Mapping epitopic regions of cholera toxin B-subunit protein.
Mapping epitopic regions of cholera toxin B-subunit protein.
复制标题
绘制霍乱毒素 B 亚基蛋白表位区域图。
DOI：
10.1016/0161-5890(91)90050-t
复制
发表时间：
1991
影响因子：
3.6
通讯作者：
Finkelstein,RA
中科院分区：
文献类型：
--
作者：
Kazemi,M;Finkelstein,RA
使用代表霍乱肠毒素免疫显性 B 亚基蛋白的完整 103 个氨基酸序列的连续重叠合成六肽来检查各种抗血清的反应性，试图检测和定义序列相关（连续）抗原区域。该方法的有效性是通过针对较长合成肽的多克隆抗血清与适当的合成六肽的反应来确定的。意想不到的交叉反应归因于 B 亚基链的两个部分中存在三个相同的氨基酸 (Gln16-Ile17-His18)，尽管顺序不同 (Gln56-His57-Ile58)。使用多克隆兔抗血清的吸附研究表明，在许多情况下，变性的 B 亚基蛋白比天然蛋白更能有效地消除与六肽的反应性。天然全毒素比天然 B 亚基更有效。来自人类霍乱康复者的血清与合成六肽具有弥漫性反应模式——主要针对反应性六肽区域，而不是针对明确定义的连续表位。在遇到的许多表位区域中，在 B 亚基氨基酸序列的高度保守区域（残基 55-58）中发现了强反应性四聚体 Ser-Gln-His-Ile (SQHI)。吸附研究表明该表位明显暴露在天然蛋白质的表面。氨基酸取代揭示了谷氨酰胺和组氨酸残基对该表位的重要性。 Gly54 不是表位的一部分，但用酸性残基 Glu 和 Asp 取代 Gly 消除了与抗体的反应性。结果表明，连续表位可能有助于天然毒素蛋白的抗原性，并且可能对肽疫苗的开发有用。
Continuous overlapping synthetic hexapeptides representing the entire 103 amino acid sequence of the immunodominant B-subunit protein of cholera enterotoxin were used to examine reactivities of a variety of antisera in attempts to detect and define sequence-related (continuous) antigenic regions. The validity of the methods was established by the reactions of polyclonal antisera raised against longer synthetic peptides with appropriate synthetic hexapeptides. An unexpected cross-reaction is attributed to the presence of three identical amino acids (Gln16-Ile17-His18)—although in different order (Gln56-His57-Ile58)—in two parts of the B-subunit chain. Adsorption studies using polyclonal rabbit antisera revealed that, in many instances, denatured B-subunit protein more effectively removed reactivity with hexapeptides than did the native protein. Native holotoxin was more effective than native B-subunit. Sera from human cholera convalescents gave diffuse patterns of reactivity with synthetic hexapeptides—primarily against regions of reactive hexapeptides rather than with clearly defined continuous epitopes. Among many epitopic regions encountered, a strongly reactive tetramer, Ser-Gln-His-Ile (SQHI), was discovered in a highly conserved region, residues 55–58, of the B-subunit amino acid sequence. Adsorption studies revealed that this epitope is apparently exposed on the surface of the native protein. Amino acid substitution revealed the essentiality of Gln and His residues to this epitope. Gly54was not part of the epitope but substitution of acidic residues Glu and Asp for Gly eliminated reactivity with antibody. The results suggest that continuous epitopes may contribute to the antigenicity of the native toxin protein and may be potentially useful for development of a peptide vaccine.