A chimeric protein PA5O8 and its applications

Abstract

The present application relates to a kind of chimeric protein PA508 and its application, belong to the prevention, diagnosis and desensitization treatment technical field of allergic disease. The fusion polypeptide of the present application is named as chimeric protein PA508, the amino acid sequence of the fusion polypeptide is as shown in SEQ ID NO:1. The fusion polypeptide of the present application includes the allergen characteristic peptide from different species, so cross-species immune response can be realized, can prevent or treat the allergic reaction caused by allergen from animal and allergen from plant.

Description

Technical Field

[0001] This invention relates to the fields of prevention, diagnosis and desensitization treatment of allergic diseases, and particularly to a chimeric protein PA5O8 and its applications. Background Technology

[0002] Common allergic diseases include allergic dermatitis, allergic rhinitis, allergic asthma, and food allergies. Among these, so-called Type I allergic reactions diagnosed by IgE account for about 36% of allergic reactions, while the rest are mostly non-IgE mediated.

[0003] When a patient develops an allergic disease, clinical symptomatic treatment involves using anti-inflammatory drugs, such as antihistamines, calcium channel blockers, and leukotriene inhibitors, and / or targeted therapies. However, the effects of these drugs are purely symptom-relieving, and their effectiveness is limited to the duration of use. In most cases, the same symptoms will recur upon re-exposure to the allergen.

[0004] Desensitization therapy, also known as allergen-specific immunotherapy (AIT), is the only method for allergic diseases that targets the cause and alters the disease course. AIT can significantly improve clinical symptoms and reduce the risk of disease progression to severe illness by inducing immune tolerance over a long period. Unlike symptomatic drug treatment, traditional AIT typically involves repeated subcutaneous or sublingual administration of a mixture of crudely extracted natural allergens after identifying the allergen, with a treatment period lasting 3-5 years or even longer. Although desensitization therapy has been proven to be effective, its widespread application is limited by several factors, including difficulties in standardizing species-based allergen extracts, poor predictability of efficacy, high risk of potential adverse reactions, poor patient compliance, high costs, and lengthy treatment cycles. Allergen diagnostic testing is the first step in conducting desensitization therapy. Currently, common methods for allergen detection include allergen skin prick tests, serological tests, and allergen challenge tests. However, given the vast number of species containing allergens, standardizing all allergen preparations is impossible; therefore, the current variety of allergen preparations is limited. Furthermore, cross-reactivity exists between different species and allergens, making it highly likely that diagnostic tests for numerous species of allergens will yield positive results. Even with reliable allergen preparations, clinicians may struggle to select a specific allergen for subsequent desensitization therapy. Moreover, since each species contains numerous allergen components, replacing species-specific allergen diagnosis with so-called allergens of different components would require far more allergen preparations than the number of species, significantly increasing the complexity of preparation and the cost and discomfort for patients. Therefore, creating desensitization therapy preparations with cross-species responses has become a crucial core issue in allergology.

[0005] Ideally, allergen formulations should contain all sensitizing components in stable, homogeneous, and sufficient quantities. However, due to the diversity and variability of the source species, and the limitations of growth cycles and conditions on biological materials, natural extracts often exhibit inconsistent quality and significant variations in protein content between different batches. In contrast, recombinant allergens offer advantages such as high controllability, high yield, low cost, and ease of standardized production. Numerous studies have shown that recombinant allergens exhibit high bioactivity consistency with their natural counterparts, thus serving as alternative materials for in vivo and in vitro challenge experiments and clinical desensitization therapy.

