Histamine oxidase protein and use thereof

Abstract

The application discloses a histamine oxidase protein and application thereof, and the amino acid sequence of the histamine oxidase protein is shown as SEQ ID NO. 1. The histamine oxidase protein PAO can be used for preparing a medicine for treating allergic rhinitis. The application can degrade histamine locally through nasal mucosa, reduce the aggregation of histamine locally in the nasal mucosa, improve the symptoms of allergic rhinitis such as sneezing, and reduce the type 2 inflammatory response of the nose, thereby achieving the therapeutic effect on allergic rhinitis, and providing a new perspective and target for the treatment of allergic rhinitis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a histamine oxidase protein and its applications. Background Technology

[0002] Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa, triggered by exposure to allergens in sensitized individuals. It is characterized by symptoms such as nasal congestion, runny nose, sneezing, and itching. Globally, AR affects 10%-40% of the population, often coexisting with asthma, severely impacting patients' quality of life and imposing a heavy socioeconomic burden.

[0003] The pathogenesis of AR involves both genetic susceptibility and environmental factors, both of which jointly promote the growth of type 2 helper T cells (T cells). H 2) Primarily an inflammatory response, characterized by the upregulation of key cytokines such as interleukin (IL)-4, IL-5, and IL-13, and significant eosinophil infiltration in the nasal mucosa. Histamine is a core mediator in allergic reactions, primarily released through IgE-dependent (e.g., allergen-linked IgE-binding FcεR1 receptors) or non-IgE-dependent (e.g., complement activation or neuropeptide stimulation) degranulation of mast cells, and exerts its effects through four G protein-coupled receptors (H1R-H4R). H1R signaling mediates key AR symptoms such as vasodilation, rhinorrhea, and immune cell recruitment. Although H1R antagonists (antihistamines) remain first-line treatments for AR, individual responses vary, and many patients do not achieve complete symptom relief, highlighting the need for more targeted strategies.

[0004] Histamine is synthesized by L-histidine decarboxylase (HDC) and primarily degraded by two enzymes: diamine oxidase (DAO, EC.1.4.3.22) and histamine N-methyltransferase (HNMT, EC.2.1.1.8). DAO, as a secretory protein, is responsible for clearing extracellular histamine, while HNMT is a cytoplasmic protein that methylates intracellular histamine, converting it into imidazole-4-acetaldehyde or 1-methylhistamine, respectively. These metabolites are further oxidized by aldehyde dehydrogenase (ALD) to produce imidazole-4-acetic acid or 1-methylimidazole-4-acetic acid. In the nasal mucosa, HNMT appears to be the major catabolic enzyme. Notably, HDC mRNA expression is increased and HNMT mRNA expression is decreased in the nasal tissue of patients with allergic rhinitis (AR), suggesting a local dysregulation of histamine metabolism. However, there are no reports on whether targeting histamine-degrading enzymes can treat patients with allergic rhinitis. Summary of the Invention

[0005] The purpose of this invention is to solve the treatment challenges of respiratory inflammatory diseases by providing a histamine oxidase protein and its applications. This invention is the first to discover that histamine oxidase (PAO) protein can reduce the accumulation of histamine in the nasal mucosa by degrading local histamine, thereby improving symptoms of allergic rhinitis such as sneezing and alleviating nasal T-cell pain. H 2. Inflammatory response, thereby achieving a therapeutic effect on allergic rhinitis.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a histamine oxidase protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the aforementioned histamine oxidase protein PAO. tynA The tynA The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant expression vector pET28a- tynA The recombinant expression vector pET28a- tynA The above is inserted into the pET28a carrier. tynA Gene.

[0009] The present invention also provides tynA Recombinant engineered bacteria E.coli-tynA The tynA Recombinant engineered bacteria E.coli- tynA For the recombinant expression vector pET28a- mentioned above tynA Engineered bacteria.

[0010] Furthermore, the engineered bacteria is Escherichia coli BL21-DE3.

