An antigen epitope peptide derived from dust mite allergen and application thereof

Abstract

The application relates to an antigen epitope peptide derived from a dust mite allergen and application thereof, and belongs to the technical field of immune detection. The antigen epitope peptide derived from allergens Der p 23 and Der f 23 is screened for the first time, the antigen epitope peptide can effectively capture IgE in serum samples of patients with respiratory allergic diseases, the positive rate of the captured IgE has no significant difference with the capture ability of complete allergens, and the antigen epitope peptide has wide application value in the diagnosis of respiratory allergic diseases. In addition, the antigen epitope peptide can induce cells to produce thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33) and interleukin-25 (IL-25), and then start and amplify the 2-type immune response, so that the antigen epitope peptide has a good application prospect in the preparation of vaccines for dust mite allergy.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to an antigenic epitope peptide derived from dust mite allergens and its applications. Background Technology

[0002] House dust mites (HDMs) are among the most prevalent sources of indoor allergens worldwide, playing a significant etiological role in allergic rhinitis and asthma. The most common sensitizing species—dry dust mites (Dermatophagoides farinae) and house dust mites (Dermatophagoides pteronyssinus)—are widespread in both temperate and tropical regions. Diagnosis of house dust mite allergy is based on clinical presentation, skin prick testing, provocation testing, and / or serum-specific IgE testing. Although 40 groups of mite allergens have been identified to date, extracts used in clinical practice for diagnosis and immunotherapy are typically standardized only for the primary allergen (such as house dust mites Der p1 and Der p2). Furthermore, the coexistence of allergen and non-allergen components in these extracts can affect diagnostic accuracy.

[0003] Advances in molecular biology have enabled the production of various recombinant allergens for serum-specific IgE detection, promoting the increasingly widespread clinical application of component-resolved diagnostics (CRD). CRD aims to differentiate between true sensitization and cross-reactivity, predict the risk of severe symptoms, and anticipate the progression of allergies. Therefore, quantitative detection of allergen-specific IgE significantly improves the diagnostic accuracy of allergic diseases. The efficacy of using purified major allergens or chemically modified allergen preparations for allergic reactions (AIT) has been confirmed in numerous studies.

[0004] While component-resolved diagnosis (CRD) represents a significant advancement over mite extract detection, epitope peptide analysis represents a further refinement. Although this technique has been less studied in mite allergies, its value in peanut allergies has been well-established—eptope-specific antibody profiling has greatly facilitated a deeper understanding of mechanisms and clinical applications. Early studies linked broader IgE epitope diversity to the severity and heterogeneity of responses. A subsequent pediatric study confirmed the correlation between epitope diversity and clinical sensitivity (r = 0.6) and showed that polyclonal peptide reactivity remained stable over time. Recent research has demonstrated that Ara h2 peptide-specific IgE improves diagnostic accuracy, and that peptide-specific IgG4 promotes immune tolerance by blocking IgE binding. These findings collectively underscore the importance of moving beyond extract detection towards epitope resolution analysis for better diagnosis, prognosis, and a deeper understanding of immune mechanisms.

[0005] House dust mite-derived peptide immunotherapy, based on recombinant technology and synthetic vaccines, is being used in basic research and clinical practice to optimize allergen-specific immunotherapy. For example, the recombinant Der p 1 peptide vaccine (DpTTDp) showed significant immunotherapeutic effects in a mouse model of house dust mite allergy, increasing allergen-specific IgG levels while decreasing IgE levels and pro-allergic cytokines. Similarly, the hyposensitizing Der p 2.1 peptide exhibits preventative potential by inhibiting airway inflammation, hyperresponsiveness, and pathogenic T cell responses, thereby preventing house dust mite-induced allergic asthma. Furthermore, the hyposensitizing Der p 23-based vaccine PreS-2XP4P5 demonstrates potent immunomodulatory properties by inducing blocking IgG antibodies to inhibit IgE binding and basophil activation, while simultaneously creating a tolerable immune microenvironment. These peptide-based vaccines represent promising advancements beyond traditional allergy immunotherapy, offering potentially safer and more effective strategies for managing house dust mite allergy. Currently, there are no reports regarding whether Der p23 and Der f 23 share linear IgE epitopes. This invention is the first to reveal the existence of this cross-species shared epitope region. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problems of insufficient accuracy and complex diagnostic process in the existing technology for allergic diseases.

