Paper mulberry pollen allergen and use thereof

Abstract

The present invention provides a paper mulberry pollen allergen and a use thereof. Specifically, the present invention provides a paper mulberry pollen allergen and a composition containing the paper mulberry pollen allergen, wherein the paper mulberry pollen allergen is an allergenic protein Brop1, and the amino acid sequence of Brop1 is as shown in SEQ ID NO: 4. Brop1 can be used in the screening, diagnosis, desensitization treatment, etc. of pollen-induced allergic hypersensitivity diseases, such as allergic rhinitis, allergic conjunctivitis, and asthma.

Description

A paper mulberry pollen allergen and its uses Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a paper mulberry pollen allergen and its uses. Background Technology

[0002] Allergic diseases are common clinical conditions, and exposure to allergens is a significant cause of their development. Pollen is one of the most widespread and abundant sources of outdoor allergens. Allergic diseases include hay fever, allergic rhinitis, allergic asthma, conjunctivitis, allergic dermatitis, and urticaria.

[0003] [Amended according to Rule 26, December 2025] Paper mulberry (scientific name: *Broussonetia papyrifera*; English name: paper mulberry, tapa cloth tree) is a plant belonging to the genus *Broussonetia* in the family Moraceae. Its distribution includes China, Japan, Korea, Southeast Asia, South Asia, Oceania, Europe, and the Americas. Paper mulberry pollen is recognized as a significant cause of allergies in several countries. As early as the early 1970s, paper mulberry pollen was identified as a cause of respiratory allergies in the United States, and the number of patients has steadily increased in the following decades. Similarly, literature reports that paper mulberry pollen is a major airborne allergen in northern India and a major source of airborne allergens in Madrid, Spain, from January to April. Paper mulberry has a strong survival and reproductive capacity, and is widespread in urban and rural wastelands, where it is a dominant species. Male paper mulberry trees produce extremely high levels of pollen; in Islamabad, Pakistan, and Chengdu, my country, paper mulberry pollen accounts for more than 50% of the total spring pollen. In Islamabad, the capital of Pakistan, paper mulberry pollen has been confirmed as a major cause of respiratory allergies. Because of the paper mulberry's survival ability and economic value (its leaves can be used as fodder and its bark can be used to make paper), my country will promote the planting of paper mulberry in underdeveloped areas as a national poverty alleviation measure, and the planting area of ​​paper mulberry is constantly increasing.

[0004] In summary, the incidence of mulberry pollen allergy is increasing, but reports on the identification of allergens in mulberry pollen and their sensitization are scarce. This not only hinders the exploration of the immunopathogenesis of mulberry pollen allergy but also impedes the development and application of precise and personalized diagnostic and treatment methods based on molecular allergy. Therefore, there is an urgent need in this field to analyze the molecular allergen spectrum of mulberry pollen and develop diagnostic and therapeutic methods based on molecular allergens and their combinations. Summary of the Invention

[0005] The purpose of this invention is to provide identification of the dominant and species-specific allergen in mulberry pollen, a method for extracting the protein from the pollen, a method for preparing a recombinant protein that is identical in sequence, structure and function to the natural protein, and the use of the protein in the diagnosis and treatment of mulberry pollen allergies.

[0006] In a first aspect of the invention, a composition containing a paper mulberry pollen allergen is provided, wherein the paper mulberry pollen allergen is a natural or recombinant allergenic protein Bro p 1; and the amino acid sequence of the allergenic protein Bro p 1 is shown in SEQ ID NO:4:

[0007] In another preferred embodiment, the allergen has one or more (preferably three) IgE epitopes selected from the group consisting of:

[0008] TPCLNYVRKR (SEQ ID NO: 5), AQKLNDNSKT (SEQ ID NO: 6), EQCGISATLPP (SEQ ID NO: 7).

[0009] In another preferred embodiment, the amino acids of the recombinant Bro p 1 also have a purification tag at the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID No. 4. Preferably, the purification tag is a His6 tag, preferably His6-Sumo or His6-ENLYFQ.

[0010] In another preferred embodiment, the paper mulberry pollen allergen may also be optionally modified by a modification method selected from the group consisting of: glutaraldehyde modification, carbamylation modification, or mannan modification.

[0011] In a second aspect of the invention, a nucleic acid or gene encoding a natural Bro p 1 mulberry pollen allergen is provided.

[0012] In another preferred embodiment, the nucleic acid or gene is selected from one or more combinations of the group consisting of single-stranded DNA, double-stranded DNA, plasmid DNA, and mRNA.

[0013] In another preferred embodiment, the nucleotide sequence of the nucleic acid or gene is as shown in SEQ ID NO: 3:

[0014] In a third aspect of the invention, an expression vector is provided, the expression vector comprising a nucleotide sequence capable of expressing a mulberry pollen allergen in the composition as described in the first aspect of the invention; preferably, the expression vector is selected from the group consisting of expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria.

[0015] In a fourth aspect of the invention, a pharmaceutical composition for the prevention or treatment of hay fever is provided, comprising a composition as described in the first aspect of the invention, or comprising nucleic acids or genes as described in the second aspect of the invention, or comprising an expression vector as described in the third aspect of the invention.

[0016] In another preferred embodiment, the hay fever includes diseases selected from the group consisting of allergic reactions to mulberry pollen: hay fever, allergic rhinitis, allergic asthma, conjunctivitis, allergic dermatitis, and urticaria.

[0017] In another preferred embodiment, the pharmaceutical composition is a single-ingredient or compound preparation.

[0018] In another preferred embodiment, the formulation comprises a topical formulation and an oral formulation.

[0019] In another preferred embodiment, the formulation includes: powder, granules, capsules, injections, inhalers, tinctures, oral liquids, tablets, lozenges, or drops.

[0020] In another preferred embodiment, the pharmaceutical composition is administered for immunotherapy via subcutaneous or intradermal administration.

[0021] In another preferred embodiment, when used for subcutaneous / intradermal administration, the pharmaceutical composition further employs aluminum hydroxide or aluminum phosphate as an adjuvant.

[0022] In another preferred embodiment, the pharmaceutical composition is administered sublingually for immunotherapy.

[0023] In another preferred embodiment, the mulberry pollen allergen in the pharmaceutical composition is an allergen modified from the group consisting of glutaraldehyde modification, carbamylation modification, or mannan modification.

[0024] In another preferred embodiment, the nucleic acid encoding the sensitizing protein Brop 1 is used as the active ingredient in the immunotherapy.

[0025] In a fifth aspect of the invention, a kit is provided for in vitro diagnosis of mulberry pollen allergy or for evaluating the therapeutic effect of mulberry pollen allergy in subjects, the kit comprising the composition as described in the first aspect of the invention, or comprising nucleic acids or genes as described in the second aspect of the invention, or comprising expression vectors as described in the third aspect of the invention.