[0011] This invention also provides a method for preparing histamine oxidase protein PAO, comprising the following steps: 1) The above tynA Recombinant engineered bacteria E.coli-tynA Transfer to LB liquid medium and culture until the OD value reaches 0.6-0.8, then add IPTG for induction; 2) After induction, centrifuge the bacterial culture to collect the precipitate, wash it, resuspend it in lysis buffer, lyse it on ice, and sonicate it. 3) Collect the disrupted bacterial culture, centrifuge, and collect the supernatant; 4) The supernatant was purified to obtain histamine oxidase protein.

[0012] Furthermore, in step 1), the induction concentration is 0.5 mM, the induction temperature is 25°C, and the induction time is 16 hours; In step 4), the purification method is His tag affinity chromatography purification.

[0013] The present invention also provides the application of the above-mentioned histamine oxidase protein PAO or the histamine oxidase protein PAO prepared by the above method in the preparation of a drug for treating allergic rhinitis.

[0014] The aforementioned histamine oxidase protein PAO possesses the biological activity of degrading histamine. It can locally degrade histamine through the nasal mucosa, reducing the accumulation of histamine in the nasal mucosa, thereby improving the symptoms of allergic rhinitis and alleviating nasal T-cell pain. H 2. Inflammatory response.

[0015] The present invention also provides a medicament for treating allergic rhinitis, the medicament containing the histamine oxidase protein PAO described above or the histamine oxidase protein PAO prepared by the above method.

[0016] Furthermore, the medication for treating allergic rhinitis contains histamine oxidase protein PAO, wherein the concentration of histamine oxidase protein PAO is 1 mg / mL.

[0017] The beneficial effects of this invention are: 1. The protein of this invention is derived from Klebsiella pneumoniae, a common symbiotic bacterium in the nasal cavity. It is more effective than bacterial preparations and is safe with fewer adverse reactions.

[0018] 2. The protein of the present invention can be prepared using Escherichia coli BL21-DE3, which is easy to obtain and can be produced in large quantities.

[0019] 3. The protein of this invention can reduce the accumulation of histamine in the nasal mucosa by degrading local histamine, thereby improving the symptoms of allergic rhinitis such as sneezing and reducing nasal T-cell inflammation. H 2. Inflammatory response, suitable for various products such as nasal sprays, nebulizers, nasal drops, and rinsing solutions, convenient for administration and easy for patients to use.

[0020] 4. Histamine oxidase (PAO) improves nasal symptoms by efficiently degrading histamine, a core inflammatory mediator in the pathogenesis of acute rhinitis (AR), while simultaneously reducing eosinophil infiltration in the nasal mucosa and alleviating T-cell irritation. H 2. Inflammatory response, thereby achieving a therapeutic effect on AR.

[0021] In summary, this invention achieves a therapeutic effect on allergic rhinitis by locally degrading histamine in the nasal mucosa, thereby reducing the accumulation of histamine in the nasal mucosa, improving symptoms of allergic rhinitis such as sneezing, and alleviating type 2 inflammatory response in the nose. This provides a new perspective and target for the treatment of allergic rhinitis. Attached Figure Description

