尘螨变应原Der f 1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 f 1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 f 1基因序列设计引物，巢式PCR扩增Der f 1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 W1.83构建分子进化树。结果：成功扩增出Der f 1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 f 1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,Der f 1

Objective： To determinate the nacleotides 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 designed 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, phylogenefic tree was constructed by Clustal W 1.83. Results： The cDNA fragments encoding Der f 1 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 reference sequence in Genbank was 99,9%, By InterProScan, three cysteine peptidase active sites were found in the sequence. And according 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 f 1 was amplified successfially, however, the evolution relationship deduced from phylogenefic tree is not consistent with morphological classification.