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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。
Objective To construct a prokaryotic expression vector for T and B cell epitope chimeric genes of der f 1 allergen. Methods Five T cell epitopes (T1 ~ T5) and six B cell epitopes (B1 ~ B6) contained in Der f 1 were expressed in B1-T1-B2-T2-B3-T3-B4- T5-B6 and B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5 ligation were used to directly synthesize the epitope chimeric genes and named Der f 1A and Der f 1B, respectively. The recombinant plasmid pET-28a (+) - Der f 1A and pET-28a (+) - Der f 1B was successfully constructed and transformed into E.coli BL21 (DE3) for expression. The expressed product was purified by SDS-PAGE and identified by Western blotting. ELISA method to detect chimeric protein dust dust mite allergy patients with serum IgE antibody binding. Results The results of double enzyme digestion and sequencing showed that the prokaryotic expression plasmids pET-28a (+) - Der f 1A and pET-28a (+) - Der f 1B were constructed successfully. SDS-PAGE analysis showed that Der f 1A and Der f 1B was induced and purified successfully. The results of Western blotting further confirmed the purification of Der f 1A and Der f 1B chimeric proteins. Compared with Der f 1, the binding ability of Der f 1A and Der f 1B chimeric proteins to serum IgE antibody was significantly decreased in patients with dust mite allergy (all P <0.05), but there was no significant difference between Der f 1A and Der f 1B Significance (P> 0.05). Conclusion The T and B cell epitopes of Der f 1 were successfully expressed, which laid the foundation for the specific immunotherapy of dermatophagoides.