[0022] Figure 1 A schematic diagram showing the location of the genes for three candidate histamine-degrading enzymes in the genome of Klebsiella pneumoniae; Figure 2 RT-PCR expression of three histamine-degrading enzyme-related genes; Figure 3 For transfer to pET28a- tynA plasmid (+ tynA In E. coli pET28a (+ control plasmid) and pET28a (+ control plasmid) tynA PCR detection of gene fragment expression; Figure 4 To verify the presence of Klebsiella pneumoniae (KA wild type) and mutant strains by PCR KA ΔtynA (KA knockout type) tynA Image showing successful gene knockout; Figure 5 This is a typical protein electrophoresis diagram of PAO enzyme isolated and purified from genetically engineered Escherichia coli; Figure 6 For liquid chromatography-tandem mass spectrometry detection, transfer to pET28a- tynA Diagram of histamine degradation by plasmids in E. coli: In the figure, a is the peak diagram of representative histamine ions, and b is the statistical graph of histamine content relative to the baseline. Figure 7 For transfer to pET28a- tynA Figure 1: Histamine degradation by plasmids in E. coli and detection of the products: In the figure, 'a' is a statistical graph of histamine levels detected by non-targeted metabolomics. b is a statistical graph of imidazole-4-acetaldehyde, a histamine metabolite detected by non-targeted metabolomics. c is a statistical graph of imidazole-4-acetic acid, a histamine metabolite detected by non-targeted metabolomics. Figure 8 Detection of knockout by liquid chromatography-tandem mass spectrometry tynA A diagram of histamine degradation by Klebsiella pneumoniae. In the figure, a represents the growth curve monitoring diagram of KA wild-type and KA knockout type Klebsiella pneumoniae. b is a representative ion peak diagram of histamine, and c is a statistical graph of the percentage of histamine content relative to the baseline. Figure 9 A statistical chart showing that the isolated and purified PAO enzyme can consume oxygen, degrade histamine, and produce imidazole-4-acetaldehyde, H2O2, and ammonia: In the figure, a is a statistical graph of histamine detected by non-targeted metabolomics. b is a statistical graph of imidazole-4-acetaldehyde detected by non-targeted metabolomics. c is a statistical graph of histamine detection by liquid chromatography-tandem mass spectrometry. Image d shows the representative ion peaks for the detection of imidazole-4-acetaldehyde by liquid chromatography-tandem mass spectrometry. e is a statistical chart showing the percentage of imidazole-4-acetaldehyde content relative to the baseline. f is a statistical graph showing the change in H2O2 production in the reaction system over time. g is a statistical graph showing the change in ammonia production in the reaction system over time. h is a statistical graph showing the change in O2 consumption in the reaction system over time. i represents the Michaelis-Menten equation curve of PAO degradation of histamine. j is a schematic diagram showing that PAO enzymes can consume oxygen, degrade histamine, and produce imidazole-4-acetaldehyde, H2O2, and ammonia. Figure 10 Nasal instillation of PAO significantly alleviated ovalbumin-induced AR symptoms in mice and reduced mucosal inflammatory response. In the figure, a shows the ovalbumin-induced AR model in BALB / c mice and the nasal instillation of histamine oxidase PAO treatment regimen. Figure b shows that intranasal administration of histamine oxidase PAO does not affect the expression of ovalbumin-specific IgE in mouse serum. Figure c shows that nasal instillation of histamine oxidase (PAO) significantly reduced histamine expression in mouse nasal wash fluid. Figure d shows the expression of AR-related nasal symptoms in mice significantly reduced by intranasal instillation of histamine oxidase PAO. e is a fluorescent staining image showing that nasal instillation of histamine oxidase (PAO) significantly reduced eosinophil infiltration in the nasal mucosa of mice. f is the statistical graph corresponding to the above fluorescent staining. Among them, Siglec-F positive cells (e.g., those indicated by red arrows) represent eosinophils; Figure 11 Nasal instillation of PAO significantly alleviated house dust mite-induced AR symptoms in mice and reduced eosinophilic inflammation of the nasal mucosa. (Figure:) In the figure, a shows the AR model induced by house dust mites in BALB / c mice and the treatment regimen of nasal instillation of histamine oxidase PAO. Figure b shows that intranasal administration of histamine oxidase PAO does not affect the expression of house dust mite-specific IgE in mouse serum. Figure c shows that nasal instillation of histamine oxidase (PAO) significantly reduced histamine expression in mouse nasal wash fluid. Figure d is a schematic diagram showing how nasal instillation of histamine oxidase PAO significantly reduced AR-related nasal symptoms in mice. e is a fluorescent staining image showing that nasal instillation of histamine oxidase (PAO) significantly reduced eosinophil infiltration in the nasal mucosa of mice. f is the statistical graph corresponding to the above fluorescent staining. Among them, Siglec-F positive cells (e.g., those indicated by red arrows) represent eosinophils. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0024] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] Example 1: Encoding of histamine oxidase protein tynA Gene screening 1) Bacterial culture: Take 100 μL of Klebsiella pneumoniae (Klebsiella pneumoniae) Klebsiella aerogenes HW2024 cryopreserved glycerol bacteria were evenly spread on Columbia blood culture dishes and thawed at 37°C for 1-2 days until obvious colonies appeared. Single colonies were picked and inoculated into 1 mL of ampicillin-resistant (10 μg / mL) broth and incubated overnight at 37°C. The next day, they were inoculated into Erlenmeyer flasks containing 50 mL of LB medium and incubated at 37°C and 220 rpm for one day until they reached the logarithmic growth phase. The bacteria were precipitated by centrifugation at 4000 rpm for 5 minutes, washed three times with PBS, resuspended with an equal volume of PBS, and aliquoted into 1 mL EP tubes for long-term storage at -80°C. The aforementioned *Klebsiella pneumoniae* is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M20252524, deposit date: November 11, 2025, address: Wuhan University, Wuhan, China. This *Klebsiella pneumoniae* (… Klebsiella aerogenes HW2024 is disclosed in Chinese invention patent publication number CN121610416A, entitled "Klebsiella pneumoniae HW2024 and its application in the preparation of medicines for treating allergic rhinitis".

