A fructose bisphosphate aldolase type pollen allergen and its application

Abstract

The present invention discloses a novel pollen allergen and diagnostic, preventive, and therapeutic drugs for allergic diseases caused by pollen using the same. The novel pollen allergen of the present invention is a fructose bisphosphate aldolase selected from the following pollen allergen proteins (a) to (c): (a) a protein comprising the amino acid sequences set forth in SEQ ID NOs. 1 to 3; (b) a protein comprising an amino acid sequence in which one or more amino acids are substituted, modified, deleted, or added to the amino acid sequences set forth in SEQ ID NOs. 1 to 3 and having allergenic activity or low allergenic activity; (c) a protein comprising an amino acid sequence that is 90% or more identical to the amino acid sequences set forth in SEQ ID NOs. 1 to 3 and having allergenic activity, or an allergen that is immunologically cross-reactive with the amino acid sequences set forth in SEQ ID NOs. 1 to 3.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a fructose bisphosphate aldolase type pollen allergen and application thereof. Background Art

[0002] Allergic diseases mediated by immunoglobulin E (IgE) are a common and frequently occurring disease worldwide, affecting more than 30% of the world's population, and have always been considered a major public health issue. It is estimated that there are more than 400 million people suffering from allergic diseases in my country, and the prevalence is still increasing year by year. Among the many allergens that cause allergic diseases, pollen is the most important outdoor allergen and has always been considered a major risk factor for allergic rhinitis and asthma. For example, Artemisia grandiflora, Artemisia annua, and Humulus japonicus pollen are the most important autumn seasonal allergens in China. The allergy rate to Artemisia pollen in patients in different regions ranges from 10.5% to 58.3%, and the allergy rate to Humulus japonicus pollen ranges from 1.6% to 21.8%.

[0003] Diagnosis of these pollen allergic diseases involves a series of skin prick tests and specific IgE binding immune response assays using pollen extracts. Treatment options include symptomatic treatments such as antihistamines and glucocorticoids, and allergen immunotherapy (desensitization therapy) using pollen extracts. However, pollen extracts are essentially crude pollen extracts and contain substances unrelated to the disease, such as non-allergenic proteins, pigments, and contaminants adhering to the raw materials. Differences in the origin, extraction process, and batches of the extracts can also affect the allergen components or major allergen content in the extracts. Therefore, current diagnostic strategies based on pollen extracts struggle to distinguish the immune response profiles of different patients to different allergen molecules, leading to a degree of diagnostic blindness. Furthermore, non-allergenic proteins and other impurities in the extracts not only interfere with the sensitivity and specificity of diagnostic methods but also impact the effectiveness and safety of immunotherapy.

[0004] Given the above challenges, identifying and understanding the true causal allergen molecules in pollen that are associated with allergic diseases is essential. The development of molecular diagnostic methods based on allergen monomers and the development of efficient allergen vaccines have been research hotspots in this field both domestically and internationally. However, the diverse and complex composition of pollen compounds significantly hinders the identification of true allergens. For example, to date, nine distinct allergen classes have been identified in Artemisia pollen, while only two have been identified in Humulus pollen. These have all been identified by the World Health Organization / International Union of Immunological Societies Allergen Nomenclature Committee and included in the relevant database (http: / / www.allergen.org). However, several serum immunological studies on Artemisia and Humulus pollen, both domestically and internationally, have hinted at the presence of new allergens in these pollens, but these have not been further characterized. Therefore, further identification of the specific allergenic protein molecules in pollen will provide precise molecular diagnostics and personalized desensitization treatments for individuals with pollen-related allergic diseases. Summary of the Invention

[0005] Purpose of the invention: In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a new type of allergen protein in pollen and its application. The pollen allergen protein provided by the present invention belongs to the fructose bisphosphate aldolase type and can be used for the diagnosis, prevention and treatment of allergic diseases, especially allergic diseases caused by pollen, and more particularly, for the diagnosis, prevention and treatment of allergic diseases caused by fructose bisphosphate aldolase in Artemisia and / or Humulus pollen.

[0006] Technical solution: In order to solve the above technical problems, the first aspect of the present invention provides a pollen allergen protein selected from the following (a) to (d):

[0007] (a) an Artemisia pollen protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3;

[0008] (b) proteins having allergenic activity or low allergenic activity, wherein the amino acid sequence in (a) has been substituted, modified, deleted or added with one or more amino acids;

[0009] (c) a protein having allergenic activity consisting of an amino acid sequence having at least 90% identity with the amino acid sequence in (a);

[0010] (d) An allergen protein that is immunologically cross-reactive with the amino acids represented by any one of SEQ ID NOs: 1 to 3 in (a).

[0011] As described in the present invention, the term "immunological cross-reactivity" is commonly understood in the industry. When an antibody can react with different antigens that are not exactly the same in molecular structure but have similar antigenic determinants, it is called cross-reactivity.

[0012] As described in the present invention, "pollen protein comprising the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3" is a new allergen protein identified, isolated and purified by the inventors from pollen extracts for the first time. It belongs to the fructose bisphosphate aldolase type, has a molecular weight of approximately 38 kDa, and its amino acid sequence is shown in SEQ ID NOs: 1 to 3.

[0013] The second aspect of the present invention also includes a nucleic acid or gene encoding the pollen allergen protein, more specifically, a polynucleotide selected from the following (2a) to (2c);

[0014] (2a) a polynucleotide having a base sequence as shown in any one of SEQ ID NOs: 4 to 6;

[0015] (2b) a polynucleotide having a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 4 to 6 in (2a) that hybridizes under stringent conditions and encodes a protein having allergenic activity;

[0016] (2c) A polynucleotide having a base sequence that is 90% or more identical to the nucleotide sequence of (2a) and encoding a protein having allergenic activity and immunological cross-reactivity.

[0017] As described in the present invention, it is not difficult to understand that the "nucleotide sequence encoding the pollen allergen as described in the first aspect" should take degenerate bases into consideration. For example, if the nucleotide sequence of the amino acid sequence shown in SEQ ID NOs: 1 to 3 includes SEQ ID NOs: 4 to 6, the scope of protection should also protect sequences that have base degeneracy with SEQ ID NOs: 4 to 6. The amino acid sequences encoded by these nucleotide sequences are still SEQ ID NOs: 1 to 3.

[0018] The third aspect of the present invention also includes an expression cassette, a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium, characterized in that it contains the nucleic acid or gene.

