关键词: 尘螨, Der p 1, 克隆, 原核表达, 生物信息学

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.