[0027] 2) Screening candidate genes by bacterial whole-genome sequencing: culturing nasal commensal bacteria—Klebsiella pneumoniae ( Klebsiella aerogenes HW2024 KA(CCTCC NO: M 20252524) to the logarithmic growth phase. Genes in *Klebsiella pneumoniae* were identified using whole-genome sequencing and compared with histamine-degrading enzymes in the histamine metabolism pathway in the KEGG database (https: / / www.genome.jp / pathway / map00340+K00546), identifying three candidate genes; respectively tynA Gene, gene3912 Genes and gene0698 Genes (such as) Figure 1 Location distribution of the three candidate histamine-degrading enzyme genes: tynA (gene2096, located at 2,215,034–2,212,767), gene3912 (located at 4,150,155–4,148,560), and gene0698 (located at 719,066–720,478) are scattered in different regions of the genome.

[0028] 3) Encoding of histamine oxidase protein tynA Gene identification and sequencing: After Klebsiella pneumoniae HW2024 was cultured to the logarithmic phase, it was incubated with histamine-PBS solution (1 mM) for 24 hours as the histamine group, and incubated with PBS solution as the control group. The expression levels of the three identified candidate histamine-degrading enzyme genes were detected by RT-PCR. Figure 2 ); like Figure 2 As shown: After incubation of Klebsiella pneumoniae with histamine (1 mM) for 24 hours, only tynA The expression of [gene name] was significantly upregulated by histamine (p=0.0007, relative expression level approximately 28-fold higher than the control group), while gene3912 (p=0.3555) and gene0698 (p=0.2392) showed no significant changes. The results indicate that: tynA The gene is a key functional gene in response to histamine and participates in histamine metabolism. The histamine oxidase protein PAO was obtained by whole-genome sequencing, and its amino acid sequence is shown in SEQ ID NO.1. The PAO coding region of histamine oxidase protein (i.e. tynA The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0029] Example 2 tynA Construction of recombinant engineered bacteria 1) The nucleotide sequence of the histamine oxidase protein coding region described above was transferred into the pET28a plasmid vector (insertion site 5'NcoI / Xhol 3') to construct the recombinant expression vector pET28a- tynA The control plasmid was the empty vector pET28a without the inserted gene. 2) The recombinant expression vector pET28a- tynA The empty vector pET28a without gene insertion was chemically transfected into Escherichia coli BL21-DE3 competent cells. The transformed bacterial culture was plated on kanamycin-resistant LB agar plates. The competent cells were picked the next day and transferred to 2 mL of liquid culture medium for culture. 3) Extract plasmids from cultured bacteria, and use upstream and downstream primers for PCR to confirm successful insertion of the target gene coding fragment, thus obtaining... tynA Recombinant engineered bacteria (containing recombinant expression vector pET28a-) tynA ); Upstream primer F: 5'-TGGACGGCGAAAACAACAC-3', as shown in SEQ ID NO.3. Downstream primer R: 5'-CCGCCGGCATAAGGGATA-3', as shown in SEQ ID NO.4; like Figure 3 As shown: Positive strains (+) tynA The target band appeared in the swim lane, while no band was observed in the control plasmid group, confirming... tynA The gene coding fragment (approximately 2300 bp) has been successfully inserted into the pET28a plasmid and transformed into E. coli BL21(DE3), validating the results. tynA Recombinant engineered bacteria E. coli-tynA Successfully built.

