虾夷扇贝过敏原tropomyosin的克隆表达、纯化及免疫学鉴定

CLONING, EXPRESSION AND PURIFICATION OF ALLERGEN TROPOMYOSIN FROM PATINOPECTEN YESSOENSIS AND IDENTIFICATION OF ITS ALLERGIC ACTIVITY

从虾夷扇贝(Patinopecten yessoensis)肌肉中提取总RNA,RT-PCR克隆虾夷扇贝中变应原原肌球蛋白的全长基因,根据序列设计带有酶切位点的特异性引物,扩增扇贝tropomyosin的完整开放阅读框,与pET-28a载体连接并转化大肠杆菌Escherichia.coli BL21(DE3),诱导表达后,Ni2+亲和层析柱纯化重组蛋白,Western-blot检测其免疫学活性。经序列测定,该基因含有长度为855bp的开放阅读框,编码284个氨基酸,其在GenBank数据库中的登录号为EU839640。SDS-PAGE检测该重组变应原在大肠杆菌中高效表达36kD的目的蛋白,且重组变应原具有良好的IgE结合活性。研究获得了具有变应原活性的重组虾夷扇贝tropomyosin,为扇贝过敏性疾病的诊断和治疗奠定了基础。

This study was undertaken to clone, express and immunocharacterize the allergen tropomyosin from Patinopecten yessoensis. Bioinformatic method was used for the comparative analysis of numerous homologous animal food tropomyosin sequences. Conservative domains among the sequences were determined for degenerate primer designing. The RT-PCR was applied to clone the full-length allergen genes from Patinopecten yessoensis and the sequences were analyzed. The specific primers were designed. The complete coding cDNA sequence including the start and the stop codons of tropomyosin of Patinopecten yessoensis was subcloned into the expression vector pET 28a. Expression of the recombinant Patinopecten yessoensis tropomyosin was carried out in Escherichia coli BL21 （DE3） and the purification of the recombinant protein was performed via affinity chromatography with Ni2＋ coupled to sepharose. Protein from E. coli lysate and purified recombinant tropomyosin were analyzed by SDS-PAGE. IgE reactivity of recombinant Patinopecten yessoensis tropomyosin was investigated by Western-blotting. The cloned cDNA ORF sequence contained 855 bp and encoded 284 amino acids. The GenBank accession number of the clones was EU839640. Sequence analysis showed that this clone shared high identities with tropomyosin from Patinopecten yessoensis. Nucleotide and amino acid comparison showed that this protein was the Patinopecten yessoensis tropomyosin. The recombinant allergen tropomyosin was highly expressed in E. coli BL21 （DE3） with the molecular weight of about 36 kD under induction of IPTG and purified by 6-His-tag purification system. And the recombinant allergen was identified as its affinity to IgE antibodies from the scallop patient sera by Western blotting method. Immunoassay showed that the recombinant allergen has good IgE binding capacity. We obtained recombinant Patinopecten yessoensis tropomyosin with good allergenicity in this study, which would be used as a base for further study on Patinopecten yessoensis related allergy.