[1]陈扬,陈献雄,欧阳春艳,等.粉尘螨过敏原Der f 35的克隆表达及免疫学鉴定[J].深圳大学学报理工版,2021,38(3):301-306.[doi:10.3724/SP.J.1249.2021.03301]
　CHEN Yang,CHEN Xianxiong,OUYANG Chunyan,et al.Cloning,expression and immunological identification of Der f 35 allergen from Dermatophagoides farinae[J].Journal of Shenzhen University Science and Engineering,2021,38(3):301-306.[doi:10.3724/SP.J.1249.2021.03301]
点击复制

粉尘螨过敏原Der f 35的克隆表达及免疫学鉴定()
分享到：

《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第38卷
期数:
2021年第3期
页码:
301-306
栏目:
生物工程
出版日期:
2021-06-14

文章信息/Info

Title:
Cloning,expression and immunological identification of Der f 35 allergen from Dermatophagoides farinae
文章编号:
202103013
作者:
陈扬; 陈献雄; 欧阳春艳; 钟永浩; 刘晓宇
深圳大学过敏反应与免疫学研究所，深圳大学医学部，广东深圳 518060
Author(s):
CHEN Yang;  CHEN Xianxiong;  OUYANG Chunyan;  ZHONG Yonghao;  and LIU Xiaoyu
Institute of Allergy and Immunology, Department of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China
关键词:
生物信息学; 免疫学; 粉尘螨; 过敏原Der f 35; 克隆表达; 过敏原性鉴定
Keywords:
bioinformatics;  immunology;  Dermatophagoides farinae;  allergen Der f 35;  cloning and expression;  allergen identification
分类号:
R384.4
DOI:
10.3724/SP.J.1249.2021.03301
文献标志码:
A
摘要:
粉尘螨是诱导过敏性疾病的重要变应原，可诱发Ⅰ型变态反应，对其免疫原性的鉴定是研究过敏性鼻炎等过敏性疾病的基础．首次对粉尘螨过敏原基因Der f 35进行克隆表达、纯化及免疫原性鉴定，提取粉尘螨总核糖核酸（ribonucleic acid, RNA），根据已知基因序列（GenBank：LC175222.1）设计引物进行反转录聚合酶链式反应扩增，经BamH I和Xho I双酶切，连接构建pET-32a-Der f 35载体，并克隆至大肠杆菌TOP 10菌株，取1 mL克隆菌液进行测序．使用试剂盒提取高纯度质粒转至感受态细胞Rosetta (DE3)中进行诱导、表达纯化及免疫学鉴定，并进行同源性比对分析、进化树构建及二级结构预测．克隆和表达结果显示，Der f 35基因的片段长约436个碱基对（base pair, bp）；重组蛋白Der f 35相对分子质量约为30 ku (1 u =1 D)，与预期结果相符．蛋白质印迹法结果证明，Der f 35与尘螨过敏性疾病患者的血清结合有明显的反应原性．生物信息学进化树结果显示，粉尘螨与屋尘螨、热带无爪螨、害嗜鳞螨、绵羊痒螨和免耳痒螨亲缘关系较近，二级结构预测显示Der f 35的氨基酸序列由2个α螺旋、6个β折叠、2个β转角和10个无规则卷曲片段组成．研究结果可为尘螨过敏性疾病的诊断与治疗提供理论依据．
Abstract:
Dermatophagoides farinae is an important allergen that induces allergic diseases and type I allergic reaction. The identification of its immunogenicity is the basis for the study of allergic rhinitis and other allergic diseases. First, primers were designed according to the Der f 35 gene sequence (GenBank: LC175222.1), and the total RNA was extracted from dust mite, use it as a template for Der f 35 reverse transcription polymerase chain reaction amplification. Then, the pET-32a vector was ligated with the Der f 35 gene and transfer it into Escherichia coli (E.coli) TOP 10 cloned bacteria, and 1 mL of the cloning bacteria liquid was aspirated for sequencing. The high purity plasmid was extracted and transferred to Rosetta (DE3) cells. Expression of the objective protein Der f 35 was induced, purified and immunologically identified. Homology comparison analysis, phylogenetic tree construction and secondary structure prediction was then performed. Results showed that the fragment of the Der f 35 gene was about 436 base pair (bp). The molecular weight of the recombinant protein Der f 35 was 30 ku (1 u =1 D), which was consistent with the expected results. The results of bioinformatics evolutionary tree showed that the dust mite was close to house dust mite, blomia tropicalis, scaly mite, sheep itchy mite and ear free mite. Secondary structure prediction indicated that Der f 35 amino acid sequence consists of two α helices, six β folds, two β turns and ten random coil fragments. Der f 35 gene was successfully cloned, and the coded protein was expressed and purified in E. coli with good reactivity. It lays a foundation for the study of allergic diseases of dust mites.

