Ito K, Ishiguro A, Kanbe T, Tanaka K, Torii S
Department of Paediatrics, Nagoya University School of Medicine, Japan.
Clin Exp Allergy. 1995 Jun;25(6):529-35. doi: 10.1111/j.1365-2222.1995.tb01090.x.
Candida albicans enolase is one of the important allergens in Candida allergy. We isolated and purified 46kDa C. albicans enolase (CAE) from C. albicans and characterized epitopes for IgE antibody by lectin-blotting and enzymatic digestion followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Lectin blotting and deglycozilation indicated that this protein did not contain polysaccharide side chains. The purified CAE and recombinant fusion protein produced from CAE gene possessed common epitopes for IgE antibody. We estimated IgE binding epitopes on the basis of reported amino acid sequences from the analysis of cDNA encoding CAE. V8 protease digestion of CAE gave six polypeptide fragments (A-F). The N-termini of each fragment were confirmed by amino acid sequence and the C-termini were estimated by molecular weights of each fragment and the specific cutting site of V8 protease. Fragment C (25.0 kDa; F-171-I-399) reacted to 90% IgE antibodies examined, whereas fragments D (21.0 kDa; F-171-I-360), E (16.2 kDa; F-171-D-317) and F (13.0 kDa; A-47-E-170) showed no IgE binding. Our results suggest that epitopes for IgE antibodies exist near the C-terminal of the protein.
白色念珠菌烯醇化酶是念珠菌过敏中的重要变应原之一。我们从白色念珠菌中分离并纯化了46kDa的白色念珠菌烯醇化酶（CAE），通过凝集素印迹和酶消化，随后进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹来鉴定IgE抗体的表位。凝集素印迹和去糖基化表明该蛋白不含多糖侧链。纯化的CAE和由CAE基因产生的重组融合蛋白具有IgE抗体的共同表位。我们根据编码CAE的cDNA分析中报道的氨基酸序列来估计IgE结合表位。CAE经V8蛋白酶消化产生六个多肽片段（A-F）。每个片段的N端通过氨基酸序列确定，C端通过每个片段的分子量和V8蛋白酶的特异性切割位点来估计。片段C（25.0 kDa；F-171-I-399）与所检测的90%的IgE抗体发生反应，而片段D（21.0 kDa；F-171-I-360）、E（16.2 kDa；F-171-D-317）和F（13.0 kDa；A-47-E-170）未显示出IgE结合。我们的结果表明IgE抗体的表位存在于该蛋白的C端附近。
