鳙鱼过敏原小清蛋白基因的克隆表达、纯化及免疫学鉴定

Cloning,expression and purification of allergen parvalbumin from Aristichthys nobilis and its allergic activity

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 identify the parvalbumin gene from Aristichthys nobilis,and investigate its allergenicity.Methods The parvalbumin gene was amplified by RT-PCR and cloned into PMD18-T for sequencing and analysis.Then the target gene was subcloned into pET-28a（＋）for expression in E.coli BL21（DE3）by IPTG induction.The recombinant protein was purified by metal（Ni2＋）chelating affinity chromatography.Its allergenicity was examined by Western-blotting assay.Results The length of gene（Accession No.FJ013047）was 330 bp,coding 109 amino acids.The E.coli strain could express a recombinant protein with a molecular weight of11 537 Da.The recombinant allergen was identified as its affinity to specific IgE antibodies from the allergic patient sera by Western-blotting.Conclusion The parvalbumin gene from Aristichthys nobilis is successfully cloned and expressed in this study,and the recombinant protein possesses good IgE-binding capacity.