[0026] In another preferred embodiment, the kit further includes instructions for use in diagnosing mulberry pollen allergens.

[0027] In another preferred embodiment, the kit further includes a solid support for adsorbing or connecting the composition as described in the first aspect of the invention.

[0028] In another preferred embodiment, the kit further includes a detection reagent that specifically recognizes IgE or IgG.

[0029] In another preferred embodiment, the detection reagent is a composition as described in the first aspect of the invention.

[0030] In another preferred embodiment, the reporter group is selected from the group consisting of biotin, peroxidase, fluorescent groups, or rare earth metal chelates.

[0031] In another preferred embodiment, the sample detected by the kit is serum or plasma.

[0032] In another preferred embodiment, the kit detects the level of IgE or IgG in the sample that specifically binds to the protein in the composition described in the first aspect of the invention.

[0033] In another preferred embodiment, the detection method is selected from the group consisting of: Western blot detection, ELISA detection or immunoCAP detection and other similar ligand binding assays.

[0034] In a sixth aspect of the invention, the use of the composition as described in the first aspect of the invention in the preparation of a pollen allergy detection reagent is provided.

[0035] In another preferred embodiment, the diagnostic reagent is selected from the group consisting of: prick solution, subcutaneous injection, tablet, capsule, aerosol, nasal preparation, patch, drop, or spray.

[0036] In another preferred embodiment, the reagent is used for a nasal provocation test.

[0037] In another preferred embodiment, the reagent is used for a skin prick test.

[0038] In another preferred embodiment, the reagent is used for skin patch testing.

[0039] In another preferred embodiment, the reagent is used for a patient basophil cell activation test.

[0040] In a seventh aspect of the invention, use is provided for a composition as described in the first aspect of the invention, a nucleic acid or gene as described in the second aspect of the invention, or an expression vector as described in the third aspect of the invention; for use in:

[0041] (1) Prepare a kit for diagnosing mulberry pollen allergy, or prepare a drug for the prevention or treatment of mulberry pollen allergy;

[0042] (2) Prepare drugs and / or reagents for detecting the content of mulberry pollen allergen-specific IgE in the serum of patients;

[0043] (3) Prepare drugs and / or reagents for detecting the degree of basophil activation in patients.

[0044] In an eighth aspect of the invention, a method for purifying allergens from paper mulberry pollen in the composition described in the first aspect of the invention is provided, comprising the following steps:

[0045] 1) Extraction of total protein from paper mulberry pollen: Defatted pollen was mixed with buffer (pH 6.5-9.0), protease inhibitor was added and incubated. After centrifugation, the supernatant was collected and filtered to obtain paper mulberry pollen extract.

[0046] 2) Purification of allergen components:

[0047] 2.1) The paper mulberry pollen extract was subjected to anion exchange chromatography to obtain a first purified extract;

[0048] 2.2) The target allergen portion of the first purified extract is subjected to gel filtration to obtain the second purified extract.

[0049] In another preferred embodiment, the paper mulberry pollen extract comprises crude paper mulberry pollen extract.

[0050] In another preferred embodiment, the defatted pollen is prepared by defatting pollen.

[0051] In another preferred embodiment, in step 1), membrane filtration is performed using a 0.22 μm membrane.

[0052] In another preferred embodiment, in step 2.1, the anion exchange is performed using a HiTrap Capto QImpRes Cytiva column.

[0053] In another preferred embodiment, in step 2.1, the anion exchange uses Tris-HCl buffer as the elution medium; preferably, the pH of the Tris-HCl buffer is 8-9.

[0054] In another preferred embodiment, step 2.1 further includes: linearly eluting the pollen extract obtained by elution and filtration using Tris-HCl and NaCl.

[0055] In another preferred embodiment, in step 2.2, the gel filtration is performed using a Superdex 200 gel filtration column.

[0056] In another preferred embodiment, in step 2.2, the gel filtration uses Tris-HCl buffer as the elution solution.

[0057] In a ninth aspect of the present invention, a method for preparing recombinant Brop 1 protein is provided, comprising the steps of:

[0058] (1) Recombinant Brop 1 protein was expressed in Escherichia coli;

[0059] (2) The expression product was purified using an affinity column to obtain the desired recombinant Brop1 protein;

[0060] The amino acid sequence of the recombinant Bro p 1 protein is shown in SEQ ID NO:4:

[0061] In another preferred embodiment, the amino acid sequence of the recombinant Bro p 1 protein is tagged with purification and / or expression-promoting tags.

[0062] In another preferred embodiment, the purification and / or expression-promoting tag is selected from the group consisting of His-SUMO and His-ENLYFQ.

[0063] In another preferred embodiment, the affinity column is selected from nickel columns.

[0064] In another preferred embodiment, step (2) includes: purifying the expression product with a nickel column, then removing the tag with a tag-removing enzyme, and then removing the unremoved protein, the tag, and the tagged enzyme with a nickel column to obtain recombinant Bro p 1 protein.

[0065] In another preferred embodiment, the protease used to cleave the tag is selected from the group consisting of His-Ulp1 enzyme and His-TEV enzyme.

[0066] In another preferred embodiment, when the purification and expression-promoting tag is His-SUMO, the tag-excision enzyme is His-Ulp1 enzyme.

[0067] In another preferred embodiment, when the purification and expression-promoting tag is His-ENLYFQ, the tag-excision enzyme is His-TEV enzyme.

[0068] In a tenth aspect of the present invention, a method for standardizing mulberry pollen extract using Bro p 1 protein prepared by the method described in the eighth or ninth aspect of the present invention is provided, comprising the steps of:

[0069] Bro p 1 protein was used as a control, and the content / concentration / potency of this protein in the mulberry pollen extract was determined by ELISA or other similar ligand binding assays.

[0070] In another preferred embodiment, the method further includes the step of: using Bro p 1 protein as a control, measuring the binding potency of the paper mulberry extract to patient serum IgE by a competitive ELISA method.

[0071] In another preferred embodiment, the method is neither diagnostic nor therapeutic.

[0072] In an eleventh aspect of the invention, a composition containing mulberry pollen allergen is provided, said composition containing the allergenic protein Bro p 1 as described in the composition of the first aspect of the invention.

[0073] In another preferred embodiment, the composition comprises a pharmaceutically acceptable carrier.

[0074] In another preferred embodiment, the composition comprises a crude extract of mulberry pollen allergens.

[0075] In another preferred embodiment, the composition is a crude extract of mulberry pollen allergens.

[0076] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0077] Figure 1 shows the detection results of total protein from paper mulberry pollen. Figure A shows the SDS-PAGE results; Figure B shows the Western blot results. Specifically, after SDS-PAGE of the total protein extracted from paper mulberry pollen, the protein was transferred to a PVDF membrane and incubated with a mixed serum pool of patients with paper mulberry allergy or a single patient's serum. Then, it was incubated with enzyme-labeled anti-human IgE secondary antibody to detect IgE that specifically binds to the allergen. The serums incubated from left to right represent: a serum pool containing 50 patients with paper mulberry allergy and a single serum sample from 11 of these patients.

