表达rFel d 1的重组大肠杆菌预防猫主要变应原Fel d 1诱发的过敏性哮喘的效果分析

doi:10.11843/j.issn.0366-6964.2025.09.032

摘要/Abstract

摘要： Felis domesticus allergen 1(Fel d 1)作为猫诱发过敏性疾病的主要变应原，其诱发的症状从轻度的过敏性鼻炎到危及生命的哮喘反应不等。本研究基于抗原递送系统构建了一种新型免疫干预策略：通过重组大肠杆菌Nissle 1917(Escherichia coli Nissle 1917，EcN)实现体内表达rFel d 1，以期能够预防rFel d 1引起的过敏反应。猫主要变应原Fel d 1基因序列号来自NCBI GenBank库，选取链1和链2的CDS序列，将链1 C端的半胱氨酸残基(Cys)与链2 N端的缬氨酸残基(Val)共价连接形成重组Fel d 1(rFel d 1)。在rFel d 1的N端引入3个重复的GS序列，C端添加一段6×His标签序列，经密码子优化后克隆至pBad载体，构成pBad-rFel d 1原核表达载体。将pBad-rFel d 1质粒转化至EcN感受态细胞中构建重组大肠杆菌EcN^{rFel d 1}。在小鼠过敏模型中，综合分析EcN^{rFel d 1}对rFel d 1诱发的过敏反应的预防效果。结果表明，预先给予体内表达rFel d 1的重组大肠杆菌EcN^{rFel d 1}能够有效预防气道高反应性和IgE水平的升高，避免肺组织发生病变以及rFel d 1特异性激活的局部或全身过敏反应，防止体内Th1/Th2和Th17/Treg失衡。本研究基于益生菌EcN的体内定植优势及局部缓释特性，构建预防性重组大肠杆菌EcN^{rFel d 1}。该疫苗通过调控Th1/Th2、Treg/Th17免疫平衡及诱导阻断性抗体的产生，从而有效预防rFel d 1诱发的过敏反应。

关键词: 过敏性哮喘, 变应原, 益生菌, 气道炎症, 蛋白疫苗

Abstract: Felis domesticus allergen 1 (Fel d 1), the major allergen responsible for cat-induced allergic diseases, can trigger symptoms ranging from mild allergic rhinitis to life-threatening asthmatic reactions. This study developed a novel immune intervention strategy based on an antigen delivery system: utilizing recombinant Escherichia coli Nissle 1917 (EcN) to achieve in vivo expression of rFel d 1, aiming to prevent allergic reactions induced by rFel d 1. The gene sequence of the major Fel d 1 was obtained from the NCBI GenBank database. The coding sequences (CDS) of Chain 1 and Chain 2 were selected, with a covalent linkage formed between the C-terminal cysteine residue (Cys) of Chain 1 and the N-terminal valine residue (Val) of Chain 2 to generate recombinant Fel d 1 (rFel d 1). A triple GS repeat sequence was introduced at the N-terminus of rFel d 1, while a 6×His tag was added to the C-terminus. After codon optimization, the construct was cloned into the pBad vector to generate the prokaryotic expression vector pBad-rFel d 1. The pBad-rFel d 1 plasmid was then transformed into EcN competent cells to construct the recombinant E. coli strain EcN^{rFel d 1}. In a murine allergy model, the preventive efficacy of EcN^{rFel d 1} against rFel d 1-induced allergic reactions was comprehensively evaluated. We found that pre-administration with the recombinant E. coli strain EcN^{rFel d 1} capable of expressing rFel d 1 in vivo could effectively prevent airway hyperresponsiveness and the elevation of IgE levels, protecte lung tissues from pathological changes and avoid local or systemic allergic reactions specifically activated by rFel d 1, and prevent the imbalance of Th1/Th2 and Th17/Treg in the body. Based on the inherent colonization advantages and sustained-release properties of probiotic EcN, this study developed a preventive recombinant E. coli strain EcN^{rFel d 1}, Which effectively prevents rFel d 1-induced allergic reactions by modulating Th1/Th2 and Treg/Th17 immune balance while inducing the production of blocking antibodies.

Key words: allergic asthma, allergens, probiotics, airway inflammation, protein vaccines

中图分类号:

S852.4

麦艳琪, 孙晓筝, 刘晓烜, 梁星烂, 黄颍, 吴晓娟, 柳贤德, 裴业春. 表达rFel d 1的重组大肠杆菌预防猫主要变应原Fel d 1诱发的过敏性哮喘的效果分析[J]. 畜牧兽医学报, 2025, 56(9): 4529-4545.

MAI Yanqi, SUN Xiaozheng, LIU Xiaoxuan, LIANG Xinglan, HUANG Ying, WU Xiaojuan, LIU Xiande, PEI Yechun. Analysis of the Preventive Effect of Recombinant E. coli Expressing rFel d 1 against Fel d 1-Induced Allergic Asthma[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4529-4545.

