【摘要】 目的构建尘螨变应原Der f1原核表达体系,并了解其分子特征。方法提取粉尘螨总RNA,用RT-PCR合成Der f1编码基因,将其克隆至pMD19-T载体,亚克隆至表达载体pET-28a(+),转化至大肠杆菌并用IPTG诱导表达。用生物信息学软件对测序结果进行分析并预测其空间结构。结果从粉尘螨总RNA中扩增获得Der f1 cDNA片段,成功构建了表达质粒pET-28a(+)-Der f1,Western blotting显示原核表达获得成功。测序结果提交GenBank,登陆号为EU095368,该基因长966 bp,与参考序列同源性达99.9%,推测其编码氨基酸321个,属疏水蛋白,位于细胞外,信号肽位于1~18氨基酸处。同源性分析提示Der f1和Eur m1相似率为88%,而Der f1和Der p1的相似率为77%,分子进化树中粉尘螨和梅氏嗜霉螨聚成一簇。Der f1的二级结构由α-螺旋(109 aa,33.96%)、延伸链(55aa,17.13%)、β-转角(18 aa,5.61%)和随机卷曲(139 aa,43.30%)组成。结论尘螨变应原Der f1原核表达获得成功,为进一步生产重组变应原奠定了基础。生物信息学分析表明粉尘螨和梅氏嗜霉螨的亲缘关系可能更近,而与屋尘螨关系稍远,此与现行的形态学分类系统并不符合。更多还原

【Abstract】 Objective To clone,express and characterize Dermatophagoides farinae allergens in order to further produce recombinant allergens for clinical applications in the future.Methods Based on the complete encoding CDS the dust mite allergen Der f1 in the Genbank,we designed primers and amplified the cDNA fragment coding for the group 1 allergen from adult D.farinae in China by nested-PCR.After purification and recovery,the cDNA fragment was cloned into the pMD19-T vector.The fragment was then sequenced,subcloned into the plasmid pET-28a(+),expressed in Escherichia coli BL21 and identified by Western blotting.Using bioinformatics softwares,the sequenced result was analyzed and the inferred amino acid sequence was used for alignment,phylogeny construction,and the predictions of secondary and three-dimensional structures.Results The cDNA coding for group 1 allergen of adult D.farinae was cloned,sequenced and expressed successfully.Sequence analysis showed the presence of an open reading frame containing 966 bp and encoding a protein of 321 amino acids,the sequenced results were submitted to GenBank and got Accession Number EU095368.Through bioinformatics analyses,the hydrophobic protein was demonstrated to be likely extracellular,with a cleavage site between positions 18 and 19.Interestingly,during homology analysis,we found that the Der p1 shared a greater than 87% amino acids sequence identity with Eur m1 but only 80% with Der f1.Phylogenetic analysis suggested that D.farinae was closer to Euroglyphus maynei than to D.pteronyssinus,even though D.pteronyssinus and D.farinae belonged to the same genus Dermatophagoides.A total of three cysteine peptidase active sites were found in the predicted amino acid sequence.These sites were 127~138(QGGCGSCWAFSG),267~277(NYHAVNIVGYG) and 284~303(YWIVRNSWDTTWGDSGYGYF).Secondary structure analysis revealed that Der f1 from China contained an alpha helix(33.96%), an extended strand(17.13%),a beta turn(5.61%),and a random coil(43.30%).With the help of SWISS-MODEL server,a simple three-dimensional model of the protein was constructed.Conclusion The cDNA coding for group 1 allergen of D.farinae were cloned,and expressed successfully,which provided a foundation for further genetic allergens product.Importantly,sequence analysis yield that D.farinae was closer to E.maynei than to D.pteronyssinus,which was not consistent with morphologic taxonomy adopted by most of acarologists.更多还原