[0078] Figure 2 shows the purification results of Brop 1 protein in one embodiment of the present invention. Figure A is a reversed-phase chromatogram; Figure B is the SDS-PAGE result of the collected fraction; Figure C is the Western blot result of the collected fraction; and F1, F2, and F3 are the three fractions of the collected fraction.

[0079] Figure 3 shows the mass spectrometry results and interpretation of the peptide (ALGQFSDI(L)DPGR) in the amino acid sequence (SEQ ID NO:4) of Bro p 1 identified in this invention (identification of b / y ions).

[0080] Figure 4 shows the analysis data of the amino acid sequence of Brop 1 protein according to this invention. Figure A shows the mRNA-encoded protein sequence, with the signal peptide, the mRNA-encoded sequence that is not ultimately present, the eight conserved cysteine ​​residues of the nsLTP protein family, and the peptides whose sequences were successfully identified based on the raw mass spectrometry data shown in Figure 3; where purple amino acid sequences represent signal peptide sequences; green amino acid sequences represent mRNA-encoded sequences that are not ultimately present in the protein; red letter C represents conserved cysteine ​​residues of the nsLTP protein family; and yellow amino acid sequences represent sequences determined by MS / MS spectrometry. Figure B shows the coverage of the Brop 1 protein sequence by mass spectrometry data after digesting the protein into peptides with trypsin and Glu-C enzyme, respectively, and performing MS / MS analysis on all peptides. Figure C shows the results of N- and C-terminal sequence confirmation using trypsin mass spectrometry data. Figure C confirms that the N-terminus of this protein is D (aspartic acid) and the C-terminus is T (serine).

[0081] Figure 5 shows the preparation of recombinant Bro p 1 protein. Figure A is an agarose gel electrophoresis image of the Bro p 1 protein gene after PCR amplification. Figure B is an image of the pET-28a plasmid expressing Bro p 1 protein. Figure C shows the SDS-PAGE results of purified Bro p 1 protein (labeled as nBro p 1) and recombinant Bro p 1 protein with a His tag (His-rBro p 1). Figure D shows the Western blot results of the above two proteins using serum from patients with mulberry pollen allergy as the primary antibody and enzyme-labeled IgE antibody as the secondary antibody.

[0082] Figure 6 shows the results of detecting Bro p 1 protein-specific IgE in the serum of patients with BP allergy and healthy individuals. Figure A shows the detection results of recombinant Bro p 1 protein-specific IgE levels in the serum of patients with BP allergy (BP Allergy) and healthy individuals (Control); Figure B shows the correlation between recombinant Bro p 1 protein (rBro p 1) and natural Bro p 1 protein (nBro p 1) specific IgE levels, r = 0.9258; Figure C shows the correlation between recombinant Bro p 1 protein (rBro p 1) and BP total protein (BP) specific IgE levels detected by ELISA, r = 0.8328; Figure D shows the correlation between recombinant Bro p 1 protein (rBro p 1) and BP total protein (BP) specific IgE levels detected by ImmunoCAP, r = 0.4466.

[0083] Figure 7 shows the results of recombinant Bro p 1 protein inhibiting the binding of natural Bro p 1 protein (nBro p 1) and total broccoli pollen protein (BP) to IgE. Figures A and B are line graphs showing the OD values ​​(rBro p 1 - nBro p 1 inhibit) and inhibition rates (rBro p 1 - nBro p 1 inhibit ratio) of different concentrations of recombinant Bro p 1 protein (rBro p 1) to IgE (different lines represent serum from different broccoli pollen allergy patients, n = 10). Figures C and D are line graphs showing the OD values ​​(rBro p 1 - BP inhibit) and inhibition rates (rBro p 1 - BP inhibit ratio) of different concentrations of recombinant Bro p 1 protein (rBro p 1) to total broccoli pollen protein (BP) (different lines represent serum from different broccoli pollen allergy patients, n = 10). Detailed Implementation

[0084] The inventors have for the first time identified the protein sequence of the major allergen in mulberry pollen, confirming that the protein is a non-specific lipid transfer protein (nsLTP), and named it Bro p 1 according to the naming conventions of the international pollen database. The inventors have provided a method for purifying this protein from mulberry pollen and for preparing a recombinant protein with the same sequence as the protein. This protein plays an important role in allergic diseases caused by mulberry pollen, such as hay fever, allergic rhinitis, allergic conjunctivitis, and asthma. Bro p 1, as a novel allergen protein, can be applied to the screening, diagnosis, desensitization treatment, diagnostic reagents, new therapeutic drugs, and the development of treatment methods for patients with pollen allergies, including those targeting allergic rhinitis, allergic conjunctivitis, and asthma. This invention is based on these findings.

[0085] the term

[0086] As used herein, the term “comprising” or its variations such as “including” or “comprises” are to be understood as including the stated elements or components without excluding other elements or other components.

[0087] As used herein, the terms "allergenic protein of the present invention," "allergen of the present invention," "Bro p 1 protein," and "the protein" are used interchangeably and all refer to the nonspecific lipid transfer protein derived from mulberry pollen identified in this invention. The protein is a naturally extracted or recombinant protein having the sequence shown in SEQ ID NO:4. It should be understood that, in addition to the main sequence, the protein may have other conventional modifications that do not affect protein function, such as purification tags located at the N-terminus or C-terminus.

[0088] allergy

[0089] An allergy is an abnormal reaction of the human body to harmless substances (allergens). An allergic reaction will only occur when a person with an allergic constitution comes into contact with an allergen. There are hundreds of allergens, such as pollen, dust, foreign proteins, chemicals, and ultraviolet rays. Some allergies are even so rare that no allergen can be found.

[0090] The incidence of allergic diseases (including allergic syndromes) is approximately 20%. They can occur at any age, from newborns to the elderly, with no obvious gender characteristics, but they have a significant heritability.

[0091] The human body contains two types of cells: mast cells and basophils. These are widely distributed in the nasal mucosa, gastrointestinal mucosa, and the capillaries surrounding the connective tissue beneath the skin, as well as in the capsules of internal organs. These cells contain allergy mediators such as histamine, leukotrienes, serotonin, and kinins. Under the stimulation of environmental factors such as chemicals, air pollution, and sunlight radiation, the body produces a large number of free radicals. If these free radicals are not promptly eliminated after oxidation, they will damage the cell membranes of mast cells and basophils, causing cell membrane degeneration and instability. When unstable cells encounter allergens, antigens and antibodies react specifically, leading to cell membrane detachment and the release of allergy mediators. These mediators can cause smooth muscle contraction, capillary dilation, increased permeability, mucus secretion, and tissue damage, thereby triggering allergic reactions. In the process of allergic reactions, allergy mediators play a direct role. Allergens are the external cause of allergic symptoms, while a weakened immune system and the oxidative damage of mast cells and basophils by a large number of free radicals are the internal causes of allergies.

