屋尘螨第十类变应原的克隆、表达、纯化及免疫原性分析

doi:10.12140/j.issn.1000-7423.2020.06.008

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (6): 723-729.doi: 10.12140/j.issn.1000-7423.2020.06.008

屋尘螨第十类变应原的克隆、表达、纯化及免疫原性分析

郭楚桐¹(), 欧阳春艳¹, 何俊贤¹, 季树宇¹, 杨礼腾², 刘晓宇¹^,^*()

1 深圳大学医学院过敏反应与免疫学研究所,深圳 518060
2 深圳大学第三附属医院变态反应科,深圳 518001

收稿日期:2020-03-25 出版日期:2020-12-30 发布日期:2021-01-12
通讯作者: 刘晓宇
作者简介:郭楚桐（2000-）,女,本科生,主要从事过敏性疾病过敏原的基础研究。E-mail: 805692234@qq.com
基金资助:
国家自然科学基金(31729002);国家自然科学基金(U1801286);国家自然科学基金(81971514);广州市科学研究计划重点项目(201804020043);深圳市孔雀计划团队项目(KQTD20170331145453160);深圳市科技计划国际科技合作项目(GJHZ2018041819053);南山区“领航团队”支持计划项目(LHTD20180007);呼吸疾病国家重点实验室开放课题(SKLRD-OP-201909)

Cloning, expression, purification and immunogenicity analysis of the tenth-class allergen of Dermatophagoides pteronyssinus

GUO Chu-tong¹(), OUYANG Chun-yan¹, HE Jun-xian¹, JI Shu-yu¹, YANG Li-teng², LIU Xiao-yu¹^,^*()

1 Allergy and Immunology Institute, Shenzhen University, School of Medicine, Shenzhen 518060, China
2 Allergy Department, The Third Affiliated Hospital of Shenzhen University, Shenzhen 518001, China

Received:2020-03-25 Online:2020-12-30 Published:2021-01-12
Contact: LIU Xiao-yu
Supported by:
National Natural Science Foundation of China(31729002);National Natural Science Foundation of China(U1801286);National Natural Science Foundation of China(81971514);Key Project of Guangzhou Scientific Research Plan(201804020043);Shenzhen Peacock Project Team Project(KQTD20170331145453160);Shenzhen Science and Technology Plan International Science and Technology Cooperation Project(GJHZ2018041819053);Nanshan District “Pilot Team” Support Project(LHTD20180007);Open Project of State Key Laboratory of Respiratory Diseases(SKLRD-OP-201909)

摘要/Abstract

摘要：

目的克隆屋尘螨（Dermatophagoides pteronyssinus）第十类变应原Der p 10基因,表达并纯化Der p 10重组蛋白,分析其免疫原性和生物学信息。方法挑取经纯培养的屋尘螨,提取总RNA,逆转录生成cDNA,RT-PCR扩增Der p 10基因。将pET-24a载体与Der p 10基因连接并转化入Top10克隆菌体中,经BamHⅠ和XhoⅠ双酶切鉴定和测序后转入大肠埃希菌BL21(DE3)中,1 mmol/L IPTG诱导后,采用12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白表达情况。经Ni⁺离子亲和层析纯化后,以尘螨过敏患者血清为一抗,蛋白质免疫印迹（Western blotting）分析重组蛋白的免疫原性。使用ProtParam Tools、HNN、SWISS MODEL、DNAStar预测Der p 10的理化性质、原肌球蛋白的二级和三级结构、B细胞抗原表位,Blastp和MEGA工具构建Der p 10系统进化树。结果 RT-PCR获得目的基因Der p 10,其开放阅读框为855 bp,可编码284个氨基酸。双酶切结果显示Der p 10已连接至载体上。SDS-PAGE分析结果显示,Der p 10重组蛋白相对分子质量（M_r）为38 000,为可溶性蛋白。Western blotting分析结果显示,Der p 10重组蛋白与尘螨过敏患者血清反应呈阳性结果。信息学分析结果显示,Der p 10蛋白不稳定,二级结构和三级结构均以α-螺旋为主。DNAStar预测到Der p 10存在1个B细胞抗原表位肽序列。Blastp和MEGA分析结果显示,与粉尘螨Der f 10亲缘关系较近,序列相似性达94.4%。结论成功构建Der p 10表达菌并纯化出与尘螨过敏患者血清中的IgE抗体结合较高及纯度较好的Der p 10重组蛋白并证实其反应原性。

关键词: 屋尘螨, Der p 10, 表达, 纯化, 免疫学鉴定, 生物信息学

Abstract:

