Cloning, expression, purification and immunoassay of Tyr p 13 from Tyrophagus putrescentiae (Schrank)

Objective In this study, Tyrp 13 gene was cloned, the recombinant protein was expressed and purified, the immunological characteristics were identified, and the information of allergen epitope was analyzed by bioinformatics software. Methods The total RNA of the mite was extracted, RT-PCR was used to amplify the cDNA. The primers were designed according to the known Tyr p 13 gene sequence (GenBank Accession No. AY710432.1). A large number of target genes was amplified by PCR. Prokaryotic expression vector pet-32a (+) - Tyr p 13 was constructed, then transformed into E.coli Rosetta™ (DE3) Competent Cells, and the target protein expression was inducted by isopropyl β-d-1-thiogalactopyranoside (IPTG); purification of the expression product by chromatography, Western blot method was used to detect the immunological activity of the purified expression product; its antigen epitope was estimated by bioinformatics software, and its evolution tree was constructed. Results The length of the cloned DNA sequence was about 400 bp, and the expressed product was soluble in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weight of about 14 kD. Western blotting showed that Tyr p 13 could specifically bind to IgE in sera of allergic patients, which indicated that Tyr p 13 had allergenicity; epitope analysis further confirmed that the allergen had immunogenicity. Conclusion High purity Tyr p 13 can be obtained by clone expression and purification, which lays a theoretical foundation for further clinical specific diagnosis and treatment. 摘要：目的 克隆腐食酪螨第 13 组变应原基因 ( Tyr p 13 ) , 表达纯化其重组蛋白, 并进行免疫学特性鉴定, 利用生 物信息学软件分析该过敏原抗原表位等信息。 方法 挑取腐食酪螨, 提取螨总 RNA。逆转录-聚合酶链式反应 (RT- PCR) 扩增 cDNA, 根据已知的 Tyr p 13 基因序列 (GeneBank 登录号 AY710432.1) 设计引物, PCR 大量扩增目的基因。构 建原核表达载体 pET-32a (+) - Tyr p 13 , 转化感受态细胞 E. coli Rosetta (DE3) , 用 IPTG (异丙基-β-D-硫代半乳糖苷) 诱 导目的蛋白表达; 层析纯化表达产物, 免疫印迹 (Western Blot) 法检测纯化后的表达产物免疫学活性; 通过生物信息学 软件推测其抗原表位、构建进化树。 结果 克隆得到的序列经基因测序可知其长度约 400 bp, 其表达产物经十二烷基 硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分子量约为 14 kD, 呈可溶性表达。免疫印迹结果显示, Tyr p 13 能够与过敏 患者血清 IgE 特异性结合, 表明其具有过敏原性; 表位分析进一步证实该过敏原具有免疫原性。 结论 经克隆表达、纯 化后得到纯度较高的腐食酪螨 Tyr p 13 , 为进一步开展临床特异性诊断和治疗提供参考。