[0030] Example 3 tynA Knockout mutant strain KA ΔtynA Construction Following previously reported methods (Yu, et al. Environ Microbiol. 2022;24:4755-4770), the suicide plasmid pDS3.0 was used to target the *Klebsiella pneumoniae* strain HW2024 (hereinafter KA wild-type). tynA Gene knockout was performed following a previously reported method (document number 35837862). The specific steps are as follows: 1) Construct the recombinant suicide plasmid pDS3.0- tynA : Amplification using the corresponding primers tynA Homologous arm sequences approximately 1000 bp upstream and downstream of the gene knockout region: upstream primer tynA -UF / tynA -UR: tynA -UF: 5'-ATGCGATATCGAGCTTAGAATCAACCGGCCACGG-3', shown in SEQ ID NO.5; tynA -UR: 5'-TGTGACCTCATCTTATTGTTGTGTTG-3', as shown in SEQ ID NO.6.

[0031] Downstream primer tynA -DF / tynA -DR: tynA -DF: 5'-CAATAAGATGAGGTCACATCTTAGCCCGCGGTGAAATCG-3', shown in SEQ ID NO.7; tynA -DR: 5'-ATTCCCGGGAGAGCTCGGGTACAAATGCGCTATGC-3, shown in SEQ ID NO.8.

[0032] The amplified product and the SacI-digested linearized pDS3.0 fragment were purified by agarose gel electrophoresis and then mixed with Gibson assembly mixture (Proteinligand Biotech, Wuhan, China, catalog number PLK011M) at a 1:1:1 molar ratio (total volume 5 μL). After incubation at 50°C for 1 hour, the ligation product was transformed into *E. coli* S17-1 λpir competent cells, plated on LB agar plates containing gentamicin, and incubated overnight at 37°C. Single clones were picked for plasmid extraction, PCR amplification, and sequencing verification.

[0033] 2) Transform the suicide plasmid into Klebsiella pneumoniae HW2024: To be cultivated to the logarithmic growth phase KA Wild-type and those carrying the recombinant suicide plasmid pDS3.0- tynA The donor bacteria, *Escherichia coli* S17-1 λpir, were mixed at a volume ratio of 3:1 and added dropwise onto a sterile filter membrane. The membrane was then placed on LB agar plates for conjugation transfer culture overnight. The mixed bacterial culture on the filter membrane was collected, aseptically washed, centrifuged, and spread onto vortex-methyl red kinetic agar plates containing gentamicin. The plates were incubated at 37°C for 36 hours. Single colonies were picked and inoculated onto VBMM plates containing 10% sucrose and vortex-methyl red kinetic agar plates containing 10 μg / mL gentamicin, respectively. Single-exchange mutant strains exhibiting sucrose resistance and gentamicin sensitivity were screened, and finally verified by PCR to obtain the mutant strains. KA ΔtynA .

