KR20210033775A - Compositions for diagnosis of allergy to Fagales plant pollen, diagnosis methods using the same, and composition for prevention or treatment of allergy to Fagales plant pollen - Google Patents

Abstract

The present invention relates to a composition for diagnosing allergy to Fagales plant pollen, a diagnosis method using the same, and a composition for preventing or treating allergy to Fagales plant pollen. More specifically, the present invention provides the composition which provides a diagnostic result with high reliability and validity for allergy to Fagales plant pollen by refining a major allergen of Quercus acutissima, one of the main causes of pollen allergy in Koreans, and thus has an excellent effect in the prevention or treatment of allergy.

Description

Compositions for diagnosis of allergy to Fagales plant pollen, diagnosis methods using the same, and composition for prevention or treatment of allergy to Fagales plant pollen}

It relates to a composition for diagnosing allergy to pollen of an oak tree plant, a method for diagnosing allergy to pollen of an oak tree plant, and a composition for preventing or treating allergy to pollen of an oak tree plant.

Allergic diseases caused by pollen are known to include allergic conjunctivitis, allergic rhinitis, allergic bronchitis, bronchial asthma, skin rash, and anaphylaxis. Representative pollen that causes allergic diseases in Korea are tree pollen such as oak, pine, and birch, and weed pollen such as round ginseng vine, mugwort, and ragweed. Pine pollen, which blows the most in Korea, has a weak allergenicity, while oak pollen, which blows the second most, has a very strong allergenicity. Therefore, oak pollen, which is detected in large quantities between the end of April and May, is known as a representative pollen of the spring season. As a result of an allergic skin test on respiratory allergy patients in Korea, 14.36% of oak trees were positive, 14.27% of pine trees, 13.57% of birch trees, 13.39% of alder trees, 10.04% of beech and mulberry trees, and 8.81% of elm trees, respectively. It was in the order of.

The treatment for pollen allergy includes drug therapy such as antihistamines, leukotriene receptor antagonists, steroids, and nasal mucosa, and immunotherapy that is injected subcutaneously or administered sublingually, starting with the minimum amount of the allergen and gradually increasing the concentration, and does not respond to drugs. Surgical therapy to induce volume reduction is being used for chronic inferior turbinate thickening. However, there are hardly any cases where allergic diseases are cured by the above treatment, and because the molecular properties of pollen are not sufficiently known, diagnosis and treatment are difficult.

Under this technical background, research has been continuously conducted to solve the above-described problems related to diagnosis and treatment of allergies (Korean Patent Registration No. 10-1302483), but it is still insufficient to solve this problem.

It provides novel pathogenic related proteins.

It provides a composition for diagnosing allergy to pollen of an oak tree plant.

Provides a method of providing information necessary for diagnosing allergy to pollen in oak tree plants.

It provides a composition for preventing or treating an allergy to pollen of an oak tree plant.

One aspect provides a novel pathogenesis related protein-10 (PR-10). The protein is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, with the amino acid sequence of SEQ ID NO: 1, Amino acid sequence having at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least 100% sequence identity It may include a polypeptide or a fragment thereof containing.

The above “polypeptide”, “peptide” and “protein” are used interchangeably and include references to polymers of amino acid residues. The terms apply to natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acid. The terms also apply to those polymers containing conservative amino acid substitutions such that the protein remains functional.

The "identity" means the percentage of identity between two polynucleotide nucleic acid sequences or between a polypeptide amino acid sequence. The identity between sequences from one moiety to another moiety can be determined by known techniques in the art. For example, the algorithm BLAST by literature [see Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA by Pearson (Methods Enzymol., 183, 63 (1990)).

The "fragment" may refer to an immunogenic polypeptide capable of inducing an antigen-antibody reaction by reacting with an antibody as a part of the polypeptide rather than the entire polypeptide.

The protein may be derived from an oak tree (Fagales) plant. The oak tree plant may include a plant in the family Fagaceae or Betulaceae. The oak family plants are the genus Quercus , for example, the oak tree ( Quercus accutisima ), the white oak tree (Q. aliena ), the oak tree ( Q. dentata ), the red oak tree (Q. mongolica, Mongolian oak), the Japanese oak tree ( Q. serrata ), oyster oak ( Q. variabilis ), etc., beech ( Fagus ), for example beech ( F. engleriana ), American beech ( F. grandifolia ), etc.), or chestnut ( Castanea ) , For example, chestnut ( C. crenata ), medicinal chestnut ( C. bungeana ) and the like plants. The birch family is Alnus , for example, A. japonica , A. sibilica , etc., Betula , for example, B. platyphylla var. japonica ), Manchurian white birch (B. platyphylla), birch (B. schmidtii), mulbakdal tree (B. davurica) or the like, Hazel (Corylus), for example, bottles hazel (C. hallaisanensis), hazel ( C. heterophylla ), seal hazel (C. sieboldiana var. manshurica ), etc., or hornbeam ( Carpinus ) plants, such as hornbeam ( C. laxiflora ), magpie ( C. cordata ), cypress ( C. turczaninowii ) and the like. The oak tree plant may be an oak tree plant native to Korea.