[0092] hay fever

[0093] When pollen contains oils and polysaccharides, it is digested by nasal secretions after inhalation, releasing more than ten types of antibodies. If these antibodies encounter invading pollen and accumulate in large quantities, it can cause skin allergies. Although the incidence of pollen allergy symptoms is relatively high, it is caused by a very small number of pollen types. The reason pollen causes allergies is that it contains abundant proteins, some of which are the main allergens. Typical clinical symptoms of pollen allergy include sneezing, clear nasal discharge, itching of the nose, eyes, ears, and palate, and may be accompanied by difficulty breathing or urticaria and eczema.

[0094] paper mulberry pollen

[0095] Paper mulberry (scientific name: Broussonetia papyrifera; English name: paper mulberry, tapa cloth tree) is a plant belonging to the genus Broussonetia in the family Moraceae. It is distributed in Asia, Europe, the Americas and Oceania.

[0096] Paper mulberry pollen is recognized as a significant cause of allergies in several countries and regions. In Pakistan, it has been identified as a leading cause of respiratory allergies. As early as the early 1970s, paper mulberry pollen was known to cause respiratory allergies in the United States, and the number of patients has steadily increased in the following decades. Similarly, literature reports that paper mulberry pollen is a major airborne allergen in northern India and a primary source of airborne allergens in Madrid, Spain, from January to April.

[0097] Nonspecific lipid transfer proteins are the main allergens in mulberry pollen.

[0098] This invention discovered that allergen molecules with a molecular weight of 10-15 kDa exist in mulberry pollen, and most patients allergic to mulberry are allergic to this allergen. This invention extracted and purified Bro p 1 protein from mulberry pollen using a three-step chromatographic process. Mass spectrometry analysis of the Bro p 1 protein showed that the purified protein was of uniform quality, and a sequence segment of the Bro p 1 protein was successfully identified. Based on this sequence, this invention successfully identified and PCR-amplified the cDNA of the Bro p 1 protein.

[0099] This invention confirmed the full-length sequence of the Bro p 1 protein after maturation through mass spectrometry analysis. The sequence showed that the signal peptide encoded by the cDNA of the Bro p 1 protein, as well as a portion of the N- and C-terminal sequences encoded by the cDNA, were absent in the mature protein. Sequence analysis revealed that: 1) Bro p 1 protein is a type of nonspecific lipid transfer protein. The nonspecific lipid transfer protein family exists only in terrestrial plants, having evolved over hundreds of millions of years and playing important roles in various plant functions. This family contains eight conserved cysteine ​​residues, forming four disulfide bonds; the sequence forms four alpha-helices, which in turn form a hydrophobic, dynamic cavity capable of accommodating different lipids. 2) This family is a common plant allergen, most notably the major allergen in peach allergies, which are widely present in the Mediterranean region of Europe. The major allergen in peaches is the nonspecific lipid transfer protein Pru p 3, which is abundant in the peel.

[0100] The Bro p1 protein shares the highest sequence homology with any known DNA / protein sequence with a theoretical protein from *Morus notabilis* (only the DNA sequence is available; there is currently no evidence of Bro p1 protein expression, with 74% homology). The allergen with the highest sequence similarity to Bro p1 is Par j 1 (45% homology, a nonspecific lipid transfer protein derived from yarrow pollen). Bro p1 shares only 31% sequence homology with Pru p3. Furthermore, Bro p1 differs from both Par j 1 and Pru p3 in the sequences of the three common IgE epitopes in the nonspecific lipid transfer protein family.

[0101] In summary, Bro p1 protein is the dominant allergen in mulberry pollen. Bro p1 protein has important value in the diagnosis and treatment of mulberry pollen allergy.

[0102] Allergy diagnosis

[0103] Allergen-specific IgE testing is an important method for diagnosing the etiology of allergic diseases. One of the characteristics of anaphylaxis is the identification of allergens, that is, the specific diagnosis of allergens. Allergen-specific diagnosis is further divided into in vivo and in vitro methods. Skin tests belong to in vivo methods, while serum specific IgE (sIgE) testing belongs to in vitro methods. In vitro allergen diagnostics in my country began in the 1990s, and common detection methods include the immunoCAP allergen detection system and group testing.

[0104] The immunoCAP allergen detection system was one of the first in vitro detection systems introduced from abroad and is now widely used. In vitro allergen detection methods can be broadly divided into two categories: selective detection and group detection. Selective detection involves selectively detecting one or more allergens based on clinical data such as medical history and skin tests; the immunoCAP system belongs to this category. Group detection involves simultaneously detecting a group of common allergens coated on a carrier; this is non-selective, and Mediwiss is an example of this type of detection.

[0105] In this invention, the provided allergen can be optionally used in the above-mentioned conventional detection methods, such as preparing kits or detection reagents, or for laboratory testing.

[0106] Pharmaceutical Compositions and Applications

[0107] The present invention also provides compositions, formulations, or products containing the combination reagents of the present invention, which can be used as medicines for treating and / or alleviating pollen allergies.

[0108] A preferred composition is a pharmaceutical composition comprising:

[0109] The pharmaceutical composition comprises Bro p 1 protein, the amino acid sequence of which is shown in SEQ ID NO:4.

[0110] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating and / or alleviating mulberry pollen allergy.

[0111] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0112] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0113] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, sustained-release formulations, sublingual tablets, or drops. For example, they are prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0114] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0115] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0116] The main advantages of this invention include:

[0117] This invention identifies the sequence of the dominant allergen in mulberry pollen. The Bro p 1 protein can be used to prepare mulberry pollen allergen detection kits or for the development and application of personalized desensitization vaccines.

[0118] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). Skin prickling, intradermal testing, and desensitization treatments are performed according to industry standard procedures. The use of chromatographic columns and resins in protein purification is performed according to the manufacturer's recommendations. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0119] Methods and Materials

[0120] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0121] Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0122] Example 1: Identification of major allergens in paper mulberry pollen

[0123] The specific experimental steps are as follows:

[0124] 1. Extraction of Broussonetia papyrifera pollen extract

[0125] Weigh 1g of defatted Broussonetia papyrifera pollen, add 10ml of 20mM pH 8.5 Tris-HCl buffer, mix well, and shake on a horizontal shaker at 150r / min for 12h at 4℃. Centrifuge the Broussonetia papyrifera pollen suspension at 15000g for 30min at 4℃, and transfer the supernatant pollen extract to a 1kDa dialysis bag. Dialyze in 2L of 20mM pH 8.5 Tris-HCl buffer at 4℃ for 2h.

[0126] The pollen extract was filtered using a 0.2 μm filter, and the protein concentration in the pollen extract was measured using the BCA method.