[0034] like Figure 4 As shown: This verifies the presence of Klebsiella pneumoniae HW2024. tynA Successful gene knockout (i.e., mutant strain) KA ΔtynA (KA knockout type). KA Compared to the wild type, the mutant strain KA ΔtynA The PCR amplification band shortened from approximately 4400 bp to approximately 2100 bp. This change in band size is consistent with the expected homologous arm-mediated gene deletion, proving that the mutant strain... KA ΔtynA Successfully built.

[0035] Example 4 tynA Expression of and purification of its encoded protein PAO 1. Expression 1) The above embodiment 2 was successfully constructed tynA Recombinant engineered bacteria E.coli-tynA Transfer to 100 mL LB liquid medium and culture until the OD value is 0.5-0.8. Add isopropyl-β-D-thiogalactoside (0.5 mM) and induce at 25℃ for 16 hours to obtain bacterial culture. 2) Centrifuge the bacterial culture (4000 rpm, 5 minutes) to collect the precipitate, wash three times with PBS, and resuspend in 20 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with NaOH solution). Lyse on ice for 30 minutes, then sonicate on ice for 10 minutes, pausing for 5 seconds after sonication.

[0036] 3) Collect the disrupted bacterial culture and centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and precipitate separately, and run 20 μL of each sample on a gel. Coomassie brilliant blue staining confirmed that the purified PAO protein showed a clear single band at approximately 85-90 kDa, consistent with the theoretical molecular weight of PAO, indicating high purity and minimal background bands, thus demonstrating successful protein expression. This also indicates that His-tag affinity chromatography purification was effective, providing high-purity protein for subsequent in vitro functional validation experiments. Figure 5 ).

[0037] 2. Purification The histamine oxidase protein PAO was purified using the Beyotime protein purification kit. The purified PAO protein was diluted to a final concentration of 1.5 μg / μL and stored at -80℃ for subsequent experiments.

[0038] Example 5 Detection of histamine and its metabolites 1. The results obtained from the above Example 2 were tested. tynA Recombinant engineered bacteria E.coli-tynA (Express tynA The content of histamine and metabolites in the supernatant after incubation with histamine (1 mM) The constructed plasmid containing the nucleotide sequence of the histamine oxidase protein PAO (hereinafter referred to as PAO protein) was inserted. E. coli Incubate with LB medium containing histamine for the corresponding time, while simultaneously incubating the empty vector pET28a plasmid without the inserted gene. E. coli As a control, the histamine content in the culture supernatant was detected by liquid chromatography-tandem mass spectrometry, referring to the methods used in previous studies (Li D, et al. Cell Host Microbe. 2022; 30:329-339; Li D, et al. Cell Metabolism. 2023; 35:685-694.).

[0039] like Figure 6 As shown: In tynA Recombinant engineered bacteria E.coli-tynA In the incubation process, histamine levels decreased with prolonged incubation time, indicating that... E.coli-tynA It can effectively degrade histamine. And in the absence of... tynA of E. coli (including expression vector pET28a, i.e.) E.coli- In the control plasmid, histamine levels did not change significantly with prolonged incubation time, indicating that... E.coli- The control plasmid could not degrade histamine.

[0040] In addition, non-targeted metabolomics analysis was performed using methods from previous studies (Li JX, et al. Journal of Allergy and ClinicalImmunology. 2022; 150:727-735).

[0041] Non-targeted metabolomics further confirmed that: in expression tynA of E. coli In this process, histamine levels decreased with prolonged incubation time. Figure 7 a), and histamine product imidazole-4-acetaldehyde was also observed ( Figure 7 b) and imidazole-4-acetic acid increased ( Figure 7 c).

[0042] 2. Detection of mutant strains KA ΔtynA The histamine content in the supernatant after incubation with histamine (1 mM) Compared to KA Wild type, knockout tynA Genes do not affect mutant strains KA ΔtynA Growth of (KA knockout) in LB medium Figure 8 a).

[0043] Following the methods used in previous studies (Li D, et al. Cell Host Microbe. 2022; 30:329-339; LiD, et al. Cell Metabolism. 2023; 35:685-694.), liquid chromatography-tandem mass spectrometry was employed to detect wild-type cells. KA and mutant strains KA ΔtynA The content of histamine in the supernatant after incubation with histamine.