Objective To clone the gene of the tenth-class allergen Der p 10 (tropomyosin) of Dermatophagoides pteronyssinus, express and purify recombinant protein Der p 10, and analyze the immunogenicity and biological information of the protein. Methods Cultured mites were picked for total RNA extraction, then cDNA was generated by reverse transcription and underwent RT-PCR to amplify the Der p 10 gene. The pET-24a vector was ligated with the Der p 10 gene and transformed into the Top10 cloned bacteria. After BamH I and Xho I double enzyme digestion and sequencing, the construct was transformed into Escherichia coli BL21 (DE3), induced by 1 mmol/L IPTG, and examined in 12% SDS-PAGE to analyze the expression of recombinant protein. After purification by Ni⁺ ion affinity chromatography, the immunogenicity of Der p 10 recombinant protein was analyzed by Western blotting using serum of patient with dust mite allergy as the primary antibody. The ProtParam Tools, HNN, SWISS MODEL, DNAStar were used to predict the physiochemical properties of Der p 10, the secondary and tertiary structure of tropomyosin, and B cell epitope; Blastpand MEGA tool were used to construct Der p 10 phylogenetic tree. Results The target gene Der p 10 was obtained by RT-PCR, with an open reading frame of 855 bp, encoding 284 amino acids. The results of double enzyme digestion and sequencing showed that Der p 10 was linked to the vector. SDS-PAGE analysis showed that the relative molecular mass of Der p 10 recombinant protein was 38 000, which was a soluble protein. Western blotting analysis showed that the Der p 10 recombinant protein reacted with the serum of dust mite allergy patient. The bioinformatics analysis indicated that the Der p 10 protein was unstable, and its secondary and tertiary structures were mainly by α-helices. DNAStar predicted the presence of one B cell epitope peptide sequence in Der p 10. Blastp alignment and MEGA analysis showed that it was closely related to Der f 10, with sequence similarity reaching 94.4%. Conclusion The Der p 10 recombinant plasmid was constructed, and the expressed soluble recombinant protein of Der p 10 can specifically bind to the IgE antibody in patient serum with dust mite allergy, demonstrating its apparent immunogenicity.

Key words: Dermatophagois pteronyssinus, Der p 10, Expression, Purification, Immunological characteristics, Bioinformatics

中图分类号:

R384.4

郭楚桐, 欧阳春艳, 何俊贤, 季树宇, 杨礼腾, 刘晓宇. 屋尘螨第十类变应原的克隆、表达、纯化及免疫原性分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(6): 723-729.

GUO Chu-tong, OUYANG Chun-yan, HE Jun-xian, JI Shu-yu, YANG Li-teng, LIU Xiao-yu. Cloning, expression, purification and immunogenicity analysis of the tenth-class allergen of Dermatophagoides pteronyssinus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(6): 723-729.

