國倫 |
指導教授: | 王宗興(Tsung-Shing Wang) |
關鍵字: | 共聚物,鐵載體,甘露糖,抗體徵招,吞噬作用, copolymer,siderophore,mannose,antibody-recruiting,phagocytosis, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 近年來,由於抗藥性細菌導致的感染在世界上越來越嚴重,越來越少抗生素可消滅抗藥性細菌,因此需要開發新的抗菌劑跟治療方法,其中專一性標記抗藥性菌屬的探針是設計的關鍵。在此我們利用共聚物(copolymer) 作為骨架,使其帶有多個辨識分子;辨識分子的多價性 (multivalency) 可增強專一性標定特定細菌的親和力。聚丙烯醯胺(polyacrylamide)具有水溶性和生物相容性 (biocompatible) 可作為聚合物骨架。我們合成帶有可點擊 (clickable) 的丙烯醯胺衍生物,利用ATRP (Atom-transfer radical-polymerization) 跟丙烯醯胺合成可改變點擊官能基密度的共聚物,調整多價性。利用點擊反應修飾上各式辨識單元,如:鐵載體 (siderophore) 和甘露糖 (mannose, Man)。鐵載體與鐵具有高度親和力,在限鐵的環境下會被細菌辨識而吸收利用,由副溶血弧菌(Vibrio parahaemolyticus)所分泌的vibrioferrin (VF) 為例子;另外大腸桿菌鞭毛可專一吸附甘露糖。配合部分點擊上螢光素 (fluorescein, FL),我們合成兩種共聚物探:PVF (Polymer+VF+FL) 與PMF (Polymer+Man+FL) 幫助標定,再利用流式細胞術 (Flow cytometry) 和螢光顯微術 (Fluorescence microscopy) , PVF及PMF可分別專一標記弧菌及大腸桿菌,但不標記其他菌種。更進一步,我們合成具有細菌辨識單元和Biotin及Dinitrophenyl的雙辨識共聚物PMB (Polymer+Man+Biotin) 和PMD (Polymer+Man+Dinitrophenyl) ,可同時辨識細菌和抗體 ,抗體可徵召巨噬細胞,加速被標記細菌的吞噬作用 (phagocytosis)。最後用帶GFP的大腸桿菌和巨噬細胞做實驗,加入PMB和anti-biotin的例子得到螢光反應最大的增幅。 In recent years, serious infections caused by drug-resistant bacteria are increasing while fewer and fewer antibiotics can eliminate drug-resistant bacteria. As a result, people need to develop new antibiotics and treat-ments. One of them is the probe specifically targeting drug-resistant bacteria. In our research, we use copoly-mers as backbone to carry multiple cargos;the multivalency can enhance the affinity to targeting bacteria. Pol-yacrylamide is a water-soluble and biocompatible polymer backbone. We synthesized clickable acrylamide de-rivatives. We prepared copolymers from clickable acrylamide derivatives and acrylamide with ATRP (Atom-transfer radical-polymerization). By tuning the ratio of two monomers, we can adjust the density of clickable functional groups, namely multivalency of copolymers. Cargos such as iron carrier (siderophore) and mannose (Man) were introduced on copolymers through CuAAC (copper(I)-catalyzed azide alkyne cycloaddition) click chemistry. Siderophores, such as vibrioferrin (VF) secreted by Vibrio parahaemolyticus, have high affinity to iron(III) and can be recognized and uptake by bacteria in iron-limited conditions;mannose can specifically bind onto E. coli’s flagella. When clickable fluorescein (FL) was introduced along with clickable cargos, we can synthesize two probes: PVF (Polymer+VF+FL) and PMF (Polymer+Man+FL) to help targeting bacteria. By Flow cytometry and Fluorescence microscopy, PVF and PMF can specifically target respectively Vibrio and E. coli, but not other strains. Furthermore, we synthesize copolymers with bacterial recognition units and biotin and dinitrophenyl, PMB (Polymer+Man+Biotin) and PMD (Polymer+Man+Dinitrophenyl), to simultaneous-ly target bacteria and antibodies. By recruiting macrophages through bound antibodies, PMB can accelerate the phagocytosis of labeled bacteria. Finally, experiment E. coli (GFP) and macrophages are added PMB and anti-biotin gave the largest increase in fluorescence response. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68134 |
DOI: | 10.6342/NTU201704365 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
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