[0019] In one embodiment of the present invention, the expression cassette and recombinant vector are recombinant expression vectors encoding the pollen allergen fructose bisphosphate aldolase gene, containing the base sequences shown in SEQ ID NOs: 4 to 6; and the recombinant cell or recombinant bacterium is Escherichia coli transformed with the recombinant expression vector.

[0020] In a preferred embodiment of the present invention, the recombinant expression vector is obtained by inserting the base sequence shown in SEQ ID NO: 4 to 6 between the NcoI and XhoI restriction sites of pET-28a(+); the recombinant cell or recombinant bacteria is Escherichia coli BL21 (DE3) into which the recombinant expression vector is transferred.

[0021] A fourth aspect of the present invention also includes the use of the pollen allergen protein, the nucleic acid or gene, the expression cassette, recombinant vector, recombinant protein, recombinant cell or recombinant bacteria in the production process of a kit for diagnosing pollen allergic diseases or a drug or pharmaceutical composition for preventing or treating pollen allergic diseases, or for content calibration and quality control in the finished product.

[0022] Wherein, the pollen is Artemisia pollen and / or Humulus pollen; specifically including Artemisia annua pollen, Artemisia macroseed pollen and Humulus pollen.

[0023] In a fifth aspect, the present invention also includes any of the following preparations (e) to (f) for preventing, diagnosing, and treating allergic diseases:

[0024] (d) a preventive or therapeutic agent containing the pollen allergen protein, nucleic acid or gene as an active ingredient;

[0025] (e) a preventive or therapeutic agent containing as an active ingredient at least one of the immunoregulatory T-cell reactive polypeptide fragments, B-cell reactive polypeptide fragments, proteins or polynucleotides constructed by fusion of the polypeptide fragments with carrier proteins derived from the pollen allergen protein;

[0026] (f) A specific therapeutic antibody derived from the pollen allergen protein, wherein the antibody comprises a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanized modified antibody.

[0027] The term "prevention" as used herein covers not only measures to prevent the disease from occurring, such as reducing risk factors, but also measures to arrest its progression and reduce its consequences once established. "Prevention" also means preventing sensitization in individuals at risk of developing an allergic disease.

[0028] As used herein, the term "treating" or grammatical equivalents include improvement and / or reversal of symptoms of a disease (e.g., an allergic disease). When the substances provided herein induce improvement in any parameter associated with a disease, the substances can therefore be considered therapeutic substances. The term "treating" refers to both therapeutic treatment and prophylactic treatment.

[0029] In a sixth aspect, the present invention also includes a drug for preventing or treating allergic diseases, characterized in that the drug is a single or compound preparation, which includes the pollen allergen protein, the nucleic acid or the gene.

[0030] In one embodiment of the present invention, the composition further includes but is not limited to other conventional ingredients used in the industry for diagnosing individual allergies to pollen, such as diluents, detergents, enzymes or fluorescently labeled anti-IgE antibodies, developers, and the like.

[0031] Wherein, the drug is an allergen vaccine.

[0032] In a seventh aspect, the present invention also includes a kit for allergen diagnosis, which includes the allergen protein, the nucleic acid or the gene.

[0033] In an eighth aspect, the present invention provides a method for preventing or treating an allergic disease caused by pollen, wherein the preventive or therapeutic agent is administered to a patient.

[0034] In a ninth aspect, the present invention provides a method for diagnosing, preventing, or evaluating the efficacy of treatment for pollen-related allergic diseases, wherein the diagnostic drug or composition is administered to a patient to detect whether the individual develops an immune response to the pollen allergen protein described in the first aspect.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0036] (1) The pollen allergen protein of the present invention belongs to the fructose bisphosphate aldolase type and can be used for the diagnosis, prevention, and treatment of allergic diseases, especially allergic diseases caused by pollen. More particularly, it is used for the diagnosis, treatment, and prevention of allergic diseases caused by the fructose bisphosphate aldolase type allergen in Artemisia pollen and / or Humulus pollen;

[0037] (2) The present invention prepares pollen allergen recombinant protein through gene cloning, protein expression, and purification. The protein has high purity, good specificity, and abundant yield. It can be used to prepare drugs for treating or preventing allergies, or to prepare kits for allergen detection.

[0038] (3) The novel pollen allergen protein provided by the present invention has no identical amino acid sequence to other known pollen allergen types. Therefore, by combining with them, more efficient diagnosis and allergy treatment can be performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A is a diagram of the anion exchange chromatography separation of the Artemisia macrophylla pollen extract. Inserted are the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the collected fraction containing natural fructose bisphosphate aldolase followed by Coomassie Brilliant Blue G-250 (CBB) staining analysis and IgE immunoblotting using the serum of patients allergic to Artemisia pollen.

[0040] Figure 1 B is Figure 1 The spectrum in A shows the fraction containing fructose bisphosphate aldolase, which was then separated by size exclusion chromatography. The inserted figure shows the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the fraction containing native fructose bisphosphate aldolase, followed by Coomassie Brilliant Blue G-250 (CBB) staining analysis, and IgE immunoblot analysis using serum from patients allergic to Artemisia pollen.

[0041] Figure 1 C is Figure 1 B shows a map of the collected fraction containing fructose bisphosphate aldolase, which was then separated by cation exchange chromatography. Inserted are the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the final purified natural fructose bisphosphate aldolase followed by Coomassie Brilliant Blue G-250 (CBB) staining analysis and IgE immunoblot analysis using serum from patients allergic to Artemisia pollen.

[0042] Figure 2 includes Figure 2A 、 2B , 2C, Figure 2A This is the cloning result of the fructose bisphosphate aldolase gene from Artemisia grandiflora pollen provided in the embodiment of the present invention; Figure 2B This is the cloning result of the fructose bisphosphate aldolase gene from Artemisia annua pollen provided in the embodiment of the present invention; Figure 2C This is the cloning result of the Humulus japonicus pollen fructose bisphosphate aldolase gene provided in the embodiment of the present invention.

[0043] Figure 3 Figure 1 shows the reduced and non-reduced SDS-PAGE electrophoresis analysis of the novel allergen fructose bisphosphate aldolase from purified pollen of Artemisia grandis, Artemisia annua, and Humulus japonicus. Art si FBA, Art an FBA, and Hum j FBA represent the novel allergen fructose bisphosphate aldolase from pollen of Artemisia grandis, Artemisia annua, and Humulus japonicus, respectively. Nat and Rec correspond to the native and recombinant forms of the allergen protein, respectively. +βME and -βME correspond to the SDS-PAGE electrophoresis analysis of the respective forms under reduced and non-reduced conditions, respectively.