[0127] 2. SDS-PAGE of total protein in Broussonetia papyrifera pollen

[0128] Based on step 1 above, 5X denaturing and reducing protein loading buffer was added to 4 times the volume of pollen extract and mixed well. The sample was boiled at 95°C for 10 min, centrifuged rapidly, and 20 μL of supernatant was added to the SDS-PAGE wells. Electrophoresis was performed at 140 V for 60 min. After electrophoresis, the gel was stained with Coomassie Brilliant Blue and then destained.

[0129] The SDS-PAGE results are shown in Figure 1A, with molecular weight standards labeled on the left. The bands in the 10-15 kDa range represent the main allergen in Broussonetia papyrifera pollen identified in this invention, namely the Bro p 1 protein.

[0130] 3. Western blot analysis of total protein in Broussonetia papyrifera pollen

[0131] After performing SDS-PAGE as described above, the protein was transferred from the gel to a PVDF membrane using a wet transfer method at 200 mA / 90 min. The PVDF membrane was then blocked in TBST + 5% skim milk for 1 h and washed three times with TBST.

[0132] Serum from 50 patients with mulberry pollen allergy (allergy to mulberry pollen had been confirmed clinically through nasal provocation and skin prick tests) was pooled to form a serum pool. The serum pool or a single patient / healthy serum sample was diluted 1:10 with TBST solution containing 5% BSA. PVDF membranes were immersed in the serum and incubated overnight at 4°C. The membranes were washed three times with TBST for 10 min each time. The membranes were then incubated with a 1:5000 dilution of HRP-anti-human IgE secondary antibody at room temperature for 1 h, followed by three washes with TBST for 10 min each time. Developing was performed using enhanced chemiluminescence (ECL) reagent.

[0133] The Western blot results are shown in Figure 1B. A distinct sensitizing protein band was observed in the 10kDa-15kDa range, and Brop 1-specific IgE was present in all tested sera. Furthermore, Brop 1-specific IgE dominated the mulberry tree-specific IgE levels in both the serum pool and individual serum assays from 11 subjects.

[0134] 4. Purification of Brop 1 protein from Broussonetia papyrifera pollen

[0135] The paper mulberry pollen extract obtained in step 1 above was separated sequentially by anion exchange, gel filtration, and reversed-phase chromatography. The specific procedures are as follows:

[0136] (1) Anion exchange: The anion exchange column (5 ml, HiTrap Capto Q ImpRes Cytiva) was equilibrated with 20 mM pH 8.5 Tris-HCl buffer and loaded with the filtered pollen extract at a flow rate of 2 ml / min, gradient from 0% to 100%. Linear elution was then performed with 20 mM pH 8.5 Tris-HCl and 500 mM NaCl. Following the methods described in Experiments 2 and 3 above, the collected flow-through and eluent were analyzed by SDS-PAGE and Western blot.

[0137] (2) Gel filtration: The fractions with the same protein distribution were further adsorbed onto a 20 mM pH 8.5 Tris-HCl buffer equilibrated gel filtration column (96 ml, Superdex 200) and eluted at a flow rate of 2 ml / min. The collected solution was then analyzed by SDS-PAGE and Western blot.

[0138] (3) Reversed-phase chromatography: Solution A was prepared using 0.1% formic acid aqueous solution, and solution B was prepared using 0.1% formic acid acetonitrile solution. The column temperature was set to 60℃. The reversed-phase column (BioResolve RP mAb) was equilibrated with 20% solution B and 20% solution A. Elution was performed using a 2.7 μm, 4.6 x 100 mm (Waters) filter at a flow rate of 0.5 mL / min. The collected solution was then analyzed by SDS-PAGE and Western blot.

[0139] This step was solely for sequence identification and characterization of the purified Bro p 1 protein; the results are detailed in Figure 2. Bro p 1 protein was enriched in fraction F2 (Figure 2A); SDS-PAGE showed the absence of other contaminating proteins (Figure 2B); Western blotting confirmed that the purified protein was the main allergen of mulberry pollen (Figure 2C). Because reversed-phase chromatography is a denaturing purification method that would destroy the protein's three-dimensional structure, the Bro p 1 protein used for the diagnosis and desensitization treatment of mulberry pollen allergy was purified only by anion exchange and gel filtration.

[0140] 5. Bro p 1 protein characterization results

[0141] The purified protein was analyzed for molecular weight, UV-Vis absorption spectroscopy, and MS / MS mass spectrometry. The results are shown in Table 1 below.

[0142] Table 1

[0143] *I and L are isomers and cannot be distinguished by mass spectrometry. The MS / MS spectra of the identified b / y ions are shown in Figure 3; among them, ALGQFSDIDPGR has the sequence number SEQ ID NO:14 and ALGQFSDLDPGR has the sequence number SEQ ID NO:15.

[0144] 6. Identification of the full-length sequence of Brop 1 protein

[0145] Total mRNA was extracted from mulberry pollen and RNA-seq was performed. In step 5 above, only one sequence of the peptide (ALGQFSDI(L)DPGR) was identified.

[0146] Total mRNA was extracted from Broussonetia papyrifera pollen and reverse transcribed into cDNA using the primer pairs shown below:

[0147] The cDNA was successfully amplified using SEQ ID NO:1 and SEQ ID NO:2, and its nucleic acid sequence was confirmed as follows:

[0148] The full-length sequence of the protein encoded by the cDNA is shown in Figure 4A. The theoretical average molecular weight after removing the signal peptide is 12507 Da, which is greater than the measured value (10271 Da). The protein was digested with the protease and analyzed by LC-MS / MS. The coverage of the mass spectrometry data to the cDNA-encoded protein sequence was analyzed (Figure 4B, data from trypsin and Glu-C enzymes, respectively), and the C-terminal and N-terminal sequences were confirmed (Figure 4C, data from trypsin). The results show that the full-length sequence of the protein is:

[0149] The sequence contains 8 cysteine ​​residues, tyrosine but no tryptophan, and has a theoretical reduced average molecular weight of 10273 Da, consistent with the characterization results. The specific steps of the above experiment are as follows:

[0150] 6.1 Enzyme digestion

[0151] The F2 solution from the reversed-phase chromatography was replaced with 20 mM pH 8.5 Tris-HCl using a 3 kDa ultrafiltration tube and concentrated to 25 μL (20 μg). 75 μL of 7 M guanidine hydrochloride (dissolved in 50 mM pH 8.0 Tris-HCl) and 2 μL of 1 MdTT were added, and the mixture was incubated at 37°C for 30 min to denature and reduce the protein. 11.3 μL of 500 mM IAM was added to each tube, and the mixture was incubated at 37°C in the dark for 30 min to block the thiol groups. The protein was then replaced with 50 mM pH 8.0 Tris-HCl using ultrafiltration and concentrated to less than 80 μL. 0.6 μg of mass spectrometry enzyme was added, and the mixture was digested overnight at 37°C.

