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目的克隆、表达与纯化鳙鱼(Aristichthys nobilis)过敏原小清蛋白,并检测免疫学活性。方法提取鳙鱼的总RNA,设计简并引物,采用RT-PCR方法扩增目的基因,克隆入T载体中进行测序和分析。将目的基因克隆到pET-28a(+)表达载体中于E.coli BL21(DE3)表达。通过Ni2+亲和层析,对重组蛋白进行纯化,采用免疫印迹(Western-blotting)方法检测其IgE结合活性。结果获得了鳙鱼过敏原小清蛋白基因,该基因被GenBank收录,登陆号FJ013047。基因开放阅读框共330个碱基(包括终止密码子),编码109个氨基酸,相对分子质量为11 537。Western-blotting结果表明,纯化后的重组蛋白能与过敏性患者血清中的特异性IgE结合。结论成功克隆、表达、纯化鳙鱼过敏原小清蛋白,此重组蛋白具有良好的免疫原性。
Objective To clone, express and purify the allergen small albumin of Aristichthys nobilis and measure its immunological activity. Methods The total RNA was extracted from minnows and degenerate primers were designed. The target gene was amplified by RT-PCR and cloned into T vector for sequencing and analysis. The target gene was cloned into pET-28a (+) expression vector for expression in E. coli BL21 (DE3). The recombinant protein was purified by Ni2 + affinity chromatography and its IgE binding activity was detected by Western-blotting. Results The allelopathrin gene was obtained from GenBank, accession number FJ013047. The open reading frame (ORF) of gene contains 330 bases (including the stop codon) and encodes 109 amino acids with a relative molecular mass of 11 537. Western-blotting results showed that the purified recombinant protein could bind with specific IgE in the serum of allergic patients. Conclusion The cloning, expression and purification of the minnow allergen was successful. The recombinant protein has good immunogenicity.