[0044] Figure 4 include Figure 4 A, 4B, Figure 4 A is the dot-blot result of the interaction between Artemisia spp. pollen extract (Art si CE), natural fructose bisphosphate aldolase (nArt si FBA), and recombinant fructose bisphosphate aldolase (rArt si FBA) and sera IgE from 20 patients allergic to Artemisia pollen; Figure 4 B is Figure 4The results of immunoblotting of serum that produced positive IgE binding spots to fructose bisphosphate aldolase in A. In the figure, Nat and Rec correspond to the natural (nArt si FBA) and recombinant (rArt si FBA) forms of the allergen protein, respectively.

[0045] Figure 5 Figure 1 shows the results of an immunoglobulin (IgE) cross-reactivity study against the novel allergen fructose bisphosphate aldolase (FBAP) in pollen from Artemisia annua, Artemisia annua, and Humulus japonicus. Figure A shows the degree of inhibition of IgE binding to an Artemisia annua pollen extract (Art an CE), Humulus japonicus pollen extract (Hum j FBA), recombinant Artemisia annua pollen novel allergen fructose bisphosphate aldolase (rArt an FBA), and recombinant Humulus japonicus pollen novel allergen fructose bisphosphate aldolase (rArt an FBA) on an ELISA plate coated with Artsi CE. Figure B shows the degree of inhibition of recombinant Artemisia annua pollen new allergen fructose bisphosphate aldolase (rArt an FBA), recombinant Artemisia annua pollen new allergen fructose bisphosphate aldolase, and recombinant Humulus ulmoides pollen new allergen fructose bisphosphate aldolase on IgE binding to the recombinant Artemisia annua pollen new allergen fructose bisphosphate aldolase (coated with rArt an FBA) adsorbed on the ELISA plate. Figure C shows the degree of inhibition of recombinant Artemisia annua pollen new allergen fructose bisphosphate aldolase, recombinant Artemisia annua pollen new allergen fructose bisphosphate aldolase, and recombinant Humulus ulmoides pollen new allergen fructose bisphosphate aldolase on IgE binding to the recombinant Artemisia annua pollen new allergen fructose bisphosphate aldolase (coated with rArt an FBA) adsorbed on the ELISA plate. Figure D shows the degree of inhibition of recombinant fructose bisphosphate aldolase from Artemisia grandiflora pollen, recombinant fructose bisphosphate aldolase from Artemisia annua pollen, and recombinant fructose bisphosphate aldolase from Humulus japonicus pollen on IgE binding to recombinant fructose bisphosphate aldolase (coated with rHum j FBA) adsorbed on the ELISA plate.

[0046] Figure 6 The expression level of CD63 on the surface of basophils after activation of patients' basophils by fructose bisphosphate aldolase, a new allergen in Artemisia grandiflora, Artemisia annua, and Humulus japonicus pollen. Figure 6 A is a representative graph of flow cytometry detection of basophils in the peripheral blood of patients after activation using phosphate buffered saline (PBS) as a negative reference, anti-IgE as a positive reference, Artemisia sibiricum pollen new allergen fructose bisphosphate aldolase (Art si FBA), Artemisia annua pollen new allergen fructose bisphosphate aldolase (Art an FBA) and Humulus japonicus pollen new allergen fructose bisphosphate aldolase (Hum j FBA). Figure 6B The ratio of activated basophils after three replicate measurements. DETAILED DESCRIPTION

[0047] Pollen allergen protein

[0048] The pollen allergen protein of the present invention is a protein selected from the following (a) to (d).

[0049] (a) a protein consisting of the amino acid sequence shown in SEQ ID NOs: 1 to 3;

[0050] (b) proteins having allergenic activity or low allergenic activity, wherein the amino acid sequence in (a) has been substituted, modified, deleted or added with one or more amino acids;

[0051] (c) a protein having allergenic activity consisting of an amino acid sequence having at least 90% identity with the amino acid sequence in (a);

[0052] (d) An allergen protein that is immunologically cross-reactive with the protein composed of amino acids represented by SEQ ID NOs: 1 to 3 in (a).

[0053] The pollen allergen protein of the present invention includes proteins comprising an amino acid sequence in which one or more amino acids are substituted, deleted, or added to the amino acid sequence of SEQ ID NOs: 1 to 3, as long as it has pollen allergen activity ((b) above). Examples of pollen proteins comprising such an amino acid sequence include isoforms of the pollen proteins having the amino acid sequences of SEQ ID NOs: 1 to 3.

[0054] The multiple amino acids to be deleted, substituted or added are, for example, 1 to 10, more preferably 1 to 5. The addition or deletion mentioned above also includes the addition or deletion of 1 to several amino acids at both ends.

[0055] Here, "pollen allergen activity" includes not only activity that binds to IgE on mast cells and / or basophils to induce immediate allergic reactions in atopic humans (De Weck, AL. et al., Int. Arch. Allergy Immunol., 146: 177-189, 2008), but also activity that only binds to IgE in serum.

[0056] Furthermore, the pollen allergen protein of the present invention includes proteins having 90% or more identity with the amino acid sequences of SEQ ID NOs: 1 to 3 when the corresponding sequences are appropriately aligned, as long as they have pollen allergen activity ((above (c)).

[0057] The identity with the amino acid sequences shown in SEQ ID NOs: 1 to 3 is preferably 95% or greater, more preferably 98% or greater. The amino acid sequence identity can be calculated, for example, using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) with optional parameters set to initial values.

[0058] Furthermore, the pollen protein of the present invention may be fused with a protein or the like to form a sequence useful for purification, such as multiple amino acid residues, to ensure stability during recombinant production.

[0059] Polynucleotide encoding pollen allergen protein

[0060] The polynucleotide of the present invention is a polynucleotide encoding the above-mentioned pollen allergen protein, and preferred examples include (2a) a polynucleotide comprising the base sequence shown in SEQ ID NOs: 4 to 6; (2b) a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NOs: 4 to 6, which hybridizes under stringent conditions and encodes a protein having Artemisia or Humulus pollen allergen activity; and (2c) a polynucleotide comprising a base sequence having 90% or greater identity with the base sequence shown in SEQ ID NOs: 4 to 6, which encodes a protein having Artemisia or Humulus pollen allergen activity and immunological cross-reactivity.

[0061] The polynucleotides (2b) and (2c) include variants of the polynucleotide (2a), including natural allele variants or naturally occurring variants that can be generated using mutagenesis techniques known in the art.