[0007] To address the aforementioned technical problems, this invention provides an antigenic epitope peptide derived from house dust mite allergens and its applications. This invention is the first to discover that house dust mite allergens Der p 23 and Der f 23 share a highly conserved, common linear epitope region in the immunoglobulin E (IgE) recognition area. This region differs from the other two mite species by only one amino acid (histidine H in Der p 23 and cysteine ​​C in Der f 23), and this single amino acid variation does not affect the epitope's IgE binding activity or epithelial cell stimulating activity. Based on this, this invention provides two variant peptides derived from this common epitope region, with amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The antigenic epitope peptide of this invention can effectively capture IgE in serum samples from patients with respiratory allergic diseases, and the capture positivity rate is not significantly different from that of intact allergens, thus having broad application value in the diagnosis of respiratory allergic diseases. In addition, the antigenic epitope peptide of the present invention can induce cells to produce thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33) and interleukin-25 (IL-25), thereby initiating and amplifying type 2 immune responses, and has good application prospects in the preparation of vaccines against house dust mite allergies.

[0008] The first objective of this invention is to provide an antigenic epitope peptide derived from a dust mite allergen and its application, wherein the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0009] Furthermore, SEQ ID NO.1: RFGYFADPKDPHKFYICSNWEA.

[0010] Furthermore, SEQ ID NO.2: RFGYFADPKDPCKFYICSNWEA.

[0011] A second objective of this invention is to provide a nucleic acid molecule encoding the aforementioned antigenic epitope peptide.

[0012] A third objective of this invention is to provide an application of the above-mentioned antigenic epitope peptides or nucleic acid molecules in the preparation of diagnostic products for respiratory allergic diseases.

[0013] Furthermore, the respiratory allergic diseases include respiratory allergic diseases caused by dust mite allergens.

[0014] Furthermore, the dust mite allergens include Der p 23 or Der f 23.

[0015] A fourth object of the present invention is to provide a product for detecting dust mite allergens, the product comprising the above-mentioned antigenic epitope peptide or the above-mentioned nucleic acid molecule.

[0016] The fifth objective of this invention is to provide a method for detecting dust mite allergens in a test sample. The method involves biotinylating the aforementioned antigenic epitope peptide, immobilizing the biotinylated antigenic epitope peptide on a streptavidin-coated microplate, adding the test sample to the microplate for co-incubation, and finally detecting it using immunoglobulin E secondary antibody.

[0017] Furthermore, the sample to be tested is plasma.

[0018] A sixth object of the present invention is to provide the use of the above-mentioned antigenic epitope peptide or the above-mentioned nucleic acid molecule in the preparation of an immune product for preventing dust mite allergy.

[0019] Furthermore, the immune product induces cells to produce thymic stromal lymphopoietin, interleukin-33, and interleukin-25.

[0020] A seventh object of the present invention is to provide a vaccine for preventing dust mite allergy, the vaccine comprising the above-described antigenic epitope peptide.

[0021] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0022] This invention is the first to screen antigenic epitope peptides derived from allergens Der p 23 and Der f 23. These epitope peptides effectively capture IgE from serum samples of patients with respiratory allergic diseases, and the capture positivity rate is not significantly different from that of intact allergens. However, the epitope peptides have a more defined compositional structure than intact allergens, exhibit high batch-to-batch consistency, are easy to synthesize, and reduce production costs, making them widely applicable in the diagnosis of respiratory allergic diseases. Furthermore, these epitope peptides not only retain their IgE binding ability but also their stimulatory effect on epithelial cells, inducing the production of thymic stromal lymphopoietin (TSLP), interleukin-33 (IL-33), and interleukin-25 (IL-25), thereby initiating and amplifying type 2 immune responses. This lays the foundation for future development of epitope-based diagnostic methods and novel immunotherapy strategies for HDM allergies. Attached Figure Description

[0023] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0024] Figure 1 The bar chart (A) and box plot (B) show the number of positive peptides in each serum sample from allergy patients (n=116) and healthy controls (n=50).

[0025] Figure 2 It is a statistical graph of unique and shared peptides identified in allergy patients and healthy controls;

[0026] Figure 3 These are the top five peptides with significantly higher enrichment levels in allergy patients compared to the control group (Fisher exact test, P < 0.05).

[0027] Figure 4 The positive rates of each dust mite allergen component were found in allergy patients (n=116) and healthy controls (n=50).

[0028] Figure 5 The positive rates of each house dust mite allergen component were found in allergy patients (n=116) and healthy controls (n=50).