[0006] However, the sheer variety of allergens remains a challenge. Studies show that a single allergen can induce multiple types of inflammation in different organs and tissues, demonstrating multi-targeting characteristics. Furthermore, different types of allergens can activate the same symptom phenotypes through the same inflammatory pathways. Clearly, different allergens share common epitopes, exhibiting convergence, which means that the quantity of allergens can be reduced based on their characteristic peptides. Therefore, obtaining characteristic allergen peptides with multi-targeting effects and the ability to activate multiple inflammatory phenotypes for detection and treatment has become crucial to addressing the problem of the vast variety of allergens. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a chimeric protein PA5O8 and its applications. This chimeric protein can be used for in vitro diagnosis of allergic diseases caused by natural allergens, and can also be used to treat allergic diseases.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a fusion polypeptide named chimeric protein PA5O8, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] This invention utilizes bioinformatics, molecular biology, and immunology techniques to construct and validate a cross-species responsive immunotherapeutic chimeric protein, PA5O8. This protein comprises a characteristic peptide from the allergen P16311 of house dust mites, a characteristic peptide from the allergen A5I875 of the giant Java frog, and a characteristic peptide from the allergen O81701 of the alder tree, with its amino acid sequence shown in SEQ ID NO: 1. Because it contains characteristic peptides from allergens of different species, it can achieve a cross-species immune response to allergens (animal and plant), and can prevent or treat allergic reactions caused by allergens from animals (e.g., house dust mites) and plants (e.g., pollen).

[0011] As a preferred embodiment of the first aspect, the nucleotide sequence of the fusion polypeptide is shown in SEQ ID NO: 2-3.

[0012] The present invention optimizes the nucleotide sequence of the chimeric protein PA5O8 to make it more suitable for expression in Escherichia coli. Its nucleotide sequence before optimization is shown in SEQ ID NO: 2, and its nucleotide sequence after optimization is shown in SEQ ID NO: 3.

[0013] Secondly, the present invention provides applications of the fusion peptide, including any one of the following c1)-c3): c1) preparing peptide drugs for treating or preventing allergic diseases; c2) preparing recombinant allergen preparations; c3) preparing reagent kits for diagnosing allergic diseases.

[0014] As a preferred embodiment of the second aspect, the polypeptide drug further includes pharmaceutical excipients.

[0015] As a preferred embodiment of the second aspect, the pharmaceutical excipients include dehydrating agents, sweeteners, lubricants, surfactants, fillers, and isotonic modifiers.

[0016] Thirdly, the present invention provides a pharmaceutical composition comprising the fusion polypeptide.

[0017] As a preferred embodiment of the third aspect, the dosage form of the drug is an ointment, spray, or external patch.

[0018] The pharmaceutical compositions of the present invention containing the chimeric protein PA5O8 can be formulated into ointments, sprays or topical patches for the prevention or treatment of allergic reactions caused by allergens from animals and allergens from plants.

[0019] Fourthly, the present invention provides a product for detecting the specific immune protein IgE in serum, the product comprising the aforementioned fusion polypeptide.

[0020] The chimeric protein PA5O8 of the present invention has a good binding epitope to the specific immune protein IgE in serum, and can be used to detect the immune protein IgE in serum.

[0021] Fifthly, the present invention provides a recombinant plasmid comprising a nucleotide sequence as shown in any one of SEQ ID NO: 2-3.

[0022] In a sixth aspect, the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium contains the recombinant plasmid described above.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention utilizes bioinformatics, molecular biology, and immunology techniques to construct and validate the allergenicity of the cross-species responsive chimeric protein PA5O8 for immunotherapy. The PA5O8 codon was optimized, and an endotoxin-free Escherichia coli strain was constructed to stably express the protein, providing a simple, efficient, and endotoxin-free method for preparing recombinant PA5O8. The binding epitopes of PA5O8 protein to T cells and B cells were predicted, and immunological experiments demonstrated its ability to activate dendritic cells (DCs) and increase related T cell activation factors. The invention also found that PA5O8 protein binds to serum IgE from patients with house dust mite allergies. The chimeric protein PA5O8 is beneficial for improving the accuracy and richness of allergic disease diagnosis and can also be prepared into an immunomodulator for desensitization prevention or treatment of allergic diseases. Therefore, this invention has significant clinical value for the development of novel immunomodulators for allergen detection and allergen immunotherapy or prevention. Attached Figure Description

[0025] Figure 1 Figure A shows the composition and 3D structure of the chimeric protein PA5O8; Figure B shows the 3D structure of the chimeric protein PA5O8.

