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  • 學位論文

鍍鋁玻片上的醣晶片研究

Development of Glycan Array Based on Aluminum Oxide Coated Glass Slide

指導教授 : 翁啟惠
共同指導教授 : 吳宗益(Chung-Yi Wu)

摘要


利用鍍鋁的玻片表面,我們研發出新型的醣晶片平台。在早年的研究中,磷酸分子被證實能夠快速與氧化鋁進行結合反應。利用這個特性,我們將數種不同的帶磷酸特別化合物,快速結合到鍍鋁玻片表面,製造出了兩種不同的醣晶片: 共價結合醣晶片與非共價結合醣晶片。 針對非共價結合醣晶片,我們使用多氟化合物製造出高疏水性但高氟親合性的表面。 接著,將帶氟的醣分子轉移到非共價結合醣晶片上。利用質譜儀,我們能夠去分析晶片上醣分子的分子量變化。擁有如此方便的特性,我們將能分析許多醣轉移酶或是醣分解酶的活性。 針對共價結合醣晶片,帶有磷酸的醣分子直接被結合到晶片表面。利用金屬表面所帶來的高度反射性,螢光訊號將能夠被高度放大,如此我們能夠分析許多以前無法分析的抗體-抗原結合反應。 利用所建立的分析平台,我們針對愛滋病毒的廣效中和性抗體,進行了深入的探討。其中PG16 和PGT141-144 抗體,皆第一次經由完整的多醣資料庫而被分析出抗原的專一性。另外針對PG9抗體,我們完整分析出非常特別的多抗原結合反應。而這些新發現的抗原組合,將能夠為將來的愛滋病毒疫苗設計,帶來相當程度的幫助。

關鍵字

醣晶片

並列摘要


A new type of glycan array covalently or non-covalently attached to aluminum-oxide coated glass (ACG) slides has been developed for studies of enzymatic reactions and antibody bindings. For non-covalent array, glycans with a poly-fluorinated hydrocarbon (-C8F17) tail are spotted robotically onto the ACG slide surface con taining a layer of poly-fluorinated hydrocarbon terminated with phosphonic acid. The samples on non-covalent array can be characterized by MS-TOF without addition of matrix. A representive cellotetraose array was developed to study the activity and specificity of different cellulases and to differentiate the exo- and endo-glucanase activities. For covalent array, glycans with a phosphonic acid tail were synthesized and printed onto the ACG slide surface. Compared to glycan arrays on glass slides and other surfaces, this method of arraying using phosphonic acid reacting with ACG is more direct, convenient, and effective. Moreover, the high reflectivity of ACG surface significantly amplified the signal of binding. With the advantage of ACG-based glycan array, the weak bindings of HIV-1 broadly neutralizing antibodies (bNAbs) PG16, PG9, and PGT141-144 have been well characterized by ACG-based glycan array. Through glycans affinity profiling, we proved that the epitope of PG16 is a tri-saccharide of α-2,6-NeuAc-Gal-GlcNAc rather than previously reported whole complex-type glycan. Moreover, we found the hetero-ligand binding effect of PG9 was proved by the combination of complex-type glycan and Man5GlcNAc glycan. Besides, the promiscuous affinity of PG9 to hybrid type glycan also expands the neutralizing range of HIV-1 bNAbs PG9. Moreover, glycan array of mixing glycans becomes critical in dissolve the epitope of bNAbs since binding assay on single glycans may yield misleading results. The results of ACG-based glycan array showed that Man3GlcNAc2 has also been discovered to be the best binding target of bNAbs PGT141, PGT142, PGT143, and PGT144. In addition, Man5GlcNAc2 and Man9GlcNAc2, both have similar core structure, could be bound by PGT 141-145 antibodies in a weaker manner. This result also explains the broad neutralizing range of isolated HIV-1 antibodies. With ACG-based glycan array, we are able to discuss, for the first time, the details of broad neutralizing ranges of many isolated HIV-1 antibodies with glycan database of diverseness.

並列關鍵字

Glycan Array

參考文獻


135. Srinivasan, A.; Viswanathan, K.; Raman, R., et al. Proc Natl Acad Sci U S A 2008, 105 (8), 2800-5.
73. Ratner, Daniel M.; Seeberger, Peter H. Current Pharmaceutical Design 2007, 13 (2), 173-183.
68. Park, Sungjin; Lee, Myung-Ryul; Shin, Injae. Chemical Communications 2008, (37), 4389-4399.
140. Liang, C. H.; Wang, S. K.; Lin, C. W., et al. Angew Chem Int Edit 2011, 50 (7), 1608-1612.
131. Huang, C. Y.; Thayer, D. A.; Chang, A. Y., et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15.

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