尘螨变应原Der f 2基因克隆及序列分析

Clone and sequence analysis of a major allergen Der f 2 from house dust mites

目的:获得尘螨变应原Derf2基因及其生物信息学资料.方法:提取粉尘螨总RNA,RT-PCR合成Derf2的cDNA片段,回收PCR产物并连接至pMD19-Tsimple,经测序验证后亚克隆入表达载体pET-28a(+),转化大肠杆菌感受态细胞后提取质粒,双酶切鉴定.用ExPaSy,EBI,NCBI网站的在线软件对测序结果进行分析.结果:RT-PCR扩增获得了Derf2cDNA片段,酶切、电泳结果表明克隆和亚克隆获得成功.与参考序列相比,测序结果多了87bp(77～163),但Blastn发现中国广州和德国Reinbek报道的序列中也含此87bp.同源性、相似性、序列比对及分子进化分析提示,该序列与中国广州和德国Reinbek报道的序列亲缘关系较近.推测该变应原由176个氨基酸组成,与附睾分泌蛋白E1具有同源性,信号肽位于1～17aa处,在6～24aa处有一跨膜螺旋.二级结构由a-螺旋(16.57%),延伸链(32.57%)和随机卷曲(50.86%)组成.结论:获得了粉尘螨变应原Derf2编码基因及其分子特征,为进一步生产基因工程变应原用于临床诊治变态反应性疾病奠定了基础.

AIM： To characterize the group 2 allergen of the house dust mite Dermatophagoides farinae from Hainan Island, a tropical region in Southeastern China. METHODS： After the total RNA of D. farinae was isolated, eDNA encoding the group 2 allergen was synthesized by RT-PCR, and the PCR products were inserted into the vector pMD19-T simple, which were subcloned into pET-28a（ ＋ ） and transformed into E. coli and identified with restriction enzyme analysis. Subsequently the sequencing result was analyzed by software in ExPaSy, EBI and NCBI web. RESULTS： The cDNA coding for Der f 2 was cloned and sequenced successfully, and we found an additional region of 87 bp （from 77 to 163 bp） in our strain but it was absent in reference sequence （GenBank AB195580）. By Blastn analysis, the same additional nucleotides were found in the strains reported from Reinbek, Germany and Guangzhou, China. A polygenetic tree, constructed using the nucleotides sequences coding for Der f 2 reported from different regions or countries showed that our sequence was clustered with the strains from Reinbek and Guangzhou. Analysis of the translated amino acid sequence suggested that the encoded peptide was hydrophobic and extracellular with a cleavage site between 17aa and 18aa and a strong transmembrane helices from 6aa to 24 aa, indicating a MD-2-related lipid recognition domain. The secondary structure of the pro-protein consisted of 16.57% alpha helix, 32.57% extended strand and 50.86% random coil. CONCLUSION： We have obtained a gene coding for Der f 2 and this gene shows significant difference with those reported previously. We can predict the molecular characteristics of this protein using bioinformatic tools.