[0026] Figure 2 A schematic diagram illustrating the linear IgE epitopes of the chimeric protein PA5O8 that can bind to B cells, as predicted by the online tool IEDB.

[0027] Figure 3 These are the RNA secondary structure diagrams of the chimeric protein PA5O8 before and after codon optimization (A is the structure diagram before codon optimization, and B is the structure diagram after codon optimization).

[0028] Figure 4 This is a PCR electrophoresis image of Rosetta-pET44a-PA5O8 colonies (lane M is DNA2000Maker; lanes 1-9 are single colonies; + lane is positive control pUC57-PA5O8 plasmid amplification; - lane is negative control ddH2O).

[0029] Figure 5Figure A shows the SDS-PAGE electrophoresis of the induced expression of the chimeric protein PA5O8 (in Figure A, lane M represents the protein molecule marker; lane RO represents the expression bacteria containing the target gene; lane RO+IPTG represents the expression protein of the recombinant bacteria after IPTG induction; lane inclusion bodies represent the expression protein in inclusion bodies; lane supernatant represents the expression protein in the supernatant; Figure B is the SDS-PAGE electrophoresis of PA5O8 purified by StrepTrapHP affinity chromatography, lane M represents the protein molecule marker; lanes 1-3 represent the solution after pre-column protein denaturation and renaturation; lane precipitation represents the precipitate precipitated during dialysis; lanes 4-5 represent the solution in the permeate that did not bind to the column; lanes 6-8 represent the elution buffer, the target protein eluted from the column using dethiobiotin).

[0030] Figure 6 This is a schematic diagram showing the results of secondary structure analysis of the purified chimeric protein PA5O8 by circular dichroism spectroscopy.

[0031] Figure 7 Schematic diagrams showing the increased expression levels of relevant cytokines detected by ELISA (Figure A shows the increased expression levels of cytokines IL-1β and IL-6; Figure B shows the increased expression levels of cytokines IL-8 and TNFα).

[0032] Figure 8 Figure A shows the binding activity of Der f1 and PA5O8 with specific IgE in the serum of patients with house dust mite allergy, as detected by ELISA (Figure A shows the binding activity of Der f1 with specific IgE in the serum of patients with house dust mite allergy; Figure B shows the binding activity of PA5O8 with specific IgE in the serum of patients with house dust mite allergy; where "other" indicates patients who tested positive in mixed food allergy tests; "d2 (case)" indicates the d2 grade of UniCAP test).

[0033] Figure 9 Schematic diagram of airway hyperresponsiveness and inflammatory response in a mouse model of house dust mite-induced asthma, using PA5O8 chimeric protein to alleviate or treat the condition (Figure A shows changes in lung compliance; Figure B shows changes in airway resistance; Figure C shows airway histopathological changes assessed by hematoxylin-eosin (H&E) staining).

[0034] Figure 10 Schematic diagram of airway hyperresponsiveness and inflammatory response in a pollen-induced mouse asthma model for the relief or treatment of PA5O8 chimeric protein (Figure A shows changes in lung compliance; Figure B shows changes in airway resistance; Figure C shows airway histopathological changes assessed by hematoxylin-eosin (H&E) staining). Detailed Implementation

[0035] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0036] Example 1: Obtaining the PA5O8 chimeric protein

[0037] 1. Filtering and Building:

[0038] In the ALLERGENIA (http: / / allergenia.gzhmu.edu.cn) allergen database, allergen sequences were divided into peptide segments of a certain length using a 25-amino acid sliding method. The resulting peptide segments were compared with non-allergen sequences, and fragments identical or similar to non-allergens were removed. Adjacent peptide segments from the same allergen were spliced ​​together to form initial allergen family characteristic peptides. A recursive feature comparison elimination method was then used to screen for AFFP combinations with minimal noise, forming 531 allergen family characteristic peptides (AFFPs). These AFFPs were compared with the amino acid sequences of 21 major representative allergens. AFFPs matching at least three major representative allergens were selected, and at least three of these AFFPs were randomly combined to form an AFFP chimeric protein, named PA5O8, with its amino acid sequence shown in SEQ ID NO: 1.

