Expression, cloning, and IgE-binding of the full-length dust mite allergen Der f 8
Expression, cloning, and IgE-binding of the full-length dust mite allergen Der f 8
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全长尘螨过敏原 Der f 8 的表达、克隆和 IgE 结合
DOI：
10.1007/s12026-014-8553-9
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发表时间：
2014-10-01
影响因子：
4.4
通讯作者：
Zhang, Cheng-bo
中科院分区：
文献类型：
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作者：
Cui, Yu-bao;Zhou, Ying;Zhang, Cheng-bo
粉尘螨是一种室内螨类，产生一些最强的过敏原，在中国和世界范围内引起过敏。我们试图克隆和表达粉尘螨第8组变应原（Der f 8），以研究其IgE结合反应性。采用RT-PCR和5′ RACE技术扩增Der f 8基因的全长cDNA，将其克隆到pCold-TF表达载体中，经测序确认后，亚克隆到pET-28 b（+）中，转染E. coliBL 21细胞进行表达。经镍亲和层析和SDS-PAGE鉴定，重组Der f 8与40.9%（9/22）的螨过敏患者血清结合。重组DNA序列的分析揭示了一个231个氨基酸的开放阅读框，编码一种衍生分子量为26.4 kDa和等电点为6.84的蛋白质。推导的氨基酸序列有9个磷酸化位点，与谷胱甘肽S-转移酶有很强的同源性，二级结构由α螺旋（45.5%）、延伸链（11.3%）和无规卷曲（43.3%）组成。通过国家生物技术信息中心数据库的BLAST和比对，鉴定了与屋尘螨、棉兰Suidasia medanensis、破坏性鳞螨、食豆螨和卵形粉螨的8组过敏原或谷胱甘肽S-转移酶的相似性（分别为64、65、53、53和50%）。第一个重组的全长Der f 8蛋白成功地与患者IgE结合，证明了Der f 8在螨过敏中的重要性。
Dermatophagoides farinae, a domestic mite species, produces some of the most potent allergens that contribute to allergy in China and worldwide. We sought to clone and express the group 8 allergen ofD.farinae(Der f 8) to investigate its IgE-binding reactivity. The full-length cDNA encoding Der f 8 was generated by using RT-PCR and 5′ RACE, cloned into pCold-TF expression vector, confirmed by nucleotide sequencing, sub-cloned into pET-28b (+), and transfected intoE. coliBL21 cells for expression. After purification by nickel affinity chromatography and identified by SDS-PAGE, the recombinant Der f 8 bound with sera from 40.9 % (9/22) of mite-allergic patients according to ELISA testing. Analysis of the recombinant DNA sequence revealed a 231 amino acid open reading frame encoding a protein with a derived molecular mass of 26.4 kDa and an isoelectric point of 6.84. The deduced amino acid sequence has nine phosphorylation sites, displaying strong homology with glutathione S-transferase, and its secondary structure comprises alpha helix (45.5 %), extended strand (11.3 %), and random coils (43.3 %). BLAST through the National Center for Biotechnology Information database and alignment identified similarity with group 8 allergens or glutathione S-transferases ofDermatophagoides pteronyssinus,Suidasia medanensis,Lepidoglyphus destructor,Glycyphagus domesticus, andAleuroglyphus ovatus(64, 65, 53, 53, and 50 %, respectively). The first recombinant Der f 8 protein produced in full length successfully bound with patient IgE, demonstrating the importance of Der f 8 in mite allergy.