[0029] Figure 6 This is a schematic diagram of the overlapping peptides covering Der p 23 and Der f 23;

[0030] Figure 7 The positive rate of IgE binding of synthetic peptides to recombinant full-length allergens, as determined by ELISA, in an independent validation cohort of children with dust mite allergies (n=41).

[0031] Figure 8 This describes the induction of type 2 epithelial cytokines (TSLP, IL-33, IL-25) in BEAS-2B cells 24 hours after stimulation with each antigen (30 μg / mL). Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0033] Example 1: Screening of peptides

[0034] 1. Sample collection

[0035] Serum samples were collected (72 positive and 50 negative from Wuxi People's Hospital affiliated with Nanjing Medical University, and 46 from Chongqing Hospital affiliated with Chongqing University) and subsequently used for PhIP-Seq analysis. Allergen-specific IgE levels were assessed using the ImmunoCAP® system (UniCAP 1000, Thermo Fisher Scientific Inc., Uppsala, Sweden), with a titer >0.35 IU / mL for house dust mite and / or dust mite extracts considered seropositive. Patients diagnosed with allergic rhinitis and / or asthma were recruited according to established clinical criteria. This is a prospective preliminary study, approved by the Institutional Review Board of Jiangnan University Affiliated Children's Hospital (Approval No.: WXCH2024-12-142), the Ethics Committee of Wuxi People's Hospital (Approval No.: KY26153), and the Medical Ethics Committee of Chongqing People's Hospital (Approval No.: KYS2025-067-01). All participants signed written informed consent forms before enrollment.

[0036] 2. PhIP-Seq Library Design and Construction

[0037] Allergen amino acid sequences were obtained from five major databases: WHO / IUIS Allergen Nomenclature Database, Allergome, AllergenOnline (including gliadin), the Allergen Protein Structure Database (SDAP), and the Allergen Family Database (AllFam). This work yielded 4,300 proteins. An additional 1,100 proteins were integrated from UniProt using the keyword "allergen".

[0038] Peptide sequences, each 56 amino acids long with 28 amino acid overlap, were designed to comprehensively cover all representative allergen proteins. Each protein sequence was progressively divided into 56-amino acid peptide segments from the N-terminus to the C-terminus, with 50% overlap between consecutive segments. If the C-terminal segment was shorter than 56 amino acids, it was extended towards the N-terminus to reach the full length. For protein sequences shorter than 56 amino acids, a repeating "GSGS" sequence was added to the N-terminus to meet the length requirement. Finally, all designed peptide sequences were deduplicated.

[0039] Next, the peptide sequence was reverse-translated into a DNA nucleotide sequence suitable for expression in *E. coli*. The amino acid sequence was converted into a codon-optimized DNA sequence using R software: high-frequency *E. coli* codons were preferentially selected, followed by further optimization to maintain GC content within the range of 40%–60%. Adapter sequences were then added to the 5′ and 3′ ends of each optimized sequence to facilitate downstream PCR amplification and cloning.

[0040] The high-throughput DNA synthesis for the designed sequences was outsourced. The synthesized DNA oligonucleotide libraries were quality controlled using high-throughput sequencing (NovaSeq 6000, Illumina).

[0041] T7 phage packaging was performed using the commercially available T7Select10-3 cloning kit (Novagen, GER, 70550-3). The synthesized oligonucleotide library was dissolved and quantified. Primers were designed based on the shared sequence, and the oligonucleotide library was amplified by PCR. The amplified products were then purified by gel electrophoresis. The purified products were double-digested with EcoRI and HindIII (NEB, R3101V and R3104V) and ligated into phage vectors using T4 DNA ligase (NEB, M0202V). The ligation products were verified by agarose gel electrophoresis. The ligation mixture was added to the packaging extract and incubated at 22°C for 2 hours. The reaction was terminated by adding 90 μL of antibiotic-free LB medium, and 10 μL was used for packaging efficiency testing. After verification, the library was amplified on large-scale plates. Phage particles were eluted, centrifuged, and titrated. The packaged phage display peptide library was finally quality controlled by deep sequencing (NovaSeq 6000, Illumina).

[0042] The 200-nucleotide sequence was synthesized by Twist Bioscience. The resulting oligonucleotide library was amplified by PCR and inserted into the T7FNS2 vector backbone via restriction cloning. The constructed library was packaged using the T7 Select packaging kit (EMD Millipore) and subsequently amplified for use as a PhIP-Seq library.