参考文献

[1] VAN HAGE M，KACK U，ASARNOJ A，et al. An update on the prevalence and diagnosis of cat and dog allergy-emphasizing the role of molecular allergy diagnostics[J]. Mol Immunol，2023，157: 1-7.
[2] YANG M S，LEE S P，KWON Y J，et al. Dog and cat allergies and allergen avoidance measures in Korean adult pet owners who participated in a pet exhibition[J]. Allergy Asthma Immunol Res，2018，10(2): 155-164.
[3] CHARPIN C，MATA P，CHARPIN D，et al. Fel d I allergen distribution in cat fur and skin[J]. J Allergy Clin Immunol，1991，88(1): 77-82.
[4] GRONLUND H，SAARNE T，GAFVELIN G，et al. The major cat allergen，Fel d 1，in diagnosis and therapy[J]. Int Arch Allergy Immunol，2010，151(4): 265-274.
[5] LUCZYNSKA C M，LI Y，CHAPMAN M D，et al. Airborne concentrations and particle size distribution of allergen derived from domestic cats(Felis domesticus). Measurements using cascade impactor，liquid impinger，and a two-site monoclonal antibody assay for Fel d I[J]. Am Rev Respir Dis，1990，141(2): 361-367.
[6] MELLERUP M T，HAHN G W，POULSEN L K，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.
[7] STRACHAN D P. Hay fever, hygiene, and household size[J]. Br Med J，1989，299(6710): 1259-1260.
[8] LEE M J，KANG M J，LEE S Y，et al. Perturbations of gut microbiome genes in infants with atopic dermatitis according to feeding type[J]. J Allergy Clin Immunol，2018，141(4): 1310-1319.
[9] THORSEN J，RASMUSSEN M A，WAAGE J，et al. Infant airway microbiota and topical immune perturbations in the origins of childhood asthma[J]. Nat Commun，2019，10(1):5001.
[10] ZIMMERMANN P，MESSINA N，MOHN W W，et al. Association between the intestinal microbiota and allergic sensitization，eczema，and asthma：A systematic review[J]. J Allergy Clin Immunol，2019，143(2): 467-485.
[11] WANG L，ZHU L M，QIN S. Gut microbiota modulation on intestinal mucosal adaptive immunity[J]. J Immunol Res，2019，2019: 4735040.
[12] LUO X Y，WANG H，LIU H X，et al. Effects of probiotics on the prevention and treatment of children with allergic rhinitis: a meta-analysis of randomized controlled trials[J]. Front Pediatr，2024，12:1352879.
[13] ANANIA C，DI MARINO V P，OLIVERO F，et al. Treatment with a probiotic mixture containing Bifidobacterium animalis subsp. Lactis BB12 and Enterococcus faecium L3 for the prevention of allergic rhinitis symptoms in children：A randomized controlled trial[J]. Nutrients，2021，13(4): 1315-1315.
[14] YAMASHITA M，MIYOSHI M，IWAI M，et al. Lactobacillus helveticus SBT2171 alleviates perennial allergic rhinitis in Japanese adults by suppressing eosinophils：A randomized，double-blind，placebo-controlled study[J]. Nutrients，2020，12(12): 3620-3620.
[15] YU X L，LIN C S，YU J，et al. Bioengineered Escherichia coli Nissle 1917 for tumour-targeting therapy[J]. Microb Biotechnol，2020，13(3): 629-636.
[16] ZHAO Z J，XU S M，ZHANG W Y，et al. Probiotic Escherichia coli NISSLE 1917 for inflammatory bowel disease applications[J]. Food Funct，2022，13(11): 5914-5924.
[17] 刘志伟. 一株产亚精胺的工程益生菌Nissle 1917的构建[D]. 长春：吉林大学，2024.
LIU Z W. Construction of a spermidine-producing engineered probiotic Nissle 1917[D]. Changchun: Jilin University，2024.(in Chinese)
[18] SARATE P J，HEINL S，POIRET S，et al. E. coli Nissle 1917 is a safe mucosal delivery vector for a birch-grass pollen chimera to prevent allergic poly-sensitization[J]. Mucosal Immunol，2019，12(1): 132-144.
[19] 彭美琪，魏春洁，郑安琪，等. 层状双氢氧化物-rFel d 1-Can f 1融合变应原对小鼠模型过敏反应的预防效果分析[J]. 畜牧兽医学报，2024，55(7): 3143-3154.
PENG M Q，WEI C J，ZHENG A Q，et al. Preventive effect of layered double hydroxide-rFel d 1-Can f 1 fusion allergens on anaphylaxis in mouse model[J]. Acta Veterinaria et Zootechnica Sinica，2024，55(7): 3143-3154.(in Chinese)
[20] 李树民，张敏，王朋，等. 小鼠无创性肺功能的测定及其意义[J]. 中国实验动物学报，2018，26(5): 548-553.