[0062] The polynucleotide of the present invention not only comprises double-stranded DNA, but also comprises various single-stranded DNA or RNA such as the sense strand and antisense strand constituting the same. The antisense strand can be used as a probe, etc. DNA comprises cDNA or genomic DNA etc. obtained by, for example, cloning or chemical synthesis technology or a combination thereof. In addition, for the polynucleotide of the present invention, in addition to the base sequence encoding the polypeptide of the present invention, the base sequence of the sequence of the untranslated region (UTR) or the vector sequence (including expression vector sequence) etc. can also be added.

[0063] Here, stringent conditions include, for example, the conditions described in Molecular Cloning: A Laboratory Manual (Second Edition, J. Sambrook et al., 1989).

[0064] Furthermore, the identity with the base sequences represented by SEQ ID NOs: 4 to 6 is preferably 95% or higher, more preferably 98% or higher.

[0065] Regarding the identity of the base sequences, for example, a method of calculating using BLAST with optional parameters set to initial settings can be applied.

[0066] Obtaining polynucleotides encoding pollen allergen proteins

[0067] The polynucleotide encoding the pollen protein of the present invention can be obtained by cloning the corresponding pollen. Examples of cloning methods include methods using known methods such as the shotgun method and the PCR method.

[0068] For example, a probe that specifically hybridizes to a portion of the base sequence of the polynucleotide of the present invention can be prepared and used to screen a genomic DNA library or a cDNA library. As such a probe, any sequence and length can be used as long as it is a probe that specifically hybridizes to at least a portion of the polynucleotide involved in the present invention or its complementary chain. Methods for artificially synthesizing polynucleotides can also be cited (Kosuri S et.al., Nature Methods 11, 499-507, 2014).

[0069] In addition, by using appropriate principles, a sequence that hybridizes with a polynucleotide comprising a portion or all of the polynucleotide of the present invention can also be obtained. For example, a PCR method using a polynucleotide comprising a portion of the polynucleotide of the present invention as a primer or a method using a polynucleotide comprising a portion of the polynucleotide of the present invention as a probe can be mentioned.

[0070] For example, with respect to methods using amplification methods such as PCR, primers are prepared from the sequences on the 5' side and 3' side of the polynucleotide of the present invention (or its complementary sequence), respectively. Using these primers, genomic DNA (or cDNA) is used as a template, and an amplification reaction such as PCR is carried out to amplify the DNA region sandwiched between the two primers, thereby being able to obtain a large amount of DNA fragments containing the polynucleotide.

[0071] Furthermore, the polynucleotide provided by the present invention can also be produced by modifying a polynucleotide comprising the base sequence shown in SEQ ID NOs: 4 to 6 by, for example, a planned or random mutagenesis method.

[0072] Among them, the planning of the variation when introducing variation in a planned manner can be carried out, for example, with reference to the characteristic sequence on the polynucleotide sequence. In addition, as a method for randomly introducing variation, for example, PCR method and method using mutagen treatment can be cited. As a method for introducing variation in a planned manner, site-directed mutagenesis method can be cited, more specifically, for example, Site-Directed Mutagenesis System Mutan-Super Express Km kit (Takara Bio) can be used. In addition, recombinant PCR method (PCR protocols, Academic Press, New York, 1990) can also be used.

[0073] Preparation of pollen allergen protein

[0074] The pollen protein of the present invention can be obtained by separation and purification of various pollen extracts. The separation and purification method is not particularly limited, and the pollen extract can be separated and purified using currently known methods such as gel filtration, ion exchange chromatography, affinity chromatography, and hydrophobic chromatography.

[0075] Alternatively, a recombinant vector prepared by inserting the polynucleotide of the present invention into an appropriate vector may be introduced into host cells to express the pollen allergen protein intracellularly or extracellularly and collect the protein.

[0076] The host is not particularly limited as long as it is a living cell that can be transformed, and examples include bacteria such as Escherichia coli and Bacillus subtilis, fungi such as yeast and filamentous fungi, cultured insect cells such as Sf9 cells, insects such as silkworms, animal cells, plants, or cells derived from plants.

[0077] The vector for inserting the polynucleotide of the present invention is not particularly limited as long as it can replicate in the above-mentioned host, and can be appropriately determined depending on the type of host to be introduced, the method of introduction, and the like.

[0078] For example, plasmid DNA, phage DNA, viral vectors, etc. can be cited. The vector DNA used for the construction of the expression vector can use a widely available and easily accessible vector. For example, pTV118N (Takara Bio), pMAMneo (Clontech), pGEX (GE Healthcare), pET160 (Thermo Fisher Scientific), pDEST (ThermoFisher Scientific), pIEx (Merck Millipore), pBacPAK (Takara Bio), etc. can be cited. In addition, as viral vectors, for example, baculovirus vectors, retrovirus vectors, lentivirus vectors such as human immunodeficiency virus (HIV), adenovirus vectors, adeno-associated virus vectors (AAV vectors), herpes virus, vaccinia virus, pox virus, polio virus, Sindbis virus, Sendai virus, simian virus-40 (SV-40) and other DNA viruses or RNA viruses can be cited.

[0079] Transformation of a host using this recombinant vector can be performed using methods such as protoplasting, competent cell methods, and electroporation. The resulting transformants can be cultured under appropriate conditions using a culture medium containing a suitable carbon source, nitrogen source, metal salt, vitamin, etc. Protein can be collected and purified from the culture solution obtained using conventional methods to obtain the pollen allergen protein of the present invention. For example, when using Escherichia coli as a host, methods described in the PET System Manual, 10th edition (Novagen) can be used.

[0080] Preventive or therapeutic agent for allergic diseases caused by pollen

[0081] The pollen allergen protein of the present invention can be used as a preventive or therapeutic agent for allergic diseases caused by pollen, and can be administered to a person (patient) in need thereof to prevent or treat allergic diseases caused by pollen.

[0082] Examples of allergic diseases caused by pollen include all allergic diseases caused by pollen-specific antigens, and specific examples include atopic bronchial asthma, allergic rhinitis, allergic conjunctivitis, and atopic dermatitis.

[0083] Such a preventive or therapeutic agent for allergic diseases caused by pollen can be used as, for example, a desensitizing therapeutic agent for allergic diseases caused by pollen.

[0084] The results of amino acid identity analysis using BLAST revealed that the pollen allergen protein of the present invention shared no identity with any of the various allergens reported to be associated with the onset of hay fever. This suggests that since the protein is a different protein from currently known allergens, more effective allergy-reducing treatment may be possible by combining it with these allergens.

