Abstract: Objective To clone, express and characterize Dermatophagoides pteronyssinus allergens. Methods Based on nucleotide sequence coding for Der p 1 in the GenBank, we designed primers and amplified the cDNA fragment coding for the group 1 allergen from adult D.pteronyssinus by RT-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in E.coli BL21 and identified by Western blotting. Results The cDNA coding for group 1 allergen of adult D.pteronyssinus was cloned, sequenced and expressed successfully. Sequence analysis showed the gene homology with Der p 1 reported in GenBank was 99.9%, the latter encoding for a protein with 222 amino acids. Homology analysis revealed that Der p 1 shared only 60% identity sequence with Der f 1, but Der f 1 shared 85% identity sequence with Eur m 1. Polygenetic analyses suggested that D.farinae was evolutionarily closer to Euroglyphus mayneithan to D.farinae, even though D.pteronyssinus and D.farinae belonged to the same genus Dermatophagoides. Secondary structure analysis revealed that Der p 1 from China contained an alpha helix (33.78%), an extended strand (21.62%) and a random coil (44.59%). Conclusion The cDNA coding for group 1 allergen of D.pteronyssinus were cloned, and expressed successfully, which provided a foundation for further genetic allergens product. Importantly, sequence analysis give out that D.farinae was evolutionarily closer to E.maynei than to D.pteronyssinus, which was not consistent with morphologic taxonomy adopted by most of acarologists.

摘要： 目的 克隆和分析屋尘螨主要变应原Der p 1,并实现原核表达。方法 分离屋尘螨总RNA,根据Gen Bank已公布的Der p 1核酸序列设计引物,用RT-PCR扩增Der p 1编码基因,克隆至pMD19-T载体、亚克隆至表达载体pET-28a(+),将表达质粒转化至E.coliBL21(DE3)并用IPTG诱导表达,并对表达产物进行免疫印迹鉴定。结果 成功构建了表达质粒pET-28a(+)-Der f 1,Western blotting显示原核表达获得成功。序列分析表明所获得的Der p 1编码基因与参考序列同源性达99.9%,推测其编码氨基酸222个。屋尘螨和粉尘螨1类变应原氨基酸序列相似率为60%,而粉尘螨与梅氏嗜霉螨1类变应原相似率为85%,分子进化分析亦提示粉尘螨和梅氏嗜霉螨亲缘关系较近。推测所获rDer p 1二级结构中,α螺旋占33.78%,延伸链占21.62%,随机线圈占44.59%。结论 尘螨变应原Der p 1原核表达获得成功,为进一步生产重组变应原奠定了基础。序列分析表明粉尘螨和梅氏嗜霉螨的亲缘关系可能更近,而与屋尘螨关系稍远,此与现行的形态学分类系统并不符合。

关键词: 尘螨, Der p 1, 克隆, 原核表达, 生物信息学