小龙虾主要过敏原精氨酸激酶的表达、纯化及 免疫原性鉴定

Expression, purification and immunological identification of the main allergen arginine kinase in Procambarus clarkii

中文关键词:  小龙虾  精氨酸激酶  重组蛋白  过敏原

英文关键词:Procambarus clarkii  arginine kinase  recombinant protein  allergen

中文摘要:

      目的 对纯化后的原核表达小龙虾主要过敏原蛋白精氨酸激酶进行免疫学鉴定。方法 将化学合成的精氨酸激酶基因克隆至pET-28a(+)表达质粒上, 转化进入大肠杆菌Rosetta(DE3)中, 使用异丙基硫代半乳糖苷进行目的蛋白的诱导表达, 用Ni2+亲和层析柱对重组过敏原蛋白进行纯化, 使用小龙虾过敏病人混合血清对纯化后的蛋白进行免疫印迹鉴定。结果 纯化得到了分子量大小约为40 kDa的重组小龙虾精氨酸激酶, 并且重组蛋白与过敏病人血清IgE有明显的特异性结合。结论 本实验建立了小龙虾精氨酸激酶的原核表达方法, 并鉴定了其免疫原性, 为小龙虾过敏的基础研究、临床诊断与治疗奠定了基础。

英文摘要:

      Objective To carry out immunological identification of the purified prokaryotic expression of allergen protein arginine kinase of Procambarus clarkii. Methods The chemically synthesized arginine kinase gene was cloned onto the pet-28a (+) expression plasmid and transformed into Rosetta (DE3) of escherichia coli, where isopropyl thioglycoside was used to induce the expression of the target protein, the recombinant allergen protein was purified by Ni2+ affinity chromatography column, and the purified protein was identified by western blotting using the mixed serum of crayfish allergy patients. Results The recombinant crayfish arginine kinase with molecular weight of about 40 kDa was purified, and the recombinant protein had obvious specific binding with serum IgE of allergic patients. Conclusion The prokaryotic expression method of arginine kinase in crayfish is established and its immunogenicity is identified, which lays a foundation for the basic research, clinical diagnosis and treatment of crayfish allergy.