屋尘螨3类变应原基因的克隆、表达及免疫原性分析

中国寄生虫学与寄生虫病杂志 ›› 2018, Vol. 36 ›› Issue (6): 627-631.

屋尘螨3类变应原基因的克隆、表达及免疫原性分析

古霞¹, 欧阳春艳², 袁如意², 袁谢芳¹, 李国平¹, 刘志刚^1,^2,^*()

1 西南医科大学附属医院,泸州 646000
2 深圳大学过敏反应与免疫学研究所,深圳 518060

收稿日期:2018-01-30 出版日期:2018-12-30 发布日期:2019-01-08
通讯作者: 刘志刚
基金资助:
国家自然科学基金（No. 31729002,91542104）;广东省科技计划项目（No. 2014B090901041,2016A020216029）;呼吸疾病国家重点实验室开放基金（No. SKLRD2016ZJ001）

Cloning and expression of house dust mite allergen Der p 3 and its antigenicity

Xia GU¹, Chun-yan OUYANG², Ru-yi YUAN², Xie-fang YUAN¹, Guo-ping LI¹, Zhi-gang LIU^1,^2,^*()

1 Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
2 Institute of Allergy and Immunology, Shenzhen University, Shenzhen 518060, China

Received:2018-01-30 Online:2018-12-30 Published:2019-01-08
Contact: Zhi-gang LIU
Supported by:
Supported by the National Natural Science Foundation of China（No. 31729002, 91542104）, Guangdong Science and Technology Project（No. 2014B090901041, 2016A020216029）, Open Fund of National Key Laboratory of Respiratory Diseases（No. SKLRD2016ZJ001）

摘要/Abstract

摘要：

目的克隆、表达屋尘螨3类变应原（Der p 3）基因,纯化Der p 3重组蛋白,对其免疫原性及生物信息学特点进行分析。方法取实验室纯培养的屋尘螨500只,提取总RNA,逆转录生成cDNA,RT-PCR扩增Der p 3基因。扩增的Der p 3基因经测序正确后,将目的条带连接至pMD-32T载体中进行扩增,并利用限制性内切酶EcoRⅠ和XhoⅠ双酶切,酶切后的片段连接至pET-32a表达载体,转化至大肠埃希菌Rosetta中,经1 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达后,蛋白质印迹（Western boltting）分析重组蛋白的免疫原性。分别采用BLAST、ProtParam、PSIPRED、SWISS-MODEL对重组蛋白的同源性、理化性质、二级结构、三级结构进行分析;通过BLAST和MEGA构建Der p 3的系统进化树;并利用IEBD和DNA Star对Der p 3重组蛋白的抗原表位进行预测。结果 RT-PCR扩增屋尘螨Der p 3基因,得到约1 000 bp的片段,经测序为Der p 3目的序列,其开放阅读框为786 bp,编码261个氨基酸。经IPTG诱导后大量表达Der p 3重组蛋白。该蛋白主要以包涵体形式存在,相对分子质量（M_r）约30 000。Western blotting分析结果显示,重组蛋白Der p 3能与10份尘螨过敏性哮喘患者血清IgE抗体特异性结合。BLAST和MEGA分析结果显示,Der p 3基因与粉尘螨3类变应原（Der f 3）的序列一致性较高,约80%。Der p 3蛋白较稳定,二级结构由螺旋、延伸链、随机线圈组成;并预测到6个抗原表位肽序列。结论纯化后的Der p 3重组蛋白能与尘螨过敏性哮喘患者血清IgE抗体特异性结合,具有较强免疫原性。

关键词: 屋尘螨, 3类变应原, 过敏原, 生物信息学

Abstract:

Objective To clone and express Der p 3, an allergen from house dust mite Dermatophagoides pteronyssinus, as a recombinant protein for its antigenicity test. Methods The total RNA was extracted from collected D. pteronyssinus and used for reverse transcription into cDNA. The coding DNA for Der p 3 was amplified from the total cDNA by PCR. The PCR products were sequenced and cloned into E. coli expression vector pET-32a by EcoRⅠand XhoⅠsites. The recombinant Der p 3 protein with His-tag was expressed in E. coli Rosetta under induction of 1 mmol/L IPTG and purified with nickel column. The antigenicity of recombinant Der p 3 was tested by Western blotting with sera from patients of asthma allergic to dust mites. The bioinformatics and structure of Der p 3 was analyzed by using BLAST ProtParam PSIPRED SWISS-MODEL and MEGA toolkit. IEBD and DNA Star was used to predict the epitopes of Der p 3 protein. Results The coding DNA for Der p 3 was amplified from D. pteronyssinus total cDNA by RT-PCR. The open reading frame of Der p 3 was 786 bp, encoding 261 amino acids. The recombinant Der p 3 protein was expressed as insoluble inclusion body in E. coli Rosetta under induction of IPTG, with size of M_r 30 000. The inclusion body was solubilized with urea and purified by nickel column chromatography. Western blotting demonstrated that the recombinant Der p 3 was strongly recognized by IgE in sera from dust mite allergic asthma patients, indicating a potential allergen for asthma. Homology analysis revealed that it shared 80% sequence identity with of Der f 3 from D. farina. Structure of Der p 3 protein was stable containing helix, extension chain and random coil. Six immunogenic epitopes were predicted. Conclusion Der p 3 is cloned from house dust mite D. pteronyssinus and expressed as recombinant protein. The expressed recombinant Der p 3 protein can strongly be recognized by IgE in sera from dust mite allergic asthma patients, indicating its potential as an allergen for asthma.

Key words: Dermatophagoides pteronyssinus, Der p 3, Allergen, Bioinformatics

中图分类号:

R384.4

古霞, 欧阳春艳, 袁如意, 袁谢芳, 李国平, 刘志刚. 屋尘螨3类变应原基因的克隆、表达及免疫原性分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(6): 627-631.

Xia GU, Chun-yan OUYANG, Ru-yi YUAN, Xie-fang YUAN, Guo-ping LI, Zhi-gang LIU. Cloning and expression of house dust mite allergen Der p 3 and its antigenicity[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2018, 36(6): 627-631.

图/表 4

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