[0085] When the preventive or therapeutic agent for allergic diseases caused by pollen of the present invention is used as a hyposensitization therapeutic agent, it is preferred that the pollen allergen protein of the present invention be used directly or dried into a powder form, or prepared as a formulation by adding conventional adjuvants and various additives such as stabilizers, excipients, solubility aids, emulsifiers, buffers, analgesics, preservatives, colorants, etc. as needed by conventional methods.

[0086] For example, powdered purified pollen allergen protein can be dissolved in phenol-added physiological saline to use as a stock solution of an antigen for desensitization therapy.

[0087] The preventive or therapeutic agent for allergic diseases caused by pollen of the present invention can be administered by conventional administration routes, such as transdermal, oral, intradermal, subcutaneous, intramuscular, and intraperitoneal administration. Furthermore, it can be used as a transdermal or transmucosal drug, such as a buccal tablet, sublingual tablet, eye drop, nasal spray, paste, cream, or lotion.

[0088] The dosage and frequency of the preventive or therapeutic agent for allergic diseases caused by pollen of the present invention vary depending on the route of administration, symptoms, etc., and are appropriately selected, for example, to be within the range of about 0.1 to 1000 μg per dose for adults, and administered approximately once to several times per week.

[0089] Diagnosis of allergic diseases caused by pollen

[0090] The pollen allergen protein of the present invention can be used as a diagnostic drug for allergic diseases caused by pollen, and can be used for diagnosing allergic diseases caused by pollen.

[0091] Such diagnostic drugs for allergic diseases caused by pollen can be used, for example, as intradermal or prick test agents for diagnosing skin reactions to pollen-related allergic diseases. Furthermore, by preparing a specific IgE antibody test agent, diagnosis using patient serum or plasma is possible (Chen H et al., Allergy, Asthma & Immunology Research. 13:177-205, 2021).

[0092] When used as a skin reaction diagnostic agent, the pollen allergen protein of the present invention obtained by the above-mentioned method can be dried into a powder, dissolved in physiological saline containing phenol or glycerol, and diluted for use. Examples of agents for detecting specific IgE antibodies include methods in which IgE antibodies in a patient's test subject are bound to the pollen allergen protein of the present invention in an aqueous phase or on a solid phase, and detection is performed based on principles such as fluorogenic enzyme immunoassay, chemiluminescent enzyme immunoassay, and enzyme immunoassay, but the detection method is not limited thereto.

[0093] Antibodies to pollen allergen proteins

[0094] The antibodies against the pollen allergen proteins of the present invention are antibodies that can specifically bind to the pollen allergen proteins of the present invention. These antibodies refer to immunoglobulins (IgA, IgD, IgE, IgG, IgM, and their Fab fragments, F(ab')2 fragments, and Fc fragments), and examples thereof include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single-chain antibodies, anti-idiotype antibodies, and humanized antibodies.

[0095] The above-mentioned antibodies can be produced using various known methods, and the production method is not particularly limited.

[0096] The antibodies can be used to identify organisms, tissues, or cells expressing the pollen allergen protein of the present invention. For example, they can be used to determine the presence of the pollen allergen protein in the atmosphere, indoor spaces, or human mucous membranes. This determination can be performed using known immunological methods, such as ELISA.

[0097] Example 1 Purification and identification of new natural allergen proteins in pollen

[0098] (a) Preparation of Artemisia grandiflora pollen extract

[0099] To 40 g of Artemisia grandis pollen, add 400 ml of extraction buffer (10 mM sodium phosphate, pH 7.2) and, simultaneously, add the protease inhibitor PMSF at a final concentration of 1 mM. Mix thoroughly and extract overnight in a vertical mixer at 4°C. The next day, centrifuge the mixture (12,000 g, 20 minutes) and recover the supernatant. Filter the supernatant through a 0.22 μm microporous filter membrane to obtain the Artemisia grandis pollen extract.

[0100] (b) Purification of natural fructose bisphosphate aldolase (nArtsiFBA) from Artemisia grandiflora pollen

[0101] The extract of Artemisia grandis pollen is used as raw material and separated and obtained through the combined chromatography steps of anion exchange, gel filtration and cation exchange. Specifically, first, 10 ml of the Artemisia grandiflora pollen extract solution was replaced with a 20 mM Tris-HCl, pH 8.4 buffer solution using 5 × 5 HiTrap Desalting columns (Cytiva Lifesciences, Catalog No. 17140801) in series, and this process was repeated several times until the 400 ml extracted in (a) was completely replaced. The collected replacement fractions were adsorbed on a HiTrap Q HP column (Cytiva Lifesciences, Catalog No. 17115301) equilibrated with the same buffer solution. The adsorbed protein was eluted with 20 mM Tris-HCl, pH 8.4, containing 1 M sodium chloride. The elution conditions were as follows: a flow rate of 5 ml / min, and a concentration of 0% to 30% of 1 M sodium chloride in 20 mM Tris-HCl, pH 8.4 was increased in a volume of 270 ml, and then increased to 100% in 25 ml. The elution results are shown in the chromatogram and SDS-PAGE detection results. Figure 1 A. The black-boxed fractions were combined and further separated on a Superdex G75 increase10 / 300GL column (Cytiva Lifesciences, Cat. No. 29148721). Elution was performed at a flow rate of 0.7 ml / min using 50 mM phosphate buffer, pH 7.2, containing 150 mM sodium chloride. The eluted fractions were collected in sections. The elution results and SDS-PAGE analysis results are shown in Figure 2. Figure 1 B. The combined solution of the black-boxed fractions was exchanged into 20 mM MES, pH 5.0 buffer. The collected fractions were adsorbed onto a RESOURCE S column (Cytiva Lifesciences, Catalog No. 17117801) equilibrated with the same buffer. The adsorbed protein was eluted with 20 mM MES, pH 5.0, containing 1 M sodium chloride. The elution conditions were: a flow rate of 4 ml / min, and a concentration of 1 M sodium chloride in 20 mM MES, pH 5.0 was increased from 0% to 50% in a 40 ml volume, followed by a 100% increase. The elution results and SDS-PAGE analysis are shown in the figure. Figure 1 C, the part marked by the black box is the purified fructose bisphosphate aldolase from Artemisia macroseed pollen, and its purity in SDS-PAGE was >90% (purity calculated using ImageJ).

