摘要
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(GenBank AY283292)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-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。
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.
出处
《现代生物医学进展》
CAS
2009年第16期3014-3019,共6页
Progress in Modern Biomedicine
基金
国家自然科学基金(No.30660166
No.30860261)
海南省卫生厅自然科学课题(琼卫2005-6号)
关键词
尘螨
变应原
基因克隆
生物信息学
House dust mite
Allergen
Gene cloning
Bioinformatics