[0039] The screening results for the chimeric protein PA5O8 are as follows: Figure 1 As shown in A, the chimeric protein PA5O8 is composed of three allergen-specific peptides: P16311, A5I875, and O81701. It contains the characteristic peptide of P16311, an allergen from dust mites; the characteristic peptide of A5I875, an allergen from giant Java frogs; and the characteristic peptide of O81701, an allergen from alder trees. Therefore, the chimeric protein PA5O8 contains allergen-specific peptides from both animals and plants, enabling an immune response induced by cross-species allergens.

[0040] The amino acid sequence of the chimeric protein PA5O8 is: MKALEKIEKKIEEKRKEIEEIRLKNLEELKQAEQEAIEQLRSKYGEKVEAKKALRNRLKELLKQIEEKLLRAGGGGSGEKLDKFKDAKIEALKAAQEKIYQKLEEKVQKAEKAAEEKIGQKVLGRKKGGGGS (SEQ ID NO:1).

[0041] 2. Structural prediction of PA5O8 chimeric protein

[0042] Online prediction of the physicochemical properties, secondary structure, tertiary structure, T-cell epitopes, and B-cell epitopes of PA5O8 chimeric proteins, specifically including:

[0043] The physicochemical properties of the protein were predicted using the Expasy online tool ProtParam (https: / / web.expasy.org / protparam / ); the secondary structure was predicted using the SOPMA online tool (https: / / npsa.prabi.ibcp.fr / cgibin / npsa_automat.pl); the tertiary structure was predicted using the Phyre2 online tool (http: / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index); the dominant binding epitopes of PA5O8 protein to HLA-DR were analyzed using the NetMHCII 4.0 Server website (http: / / www.cbs.dtu.dk / services / NetMHCIIpan / ); and the B-cell linear IgE epitopes of the chimeric protein PA5O8 were predicted using the ABCpred Prediction Server (https: / / webs.iiitd.edu.in / raghava / abcpred / ) and the IEDB (http: / / tools.immuneepitope.org / main / ) online analysis servers.

[0044] According to the prediction results of the online tool ProtParam, PA5O8 is composed of 196 amino acids, with a molecular weight of 22089.29 Da and a theoretical isoelectric point of 5.46. Alanine is the most abundant amino acid (24 amino acids, accounting for 12.2%), and it contains 28 negatively charged amino acids (aspartic acid and glutamic acid) and 23 positively charged amino acids (arginine and lysine). Its instability coefficient is 44.59, classifying it as an unstable protein. The protein's fatty acid coefficient is 77.30, and its average hydrophilicity is -0.228, classifying it as a hydrophilic protein.

[0045] According to SOPMA predictions, the secondary structure of PA5O8 consists of approximately 56.12% α-helices, approximately 3.06% extended chains, approximately 5.1% β-turns, and approximately 35.71% random coils. A summary of the SOPMA-predicted secondary structures is shown in Table 1.

[0046] Table 1 Overview of SOPMA Predicted Secondary Structure

[0047]

[0048] The Phyre2 online website predicts the tertiary structure of PA5O8: using a single highest-scoring template d1xkga1, 160 residues (representing 82% of the sequence) were modeled with 100.0% confidence, resulting in the following... Figure 1The 3D structure of the PA5O8 chimeric protein shown in Figure B is clearly visible, with α-helices, β-sheets, turns, and coils.

[0049] Using the NetMHCII 4.0 Server website, the dominant binding epitopes of PA5O8 and HLA-DR were analyzed. The dominant epitopes were mainly concentrated at protein amino acid sites 51-65, 83-97, and 81-195. Among them, 51-65: LESLKYVEANKGAIN is the epitope that can bind the most MHC molecules, as shown in Table 2 below.

