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目的克隆粉尘螨变应原Der f 2编码基因,并构建毕氏酵母表达体系,对表达产物进行圆二色分析。方法提取粉尘螨总RNA,根据GenBank中的序列设计并合成简并引物,PT-PCR扩增Der f 2全长基因,与pGAPZα-A载体连接后转化至E.coli Top10F’,取阳性克隆并测序;将pGAPZα-A-Der f 2用BlnⅠ进行线性化,电转化至GS115感受态细胞,用IPTG进行诱导表达,采用SDS-PAGE验证表达产物,用His Trap HP亲和柱纯化重组蛋白Der f 2,进行圆二色谱扫描分析。结果 PCR扩增获得Der f 2编码基因,成功构建表达质粒pGAPZα-A-Der f 2,SDS-PAGE验证表明该质粒在GS115感受态细胞中正常表达,表达产物分子质量单位为14.9ku,重组蛋白Der f 2纯化后进行圆二色谱数据分析,其二级结构α-螺旋占41.2%,转角占10.3%,β-折叠占26.8%,不规则卷曲占21.7%。结论尘螨变应原Der f2在毕氏酵母中成功表达,为该变应原的进一步研究及规模生产和应用奠定了基础。
Objective To clone the Der f 2 coding gene of dust mite allergen and construct the expression system of Pichia pastoris. The expression products were analyzed by circular dichroism. Methods Total RNA was extracted from Dust mite. Degenerate primers were designed and synthesized according to the sequences in GenBank. The full-length Der f 2 gene was amplified by PT-PCR and ligated with pGAPZα-A vector and then transformed into E. coli Top10F ’ The pGAPZα-A-Der f 2 was linearized with BlnⅠ and electrotransformed into GS115 competent cells. The recombinant plasmid pGAPZα-A-Der f 2 was induced by IPTG. The expressed product was verified by SDS-PAGE and purified with His Trap HP affinity column Der f 2, circular dichroism scan analysis. Results The gene encoding Der f 2 was obtained by PCR. The recombinant plasmid pGAPZα-A-Der f 2 was successfully constructed. SDS-PAGE analysis showed that the plasmid was expressed normally in GS115 competent cells. The molecular weight of recombinant plasmid was 14.9ku. Der f 2 purified circular dichroism data analysis, the secondary structure of α-helix accounted for 41.2%, corner 10.3%, β-sheet 26.8%, irregular curly accounted for 21.7%. Conclusion Dust mite allergen Der f2 was successfully expressed in Pichia pastoris, which laid the foundation for the further study of the allergen and its scale production and application.