Chin J Schisto Control ›› 2014, Vol. 26 ›› Issue (4): 420-.

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Construction and expression of a chimeric gene with T-/B-cell epitopes of major allergen group 1 from Dermatophagoides farina

XU Hai-feng| XU Peng-fei| WANG Ke-xia* |LI Chao-pin   

  1. Department of Pathogeny| Biology of Anhui University of Science and Technology| Huainan 232001| China
  • Online:2014-08-15 Published:2014-08-12
  • Contact: WANG Ke?xia

粉尘螨1类变应原T和B细胞表位嵌合基因的构建与表达

徐海丰|徐朋飞|王克霞*|李朝品   

  1. 安徽理工大学医学院病原生物学教研室(淮南 232000)
  • 通讯作者: 王克霞
  • 作者简介:徐海丰| 男| 硕士研究生。研究方向: 感染性疾病
  • 基金资助:
    国家自然科学基金 (81270091、 30872367); 安徽省自然 科学基金 (070413088)

Abstract: Objective 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 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 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 Conclusion Two chimeric proteins are expressed successfully,which contain T?/B?cell epitopes of Der f 1 and provide a basis for specific immunotherapy.

Key words: Dermatophagoides farina;Group 1 allergen derived from Dermatophagoides farina(Der f 1);T?cell epitope; B?cell epitope; Western blotting

摘要: 目的 目的 构建粉尘螨1类变应原 (Der f 1) 的T和B细胞表位嵌合基因原核表达载体。方法 方法 将Der f 1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Der f 1A和Der f 1B。构建原核表达重组质粒pET?28a (+) ?Der f 1A和pET?28a (+) ?Der f 1B, 双酶切及测序验证的阳性克隆转化到E.coli BL21 (DE3) 后诱导表达。SDS?PAGE分析表达 产物后进行纯化, 并进行Western blotting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 结 果 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 (+) ?Der f 1A和pET?28a (+) ?Der f 1B; SDS?PAGE分 析表明, Der f 1A和Der f 1B诱导并纯化成功, Western blotting结果进一步证实纯化了Der f 1A和Der f 1B嵌合蛋白。与 Der f 1相比, Der f 1A和Der f 1B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Der f 1A 和Der f 1B间差异无统计学意义 (P>0.05)。结论 结论 成功表达了Der f 1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。

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

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