[0050] Table 2. Advantageous combination of P1A5O8 and HLA-DR epitopes

[0051]

[0052] The ABCpred Prediction Server results indicate that the PA5O8 chimeric protein binds to linear IgE epitopes on B cells. Using the IEDB online analysis server, seven methods were employed to obtain... Figure 2 The diagram shows the predicted linear IgE epitopes for PA5O8-bound B cells. Combining the two prediction methods, the dominant linear IgE epitopes for PA5O8-bound B cells were found to be concentrated at protein amino acid sites 20-35, 117-132, 123-138, 168-183, and 179-194.

[0053] Example 2: Cloning, expression, and purification of PA5O8 chimeric protein:

[0054] (1) Cloning: Based on the codon table of E. coli K12, the "Principles of Codon Optimization", and by adjusting the GC content, some optimal codons were replaced with better codons. At the same time, the same base was avoided from appearing more than 5 times consecutively. Rare codons were avoided throughout the process. The tag fusion expression of the cross-species response immunotherapy chimeric protein PA5O8 was optimized to obtain a gene that can be efficiently expressed in E. coli. Its gene sequence is shown in SEQ ID NO: 3, and its RNA secondary structure results are as follows: Figure 3 As shown, the gene sequence was cloned into the E. coli expression vector pET-44a to obtain the recombinant plasmid pET44a-PA5O8.

[0055] Nucleotide sequence of chimeric protein PA5O8 before optimization: ATGAAAGCGCTGGAAAAAATTGAAAAAAAAATTGAAGAAAAACGCAAAGAAATTGAAGAAATTCGCCTGAAAAACCTGGAAGAACTGAAACAGGCGGAACAGGAAGCGATTGAACAGCTGCGCAGCAAATATGGCGAAAAAGTGGAAGCGAAAAAAGCGCTGCGCAACCGCCTGAAAGAACTGCTGAAACAGATTGAAGAAAAACTGCTGCGCGCGGGCGGCGGCGGCAGCGGCGAAAAACTGGATAAATTTAAAGATGCGAAAATTGAAGCGCTGAAAGCGGCGCAGGAAAAAATTTATCAGAAACTGGAAGAAAAAGTGCAGAAAGCGGAAAAAGCGGCGGAAGAAAAAATTGGCCAGAAAGTGCTGGGCCGCAAAAAAGGCGGCGGCGGCAGC (SEQ ID NO:2).

[0056] Nucleotide sequence of chimeric protein PA5O8 after optimization: ATGAAAGCGCTGGAAAAAATTGAAAAAAAAATTGAAGAAAAACGCAAAGAAATTGAAGAAATTCGCCTGAAAAACCTGGAAGAACTGAAACAGGCGGAACAGGAAGCGATTGAACAGCTGCGCAGCAAATATGGCGAAAAAGTGGAAGCGAAAAAAGCGCTGCGCAACCGCCTGAAAGAACTGCTGAAACAGATTGAAGAAAAACTGCTGCGCGCGGGCGGCGGCGGCAGCGGCGAAAAACTGGATAAATTTAAAGATGCGAAAATTGAAGCGCTGAAAGCGGCGCAGGAAAAAATTTATCAGAAACTGGAAGAAAAAGTGCAGAAAGCGGAAAAAGCGGCGGAAGAAAAAATTGGCCAGAAAGTGCTGGGCCGCAAAAAAGGCGGCGGCGGCAGCTGGAGCCATCCGCAGTTTGAAAAATAA (SEQ ID NO:3).

[0057] (2) Expression: The above SEQ ID NO:3 sequence was recombined with the pET44a plasmid to obtain the recombinant plasmid pET44a-PA5O8 of the present invention, and it was transformed into the endotoxin-free Escherichia coli expression strain BL21 (DE3 Clearcoli) to obtain the recombinant strain Rosetta-pET44a-PA5O8. Single colonies were selected and inoculated into LB liquid medium containing 50ug / mL ampicillin and cultured overnight. The next day, 10% of the inoculum was inoculated into fresh culture medium and incubated at 37ºC, 200 rpm for 3 h. Then, 500 μL of 1mM isopropyl thiogalactoside (IPTG) was added and incubated at 37ºC, 200 rpm for 10 h for induction.