参考文献/References:

［1］ 朱永烽, 刘志刚, 高波,等. 粉尘螨Der f18变应原的克隆表达,纯化及免疫学特性鉴定［J］. 寄生虫与医学昆虫学报, 2008, 15(3):162-166.
ZHU Yongfeng, LIU Zhigang, GAO Bo, et al. Cloning, expression, purification and immunological identification of the Der f18 allergen of mites dust mites［J］. Journal of Parasites and Medical Insects, 2008, 15(3): 162-166.（in Chinese）
［2］ 朱健琦,刘志刚,高波,等. 粉尘螨Ⅰ类变应原 (Der fⅠ)的克隆表达、纯化及免疫学特性［J］.昆虫学报,2006,49(2):213-218.
ZHU Jianqi, LIU Zhigang, GAO Bo, et al. Cloning, expression, purification and immunological characteristics of Der f Ⅰ from Dermatophagoides farinae［J］. Acta Entomologica Sinica, 2006,49(2): 213-218.（in Chinese）
［3］ 李平, 黄娜娜, 常可欣,等. 屋尘螨变应原Der p9基因的克隆表达,纯化及反应原性鉴定［J］. 南昌大学学报医学版, 2018, 58(6):1-4.
LI Ping, HUANG Nana, CHANG Kexin, et al. Cloning, expression, purification and identification of allergen Der P9 gene of house dust mite［J］. Journal of Nanchang University Medical Edition, 2018, 58(6): 1-4.（in Chinese）
［4］ 孙金霞, 滕飞翔, 崔玉宝. 尘螨变应原单克隆抗体的制备及应用研究进展［J］. 中国病原生物学杂志, 2017, 12(3):1-4.
SUN Jinxia, TENG Feixiang, CUI Yubao. The preparation and application of single clone antibody of dust mite allergen［J］. Chinese Journal of Pathogenic Biology, 2017, 12(3): 2-4.（in Chinese）
［5］ 古霞,欧阳春艳,袁如意,等. 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.
GU Xia, OUYANG Chunyan, YUAN Ruyi, et al. Cloning, expression and immunogenicity analysis of three allergen genes of house dust mite［J］. Chinese Journal of Parasitology and Parasitic Diseases, 2018,36(6): 627-631.（in Chinese）
［6］ FUJIMURA T, AKI T, ISOBE T, et al. Der f 35: an MD-2-like house dust mite allergen that cross-reacts with Der f 2 and Pso o 2［J］.Allergy, 2017,72(11): 1728-1736.
［7］ 陈德盛, 胡天城, 关丽,等. 屋尘螨第21类过敏原原核表达和过敏原性鉴定［J］. 中国热带医学, 2019, 19(2):7-10.
CHEN Desheng, HU Tiancheng, GUAN Li, et al. Prokaryotic expression and allergen identification of 21 allergens of house dust mites［J］. Tropical Medicine, 2019, 19(2): 7-10.（in Chinese）
［8］ 江贝, 肖小军, 欧阳春艳,等. 粉尘螨过敏原Der f 32的克隆,表达,免疫学鉴定及其对树突状细胞的调节作用研究［J］. 中国寄生虫学与寄生虫病杂志, 2019(3):279-285.
JIANG Bei, XIAO Xiaojun, OUYANG Chunyan, et al. Cloning, expression, immunological identification of Der f 32 from Dermatophagoides farinae and its regulation on dendritic cells［J］. Chinese Journal of Parasitology and Parasitic Diseases, 2019 (3): 279-285.（in Chinese）
［9］ COMPALATI E, BRAIDO F, WALTER C G. Sublingual immunotherapy: recent advances［J］. Allergology International, 2013, 62(4):415-423.
［10］ 胡传翠,李朝品.尘螨与变应性疾病的研究进展［J］.医学综述,2009,15(7):1054-1056.
HU Chuancui, LI Chaopin. Research progress of dust mites and allergic diseases［J］. Medical Review, 2009,15(7): 1054-1056.（in Chinese）
［11］ LIPWORTH B.Clinical relevance of house dust mite immuno-therapy［J］. Journal of Allergy and Clinical Immunology, 2015, 135(1):288.
［12］ 王媛媛,林建立,黄娜娜,等.粉尘螨过敏原Der f 13基因克隆表达与免疫原性鉴定［J］.南昌大学学报医学版,2016(1):21-25.
WANG Yuanyuan, LIN Jianshe, HUANG Nana, et al. Cloning, expression and immunogenicity identification of dust mite allergen Der f 13 gene［J］. Journal of Nanchang University Medical Edition, 2016(1): 21-25.（in Chinese）
［13］ 李喆，张影，徐颖华,等. 新型尘螨过敏原疫苗的研究进展［J］.中国生物制品学杂志,2016,29（3）:312-314.
LI Zhe, ZHANG Ying, XU Yinghua, et al. Research progress of novel dust mite allergen vaccine［J］. Chinese Journal of Biological Products, 2016,29(3): 312-314.（in Chinese）
［14］ BARNES P J. New therapies for asthma: is there any progress［J］. Trends in Pharmacological Sciences, 2010, 31(7):335-343.
［15］ 郑蔓茵,邬玉兰,闫浩, 等.粉尘螨第7类变应原(Der f 7)基因的克隆表达及免疫学特性鉴定［J］.中国寄生虫学与寄生虫病杂志,2013,31(5):363-366.
ZHENG Manyin, WU Yulan, YAN Hao, et al. Cloning, expression and immunological characterization of Der f 7 gene of Dermatophagoides farinae［J］. Chinese Journal of Parasitology and Parasitic Diseases, 2013,31(5): 363-366.（in Chinese）
［16］ 黄娜娜, 陈德盛, 常可欣,等. 屋尘螨Der p7基因的克隆表达、纯化及过敏原性鉴定［J］. 南昌大学学报医学版, 2018, 58(6):13-18.
HUANG Nana, CHEN Desheng, CHANG Kexin, et al. Cloning, expression, purification and identification of allergen of Der p7 gene of house dust mite［J］. Journal of Nanchang University Medical Edition, 2018, 58(6): 13-18.（in Chinese）
［17］ CALDER N M A, LINNEBERG A, KLEINE-TEBBE J, et al. Respiratory allergy caused by house dust mites: what do we really know［J］. Journal of Allergy and Clinical Immunology, 2015,136(1):38-48.

