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

doi:10.12140/j.issn.1000-7423.2020.06.008

Abstract

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

CLC Number:

R384.4

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.

Figures/Tables 5

References 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, Math? 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)
[4]	( 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.
[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.
[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)
[8]	( 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)
[9]	( 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)
[10]	( 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.
[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)
[12]	( 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)
[13]	( 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)
[14]	( 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)
[15]	( 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.
[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.
[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)
[18]	( 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.
[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.
[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.