Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli
Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli
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主要屋尘螨过敏原 Der f 1 在大肠杆菌中的 cDNA 编码的克隆、序列分析和表达。
DOI：
10.1590/s0100-879x2008000500006
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发表时间：
2008-05-01
影响因子：
2.3
通讯作者：
Liu, L.
中科院分区：
文献类型：
--
作者：
Cui, Y.;Zhou, P.;Liu, L.
我们的目标是克隆、表达和表征成年粉尘螨第 1 组 (Der f 1) 过敏原，以进一步生产用于未来临床应用的重组过敏原，从而消除螨虫粗提物的副反应。基于GenBank数据，我们设计了引物并通过巢式PCR扩增了编码Der f 1的cDNA片段。纯化和回收后，将cDNA片段克隆到pMD19-T载体中。然后对该片段进行测序，亚克隆到质粒 pET28a(+) 中，在大肠杆菌 BL21 中表达，并通过蛋白质印迹进行鉴定。编码 Der f 1 的 cDNA 已成功克隆、测序并表达。序列分析显示存在一个包含 966 bp 的开放阅读框，编码 321 个氨基酸的蛋白质。有趣的是，同源性分析表明，Der p 1 与 Eur m 1 的氨基酸序列有超过 87% 的同一性，但与 Der f 1 的氨基酸序列同一性仅为 80%。此外，系统发育分析表明，尽管 D. pteronyssinus 和 D. farinae 属于同一个 Dermatophagoides 属，但 D. pteronyssinus 在进化上更接近 Euroglyphus maynei，而不是 D. farinae。在预测的氨基酸序列中总共发现了3个半胱氨酸肽酶活性位点，包括127-138（QGGCGSCWAFSG）、267-277（NYHAVNIVGYG）和284-303（YWIVRNSWDTTWGDSGYGYF）。此外，二级结构分析显示，Der f 1 含有 a 螺旋（33.96%）、延伸链（17.13%）、ss 转角（5.61%）和无规卷曲（43.30%）。使用瑞士模型服务器构建了该蛋白质的简单三维模型。编码 Der f 1 的 cDNA 已成功克隆、测序并表达。比对和系统发育分析表明，D. pteronyssinus 在进化上与 E. maynei 比与 D. farinae 更相似。
Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 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 pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ss turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.