相似文献/References:

[1]夏立新,闫浩,汤慕瑾,等.花生过敏原Ara h2与Ara h6的生物信息学比较研究[J].深圳大学学报理工版,2010,27(2):241.
　XIA Li-xin,YAN Hao,TANG Mu-jin,et al.Bioinformatics comparison of peanut allergen Ara h2 and Ara h6[J].Journal of Shenzhen University Science and Engineering,2010,27(3):241.
[2]唐玉林,曹雁,欧忠华,等.非生物胁迫因子对大豆Sali3-2基因的调控作用[J].深圳大学学报理工版,2012,29(No.1(001-094)):73.[doi:10.3724/SP.J.1249.2012.01073]
　TANG Yu-lin,CAO Yan,OU Zhong-hua,et al.Regulatable gene expression controlled by the promoter of Sali3-2 under different abiotic stresses[J].Journal of Shenzhen University Science and Engineering,2012,29(3):73.[doi:10.3724/SP.J.1249.2012.01073]
[3]刘志刚,杨成彬,闫浩,等.Balb/c小鼠花生过敏模型的建立及发病机理[J].深圳大学学报理工版,2012,29(No.2(095-188)):165.[doi:10.3724/SP.J.1249.2012.02165]
　LIU Zhi-gang,YANG Cheng-bin,YAN Hao,et al.Establishment and pathogenesis of mouse peanut allergy model[J].Journal of Shenzhen University Science and Engineering,2012,29(3):165.[doi:10.3724/SP.J.1249.2012.02165]
[4]毕得,肖华军,周翠红,等.心肺细胞缺氧效应生物信息学分析[J].深圳大学学报理工版,2012,29(No.6(471-580)):541.[doi:10.3724/SP.J.1249.2012.06541]
　BI De,XIAO Hua-jun,ZHOU Cui-hong,et al.Bioinformatic analysis of ?cardiac and pulmonary hypoxia[J].Journal of Shenzhen University Science and Engineering,2012,29(3):541.[doi:10.3724/SP.J.1249.2012.06541]
[5]陈梦竹,王秀杰,夏立新.肺腺癌和肺鳞癌中mRNA可变剪接特征的比较[J].深圳大学学报理工版,2017,34(1):33.[doi:10.3724/SP.J.1249.2017.01033]
　Chen Mengzhu,Wang Xiujie,and Xia Lixin.Comparison of alternative splicing in lung adenocarcinoma and lung squamous carcinoma[J].Journal of Shenzhen University Science and Engineering,2017,34(3):33.[doi:10.3724/SP.J.1249.2017.01033]
[6]曹会,蔡德丰,杨平常,等.家蚕MBP-Bmlp7蛋白的克隆表达及免疫学鉴定[J].深圳大学学报理工版,2017,34(2):117.
　Cao Hui,Cai Defeng,Yang Pingchang,et al. Expression and characterization of allergenicity of MBP-Bmlp7 from Bombyx mori[J].Journal of Shenzhen University Science and Engineering,2017,34(3):117.
[7]娄素琳,朱秀兰,曾志勇,等.盐藻microRNA高通量测序和生物信息学分析[J].深圳大学学报理工版,2018,35(4):331.[doi:10.3724/SP.J.1249.2018.04331]
　LOU Sulin,ZHU Xiulan,ZENG Zhiyong,et al.Bioinformatics analysis of Dunaliella microRNAs by high-throughput sequencing[J].Journal of Shenzhen University Science and Engineering,2018,35(3):331.[doi:10.3724/SP.J.1249.2018.04331]