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参考文献 22

[1]	Calderón MA, Linneberg A, Kleine-Tebbe J, et al. Respiratory allergy caused by house dust mites: What do we really know?[J]. J Allergy Clin Immunol, 2015,136(1):38-48.
[2]	Romagnani S. Immunologic influences on allergy and the TH1/TH2 balance[J]. J Allergy Clin Immunol, 2004,113(3):395-400.
[3]	Halim TYF, Steer CA, Mathä L, et al. Group 2 innate lymphoid cells are critical for the initiation of adaptive T helper 2 cell-mediated allergic lung inflammation[J]. Immunity, 2014,40(3):425-435.
[4]	Zhao YN, Hong Y, Li CP. Specific immunotherapeutic effect of recombinant Der p 1 T cell epitope fusion protein on asthma in a mouse model[J]. Chin J Parasitol Parasit Dis, 2020,38(1):74-79. (in Chinese)
	( 赵亚男, 洪勇, 李朝品. Der p 1 T细胞表位融合蛋白对哮喘小鼠的特异性免疫治疗效果[J]. 中国寄生虫学与寄生虫病杂志, 2020,38(1):74-79.)
[5]	Wong L, Huang CH, Lee BW. Shellfish and house dust mite allergies: is the link tropomyosin?[J]. Allergy Asthma Immunol Res, 2016,8(2):101. pmid: 26739402
[6]	Voorhorst R, Spieksma-Boezeman MI, Spieksma FT. Is a mite (Dermatophagoides sp.) the producer of the house-dust allergen?[J]. Allerg Asthma (Leipz), 1964,10:329-334.
[7]	Verhoeckx KCM, van Broekhoven S, den Hartog-Jager CF, et al. House dust mite (Der p 10) and crustacean allergic patients may react to food containing Yellow mealworm proteins[J]. Food Chem Toxicol, 2014,65:364-373. doi: 10.1016/j.fct.2013.12.049 pmid: 24412559
[8]	Chen DS, Hu TC, Guan L, et al. Cloning and expression of Der p 21 gene from Dermatophagoides pteronyssinus and identification of its immunological characteristics[J]. Chin Trop Med, 2019,19(2):103-106. (in Chinese)
	( 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):103-106.)
[9]	Wang N, Teng FX, Yu LL, et al. Cloning and expression of the full-length gene of group 8 allergen of Dermatophagoides farinae[J]. Chin J Parasitol Parasit Dis, 2017,35(4):351-356. (in Chinese)
	( 王楠, 滕飞翔, 俞黎黎, 等. 粉尘螨变应原Der f 8全长基因克隆及表达[J]. 中国寄生虫学与寄生虫病杂志, 2017,35(4):351-356.)
[10]	Hu JY. Preliminary screening of new allergens of dust mites and analysis of Der f 23 protein structure[D]. Shenzhen: Shenzhen University, 2018. (in Chinese)
	( 胡葭耘. 粉尘螨新过敏原初筛及其Der f 23蛋白结构解析[D]. 深圳: 深圳大学, 2018.)
[11]	Yi FC, Cheong N, Shek LP, et al. Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus[J]. Clin Exp Allergy, 2002,32(8):1203-1210. doi: 10.1046/j.1365-2745.2002.01449.x pmid: 12190660
[12]	Li Z, Zhang Y, Xu YH, et al. Advance in research on novel vaccine against dust mite allergens[J]. Chin J Biol, 2016,29(3):312-314. (in Chinese)
	( 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.)
[13]	Xu ZQ, Wei JF. Allergen component-resolved diagnostics in allergic diseases: an overview[J]. Clin Med J, 2019,17(1):18-22. (in Chinese)
	( 许志强, 魏继福. 过敏原组分解析诊断在过敏疾病中的应用进展[J]. 临床药物治疗杂志, 2019,17(1):18-22.)
[14]	Lin JL, Ba JG, Liu ZG, et al. Bioinformatics analysis of dust mite allergen der f 21[J]. J Nanchang Univ Med Sci, 2016,56(3):1-5. (in Chinese)
	( 林建立, 巴金戈, 刘志刚, 等. 粉尘螨过敏原Der f 21的生物信息学分析[J]. 南昌大学学报(医学版), 2016,56(3):1-5.)
[15]	Gu X, Ouyang CY, Yuan RY, et al. Cloning and expression of house dust mite allergen Der p 3 and its antigenicity[J]. Chin J Parasitol Parasit Dis, 2018,36(6):627-631. (in Chinese)
	( 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.)
[16]	Santiago HC, Bennuru S, Boyd A, et al. Structural and immunologic cross-reactivity among filarial and mite tropomyosin: Implications for the hygiene hypojournal[J]. J Allergy Clin Immunol, 2011,127(2):479-486. pmid: 21185070
[17]	Mellerup MT, Hahn GW, Poulsen LK, et al. Safety of allergen-specific immunotherapy. Relation between dosage regimen, allergen extract, disease and systemic side-effects during induction treatment[J]. Clin Exp Allergy, 2000,30(10):1423-1429. doi: 10.1046/j.1365-2222.2000.00910.x pmid: 10998019
[18]	Zheng PY, Sun BQ. Clinical significance of Der p 1, Der p 2, and Der p 10 detection[J]. Chin J Allergy Clin Immunol, 2014,8(2):150-154. (in Chinese)
	( 郑佩燕, 孙宝清. 屋尘螨致敏蛋白组分 Der p 1、Der p 2和 Der p 10检测的临床意义[J]. 中华临床免疫和变态反应杂志, 2014,8(2):150-154.)
[19]	Tagami K, Nakayama S, Furuta T, et al. Pen a 1-specific IgE does not improve the accuracy of a shrimp allergy diagnosis among Japanese children due to cross-reactivity with Der p 10[J]. Allergol Int, 2020,69(2):290-292. pmid: 31685384
[20]	Wong L, Huang CH, Lee BW. Shellfish and house dust mite allergies: is the link tropomyosin?[J]. Allergy Asthma Immunol Res, 2016,8(2):101. doi: 10.4168/aair.2016.8.2.101 pmid: 26739402
[21]	Sidenius KE, Hallas TE, Poulsen LK, et al. Allergen cross-reactivity between house-dust mites and other invertebrates[J]. Allergy, 2001,56(8):723-733.
[22]	Resch Y, Weghofer M, Seiberler S, et al. Molecular characterization of Der p 10: a diagnostic marker for broad sensitization in house dust mite allergy[J]. Clin Exp Allergy, 2011,41(10):1468-1477. pmid: 21711470

屋尘螨第十类变应原的克隆、表达、纯化及免疫原性分析

Cloning, expression, purification and immunogenicity analysis of the tenth-class allergen of Dermatophagoides pteronyssinus

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