[0058] The recombinant strain Rosetta-pET44a-PA5O8 was identified by PCR, and the results are as follows: Figure 4 As shown, the recombinant bacteria Rosetta-pET44a-PA5O8 constructed in this invention has been successfully cloned into the recombinant plasmid pET44a-PA5O8.

[0059] (3) Purification: Centrifuge 8000 g of the induced recombinant bacteria at 4ºC for 10 min, discard the supernatant, and retain the bacterial pellet. Resuspend the bacterial pellet in 1× binding buffer (150 mM NaCL, 70 Mm Tris-HCl, 1 Mm EDTA), and sonicate on an ice-water mixture for 30 min.

[0060] Inclusion bodies were resuspended in washing buffer (1× binding buffer with 0.5% Triton X-100) and stirred for 1 h, repeated 4 times. The mixture was then centrifuged at 8000 g, 4ºC for 20 min. The supernatant was discarded, and the washed inclusion body proteins were dissolved in denaturing buffer (1× binding buffer with 8 mM DTT and 5 M guanidine hydrochloride) with stirring. The solution was then placed in a beaker containing renaturing buffer (1× binding buffer with 4 M urea) and stirred at 4ºC for protein renaturation. The denatured and renatured inclusion body protein solution was filtered through a 0.22 μm filter and prepared for Strep Trap testing. HP Pre-packed columns were used to purify PA5O8 protein containing the StrepII tag. SDS-PAGE analysis showed a purity of 95%.

[0061] Purification results are as follows Figure 5 As shown in lane A, the RO+IPTG lane shows that the recombinant bacteria expressed the target protein after IPTG induction; the inclusion body lane shows that the target protein was expressed in inclusion bodies. Figure 5 As shown in B, after PA5O8 was purified by StrepTrapHP affinity chromatography, lanes 1-3 show the target protein in the pre-column denaturation and renaturation solution, and lanes 6-8 show the target protein eluted with dethiobiotin and bound to the column.

[0062] Figure 5 The results show that the theoretical molecular weight of the StrepII-tagged fusion protein PA5O8 is approximately 22 kDa. SDS-PAGE electrophoresis confirmed that the molecular weight of the fusion protein PA5O8 is consistent with the theoretical value and that it is expressed as inclusion bodies.

[0063] Example 3: Identification of purified PA5O8 chimeric protein

[0064] The secondary structure and thermal stability of the purified chimeric protein PA5O8 were analyzed using a Bio-Logic MOS-500 multi-functional circular dichroism spectrometer (BDI).

[0065] The results are as follows Figure 6 As shown, with increasing temperature, starting from 40℃, the α-helix of PA5O8 protein gradually unwinds into an irregular coil, resulting in a denaturation temperature of 327 K (53.85℃).

[0066] Example 4: Verification of the ability of PA5O8 chimeric protein

[0067] 1. Validation of the antigen-presenting ability of the PA5O8 chimeric protein:

[0068] THP-1 (human acute monocytic leukemia cells) induced dendritic cells (DCs) were stimulated with chimeric protein PA5O8 at concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 μg / mL, respectively. After 24 h, the cells were centrifuged at 500 g for 5 min, and the supernatant was collected. The content of inflammatory cytokines in the supernatant was detected by ELISA.

[0069] ELISA test results as follows Figure 7 As shown in Figure A, stimulation of DCs with different concentrations of the chimeric protein PA5O8 for 24 h significantly increased the secretion and expression levels of inflammatory cytokines IL-1β and IL-6 in the cell culture supernatant; Figure 7 As shown in Figure B, stimulation of DCs with different concentrations of the chimeric protein PA5O8 for 24 h significantly increased the secretion and expression levels of inflammatory cytokines IL-8 and TNFα in the cell culture supernatant. These results indicate that the PA5O8 chimeric protein of this invention possesses a certain antigen-presenting ability and a certain immune response capability.