[0152] 6.2 Mass Spectrometry Analysis

[0153] The samples before (after denaturation and reduction) and after enzyme digestion were analyzed by LC-MS / MS to obtain primary and secondary spectra.

[0154] 7. Brop 1 protein is the main allergen in mulberry pollen.

[0155] Analysis of the Bro p 1 protein sequence showed that:

[0156] 1) Bro p 1 protein is a type of nonspecific lipid transfer protein. The nonspecific lipid transfer protein family exists only in terrestrial plants, where it has evolved over hundreds of millions of years and plays an important role in various plant functions. This family contains eight conserved cysteine ​​residues forming four disulfide bonds; the sequence forms four alpha-helices, which in turn form a hydrophobic, dynamic cavity capable of accommodating different lipids.

[0157] 2) This family of allergens is a common plant allergen, most notably the main allergen in peach allergies, which are prevalent in the Mediterranean region of Europe. The main allergen in peaches is the nonspecific lipid transfer protein Pru p3, which is abundant in the fruit peel.

[0158] The Bro p1 protein has the highest homology with all known DNA / protein sequences to a theoretical protein from *Morus notabilis* (a relative of *Broussonetia papyrifera*, with only a DNA sequence and currently no evidence of Bro p1 protein expression, showing 74% homology). The allergen with the highest sequence similarity to Bro p1 is Par j1 (45% homology, a non-specific lipid transfer protein derived from yarrow pollen). Bro p1 has only 31% sequence homology with Pru p3. Furthermore, Bro p1 differs from both Par j1 and Pru p3 in the sequences of the three common IgE epitopes in the non-specific lipid transfer protein family. A comparison of the three IgE epitopes is shown in Table 2 below.

[0159] Table 2

[0160] In summary, Brop 1 protein is the main allergen in mulberry pollen.

[0161] Example 2: Expression and purification of recombinant Brop 1 protein

[0162] 2.1 Carrier Construction

[0163] The Bro p1 protein gene was amplified from cDNA (Figure 5A), and the gene was inserted into the pET28a vector (a gene with a His6 tag at the N-terminus) to obtain a recombinant plasmid (Figure 5B).

[0164] 2.2 Protein Expression

[0165] Thaw competent *E. coli* BL21(DE3) on ice. Add 1 μL of plasmid to 100 μL of competent *E. coli*, incubate on ice for 30 min, heat shock at 42°C for 1 min, and incubate on ice for 2-5 min. Add 400 μL of antibiotic-free LB medium and incubate at 37°C and 220 rpm for 1 h. Spread the bacterial culture onto LB agar plates containing 0.1% kanamycin and incubate upside down at 37°C overnight. Pick single colonies and incubate in 100 mL of 0.1% kanamycin LB liquid medium and incubate at 37°C and 220 rpm for 12 h. Inoculate 20 mL of the bacterial culture into 1 L of 0.1% kanamycin LB liquid medium and incubate at 37°C and 220 rpm for 3 h. When the OD value is 0.6-0.8, add 0.2 mM IPTG to induce expression and incubate at 16°C and 220 rpm for 12 h.

[0166] 2.3 Protein purification

[0167] The bacteria were collected by centrifugation. *E. coli* were resuspended in 20 mM pH 8.0 Tris-HCl and 500 mM NaCl buffer A containing 20% ​​glycerol. The cells were then lysed at low temperature using a high-pressure cell disruptor. The lysed suspension was collected and centrifuged at 15000 g for 30 min at 4°C. The supernatant was collected. The Ni-NTA column was equilibrated with buffer A, and the collected supernatant was added to the Ni-NTA column. Ten column volumes were washed with buffer A containing 20 mM imidazole. Proteins were eluted with buffer A containing 250 mM imidazole. SDS-PAGE was performed using the method described in Example 1. Figure 5C shows the recombinant Bro p 1 protein (labeled His-rBro p 1) and the native Bro p 1 protein (nBro p 1).

[0168] 2.4 Validation of Bro p 1 Recombinant

[0169] (1) Western blotting method to verify recombinant Brop 1 protein

[0170] Western blotting experiments were performed according to the method described in Example 1. The results are shown in Figure 5D. The recombinant Bro p 1 protein (labeled rBro p 1) showed similar signal intensities to the natural Bro p 1 protein (labeled nBro p 1) and sIgE binding in the serum of patients with mulberry pollen allergy. This demonstrates that the recombinant Bro p 1 protein can replace the natural Bro p 1 protein for sIgE detection.

[0171] (2) ELISA method to verify recombinant Brop 1 protein

[0172] Dilute recombinant Bro p 1 protein to 0.6 μg / ml with carbonate buffer (pH 9.6), add 100 μl / well to each well of an ELISA plate, and coat overnight at 4°C. Aspirate the coating solution and wash the plate 5 times with TBST wash buffer (300 μl / well each time). Add 100 μl of 2% BSA TBST blocking buffer to each well and incubate at room temperature for 1 h. Wash the plate 5 times. Add 100 μl of 1:10 diluted serum from patients with mulberry hay fever or controls (serum source see Example 1) to each well and incubate at 37°C for 1.5 h. Wash the plate 5 times. Add 1:5000 diluted HRP-anti-human IgE secondary antibody to each well and incubate at 37°C for 1 h. Wash the plate 5 times. Add 100 μl of TMB substrate to each well and incubate at room temperature in the dark for 30 min. Add 50 μl of stop solution to each well. Measure the OD value at 450 nm.

[0173] The ELISA results are shown in Figure 6. Compared with the control group, the positive rate of recombinant Bro p 1 protein-specific IgE in patients with broccoli hay fever (BP Allergy) was 77.5% (rBro p 1-sIgE OD value was greater than 3 times the average OD value of the negative control) (Figure 6A). Furthermore, the recombinant Bro p 1 protein-specific IgE level (rBro p 1-sIgE) was strongly correlated with the natural Bro p 1 protein-specific IgE level (nBro p 1-sIgE) (r = 0.9258, p < 0.0001, Figure 6B); ELISA detection revealed a strong correlation between rBro p 1-sIgE and the total sIgE of Broussonetia papyrifera pollen (BP-sIgE) (r = 0.8328, p < 0.0001, Figure 6C); similarly, ImmunoCAP detection revealed a correlation between rBro p 1-sIgE and the total sIgE of Broussonetia papyrifera pollen (BP-sIgE) (r = 0.4466, p = 0.0326, Figure 6D).

[0174] The above results demonstrate that recombinant Bro p 1 protein can replace natural Bro p 1 protein or total protein from mulberry pollen for sIgE detection.

