摘要
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p ET-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 k D,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Ig E抗体(s Ig E)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。
Objective To express the cashew nut allergen Ana o 2 and identify its immunological activity.Methods Ana o 2 gene was synthesized and inserted into the p ET-28 a(+)expression vector.The recombinant plasmid p ET-28 a(+)-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 k D,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(s Ig E)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.
作者
康晨
闫娟娟
姜琛
张嘉懿
谢万珍
毕宏晨
李会强
KANG Chen;YAN Juanjuan;JIANG Chen;ZHANG Jiayi;XIE Wanzhen;BI Hongchen;LI Huiqiang(College of Medical Laboratory,Tianjin Medical University,Tianjin 300203,China;Department of Medical Laboratory,Tianjin Children's Hospital,Tianjin 300074,China)
出处
《中国食品卫生杂志》
CSCD
北大核心
2020年第1期10-15,共6页
Chinese Journal of Food Hygiene
基金
国家自然科学基金项目(81772259).
