摘要
目的：构建粉尘螨1类变应原（Derf1）的T和B细胞表位嵌合基因原核表达载体。方法将Derf1包含的5个T细胞表位（T1～T5）和6个B细胞表位（B1～B6），以B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6和B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5连接方式直接合成表位嵌合基因，并分别命名为Derf1A和Derf1B。构建原核表达重组质粒pET-28a（＋）-Derf1A和pET-28a（＋）-Derf1B，双酶切及测序验证的阳性克隆转化到E.coliBL21（DE3）后诱导表达。SDS-PAGE分析表达产物后进行纯化，并进行Westernblotting鉴定。ELISA法检测嵌合蛋白对粉尘螨过敏患者血清IgE抗体的结合力。结果双酶切及测序结果表明，成功构建了原核表达重组质粒pET-28a（＋）-Derf1A和pET-28a（＋）-Derf1B；SDS-PAGE分析表明，Derf1A和Derf1B诱导并纯化成功，Westernblotting结果进一步证实纯化了Derf1A和Derf1B嵌合蛋白。与Derf1相比，Derf1A和Derf1B嵌合蛋白与粉尘螨过敏患者血清IgE抗体的结合能力显著降低（P均＜0.05），但Derf1A和Derf1B间差异无统计学意义（P＞0.05）。结论成功表达了Derf1的T和B细胞表位嵌合蛋白，为粉尘螨过敏的特异性免疫治疗奠定了基础。
Objective To construct and express a chimeric gene with T-/B-cell epitopes of the major allergen group 1 from Dermatophagoides farina（Der f 1）. Methods Two chimeric genes,Der f 1A and Der f 1B,were synthesized as B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6 and B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5 pattens. Two recombinant vectors,pET-28a（＋）-Der f 1A and pET-28a（＋）-Der f 1B,were constructed for prokaryotic expression. These chimeric genes were induced by 1 mmol/L IPTG （final concentration）,digested with restriction enzymes and sequenced. The chimeric proteins were analyzed by SDS-PAGE and Western blotting. Results After digestion by restriction enzymes and sequencing,the recombinant vectors were constructed successfully. The specific bands for chimeric proteins were visible by SDS-PAGE and Western blotting,suggesting that these proteins were purified successfully. Further analyses were performed for IgE-binding properties of Der f 1A and Der f 1B to sera from patients sensitized to house dust mite. Compared with the parental allergens Der f 1,Der f 1A and Der f 1B had reduced IgE-binding capacity（both P〈0.05）,whereas the difference between Der f 1A and Der f 1B was not statistically significant（P〉0.05）. Conclusion Two chimeric proteins are expressed successfully,which contain T-/B-cell epitopes of Der f 1 and provide a basis for specific immunotherapy.
出处
《中国血吸虫病防治杂志》
CAS
CSCD
2014年第4期420-424,共5页
Chinese Journal of Schistosomiasis Control
基金
国家自然科学基金(81270091、30872367)
安徽省自然科学基金(070413088)
关键词
粉尘螨
粉尘螨1类变应原
T细胞表位
B细胞表位
免疫印迹试验
Dermatophagoides farina
Group 1 allergen derived from Dermatophagoidesfarina （Der f 1 ）
T-cell epitope
B-cell epitope
Western blotting
