摘要
目的:获得粉尘螨变应原第6组分编码基因并了解其分子特征。方法:根据GeneBank已公布的Derf6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:获得的Der f6 cD-NA全长为840 bp,与参考序列同源性达96.8%,含1个完整的开放读码框。推测编码蛋白由279个氨基酸组成,信号肽序列位于1～19 aa,亲水性指数为-0.139,跨膜区域位于1～19 aa,二级结构由α-螺旋(7.17%)、延伸主链(36.56%)和无规卷曲(56.27%)组成;亚细胞定位于细胞外,N-端第6位和第11位的亮氨酸有明显的序列核输出信号;可能为糜蛋白酶,具有cAMP和cGMP依赖的蛋白激酶磷酸化位点、蛋白激酶C磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点、N端酰基化位点等。结论:获得了Derf6基因全长,其编码的细胞外疏水性蛋白可能具有糜蛋白酶活性。
Objective ： To obtain the gene enceding the group 6 allergen of Dematophagoh/esfar/nae （Derf6）,and to explore its molecular characteristics. Methods： The total RNA of Dermatophagoides farinae was extracted, and the full-length Der f 6 was amplified by RTPCR with the primers designed according to previous sequence published in GeneBank. The target gene was linked into pMD19-T plasmid, sequenced and analyzed by bioinfonnatics software.Results： The cDNA for Derf 6 was obtained with 840 bp in full-length,one open reading fragment （ORF）,and 96.8% homology with the reference sequence. The protein encoded by this sequence was deduced to be consisted with 279 amino acids,both signal sequence and transmembrane regions from 1 to 19 position, whose grand average of hydropathicity （GRAVY） was computed to be -0. 139, and the secondary structure was composed by alpha helix （7.17%）,extended strand （36.56%） and random coil （56. 27%） .According to NetNKS 1.1 Server, the most possible nuclear export signals should be at 6 and 11 position amino acid.This protein may be chymotrypsin, and had cAMP- and cGMP- dependent protein kinase phosphorylafon site, Protein kinase C phosphorylation site, Casein kinase 11 phosphorylation site, N-myristoylation site and some other sites. Conclusions： The full-length cDNA for Derf 6 is obtained, which should encode a hydrophobic extracellular protein associated with chymotrypsin.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2009年第7期641-646,共6页
Chinese Journal of Immunology
基金
国家自然科学基金(No.30660166,No.30860261)
盐城市科技发展计划(No.YK2008079)
海南省卫生厅自然科学课题(琼卫2005-6号)
关键词
粉尘螨
变应原
克隆
生物信息学
Dermatophago/des farinae
AUegen
Cloning
Bioinformatics
