粉尘螨变应原第7组分全长基因序列测定与分析

目的：获得粉尘螨变应原第7组分编码基因并了解其分子特征。方法：根据GenBank已公布的Derf7核酸序列设计引物，用RT—PCR扩增获得其编码基因，插入pMD19-T载体进行序列测定和生物信息学分析。结果：获得Derf7全长基因约642bp，与参考序列（GenBankAY283292）同源性达99．7％％，仅在249位”A→G”和439位“C→T”发生点突变，含1个完整的开放读码框。推测编码蛋白由213个氨基酸组成，信号肽序列位于1-17aa，亲水性指数为0．031，跨膜区域位于171—190aa，二级结构由一螺旋（57．28％）、延伸主链（6．57％）和无规卷曲（36．15％）组成；亚细胞定位于细胞质，含有N-糖基化位点1个（151-154aa），蛋白激酶c磷酸化位点1个（193-195aa），酪蛋白激酶Ⅱ磷酸化位点2个（155—158aaand173．176aa）。N端酰基化位点1个（97—102aa）。粉尘螨和屋尘螨变应原第7组分氨基酸序列相似度为86％，二者在螨类第7组分氨基酸序列构建出的分子进化树中聚成一簇。结论：获得了Derf7全长基因，为进一步获得其基因工程制品用于临床和实验研究奠定了基础。

Sequencing and Analysis of Full-length Gene Encoding the Group 7 Allergen from Dermatophagoides farinae

Objective： To obtain the gene encoding the group 7 allergen of Dermatophagoides farinae and its molecular character- istics. Methods： The total RNA of Dermatophagoides farinae were extracted, and the gene encoding Der f 7 was amplified by RT-PCR with the primers designed according to previous sequence published in GenBank. The target gene was linked into pMD 19-T plasmid, sequenced and analyzed by bioinformatics software. Results： The cDNA encoding Der f 7 was amplified with 642 base pair （bp） in full-length, one open reading fragment （ORF）, and 99.7% homology with the reference sequence （GenBank AY 283292）. The protein encoded by this sequence was deduced to be consisted with 213 amino acids, signal peptide sequence from 1 to 17 position, and transmembrane regions from 171 to 190 position, whose grand average of hydropathicity （GRAVY） is computed to be 0.03 I, secondary structure is composed by alpha helix （57.28%）, extended strand （6.57%） and random coil （36.15%）, subcellular localization was cytoplasmic by CELLO v.2.5. There are some function sites predicted by PROSCAN software, such as one N-glycosylation site （151-154aa）, one protein kinase C phospborylation site （193-195aa）, two casein kinase Ⅱ phosphorylation sites （155-158aa and 173-176aa）, and one N-myristoylation site （97-102aa）. The similarity of the group 7 allergens between Dermatophagoides farinae and D. pteronyssinus were predicted for 86%, and they were clustered at the phylogenetic tree constructed with the group 7 allergens from different mite-species. Conclusions： The gene encoding Der f 7 was obtained, which provided a foundation for further genetic allergen production used in clinical and experimental study.