[0175] (3) Competitive ELISA method to verify recombinant Brop 1 protein

[0176] The recombinant Bro p 1 protein (rBro p 1) was verified to compete with natural Bro p 1 protein (nBro p 1) and total broccoli pollen protein (BP) for IgE binding using a competitive ELISA method. Natural Bro p 1 protein (0.06 μg / well) or total broccoli pollen protein (4 μg / well) was coated onto an ELISA plate. Serum from patients with broccoli hay fever was diluted 1:5 (using the blocking buffer from Example 1) and then mixed with equal volumes of recombinant Bro p 1 protein (rBro p 1) solutions at concentrations of 0 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml, 50 μg / ml, and 100 μg / ml. The mixture was incubated at 37°C for 1 h as the primary antibody. The remaining steps were as described in (2) above.

[0177] The results of the competitive ELISA assay are shown in Figure 7. Recombinant Bro p 1 protein competitively binds to IgE with both native Bro p 1 protein and total Broussonetia papyrifera pollen protein (Figure 7), and the inhibition ratio is positively correlated with the concentration of recombinant Bro p 1 protein. When the concentration of recombinant Bro p 1 protein is 100 μg / ml, the inhibition efficiency against native Bro p 1 protein (Figures 7A-7B) and total Broussonetia papyrifera pollen protein (Figures 7C-7D) is generally greater than 90%.

[0178] The above results demonstrate that recombinant Bro p 1 protein can replace natural Bro p 1 protein or total mulberry pollen protein with comparable immunogenicity. Recombinant or natural Bro p 1 protein can be used to replace total mulberry pollen protein for specific immunotherapy.

[0179] (4) Preparation of recombinant Brop1 protein (r Brop1) with the same sequence as the natural Brop1 protein

[0180] To facilitate purification, the His tag was not removed from the recombinant Bro p 1 protein. This invention also discloses a method for preparing a recombinant Bro p 1 protein with a sequence completely identical to the natural Bro p 1 protein.

[0181] His6-Sumo was preferred as the purification tag (Sumo stands for small ubiquitin-like modifier, i.e., a ubiquitin-like protein modification molecule, such as the SMT3 protein in yeast). Protein expression and purification were performed as described above. The His tag was removed using the Drosophila Ulp 1 enzyme.

[0182] His6-ENLYFQ was selected as the purification tag. Protein expression and purification were performed as described above. The His tag was removed using a modified TEV enzyme.

[0183] Example 3: Recombinant or natural Brop 1 protein for the diagnosis or treatment of mulberry pollen allergy.

[0184] 3.1 Recombinant or natural Bro p 1 protein for skin prick / intradermal testing / nasal provocation / skin patch testing:

[0185] When serum contains IgE antibodies specific to an allergen extract / molecule, the allergic patient is sensitized to that allergen extract / molecule. However, whether this allergen extract / molecule is the cause of the patient's immediate-type allergic symptoms needs to be confirmed through nasal provocation (inhaled allergens) and skin prick / intradermal tests. The cause of delayed-type allergic symptoms is diagnosed / confirmed through skin patch testing. Because Bro p1 protein is the dominant allergen in mulberry pollen and is species-specific, using recombinant or natural proteins for the above tests has better specificity than extracts and does not expose the patient to other components in the extract, thus offering better safety.

[0186] The above trials were conducted according to standard clinical procedures, as follows:

[0187] (1) Skin prick / intradermal test: A prick needle containing Brop 1 protein solution (dissolved in PBS or physiological saline, containing 0-40% (v / v) glycerol, protein concentration less than 100 μg / mL) is used to prick the skin of the patient's upper arm or back. Wait 15-30 minutes and observe the size of the wheals. Based on the prick results, the protein concentration can be optimized, or 2-5 consecutively diluted concentrations can be pricked. The intradermal test involves injecting the above solution intradermally (volume 0.01-1 mL) using a syringe (usually a 26-gauge needle or other similar needle). Observe the size of the wheals / erythema.

[0188] (2) The nasal cavity provocation test is as follows:

[0189] Bro p 1 protein solution (in water, physiological saline, PBS, or other non-irritating aqueous solutions) can be directly dripped into the nasal cavity, sprayed into the nasal cavity, or absorbed onto filter paper and placed on the inferior turbinate. Different concentrations can be tested, with one nostril using the carrier solution and the other using the Bro p 1 protein solution to rule out non-specific nasal stimulation. After stimulation, wait 15-30 minutes and observe all nasal symptoms.

[0190] (3) The skin patch test is as follows:

[0191] Bro p 1 protein is directly coated onto a polyester film and adhered to adhesive tape, then protected with an outer masking layer. For testing, simply remove the masking layer and apply directly. Alternatively, place a filter paper pad inside the plaque applicator, then add Bro p 1 protein aqueous solution until it does not overflow. Apply the plaque applicator to the subject's skin (first the upper back, along the spine, twice), mark the area with a marker, and leave it on for 48 hours.

[0192] 3.2 Brop 1 protein used in in vitro basophil activation assay

[0193] The in vitro basophil activation assay is a supplementary test to the clinical history, the in vivo tests mentioned above, and the in vitro specific IgE detection. It is used to assess / confirm the ability of allergen molecules to induce degranulation of basophils in patients in vitro. Specifically, Brop 1 protein is added to the whole blood of patients allergic to mulberry pollen. The proportion of activated basophils is analyzed using flow cytometry (common activation markers are CD63 and CD203c).

[0194] 3.3 Pharmacodynamic testing of formulations containing Brop 1 protein

[0195] When allergens cannot be avoided, specific immunotherapy can specifically induce immune tolerance to the allergen. This invention has identified the sequence of the major allergen in mulberry pollen. This invention discloses a method for purifying Brop 1 protein and a method for preparing recombinant Brop 1 protein. Based on the above, preparations containing recombinant or natural Brop 1 protein can be used for mulberry pollen-specific immunotherapy. The specific method is as follows:

[0196] 1. Immunotherapy with the recombinant or natural protein via subcutaneous or intradermal administration.

[0197] 2. Administer the recombinant or natural protein via sublingual administration for immunotherapy.

[0198] 3. Modify the recombinant or natural protein: such as glutaraldehyde modification, carbamylation modification, and mannan modification before immunotherapy.

[0199] 4. For subcutaneous / intradermal administration, aluminum hydroxide or aluminum phosphate is used as an adjuvant. Dosage is increased from low to high concentrations, with a dosing frequency ranging from twice a week to once every three months, and a dosing period from three months to three years. For high-concentration administration, the dose is 0.01-10 μg Bro p 1 protein per dose.

[0200] 5. Use nucleic acids encoding the Brop 1 protein sequence as active ingredients for immunotherapy.

[0201] 3.4 Animal Model Drug Efficacy Experiments

[0202] (1) Preparation of the formulation

[0203] 1) After filtering and sterilizing the solution containing recombinant or natural Brop 1 protein, the protein concentration is determined using the BCA method. The allergen preparation is prepared by mixing the solution with diluent and / or adjuvant in a specific ratio. The preparation is then diluted to different concentrations according to the required therapeutic concentration and dispensed into containers.