[0070] 2. Binding of PA5O8 chimeric protein to specific IgE in patients with house dust mite allergy:

[0071] Dilute the PA5O8 chimeric protein to 1-10 μg / mL with coating buffer, add 50 μL of the PA5O8 protein dilution to each well, and incubate overnight at 4°C. The next day, discard the uncoated PA5O8 protein, add 5% BSA, and incubate on a shaker at room temperature for 1 hour. Dilute the serum of 285 patients with house dust mite allergy 10-fold with 0.3% BSA (dissolved in PBST), add to the above wells, and incubate at 37°C for 2 hours. After washing, add mouse human IgE-HPR to the above wells and incubate at 37°C for 1.5 hours. After washing, add chromogenic buffer and stop the incubation after 30 minutes, then detect the OD450 using a microplate reader. Der f1 protein is currently the diagnostic protein for clinical detection of house dust mite allergy and is used as a control.

[0072] The results are as follows Figure 8 As shown in A, the number of patients with house dust mite allergy whose serum specific IgE can bind to Der f1 is relatively small; while Figure 8 Figure B shows that a higher number of patients with house dust mite allergy had positive serum IgE levels that could bind to PA5O8. This indicates that among the 285 patients with house dust mite allergy, 256 were indeed allergic to house dust mites, 21 had no allergic symptoms and total IgE ≤10, 1 was positive for mixed food allergy testing, and 7 were positive for mixed house dust allergy testing. The results showed that the number of patients with positive serum IgE levels that could bind to PA5O8 was greater than the number of patients with positive levels that could bind to Der f 1, and 10 patients were positive for both. However, among the 21 patients with no allergic symptoms and total IgE ≤10, 3 were positive. Furthermore, the binding capacity of PA5O8 to serum IgE, as indicated by the OD value, was relatively low, suggesting high safety and accuracy in clinical testing.

[0073] 3. The therapeutic effects of PA5O8 protein dust mites and pollen allergies:

[0074] SPF-grade C57BL / 6J mice aged 6-8 weeks and weighing 18-22 grams were randomly divided into a healthy control group, a house dust mite sensitization model group, a pollen sensitization model group, a PA5O8 house dust mite sensitization treatment group, and a PA5O8 pollen sensitization treatment group. The sensitization and challenge protocols are as follows:

[0075] Mice were intraperitoneally injected with 100 μL of sensitization solution (sensitization solution: 50 μg house dust mite or pollen + 2 mg Al(OH)3 diluted to PBS buffer) on days 0, 7, and 14. The control group was injected with 100 μL of PBS. From day 21 onwards, for four consecutive days, mice were challenged by intranasal instillation of 30 μL of house dust mite or pollen challenge solution (1.7 mg / mL). Healthy control mice were instilled with an equal volume of PBS concurrently as a control. Starting one day before challenge, mice in the treatment group received subcutaneous injections of PA5O8 chimeric protein 5 and 10 μg / kg once daily for five consecutive days, while mice in the model group and control group received an equal volume of PBS intraperitoneally.

[0076] Airway hyperresponsiveness was assessed 24 hours after the final stimulation: Mice were anesthetized and tracheotomized, connected to a small animal respiratory function testing device, and stimulated with methacholine at concentration gradients of 0, 6.25, 12.5, 25, and 50 mg / mL. Lung compliance and airway resistance data were recorded. The results were compared with the baseline values ​​at 0 concentration stimulation for each group, and the proportion of the stimulated data to the baseline value was calculated.

[0077] Lung tissue was harvested immediately after airway resistance testing, fixed in 4% paraformaldehyde for 24 hours, routinely embedded in paraffin and sectioned (4 μm thick), and stained with hematoxylin and eosin. The airway wall thickness, inflammatory cell infiltration and tissue structure changes were observed under an optical microscope. The degree of inflammation was semi-quantitatively assessed using a double-blind scoring method.

