腰果主要过敏原Ana o 2原核表达及免疫学鉴定

目的 构建Ana o 2重组表达质粒,原核表达重组蛋白并评价其免疫活性。方法 将Ana o 2基因构建到pET-28a(+)载体中,测序正确的重组质粒转化Rosetta(DE3)感受态细胞,诱导表达目的蛋白并进行质谱鉴定。利用腰果过敏阳性血清,通过酶联免疫吸附试验(ELISA)和蛋白质免疫印记(Western blot)法评价重组Ana o 2的免疫活性。结果 重组蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)表明分子量为54 kD,与理论值相符,经质谱鉴定为Ana o 2。ELISA结果显示,用重组Ana o 2检测腰果过敏阳性血清与阴性血清特异性IgE抗体(sIgE)水平差异有统计学意义(t=2.44,P<0.05)。Western blot结果表明,重组Ana o 2与腰果过敏患者血清反应性良好。结论 利用原核系统表达了Ana o 2,并且重组Ana o 2与腰果过敏患者血清具有良好的反应性。

Prokaryotic expression and identification of recombinant cashew nut allergen Ana o 2

Objective To express the cashew nut allergen Ana o 2 and identify its immunological activity. MethodsAna o 2 gene was synthesized and inserted into the pET-28a(+) expression vector. The recombinant plasmid pET-28a-Ana o 2 was constructed and sequenced. The correct recombinant plasmid was transformed into Rosetta (DE3), and the recombinant Ana o 2 protein was expressed and purified. The purified recombinant protein was identified by mass spectrometry. The immunological activity of the recombinant Ana o 2 protein was evaluated by enzyme-linked immunosorbent assay(ELISA) and protein immunoblot(Western blot). Results As determined by sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE), the molecular weight of the recombinant protein was approximately 54 kD, which was consistent with the theoretical value. The recombinant protein was identified as Ana o 2 by mass spectrometry. ELISA result showed that the level of specific immunoglobulin E (sIgE) in cashew-allergic sera was significantly different from that in negative sera (t=2.44,P<0.05). Western blot result showed that recombinant Ana o 2 had good reactivity with cashew-allergic sera. Conclusion A recombinant expression vector of Ana o 2 had been successfully constructed. Recombinant Ana o 2, which was expressed by prokaryotic system, had good reactivity with cashew-allergic sera.