LI S M，ZHANG M，WANG P，et al. The significance of a non-invasive measurement of lung function in mice[J]. Acta Laboratorium Animalis Scientia Sinica，2018，26(5): 548-553.(in Chinese)
[21] 罗承慧，高江瑞，陈俊威，等. 尘螨诱导特应性皮炎小鼠模型和哮喘小鼠模型的构建[J]. 畜牧兽医学报，2024，55(3): 1257-1267.
LUO C H，GAO J R，CHEN J W，et al. Construction of mouse model of dust mite induced atopic dermatitis and asthma[J]. Acta Veterinaria et Zootechnica Sinica，2024，55(3): 1257-1267.(in Chinese)
[22] RADU M，CHERNOFF J. An in vivo assay to test blood vessel permeability[J]. J Vis Exp，2013，73: e50062.
[23] 魏春洁，徐琳皓，高江瑞，等. 猫、犬融合变应原Fel d 1-Can f 1过敏性哮喘小鼠模型的建立[J]. 热带生物学报，2024，15(5): 558-566.
WEI C J，XU L H，GAO J R，et al. Construction of a mouse model of allergic asthma induced by fusion of allergen Fel d 1-Can f 1 of cats and dogs[J]. Journal of Tropical Biology，2024，15(5): 558-566.(in Chinese)
[24] PRANGTAWORN P，CHAISRI U，SEESUAY W，et al. Tregitope-linked refined allergen vaccines for immunotherapy in cockroach allergy[J]. Scientific Reports，2018，8(1): 0-0.
[25] 罗应，彭美琪，肖正泮，等. 热带无爪螨主要变应原Blo t 5和Blo t 21融合表达质粒诱导小鼠产生IgG和调节性T细胞[J]. 畜牧兽医学报，2022，53(4): 1220-1230.
LUO Y，PENG M Q，XIAO Z P，et al. Expression of Blomia tropicalis major allergens Blo t 5 and Blo t 21 induced the production of IgG and regulatory T cells in mice[J]. Acta Veterinaria et Zootechnica Sinica，2022，53(4):1220-1230.(in Chinese)
[26] HEDLIN G，GRAFF-LONNEVIG V，HEILBORN H，et al. Immunotherapy with cat- and dog-dander extracts. V. Effects of 3 years of treatment[J]. J Allergy Clin Immunol，1991，87(5): 955-964.
[27] HEDLIN G，GRAFF-LONNEVIG V，HEILBORN H，et al. Immunotherapy with cat- and dog-dander extracts. II. In vivo and in vitro immunologic effects observed in a 1-year double-blind placebo study[J]. J Allergy Clin Immunol，1986，77(3): 488-496.
[28] JUNIPER E F，STAHL E，DOTY R L，et al. Clinical outcomes and adverse effect monitoring in allergic rhinitis[J]. J Allergy Clin Immunol，2005，115(3 Suppl 1): S390-413.
[29] SHAMJI M H，VALENTA R，JARDETZKY T，et al. The role of allergen-specific IgE, IgG and IgA in allergic disease[J]. Allergy，2021，76(12): 3627-3641.
[30] VITTE J，VIBHUSHAN S，BRATTI M，et al. Allergy，anaphylaxis and non-allergic hypersensitivity：IgE，mast cells and beyond[J]. Med Prin Pract，2022，31(6): 501-515.
[31] ZHOU X L，SONG X Z，SHU T，et al. Prevention and alleviation of allergic rhinitis by oral administration of Lacticaseibacillus paracasei GOLDGUT-Lpc969[J]. Front Immunol，2024，15: 1444778.
[32] REN J J，ZHAO Y，HUANG S，et al. Immunomodulatory effect of Bifidobacterium breve on experimental allergic rhinitis in BALB/c mice[J]. Exp Ther Med，2018，16(5): 3996-4004.
[33] JOHNATHAN M，MUHAMAD S A，GAN S H，et al. Lignosus rhinocerotis cooke ryvarden ameliorates airway inflammation，mucus hypersecretion and airway hyperresponsiveness in a murine model of asthma[J]. PLoS One，2021，16(3): e0249091-e0249091.
[34] NAKAMURA T，MURATA T. Regulation of vascular permeability in anaphylaxis[J]. Br J Pharmacol，2018，175(13): 2538-2542.
[35] BAO C，CHEN O，SHENG H，et al. A mast cell-thermoregulatory neuron circuit axis regulates hypothermia in anaphylaxis[J]. Sci Immunol，2023，8(81): eadc9417.
[36] AITORO R，SIMEOLI R，AMOROSO A，et al. Extensively hydrolyzed casein formula alone or with L-rhamnosus GG reduces-lactoglobulin sensitization in mice[J]. Pediatr Allergy Immunol，2017，28(3): 230-237.
[37] HANIDZIAR D，KOULMANDA M. Inflammation and the balance of Treg and Th17 cells in transplant rejection and tolerance[J]. Curr Opin Organ Transplant，2010，15(4): 411-415.
[38] WANG K，DONG H，QI Y，et al. Lactobacillus casei regulates differentiation of Th17/Treg cells to reduce intestinal inflammation in mice[J]. Can J Vet Res，2017，81(2): 122-128.