[0043] The library contains a total of 1,144 short peptides derived from house dust mites. Of these, 594 peptides are from the farinae house dust mite (Dermatophagoides farinae), covering allergens Derf1-11, Derf13-16, Derf18, Derf20, Derf21-32, Derf34-37, Derf39, and DerfEF. The remaining 504 peptides are from the pteronyssinus house dust mite (Dermatophagoides pteronyssinus), covering allergens Derp1-11, Derp13-15, Derp18, Derp20, Derp21, Derp23-26, Derp28-30, Derp32-33, and Derp36-38.

[0044] 3. PhIP-Seq Sample Processing and Detection

[0045] Perform the following steps sequentially for each sample. First, centrifuge the serum at >10,000 rpm for 10 minutes at 4°C. Collect the supernatant and dilute it 1:1,000 with PBST buffer to a final volume of 1 mL, then add it to a pre-blocked 96-well plate. Include negative control, blank control, and positive control wells in each 96-well plate. Next, add the diluted and centrifuged phage library to each well, ensuring a 16,000-fold diversity of phage per well. Seal the plate tightly and incubate overnight at 4°C with rotation. After incubation, add Protein G magnetic beads (IgG enrichment is used here as an example) or other magnetic beads and incubate at 4°C for 4 hours. Then centrifuge at 500×g for 3 minutes and capture the magnetic beads on a magnetic rack. Discard the supernatant and add 200 μL of wash buffer to each well. Resuspend the magnetic beads by pipetting and transfer to a new 96-well PCR plate. Repeat the washing process twice. Finally, the magnetic beads were resuspended in 40 μL of sterile water and transferred to a new 96-well PCR plate. The plate was then sealed, briefly centrifuged for 10 seconds, and then heated in a thermal cycler at 95°C for 10 minutes. Sequencing library preparation involved two rounds of PCR. The first round used universal primers matching the phage sequence, and the second round used primers containing barcodes and sequencing adapters. The amplification products were quality-checked by gel electrophoresis. PCR products from 1–3 96-well plates were mixed, and the target bands were recovered by gel electrophoresis and gel purification. Finally, sequencing was performed on the Illumina platform according to the manufacturer's protocol. Paired-end sequencing of 150 bp was used, with at least 3 M reads required per sample. Full-length sequences were obtained from the paired-end reads, and read counts for each peptide were used as the primary data.

[0046] 4. Data Standardization and Statistical Analysis

[0047] Data standardization was performed to correct for differences in sequencing depth among samples. The read count for each sample was scaled to 1.25 million reads to obtain the normalized reads (NR) for each peptide. The enrichment factor (EF) for each peptide was then calculated, defined as the ratio of the NR of that peptide in the test sample to the NR in a simulated immunoprecipitation control (magnetic beads without patient samples and subjected to the same phage incubation and processing), to correct for background signal. A generalized Poisson distribution model was then applied to simulate the expected distribution of peptides with similar abundance after immunoprecipitation, and a p-value was calculated for each peptide to quantify the significance of its enrichment.

[0048] Enriched peptides in each sample were identified based on the following threshold criteria: Normalized Read Count (NR) > 15, Enrichment Factor (EF) > 2, and p-value < 0.05. The thresholds for NR and EF were determined according to generally accepted empirical standards from the literature. The p-value threshold was set to 0.05 and calibrated based on the maximum background noise level observed in the simulated immunoprecipitation control. Peptides meeting all the above criteria were defined as positive hits.

[0049] Enrichment fold (EF), reflecting the relative strength of the antibody response, was used as the primary data for statistical analysis. First, EF values ​​were corrected to facilitate subsequent analysis. Based on a pre-defined threshold, signals above the threshold (positive) in any sample retained their original EF values, while signals below the threshold (negative) were assigned a value of 1. The corrected EF values ​​were then used for statistical comparisons between cohorts. Subsequently, all enriched peptides (defined as peptides that were positive in at least one sample in the cohort) were pooled, and the corresponding EF data for these peptides were extracted and analyzed using various statistical methods.

[0050] The analysis in this embodiment covers a total of 1,144 mite-derived short peptides, of which 594 peptides are derived from house dust mite allergens (Der f 1-11, 13-16, 18, 20, 21-32, 34-37, 39 and EF), and 504 peptides are derived from house dust mite allergens (Der p1-11, 13-15, 18, 20, 21, 23-26, 28-30, 32-33 and 36-38).