[0102] Tracking IgE binding activity during purification: Fractions from each purification step (anion exchange chromatography, size exclusion chromatography, and cation exchange chromatography) were separated by Laemmli polyacrylamide gel electrophoresis (SDS-PAGE) at 70 V for 30 min, followed by 110 V until the end of the electrophoresis. The gel was stripped and transferred to a PVDF membrane (Merck Millipore, Cat. No. IPVH00010) using the Towbin transfer system described in the Protein Blotting Guide (Bio-Rad). Following transfer, the membrane was blocked with 5% nonfat dry milk in phosphate saline for 2 h at room temperature. The membrane was then incubated with patient serum (100 μl of serum diluted in 900 μl of phosphate saline containing 1% BSA and 0.05% Tween 20) and hybridized overnight at 4°C with shaking. On the second day, the incubated strips were removed and washed three times in phosphate-buffered saline (PBST) containing 0.05% Tween 20 for 10 min each time. 2 μl of horseradish peroxidase-labeled anti-human IgE antibody (Seracare, Catalog No. 5210-0158) was then diluted to 20 ml of phosphate-buffered saline containing 1% BSA and 0.05% Tween 20. After incubation at room temperature for 1 h, the strips were washed three times with PBST and developed using Immobilon Western HRP substrate (Merck Millipore, Catalog No. WBKLS0500) on a Tianneng 5200 multifunctional imaging system.

[0103] (c) Identification of partial internal amino acid sequences

[0104] First, the purified Artemisia selengensis pollen fructose bisphosphate aldolase was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue G250 staining. Then, in order to clarify the partial amino acid sequence inside the protein of the present invention, the fragments obtained by restriction digestion with protease were measured by mass spectrometry using Orbitrap Fusion (Thermo Fisher Scientific, MA, USA). The mass spectrometry results were matched by pFind Studio in combination with the Artemisia selengensis pollen transcript data. The transcript data was extracted by extracting Artemisia selengensis pollen mRNA, reverse transcribed into cDNA and RNA-seq was performed using Illumina Hiseq2000, and protein data was constructed based on the results to match the mass spectrometry results. The obtained peptides are consistent with the gene and amino acid sequences of Artemisia selengensis pollen fructose bisphosphate aldolase as shown in Figure 2. Figure 2A As shown, the internal peptide coverage was 86.83%.

[0105] Subsequently, the internal peptide identification of fructose bisphosphate aldolase in the extracts of Artemisia annua pollen and Humulus japonicus pollen was further carried out. Artemisia annua pollen extract and Humulus japonicus pollen extract were prepared according to the pollen extract preparation method as described above, and were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, and the protein bands near 38kDa were cut out for mass spectrometry. The transcript data were obtained by extracting the mRNA of Artemisia annua and Humulus japonicus pollen respectively, reverse transcribed into cDNA and using Illumina HiSeq2000 for RNA-seq, and protein data were constructed based on the results to match the mass spectrometry results. The peptides obtained from the fructose bisphosphate aldolase in Artemisia annua pollen are consistent with its gene and amino acid sequences as shown in Figure 2. Figure 2B As shown in Figure 2, the internal peptide coverage rate is 68.07%. The peptides obtained from the fructose bisphosphate aldolase from Humulus pollen are compared with its gene and amino acid sequence as shown in Figure 2. Figure 2C As shown, the internal peptide coverage was 23.74%.

[0106] Example 2 Cloning of polynucleotides encoding pollen allergen proteins

[0107] RNA was extracted from pollen of Artemisia grandis, Artemisia annua, and Humulus ulmoides using the MiniBEST Plant RNA Extraction Kit (Baoriyi Biotechnology Co., Ltd., Cat. No. 9767) according to the manufacturer's manual. Reverse transcription was performed to obtain cDNA using PrimeScript RT Master Mix (Baoriyi Biotechnology Co., Ltd., Cat. No. RR036Q) according to the manufacturer's manual. Based on the theoretical gene sequence of the novel allergen fructose bisphosphate aldolase from pollen of Artemisia grandis, Artemisia annua, and Humulus ulmoides, PCR amplification of the sequence was performed using Takara ExTaq amplification enzyme (Baoriyi Biotechnology Co., Ltd., Cat. No. RR001A) and the manufacturer's manual in a 25 μL system: ExTaq enzyme 5 U / μL (0.125 μL), 10× ExTaq buffer (2.5 μL), dNTP mix (2 μL), pollen cDNA product (1 μL), upstream and downstream primer mix (1 μL), and sterile distilled water to 25 μL. The upstream primer used for polynucleotide amplification of Artemisia grandiflora pollen allergen protein is 5'-TCAACCAGCCAACCAGCCAC-3', and the downstream primer is 5'-CCTTCACAAAACACGGAACACAGTG-3'; the upstream primer used for polynucleotide amplification of Artemisia annua pollen allergen protein is 5'-TTCTCCTCCTCCTCTTTCAATAC-3', and the downstream primer is 5'-AAACACGGAACACAGTGAAG-3'; the upstream primer used for polynucleotide amplification of Humulus japonicus pollen allergen protein is 5'-CGCCACCAAAAGTTCTTTCACTG-3', and the downstream primer is 5'-GACATGAAAGATAATCCCCAAAGAGC-3'. All the above primers were commissioned to be synthesized by GenScript Biotech. The PCR conditions were 98°C pre-deformation / 5s (1 cycle), 98°C denaturation / 10s, 55°C annealing / 30s and 72°C extension / 1min (30 cycles), 72°C re-extension / 10min (1 cycle). The PCR amplification product was recovered, purified and ligated into the pCE2 TA / Blunt-zero plasmid vector (Novozymes Biotech Co., Ltd., Catalog No. C601-01), and transformed into JM109 competent cells (Baori Biotech Co., Ltd., Catalog No. 9022). Positive clones were screened on Luria-Bertani (LB) plates containing 100 μg / mL kanamycin and confirmed by DNA sequencing. Final results: The polynucleotide encoding the pollen allergen protein of Artemisia grandiflora pollen contains a 1074 bp open reading frame, encoding 357 amino acids. Its nucleotide sequence (SEQ ID NO.4) and amino acid sequence (SEQ ID NO.1) are shown in Figure 4. Figure 2AThe polynucleotide encoding the pollen allergen protein of Artemisia annua contains a 1074 bp open reading frame encoding 357 amino acids. Its nucleotide sequence (SEQ ID NO.5) and amino acid sequence (SEQ ID NO.2) are shown in Figure 2B The polynucleotide encoding the pollen allergen protein of Humulus truncatus contains an open reading frame of 1077 bp, encoding 358 amino acids, and its nucleotide sequence (SEQ ID NO.6) and amino acid sequence (SEQ ID NO.3) are shown in FIG. Figure 2C shown.