[0044] wild type KA The histamine content in the supernatant decreased significantly with prolonged incubation time, while that in the mutant strain... KA ΔtynA Histamine levels decreased slowly with prolonged incubation, indicating that the wild-type... KA Histamine degradation tynA ( Figure 8 b-8c).

[0045] 3. Detect the histamine and metabolite content in the supernatant after incubation of PAO protein solution 1 with histamine (1 mM). The PAO protein solution with a concentration of 1.5 μg / μL obtained in Example 4 was diluted to a PAO protein solution with a concentration of 1 μg / mL.

[0046] Untargeted metabolomics analysis was performed using methods from previous studies (Li JX, et al. Journal of Allergy and Clinical Immunology. 2022; 150:727-735).

[0047] The test revealed that, compared to the control group without PAO protein, the histamine level in PAO protein solution 1 decreased significantly after incubation with histamine for 12 hours. Figure 9 a), and an increase in the histamine metabolite imidazole-4-acetaldehyde was also observed ( Figure 9 b).

[0048] Following the methods used in previous studies (Li D, et al. Cell Host Microbe. 2022; 30:329-339; LiD, et al. Cell Metabolism. 2023; 35:685-694.), liquid chromatography-tandem mass spectrometry was employed for detection.

[0049] The test results showed that after PAO protein solution 1 was incubated with histamine, histamine consumption increased with prolonged incubation time. However, in the control group without PAO, histamine levels did not change significantly with prolonged incubation time, indicating that PAO can effectively degrade histamine. Figure 9c). Further analysis using liquid chromatography-tandem mass spectrometry revealed that the histamine metabolite imidazole-4-acetaldehyde increased with prolonged incubation time, while imidazole-4-acetaldehyde was not detected in the control group without PAO. Figure 9 d-9e).

[0050] In addition, referring to previous research methods (Toshiki, et al. Nature Communications 2019;10:413; Pei, et al. Microorganisms 2023;11), the ELISA method was used to detect the production of H2O2 and ammonia in the reaction system.

[0051] The test revealed that the production of H2O2, another product of histamine degradation, increased in the reaction system of PAO protein solution 1 and histamine (1 mM). Figure 9 f) Increased ammonia production ( Figure 9 g).

[0052] In addition, referring to the previous research method (Toshiki, et al. Nature Communications 2019;10:413), the oxygen electrode method was used to detect the O2 content during histamine degradation.

[0053] The test revealed that O2 consumption increased during histamine degradation. Figure 9 Finally, PAO protein solution 1 was incubated with different concentrations of histamine, and the generation of H2O2 in the reaction system was monitored in real time. Based on this, the enzymatic kinetic reaction equation for the degradation of histamine by PAO was constructed (h). Figure 9 i). Therefore, PAO can consume oxygen, convert histamine into imidazole-4-acetaldehyde, and produce H2O2 and ammonia ( Figure 9 j).

[0054] Example 6: Preparation of a drug for treating allergic rhinitis The medication for treating allergic rhinitis contains histamine oxidase protein PAO, with a concentration of 1 mg / mL; it is prepared by dissolving purified histamine oxidase protein PAO in PBS.

[0055] Example 7: Study on PAO's improvement of AR symptoms and relief of eosinophilic inflammation of the nasal mucosa in mice. 1. Establishment of a mouse AR model: Histamine oxidase treatment: Based on previous studies (Liu Y, et al. Journal of Allergy and Clinical Immunology 2013;131:387-97; Wang H, et al. American Journal of Respiratory and CriticalCare Medicine 2010; 181:908-16), this example uses two mouse AR models induced by ovalbumin (OVA) and house dust mite (HDM), and the methods are as follows: OVA-induced mouse model construction as follows Figure 10 As shown in a: ① Sensitization phase: On days 0, 7, and 14, 50 μg of OVA (dissolved in PBS containing 2 mg aluminum hydroxide, final volume 200 μL) was administered subcutaneously; the control was PBS. ② Activation phase: From day 21 to day 27, 1.0 mg of OVA solution was instilled (10 μL per nostril, once daily). From day 21 to day 27, 20 μL of the drug prepared in Example 6 above was instilled; PBS was used as a control.

