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粉尘螨变应原Der f 9:克隆、表达、纯化、特性鉴定及在特应性哮喘患儿中的IgE结合情况

Dermatophagoides farinae allergen Der f 9: Cloning, expression, purification, characterization and IgE-binding in children with atopic asthma.

作者信息

Cui Yubao, Teng Feixiang, Yu LiLi, Zhou Ying, Zhang Chengbo, Yang Li

机构信息

Department of Clinical Laboratory, Affiliated Yancheng Hospital, School of Medicine, Southeast University, Yancheng, 224001, Jiangsu Province, P. R. China.

Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng, 224006, Jiangsu Province, P. R. China.

出版信息

Pediatr Pulmonol. 2017 Mar;52(3):282-292. doi: 10.1002/ppul.23526. Epub 2016 Jul 19.

Abstract

BACKGROUND

The house dust mite species Dermatophagoides farinae releases allergens that cause allergies and asthma worldwide. This study sought to clone and express the full-length cDNA encoding the group 9 allergen of D. farinae (Der f 9).

METHODS

The published sequence of Der f 9 was used to design primers for RT-PCR and RACE to obtain the full-length cDNA encoding Der f 9. After removal of signal peptide sequence, Der f 9 was then sub-cloned into plasmid pET-28b (+), and the plasmid was transformed into Escherichia coli BL21 (DE3) cells for expression. The recombinant protein was purified by Nickel affinity chromatography, identified by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from children with asthma. Bioinformatics analyses were used to identify features of Der f 9.

RESULTS

By RT-PCR, 3'-RACE, and 5'-RACE, the full-length sequence of Der f 9 was generated, which was confirmed by nucleotide sequencing. The mature Der f 9 was expressed successfully in E. coli, which was identified by SDS-PAGE. The recombinant allergen was purified by chromatography and confirmed by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF. Sera from 56.7% (17/30) of mite-allergic patients reacted with the purified recombinant Der f 9.

CONCLUSIONS

The successful production of recombinant Der f 9 protein revealed the importance of Der f 9 in mite allergy, and provides a foundation for further study of this allergen in diagnosis and treatment of symptoms. Pediatr Pulmonol. 2017;52:282-292. © 2016 Wiley Periodicals, Inc.

摘要

背景

屋尘螨种类中的粉尘螨会释放变应原,在全球范围内引发过敏和哮喘。本研究旨在克隆并表达编码粉尘螨第9组变应原(Der f 9)的全长cDNA。

方法

利用已发表的Der f 9序列设计用于逆转录聚合酶链反应(RT-PCR)和快速扩增cDNA末端(RACE)的引物,以获得编码Der f 9的全长cDNA。去除信号肽序列后,将Der f 9亚克隆到质粒pET-28b(+)中,然后将该质粒转化到大肠杆菌BL21(DE3)细胞中进行表达。重组蛋白通过镍亲和层析进行纯化,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质免疫印迹法、斑点印迹法和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)进行鉴定,并通过酶联免疫吸附测定(ELISA)检测其与哮喘患儿血清的IgE反应性。利用生物信息学分析来确定Der f 9的特征。

结果

通过RT-PCR、3'-RACE和5'-RACE产生了Der f 9的全长序列,经核苷酸测序确认。成熟的Der f 9在大肠杆菌中成功表达,通过SDS-PAGE进行鉴定。重组变应原通过层析进行纯化,并通过SDS-PAGE、蛋白质免疫印迹法、斑点印迹法和MALDI-TOF进行确认。56.7%(17/30)的螨过敏患者血清与纯化的重组Der f 9发生反应。

结论

重组Der f 9蛋白的成功制备揭示了Der f 9在螨过敏中的重要性,并为进一步研究该变应原在症状诊断和治疗中的作用奠定了基础。《儿科肺病学》。2017年;52:282 - 292。©2016威利期刊公司。

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