[0108] Example 3 Purification of Novel Recombinant Allergen Protein from Pollen

[0109] Using the ClonExpress II One-Step Cloning Kit (Novozymes Biotech, Cat. No. C112-01) and following the manufacturer's instructions, the nucleotide sequences encoding the fructose bisphosphate aldolase enzymes from Artemisia grandis, Artemisia annua, and Humulus truncatula pollen were cloned and ligated between the NcoI and XhoI sites of the pET28a+ plasmid. The recombinant allergen plasmids were heat-shock transformed into BL21(DE3) host bacteria (Novozymes Biotech, Cat. No. C504-02) for plating. Single colonies were selected and cultured in liquid medium at 37°C. IPTG (Bao Ri Yi Yi Biotechnology Co., Ltd., Cat. No. 9030) was added to a final concentration of 1 mM and cultured at 37°C for 6 h. The cells were then collected by centrifugation and lysed using a 70% power ultrasonic disruptor. The lysate was purified using a HisTrap column (Cytiva Lifesciences, Catalog No. 17524801). The purified product was further purified using an anion exchange column HiTrapQHP. The purified recombinant allergen protein was analyzed by SDS-PAGE. The results are shown in Figure 2. Figure 3 ArtsiFBA is a new allergen fructose bisphosphate aldolase from Artemisia grandiflora pollen, ArtanFBA is a new allergen fructose bisphosphate aldolase from Artemisia annua, HumjFBA is a new allergen fructose bisphosphate aldolase from Humulus japonicus pollen, +βME indicates that the protein sample was treated with β-mercaptoethanol, -βME indicates that the protein sample was not treated with β-mercaptoethanol, Nat(n) corresponds to the natural form of the protein, and Rec(r) corresponds to the recombinant form of the protein.

[0110] Example 4 Detection of the Allergenic Activity of the Refined Novel Allergen Protein

[0111] (a) Dot blot assay of the binding activity of serum IgE from pollen allergy patients to novel allergen proteins from Artemisia grandiflora pollen

[0112] Artemisia grandis pollen extract (ArtsiCE), native fructose bisphosphate aldolase from Artemisia grandis pollen (nArtsiFBA), recombinant fructose bisphosphate aldolase from Artemisia grandis pollen (rArtsiFBA), and bovine serum albumin (BSA) were spotted onto nitrocellulose membranes using a microarray format, at a protein concentration of 1 mg / ml at 2 μl / spot. The samples were then allowed to dry naturally at room temperature. The spotted nitrocellulose membranes were then blocked with 1 mg / ml BSA in phosphate buffer and gently shaken for 2 hours at room temperature. Twenty sera from patients allergic to Artemisia pollen (from the First Affiliated Hospital of Nanjing Medical University) were diluted 10-fold in 1 mg / ml BSA in 0.05% Tween 20 in phosphate buffer. The spotted nitrocellulose membranes were cut and numbered, and hybridized with the diluted serum samples at 4°C. The following day, the hybridized nitrocellulose membranes were washed and incubated with a 1:10,000 dilution of goat anti-human IgE-horseradish peroxidase conjugate at room temperature for 1 hour. After washing three times, specific detection was performed using the Tianneng 5200 multifunctional imaging system. Figure 4 A. Of the sera from 20 patients with Artemisia allergy, 20 showed positive IgE binding activity to Artemisia grandis pollen extracts, 10 to native fructose bisphosphate aldolase from Artemisia grandis pollen, and the same 10 to recombinant fructose bisphosphate aldolase from Artemisia grandis pollen. Therefore, 50% of the 20 patients with Artemisia pollen allergy were allergic to the novel allergen fructose bisphosphate aldolase, and the purified natural and recombinant forms of the allergen showed high concordance in terms of IgE binding activity.

[0113] (b) Western Blot analysis of the binding activity of serum IgE from pollen allergy patients to the novel allergen protein from Artemisia grandiflora pollen

[0114] 400 μl of natural fructose bisphosphate aldolase from Artemisia grandis pollen (nArtsiFBA) and 400 μl of recombinant fructose bisphosphate aldolase from Artemisia grandis pollen (rArtsiFBA) were mixed with 100 μl of reducing loading buffer and treated in a boiling water bath for 5 minutes. 10 μl of each of the two treated samples were added to a 13% SDS-PAGE gel for electrophoresis. After the electrophoresis, the proteins in the gel were transferred to a PVDF membrane and then blocked with 5% skim milk powder. The serum that had a positive reaction with the new allergen in (a) was diluted in the same way and incubated with the PVDF membrane transferred with the allergen protein at 4°C for hybridization. The remaining processing steps were the same as those shown in (a). For test results, see Figure 4B. Of the 10 sera, 9 showed positive IgE binding bands to native and recombinant fructose bisphosphate aldolase from Artemisia grandiflora pollen, while only 1 showed a negative signal. Therefore, the IgE binding activity of the novel allergen fructose bisphosphate aldolase depends not only on conformational epitopes but also on linear epitopes. (c) Cross-sensitization activity assay of purified novel allergen proteins

[0115] Artemisia grandis pollen extract (ArtsiCE) was diluted to 10 μg / ml using coating solution (15 mM sodium carbonate + 35 mM sodium bicarbonate, pH 9.6) and added to a 96-well adsorbable microtiter plate, 100 μl per well, with three replicates. The serum pools were prepared by mixing the sera with positive IgE reactions to the new allergen in (a) and adding gradient final concentrations (0, 10 -2 , 10 -1 Artemisia annua pollen extract (ArtanCE), Humulus japonicus pollen extract (HumjCE), recombinant fructose bisphosphate aldolase from Artemisia annua pollen (rArtanFBA), and recombinant fructose bisphosphate aldolase from Humulus japonicus pollen (rHumjFBA) were pre-incubated with serum pools at 1, 1, and 10 μg / ml, respectively. The plates were then grouped and added to a blocked ELISA plate. After incubation at 37°C for 1 hour, the plates were washed with phosphate buffer containing 0.05% Tween 20. A 1:2500 dilution of HRP-conjugated goat anti-human IgE antibody was then added. The plates were incubated at 37°C for 1 hour, washed with phosphate buffer containing 0.05% Tween 20, and 150 μl of TMB colorimetric solution (Biyuntian Biotechnology Co., Ltd., Cat. No. P0209) was added for 20 minutes. The reaction was terminated by adding 50 μl of 2 M sulfuric acid solution. The absorbance of each well was read using a Multiskan microplate reader (Thermo Fisher Scientific). The inhibition rate is calculated as follows: (absorbance value of the uninhibited group - absorbance value of the different concentrations of the inhibition group) / (absorbance value of the inhibition group - absorbance value of the healthy human control serum group). Figure 5As shown in A: Artemisia annua pollen and Humulus japonicus pollen extracts (ArtanCE, HumjCE) both inhibit the binding of IgE to Artemisia grandis pollen extract in a concentration-dependent manner. Similarly, new types of allergen proteins from Artemisia annua and Humulus japonicus pollen (rArtanFBA, rHumjFBA) also produce inhibitory effects in a concentration-dependent manner. At 1μg / ml: the inhibition rate produced by Artemisia grandiflora extract was 94.39%; the inhibition rate produced by Artemisia annua pollen extract was 96.91%, and its new type allergen protein monomer was 42.53%; the inhibition rate produced by Humulus japonicus pollen extract was 34.26%, and its new type allergen protein monomer was 28.42%; at 10μg / ml: the inhibition rate produced by Artemisia grandiflora extract was 98.34%; the inhibition rate produced by Artemisia annua pollen extract was 100.75%, and its new type allergen protein monomer was 38.63%; the inhibition rate produced by Humulus japonicus pollen extract was 69.72%, and its new type allergen protein monomer was 22.58%.