[0051] Serological testing showed that these mite-derived peptides were detectable in 91 patients (positive count range: 1-71), but not in the remaining patients. In the healthy control group, 11 individuals tested positive, with counts ranging from 1 to 12. Figure 1 In addition, 1,014 and 40 peptides were identified in the patient group and healthy control group, respectively. Figure 2 Notably, five of the short peptides showed a significant increase ( ). Figure 3 The sequences of the five short peptides are shown in Table 1.

[0052] Table 1

[0053]

[0054] Example 2: PhIP-Seq profiling reveals specific IgE responses to mite allergen components

[0055] The positive rates of each house dust mite (Der f) allergen component were compared between allergy sufferers (n=116) and healthy controls (n=50). Results are shown in [Table missing]. Figure 4Of the 26 components tested, Der f 23.0201 and Der f 23.0101 had the highest positive rates in the allergy group, with 65 and 57 positive cases respectively, while only 1 case of each was positive in the control group. Other components with significant positive rates in allergy patients included Der f 37 (20 cases), Der f 2 (10 cases), Der f 1 (7 cases), Der f 39 (5 ​​cases), Der f 10 (5 cases), and Der f 35 (6 cases). In contrast, most components had very low or zero positive rates in healthy controls, with only Der f 34 (4 positive controls) and a few other components showing single positive cases. These results indicate that the Der f 23 variant, as well as Der f 37, Der f 2, Der f 1, Der f 39, Der f 10, and Der f 35, are highly specific for allergy sufferers.

[0056] like Figure 5 As shown, the positivity rates of various house dust mite (Der p) allergen components were compared between allergy sufferers (n=116) and healthy controls (n=50). Of the 20 components tested, Der p 23 had the highest positivity rate in the allergy group, with 66 positive cases, compared to only 1 positive case in the control group. Other components with significant positivity rates in allergy sufferers included Der p 36 (16 cases), Der p 37 (15 cases), Der p 2 (8 cases), Der p 29 (9 cases), Der p 10 (6 cases), Der p 1 (5 cases), and Der p 14 (4 cases). Most components had extremely low or zero positivity rates in healthy controls, with only a few scattered positive cases (e.g., Der p 14, Der p 10, Der p 11, Der p 7, Der p 9). The above results indicate that Der p 23, as well as Der p 36, Der p 37, Der p2, Der p 29, Der p 10, Der p 1 and Der p 14, are highly specific for allergic individuals.

[0057] Example 3: Selection and Synthesis of Peptides and Allergens

[0058] Based on the high positive rate observed in single-allergen component detection, we focused our analysis on peptide sequences of Der p 23 and Derf 23. Among the six short peptides derived from Der p 23 (ID4642, ID25601, ID25608, ID25607, ID33599, and ID33600), we identified a highly conserved common sequence: RFGYFADPKDPHKFYICSNWEA ( Figure 6Similarly, a highly homologous shared sequence was found in four short peptides of Der f 23 (ID25604, ID31011, ID27960, and ID33454): RFGYFADPKDPCKFYICSNWEA ( Figure 6 It is noteworthy that these two sequences differ only in a single amino acid residue (H→C), belonging to two species variants of the same shared epitope region, suggesting that they share a key structural motif and possess considerable immunogenicity and cross-reactivity potential.

[0059] Both shared peptides were chemically synthesized, coupled to bovine serum albumin (BSA) using a heterobifunctional cross-linking agent, and purified by reversed-phase HPLC.

[0060] The genes encoding Der p 23.0101 (GenBank: EU414751.1), Der f 23.0101 (GenBank: KU166910), and Der f 23.0201 (GenBank: KM009999) were synthesized and cloned into the pSumo-1 vector. The recombinant plasmids were transformed into *E. coli* for protein expression. After induction, the three recombinant proteins were purified by affinity chromatography—Der p23.0101 contained an N-terminal His-Sumo tag, while Der f 23.0101 and Der f 23.0201 both contained N-terminal His tags. Purity was confirmed as >90% by SDS-PAGE and Coomassie Brilliant Blue staining, and endotoxin levels were <0.05 EU / μg protein by LAL assay. Protein concentration was determined using the micro-BCA protein assay. The purified proteins were stored at -80°C in appropriate buffer systems (Der p23.0101 was stored in PBS; the two Der f 23 variants were stored in a buffer containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 10% glycerol) to obtain five recombinant allergens.