[0056] Construction of HDM-induced mouse models, as follows Figure 11 As shown in a: ① Sensitization phase: On days 0, 1, and 2, 10 μg of HDM (dissolved in PBS) was administered via nasal drops; saline solution was added to PBS. ② Activation phase: From day 8 to day 13, instill 50 μg of HDM solution (10 μL per nostril, once daily). From day 8 to day 13, instill 20 μL of the drug prepared in Example 6 above, or PBS.

[0057] 2. Collection of materials: After deep anesthesia, nasal lavage fluid was collected from mice and stored at -80°C. Some mice were sacrificed, their heads were separated, and the scalp skin and soft tissue were removed. The nasal cavity and sinuses were then separated, and after decalcification, fixation, and paraffin embedding, 4 μm thick coronal serial sections were prepared. The nasal cavity and sinuses of some mice were dissected under a microscope, and the nasal and sinus mucosa was obtained and stored at -80°C. After obtaining these specimens, immunofluorescence was used to detect changes in the expression of eosinophils and other cells in the mouse nasal cavity.

[0058] The flowchart for OVA-induced mouse AR model and PAO treatment is shown below. Figure 10 a. The test found that nasal instillation of PAO did not affect the level of OVA-specific IgE in mouse serum ( Figure 10 (b), but it can reduce the concentration of histamine in nasal wash ( Figure 10c), alleviates ovalbumin-induced AR symptoms in mice ( Figure 10 d), reduce mucosal eosinophilic inflammation ( Figure 10 e and Figure 10 f).

[0059] The flowchart for HDM-induced AR model in mice and PAO treatment is shown below. Figure 11 a. The test found that nasal instillation of PAO did not affect the level of serum HDM-specific IgE in mice. Figure 11 (b), but it can reduce the concentration of histamine in nasal wash ( Figure 11 c), alleviated house dust mite-induced AR symptoms in mice ( Figure 11 d), reduce mucosal eosinophilic inflammation ( Figure 11 e and Figure 11 f).

[0060] In summary( Figure 1 to Figure 11 This invention isolates, purifies, and demonstrates that a novel histamine oxidase PAO from Klebsiella pneumoniae can degrade histamine, a core inflammatory mediator in the pathogenesis of acute rheumatoid arthritis (AR). In vivo animal experiments confirmed that the purified histamine oxidase PAO can significantly improve AR symptoms and reduce eosinophil infiltration.

[0061] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of histamine oxidase protein PAO prepared by a method thereof in the preparation of drugs for treating allergic rhinitis, characterized in that: The amino acid sequence of the histamine oxidase protein PAO is shown in SEQ ID NO.1; The method includes the following steps: 1) tynA Recombinant engineered bacteria E.coli-tynA The samples were transferred to LB liquid medium and cultured until the OD value reached 0.6-0.

8. Then, IPTG (0.5 mM) was added for induction at 25°C for 16 hours. tynA Recombinant engineered bacteria E.coli-tynA For containing the recombinant expression vector pET28a- tynA Escherichia coli BL21-DE3, the recombinant expression vector pET28a- tynA Inserted on pET28a carrier tynA Genes, and the stated tynA The nucleotide sequence of the gene is shown in SEQ ID NO.2; 2) After induction, centrifuge the bacterial culture to collect the precipitate, wash it, resuspend it in lysis buffer, lyse it on ice, and sonicate it. 3) Collect the disrupted bacterial culture, centrifuge, and collect the supernatant; 4) The supernatant was purified by His-tag affinity chromatography to obtain histamine oxidase protein.

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