[8]张尉欣,刘阳,刘 帅,等.镉胁迫下菜心的转录组分析[J].深圳大学学报理工版,2018,35(5):543.[doi:10.3724/SP.J.1249.2018.05543]
　ZHANG Weixin,LIU Yang,LIU Shuai,et al.Transcriptome analysis of Brassica rapa ssp. chinensis var. parachinensis under cadmium stress[J].Journal of Shenzhen University Science and Engineering,2018,35(3):543.[doi:10.3724/SP.J.1249.2018.05543]
[9]钟姗,等.食管鳞癌中3个新miRNA的分子功能预测[J].深圳大学学报理工版,2019,36(4):347.[doi:10.3724/SP.J.1249.2019.04347]
　ZHONG Shan,WANG Yun,et al.The prediction of molecular functions for three novel miRNAs in esophageal squamous cell carcinoma[J].Journal of Shenzhen University Science and Engineering,2019,36(3):347.[doi:10.3724/SP.J.1249.2019.04347]
[10]须周恒,朱营利,孙孟菲,等.小鼠纹状体与视网膜的比较转录组学分析[J].深圳大学学报理工版,2020,37(1):17.[doi:10.3724/SP.J.1249.2020.01017]
　XU Zhouheng,ZHU Yingli,SUN Mengfei,et al.Comparative transcriptomic analysis of mouse striatum and retina[J].Journal of Shenzhen University Science and Engineering,2020,37(3):17.[doi:10.3724/SP.J.1249.2020.01017]

备注/Memo

备注/Memo:
Received：2020-07-20；Accepted：2020-12-09
Foundation：National Natural Science Foundation of China (31729002, U1801286, 81971514); Key Project of Guangzhou Scientific Research Plan (201804020043); Shenzhen Peacock Project Team Project (KQTD20170331145453160)
Corresponding author：Assistant professor LIU Xiaoyu. E-mail:lxy0901@szu.edu.cn
Citation：CHEN Yang, CHEN Xianxiong, OUYANG Chunyan, et al. Cloning, expression and immunological identification of Der f 35 allergen from Dermatophagoides farinae［J］. Journal of Shenzhen University Science and Engineering, 2021, 38(3): 301-306.（in Chinese）
基金项目：国家自然科学基金资助项目（31729002，U1801286，81971514）；广州市科学研究计划重点资助项目（201804020043）；深圳市孔雀计划团队资助项目（KQTD20170331145453160）；深圳市科技计划国际科技合作资助项目（GJHZ2018041819053）；南山区“领航团队”支持计划资助项目（LHTD20180007）；呼吸疾病国家重点实验室开放课题资助项目（SKLRD-OP-201909）
作者简介：陈扬(2000—) ，深圳大学本科生.研究方向：过敏性疾病及过敏原的研究. E-mail：2018221037@email.szu.edu.cn
引文：陈扬，陈献雄，欧阳春艳，等. 粉尘螨过敏原Der f 35的克隆表达及免疫学鉴定［J］. 深圳大学学报理工版，2021，38(3)：301-306.
更新日期/Last Update: 2021-05-30