[0061] Example 3: Detection of IgE binding capacity of synthetic common peptides derived from Der p 23 and Der f 23 by ELISA

[0062] The practicality of peptide ELISA for IgE binding was assessed. In the peptide ELISA, biotinylated peptides were first immobilized on streptavidin-coated microplates (Thermo Scientific Nunc Immobilizer Streptavidin Plates, catalog number 436014) and then incubated with serum samples diluted 1:1,000. Specific IgE binding was detected using horseradish peroxidase (HRP)-labeled goat anti-human IgE secondary antibody (Southern Biotech, catalog number 9250-05), followed by color development with TMB substrate. Serum samples from 41 donors with corresponding PhIP-Seq data were used to detect each peptide. Subsequently, IgE-ELISA was performed on another set of independently collected serum samples (n=41, from Wuxi People's Hospital Affiliated to Nanjing Medical University) using the five antigens mentioned above.

[0063] Serum IgE testing in 41 patients with respiratory allergic diseases showed no statistically significant difference in the positive rates among Der f 23 peptide, Der f 23.0101, and Der f 23.0201. Figure 7 Similarly, no significant difference was observed between the Der p 23 peptide and Der p 23.0101. Figure 7 The above results indicate that the synthesized peptide antigens effectively capture the IgE reactivity observed in their corresponding full-length allergen components, supporting their potential as simplified yet representative diagnostic biomarkers.

[0064] Example 5: In vitro stimulatory effect of a synthetic common peptide derived from Der p 23 and Der f 23 on BEAS-2B cells

[0065] In vitro stimulation experiments were performed on BEAS-2B cells (Fenghui Biotechnology Co., Ltd., catalog number CL0044) using three recombinant allergens (Der p 23.0101, Der f 23.0101, and Der f 23.0201) and two synthetic short peptides. Cells were cultured in BEGM... TM BulletKit TMCells were cultured in culture medium under standard conditions (37°C, 5% CO2). At 50%–60% confluence, cells were stimulated for 24 hours at a concentration of 30 μg / mL. This optimal concentration was determined through preliminary dose-response experiments. After stimulation, cell culture supernatant was collected, and the protein levels of epithelial-derived cytokines—IL-25 (catalog number XFH10534), IL-33 (catalog number XFH10548), and TSLP (catalog number XFH13053)—were detected using commercially available ELISA kits, following the manufacturer's instructions. All experiments were performed in biological triple replicates, with appropriate negative controls (unstimulated cells).

[0066] Preliminary dose-response experiments determined the optimal stimulation conditions to be 30 µg / mL for 24 hours (data not shown). Subsequently, BEAS-2B cells were stimulated for 24 hours with 30 µg / mL of full-length allergens (Der p 23, Der f 23.0101, Der f 23.0201) or their corresponding short peptides. ELISA analysis showed that, compared with the unstimulated control group, all treatments significantly increased the secretion of TSLP, IL-33, and IL-25 (two-way ANOVA, Šidák corrected; except for Der p 23 peptide-induced IL-25, all P < 0.0001; P = 0.0015 for Der p 23 peptide-induced IL-25). Figure 8 These results indicate that full-length allergens and their synthetic peptide derivatives can induce BEAS-2B cells to produce type 2 epithelial cytokines TSLP, IL-33, and IL-25.

[0067] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An antigenic epitope peptide derived from house dust mite allergens and its application, characterized in that, The amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A nucleic acid molecule encoding the antigenic epitope peptide of claim 1.

3. The use of the antigenic epitope peptide of claim 1 or the nucleic acid molecule of claim 2 in the preparation of diagnostic products for respiratory allergic diseases.

4. The application according to claim 3, characterized in that, The respiratory allergic diseases include those caused by dust mite allergens.

5. The application according to claim 4, characterized in that, The dust mite allergens include Der p 23 or Der f23.

6. A product for detecting dust mite allergens, characterized in that, The product comprises the antigenic epitope peptide of claim 1 or the nucleic acid molecule of claim 2.

7. A method for detecting dust mite allergens in a test sample, characterized in that, The method involves biotinylating the antigenic epitope peptide described in claim 1, then immobilizing the biotinylated antigenic epitope peptide on a streptavidin-coated microplate, adding the test sample to the microplate for co-incubation, and finally detecting it using immunoglobulin E secondary antibody.

8. The use of the antigenic epitope peptide of claim 1 or the nucleic acid molecule of claim 2 in the preparation of an immune product for preventing house dust mite allergy.

9. The application according to claim 8, characterized in that, The immune product induces cells to produce thymic stromal lymphopoietin, interleukin-33, and interleukin-25.

10. A vaccine for preventing dust mite allergy, characterized in that, The vaccine includes the antigenic epitope peptide as described in claim 1.

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