[0116] In the same way, the ELISA plate was coated with recombinant fructose bisphosphate aldolase from Artemisia grandis (rArtsiFBA). Recombinant fructose bisphosphate aldolase from Artemisia grandis (rArtsiFBA), recombinant fructose bisphosphate aldolase from Artemisia annua (rArtanFBA), and recombinant fructose bisphosphate aldolase from Humulus japonicus (rHumjFBA) all inhibited IgE binding to rArtsiFBA monomers in a dose-dependent manner, with maximum inhibition rates of 86.16%, 80.71%, and 65.63%, respectively. Figure 5 B.

[0117] In the same way, the enzyme-labeled plate was coated with recombinant fructose bisphosphate aldolase from Artemisia annua (rArtanFBA), recombinant fructose bisphosphate aldolase from Artemisia grandis (rArtsiFBA), recombinant fructose bisphosphate aldolase from Artemisia annua (rArtanFBA), and recombinant fructose bisphosphate aldolase from Humulus japonicus (rHumjFBA), all of which inhibited IgE binding to rArtsiFBA monomers in a dose-dependent manner, with maximum inhibition rates of 96.32%, 84.96%, and 86.37%, respectively. Figure 5 C.

[0118] In the same way, the enzyme-labeled plate was coated with recombinant fructose bisphosphate aldolase from Humulus japonicus (rHumjFBA), recombinant fructose bisphosphate aldolase from Artemisia grandis (rArtsiFBA), recombinant fructose bisphosphate aldolase from Artemisia annua (rArtanFBA), and recombinant fructose bisphosphate aldolase from Humulus japonicus (rHumjFBA). All of them inhibited IgE binding to rArtsiFBA monomers in a dose-dependent manner, with the maximum inhibition rates being 71.59%, 62.99%, and 76.15%, respectively. Figure 5 D.

[0119] This shows that there is a high degree of IgE cross-reactivity between the refined Artemisia annua, Artemisia annua, and Humulus japonicus new allergen proteins.

[0120] (d) Extent of activation of basophils in patients with pollen allergy by purified novel allergen protein

[0121] It is known that CD63 expression is enhanced on the surface of basophils activated by allergen stimulation (Hoffmann HJ et al., Allergy, 70:1393-405, 2015). Based on this principle, purified novel allergen proteins were used to analyze basophil activation in the blood of pollen-allergic patients. Specifically, purified novel Artemisia grandis allergen proteins (ArtsiFBA), Artemisia annua allergen proteins (ArtanFBA), and Humulus japonicus allergen proteins (HumjFBA) were added to peripheral blood cells of pollen-allergic patients treated with red blood cell lysate at final concentrations of 1 μg / ml and 10 μg / ml, respectively. A blank solution (PBS) was used as a negative control, and anti-IgE was used as a positive control. The stimulation system was incubated at 37°C for 30 minutes, and the activation reaction was terminated by adding cold EDTA-PBS. After the reaction, FITC-conjugated anti-CD63 mAb (Biolegend, CA, USA) and PE-conjugated anti-CCR3 mAb (Biolegend, CA, USA) were incubated with the stimulated cells on ice for 20 minutes. After washing, the CCR3-positive cell population was identified by flow cytometry and the expression intensity of CD63 on the cell surface was analyzed. The results showed that ( Figure 6 Compared to a blank solution (PBS), the novel allergen proteins from Artemisia grandis, Artemisia annua, and Humulus japonicus at 1 μg / ml induced the production of 16.93%, 11.03%, and 8.97% of CCR3- and CD63-positive cells, respectively, representing 2.30-, 1.50-, and 1.22-fold increases compared to the blank solution control group. At 10 μg / ml, they induced the production of 22.67%, 20.67%, and 15.27% of CCR3- and CD63-positive cells, respectively, representing 3.08-, 2.80-, and 2.07-fold increases compared to the blank solution control group. This demonstrates the ability of these novel allergen proteins to activate basophils in patients with pollen allergies.

Claims

1. A pollen allergen protein, characterized in that: The amino acid sequence of the pollen allergen protein is shown in SEQ ID NO.

3.

2. A nucleic acid or gene encoding the pollen allergen protein according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid or gene is shown in SEQ ID NO.

6.

3. An expression cassette, a recombinant vector, a recombinant protein, or a recombinant cell, characterized in that: It comprises the nucleic acid or gene according to claim 2.

4. A recombinant bacterium, characterized in that It comprises the nucleic acid or gene according to claim 2.

5. Use of the pollen allergen protein according to claim 1, the nucleic acid or gene according to claim 2, the expression cassette, recombinant vector, recombinant protein, recombinant cell or recombinant bacterium according to claim 3 in the production process or in the finished product for preparing a kit for diagnosing pollen allergic diseases or a drug or pharmaceutical composition for preventing or treating pollen allergic diseases, for content calibration and quality control.

6. The use according to claim 5, characterized in that The pollen is Artemisia pollen and / or Humulus pollen.

7. Any preparation for preventing, diagnosing or treating an allergic disease comprising the pollen allergen protein of claim 1 or the nucleic acid or gene of claim 2 as an active ingredient.

8. A drug for preventing or treating allergic diseases, characterized in that: The medicine is a single or compound preparation, which comprises the pollen allergen protein according to claim 1 and the nucleic acid or gene according to claim 2.

9. The drug according to claim 8, characterized in that The drug is an allergen vaccine.

10. A kit for allergen diagnosis, characterized in that: The kit comprises the allergen protein according to claim 1 and the nucleic acid or gene according to claim 2.

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