尘螨变应原Derf1核酸序列测定及其分子进化分析

目的:对尘螨主要变应原Der f1进行核酸序列测定,探讨其系统进化信息。方法:根据Genbank公布的Der f1基因序列设计引物,巢式PCR扩增Der f1的cDNA,纯化、回收、克隆至pMD19-T simple后进行序列测定,序列比对后用Clustal W 1.83构建分子进化树。结果:成功扩增出Der f1的cDNA片段,测序表明该基因含ORF1个,长度966bp,与参考序列同源性达99.9%。该变应原具半胱氨酸蛋白酶活性,与果蝇进化关系最远,与梅氏嗜霉螨进化关系最近。结论:成功获得了尘螨变应原Der f1基因片段,根据其编码的氨基酸序列构建出的系统进化树与形态学分类不一致。

Sequencing and Polygenetic Analysis of the Major House Dust Mite Allergen, Derf1

Objective:To determinate the nucleotides sequence of Der f 1 and get know of information on its molecular evolution.Methods:According to the complete CDS encoding the major house dust mite allergen Der f 1 in Genbank,we de- signed the primers and amplified the gene by nest PCR.After purification and recycling,the cDNA fragment was cloned into pMD19-T vector and transformed into E.coli JM109.Then the positive clones were screened and sequenced.After sequence blast, phylogenetic tree was comtructed by Clustal W 1.83.Results:The cDNA fragments encoding Der f1 were amplified successfully, and one open reading fragment(ORF)was found in the sequence with full length of 966 bp.The nucleotide identity with the ref- erence sequence in Genbank was 99.9%.By InterProScan,three cysteine peptidase active sites were found in the sequence.And ac- cording to the phylogenetic tree,Dermatophagodes farinae in this study is nearer related to Euroglyphus maynei and farthest from Drosophila pseudoobscura.Conclusion:The gene fragment encoding the major dust mite allergen Der f1 was amplified success- fully,however,the evolution relationship deduced from phylogenetic tree is not consistent with morphological classification.