[0078] Experimental data are expressed as mean ± standard deviation (Mean ± SD). Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA with Bonferroni multiple correction was used for comparisons among multiple groups. A p-value less than 0.05 was considered statistically significant.

[0079] The results are as follows Figure 9 As shown, Figure 9 A in Figure 9 Figure B shows the response curves of mice in different groups under different concentrations of methacholine stimulation, compared with the dust mite sensitization model group. Figure 9 In mice treated with PA5O8 chimeric protein A (5 and 10 μg / kg), lung compliance was significantly reduced (*P<0.05, **P<0.01). Figure 9 In mice treated with PA5O8 chimeric protein B (5 and 10 μg / kg), airway hyperresponsiveness was significantly reduced (*P<0.05, **P<0.01). Figure 9C in the figure shows the airway histopathological changes assessed by hematoxylin-eosin (H&E) staining. The healthy control group showed intact lung tissue structure, thin airway walls, and no obvious inflammatory cell infiltration. The house dust mite group showed obvious airway epithelial thickening, airway space narrowing, and a large number of inflammatory cell infiltrations. The PA5O8 treatment group (10 μg / kg) showed significantly reduced airway wall thickening and inflammation.

[0080] The results are as follows Figure 10 As shown, Figure 10 A in Figure 10 Figure B shows the response curves of mice in different groups under different concentrations of methacholine stimulation, compared with the pollen sensitization model group. Figure 10 The lung compliance of mice treated with PA5O8 chimeric protein A (5 μg / kg, 10 μg / kg) was significantly reduced (*P<0.05, **P<0.01). Figure 10 In mice treated with PA5O8 chimeric protein B (5 μg / kg, 10 μg / kg), airway hyperresponsiveness was significantly reduced (*P<0.05, **P<0.01). Figure 10 The C in the figure shows the airway histopathological changes assessed by hematoxylin-eosin (H&E) staining. The healthy control group showed intact lung tissue structure, thin airway walls, and no obvious inflammatory cell infiltration. The house dust mite group showed obvious airway epithelial thickening, airway space narrowing, and a large number of inflammatory cell infiltrations. The PA5O8 treatment groups (5μg / kg, 10μg / kg) showed significantly reduced airway wall thickening and inflammation.

[0081] In summary, through Figure 9 and Figure 10 The results showed that the chimeric protein PA5O8 of the present invention has the effect of alleviating or treating allergic diseases and inflammation-related diseases in animals and plants.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A fusion polypeptide, characterized in that, The fusion polypeptide is named chimeric protein PA508, and the amino acid sequence of the fusion polypeptide is shown as SEQ ID NO:

1.

2. The fusion polypeptide of claim 1, wherein, The nucleotide sequence of the fusion polypeptide is shown as SEQ ID NO:

2.

3. The use of a fusion polypeptide according to claim 1, characterized in that Any one of the following c1) - c3) is included: c1) preparing a polypeptide drug for treating or preventing allergic diseases; c2) preparing a recombinant allergen preparation; c3) preparing a kit for diagnosing allergic diseases; Wherein, the allergic diseases are dust mite allergy or pollen allergy; the allergen is dust mite or pollen.

4. Use of a fusion polypeptide according to claim 3, characterized in that The polypeptide drug further includes pharmaceutical excipients.

5. Use of a fusion polypeptide according to claim 4, characterized in that The pharmaceutical excipients include dehydrating agents, sweeteners, lubricants, surfactants, fillers and isotonicity adjusting agents.

6. A pharmaceutical composition, characterized by, The product includes the fusion polypeptide of claim 1 or 2.

7. The pharmaceutical composition of claim 6, wherein The dosage form of the drug is ointment, spray or external patch.

8. A product for detecting specific immune protein IgE in serum, characterized by, The product includes the fusion polypeptide of claim 1 or 2.

9. A recombinant plasmid, characterized in that, The plasmid contains a nucleotide sequence as shown in any one of SEQ ID NO: 2-3.

10. A genetically engineered bacterium, characterized by, The genetically engineered bacteria contain the recombinant plasmid of claim 9.

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