中文摘要：鞭毛(FliC)可作为载体蛋白用于制备结合疫苗，诱导产生T细胞独立免疫反应且能够作为一种固有佐剂激活TLR5从而增强疫苗的活性。为充分利用FliC自佐剂载体的这一特性，研究探索了FliC D2和D3区域相关氨基酸残基的位点选择性修饰方法，该方法不依赖于具有TLR5绑定和激活作用的D0和D1区域的过度修饰。在高浓度的Na2SO4溶液中，单个鞭毛形成了鞭毛丝，其中D0和D1区域位于管状结构的内侧；因而，D2和D3区域的赖氨酸残基（K219、K224、K324和K331）就通过与咪唑-1-磺酰叠氮盐酸盐重氮基转移反应进行了选择性修饰。通过MALDI-TOF-MS、ESI-TOF-MS和LC-MS/MS分析和定量分析能够确认这些位点的叠氮修饰。叠氮修饰后的鞭毛丝能够溶解为鞭毛单体，从而可以通过点击化学反应与炔链糖类结合。结果表明，修饰后的鞭毛单体保留了与TLR5结合以及形成鞭毛丝的能力。该研究通过FliC D0区和D1区空间自我保护作用确立了一种FliC位点选择性修饰的方法。
外文摘要：Flagellin (FliC) can act as a carrier protein in the preparation of conjugate vaccines to elicit a T-cell-dependent immune response and as an intrinsic adjuvant to activate the toll-like receptor5 (TLR5) to enhance vaccine potency. To enable the use of FliC as a self-adjuvanting carrier, an effective method for site-selective modification (SSM) of pertinent amino-acid residues in the D2 and D3 domains of FliC is explored without excessive modification of the D0 and D1 domains, which are responsible for activating and binding with TLR5. In highly concentrated Na2SO4 solution, FliC monomers form flagellar filaments, in which the D0 and D1 domains are situated inside the tubular structure. Thus, the lysine residues (K219, K224, K324, and K331) in the D2 and D3 domains of flagellin are selectively modified by a diazo-transfer reaction with imidazole-1-sulfonyl azide. The sites with azido modification are confirmed by MALDI-TOF-MS, ESI-TOF-MS, and LC-MS/MS analyses along with label-free quantitation. The azido-modified filament dissolves to give FliC monomers, which can conjugate with alkyne-hinged saccharides by the click reaction. Transmission electron microscopy imaging, dynamic light scattering measurements, and the secreted embryonic alkaline phosphatase reporter assay indicate that the modified FliC monomers retain the ability either to bind with TLR5 or to reassemble into filaments. Overall, this study establishes a feasible method for the SSM of FliC by steric self-protection of the D0 and D1 domains.
外文关键词：antigens, conjugate vaccines, flagellins, self-assembly, steric hindrance
作者：Peng, CJ; Chen, HL; Chiu, CH; Fang, JM
作者单位：中国台湾大学
期刊名称：CHEMBIOCHEM
期刊影响因子：2.847
出版年份：2018
出版刊次：4
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