粉尘螨变应原Der f 2在毕氏酵母中表达及圆二色谱分析

Expression of Dermatophagoides farinae allergen 2 in Pichia pastoris and its analysis using circular dichroism

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 gene coding for a group 2allergen（Derf2）of Dermatophagoides farinae in China,to construct a yeast expression vector,and to analyze the expressed product using circular dichroism spectroscopy.Methods Total RNA was extracted from D.farinae.The Derf2 gene was amplified with RT-PCR and cloned into a pGAPZα-A vector.pGAPZα-A-Derf2 was transformed into GS115-competent cells and expression of the target protein was induced with IPTG.The protein was then identified with SDS-PAGE.The target protein was purified using a Histrap HP affinity column and the recombinant protein Derf2 was analyzed using circular dichroism spectroscopy. Results The gene coding for Derf2 was obtained using PCR and successfully expressed in GS115-competent cells using the plasmid pGAPZα-A-Derf2.The molecular weight of the expressed product was 14.9ku.The recombinant protein Derf2 was purified and analyzed using circular dichroism spectroscopy.α-helices accounted for 41.2% of the secondary structure of the protein,β-turns accounted for 10.3%,β-folds accounted for 26.8%,and random coils accounted for 21.7%. Conclusion cDNA coding for the Derf2 allergen of D.farinae was cloned and Derf2 protein was successfully expressed,providing a foundation for further research and large-scale production of this protein for clinical treatment of allergic disorders.