[0204] 2) The solution containing recombinant or natural Brop 1 protein is prepared into tablets, capsules, sublingual preparations, and other dosage forms according to conventional formulation methods.

[0205] 3) The solution containing recombinant or natural Brop 1 protein is mixed with glutaraldehyde-modified, carbamoyl-modified, or mannan-modified proteins, and then formulated into dosage forms such as injections, tablets, capsules, and sublingual preparations according to conventional formulation methods.

[0206] (2) Pharmacodynamic experiments of the formulation

[0207] 1) Effects of the formulation on allergic guinea pigs

[0208] Grouping:

[0209] Guinea pigs were divided into three groups: a normal group, a model group, and a desensitization group.

[0210] Sensitization:

[0211] For the model group and the desensitization group, guinea pigs were sensitized by subcutaneous injection and intraperitoneal injection after mixing mulberry pollen extract with aluminum hydroxide gel. Sensitization was repeated every 10 days for a total of 3-5 times.

[0212] For the normal group, an equal volume of physiological saline was administered.

[0213] Antigen attack:

[0214] The desensitized group and the model group were subjected to antigen challenge according to the designed protocol.

[0215] Administration:

[0216] For the desensitization group, the preparation was injected according to the designed dosage and course of treatment; for the model group, physiological saline was injected according to the designed dosage and course of treatment; the normal group was not treated.

[0217] Efficacy:

[0218] (1) Effects of the formulation on the lung function of allergic guinea pigs

[0219] (2) Effects of the formulation on airway inflammatory cell aggregation and infiltration in allergic guinea pigs

[0220] (3) Effects of the formulation on serum IgG and IgE in allergic guinea pigs

[0221] (4) Effects of the formulation toxic to guinea pigs on the effects of bronchoalveolar lavage fluid on cytokines

[0222] 2) Effects of the formulation on allergic rats

[0223] Grouping:

[0224] Rats were divided into three groups: normal group, model group, and desensitization group.

[0225] Sensitization:

[0226] For the model group and the desensitization group, mulberry pollen extract was mixed with aluminum hydroxide gel and then sensitized to rats via subcutaneous and intraperitoneal injection. Sensitization was repeated every 10 days, for a total of 3-5 sensitizations.

[0227] For the normal group, an equal volume of physiological saline was administered.

[0228] Antigen attack:

[0229] The desensitized group and the model group were subjected to antigen challenge according to the designed protocol.

[0230] Administration:

[0231] For the desensitization group, the preparation was injected according to the designed dosage and course of treatment; for the model group, physiological saline was injected according to the designed dosage and course of treatment; the normal group was not treated.

[0232] Efficacy:

[0233] (5) Effects of the formulation on lung function in allergic rats

[0234] (6) Effects of the formulation on airway inflammatory cell aggregation and infiltration in allergic rats

[0235] (7) Effects of the formulation on serum IgG and IgE in allergic rats

[0236] (8) Effects of the formulation toxicity on cytokines in bronchoalveolar lavage fluid of rats.

[0237] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A composition containing mulberry pollen allergen, characterized in that, The mulberry pollen allergen in the composition is a natural or recombinant sensitizing protein, Bro p 1; and the amino acid sequence of the sensitizing protein Bro p 1 is shown in SEQ ID NO:4:

2. The composition according to claim 1, characterized in that, The allergen has one or more IgE epitopes selected from the group consisting of: TPCLNYVRKR (SEQ ID NO: 5), AQKLNDNSKT (SEQ ID NO: 6), EQCGISATLPP (SEQ ID NO: 7).

3. The composition according to claim 1, characterized in that, The recombinant allergenic protein Bro p 1 also has a purification tag at the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO:

4. Preferably, the purification tag is a His6 tag, and more preferably His6-Sumo or His6-ENLYFQ.

4. The composition according to claim 1, characterized in that, The mulberry pollen allergen can be optionally modified by a method selected from the group consisting of: glutaraldehyde modification, carbamylation modification, or mannan modification.

5. Nucleic acid or gene encoding the natural Bro p 1 mulberry pollen allergen.

6. An expression carrier, characterized in that, The expression vector comprises a nucleotide sequence capable of expressing the mulberry pollen allergen in the composition as described in claim 1; preferably, the expression vector is selected from the group consisting of: expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria.

7. A pharmaceutical composition for the prevention or treatment of hay fever, characterized in that, It includes the composition as described in claim 1, or the nucleic acid or gene as described in claim 5, or the expression vector as described in claim 6.

8. The pharmaceutical composition according to claim 7, characterized in that, The hay fever includes diseases selected from the group consisting of mulberry pollen allergy: hay fever, allergic rhinitis, allergic asthma, conjunctivitis, allergic dermatitis, and urticaria.

9. A kit for in vitro diagnosis of mulberry pollen allergy or for evaluating the efficacy of mulberry pollen allergy treatment in subjects, characterized in that, The kit comprises the composition of claim 1, or the nucleic acid or gene of claim 5, or the expression vector of claim 6.

10. The kit according to claim 9, characterized in that, The kit also includes detection reagents that specifically recognize IgE or IgG.

11. Use of the composition according to claim 1 in the preparation of a pollen allergy detection reagent.

12. Use of the composition of claim 1, the nucleic acid or gene of claim 5, or the expression vector of claim 6; characterized in that, Used for: (1) Prepare a kit for diagnosing mulberry pollen allergy, or prepare a drug for the prevention or treatment of mulberry pollen allergy; (2) Prepare drugs and / or reagents for detecting the content of mulberry pollen allergen-specific IgE in the serum of patients; (3) Prepare drugs and / or reagents for detecting the degree of basophil activation in patients.

13. A method for purifying the allergens from mulberry pollen in the composition of claim 1, characterized in that, Includes the following steps: 1) Extraction of total protein from paper mulberry pollen: Defatted pollen was mixed with buffer (pH 6.5-9.0), protease inhibitor was added and incubated. After centrifugation, the supernatant was collected and filtered to obtain paper mulberry pollen extract. 2) Purification of allergen components: 2.1) The paper mulberry pollen extract was subjected to anion exchange chromatography to obtain a first purified extract; 2.2) The target allergen portion of the first purified extract is subjected to gel filtration to obtain the second purified extract.

14. A method for preparing recombinant Brop 1 protein, characterized in that, Including the following steps: (1) Recombinant Brop 1 protein was expressed in Escherichia coli; (2) The expression product was purified using an affinity column to obtain the desired recombinant Brop1 protein; The amino acid sequence of the recombinant Bro p 1 protein is shown in SEQ ID NO:4:

15. A method for standardizing mulberry pollen extract using the Bro p 1 protein prepared by the method of claim 14, characterized in that, Including the following steps: Bro p 1 protein was used as a control, and the content / concentration / potency of this protein in the mulberry pollen extract was determined by ELISA or other similar ligand binding assays.

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