The protein may have a sequence structure and activity similar to that of pathogenesis related protein (PR)-10.

The "PR" is a protein expressed when a plant is subjected to biological and abiotic stress, and is a signal that induces a defense response. It was classified into 11 types by Van Loon (1994), and recently, the type is gradually increasing. The PR may mean a protein whose concentration rapidly increases when attacked by a pathogen, and has antibacterial activity, stress resistance activity, RNase activity, and the like. Among them, PR-10 is known to be expressed in parsley, peas, potatoes, soybeans, asparagus, or peppers, and Bet v 1-related proteins homologues, ribonucleases, etc. This belongs to the above classification.

The protein may be derived from pollen of an oak tree plant. The protein may be an allergen that causes an allergy to pollen of an oak tree plant.

The "allergen" may mean an antigen that causes allergy or an antigen that causes an allergic disease. When an antibody is produced in vivo due to ingestion or contact of a substance including an allergen substance, an antigen-antibody reaction may occur when the same substance is re-ingested or re-contacted.

Another aspect provides a composition for diagnosing allergy to pollen of an oak tree plant comprising the protein.

The protein, polypeptide or fragment thereof, oak plant, allergen, etc. are as described above.

In the composition, the protein is a polypeptide or fragment thereof comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, and an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 Consisting of a polypeptide or a fragment thereof, or a polypeptide comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 isolated directly from an oak plant, or a fragment thereof. In addition, in the composition, the protein is recombinantly produced using an animal cell, and a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, or a bacterium It may include a polypeptide or a fragment thereof that is recombinantly prepared and includes an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. The protein may include an additional amino acid sequence. The protein may include an additional amino acid sequence at the N-terminus and/or C-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. The additional amino acid sequence may be added to purify the recombinantly produced protein or to apply to affinity column chromatography. The additional amino acid sequence may include histidine. The additional amino acid sequence may include 2 to 18, 3 to 12, 4 to 9, or 6 to 8 histidines. Alternatively, it may include a sequence of 10 to 40, 15 to 30, 18 to 25, or 22 amino acids.

The bacterium may be a microorganism of the genus Escherichia , and may be E. coli . In the above composition, when the protein is recombinantly produced and expressed in bacteria, a nucleotide sequence encoding a polypeptide or a fragment thereof comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1 It can be obtained by introducing a polynucleotide having a bacterium. Or after codon optimization of a nucleotide sequence encoding a polypeptide or a fragment thereof comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, a polynucleotide comprising the nucleotide sequence It can be obtained by introducing into bacteria. The protein may be a recombinant protein obtained by codon-optimizing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 1, and then introducing a polynucleotide containing the nucleotide sequence into E. coli.

The introduction of the polynucleotide into the bacteria may be the introduction of the polynucleotide itself or in the form of an expression cassette. The expression cassette may include all elements necessary for self-expression of the polynucleotide. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. As the vector, an expression vector known in the art may be used. The introduction of the polynucleotide itself may be operably linked to a sequence required for expression in the introduced host cell. The bacterium into which the polynucleotide has been introduced may be a transduced bacterium.

Cultivation of the bacteria for producing the recombinant protein may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those of ordinary skill in the art according to the selected microorganism. The culture method may include batch, continuous, and fed-batch culture, but is not limited thereto. Various methods of culturing microorganisms are disclosed, for example, in Biochemical Engineering, James M. Lee, Prentice-Hall International Editions.

The medium contains various carbon sources, nitrogen sources, and trace element components. Carbon sources that can be used in the culture medium for microorganisms include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, fats such as soybean oil, sunflower oil, castor oil, and coconut oil, palmitic acid, stearic acid, and linoleic acid. It may include fatty acids, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, but is not limited thereto. The carbon sources may be used alone or in combination. Nitrogen sources available for the culture medium for microorganisms include organic nitrogen sources and urea such as peptone, yeast extract, broth, malt extract, corn steep liquor, and soybean meal, and inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. It may include, but is not limited thereto. The nitrogen source may be used alone or in combination. The culture medium for microbial culture may be one containing potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a corresponding sodium-containing salt as a source of phosphorus. In addition, it may contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included in the medium. The medium or individual components for microbial culture may be added to the culture medium in a batch or continuous manner.

In the composition, the protein is an allergen that causes an allergy to pollen of the oak tree plant, so in a biological sample isolated from an individual, the antigen-antibody reaction between the IgE antibody and the protein is confirmed, Pollen allergy can be diagnosed.

The "diagnosis" means identifying the presence or characteristics of a pathological condition. Therefore, the diagnosis of allergy to pollen of the oak tree plant may mean confirming or predicting the possibility of having an allergy to pollen of the oak tree plant or the possibility of having an allergy to pollen of the oak tree plant. .

The "antibody" is a term known in the art and refers to a specific polypeptide or fragment thereof directed against an antigenic site. The IgE antibody is a sensitizing antibody, produced by allergens, produced by plasma cells, and can be attached to receptors on the surface of mast cells or basophils. IgE antibodies may be those that cause anaphylaxis (specific symptoms resulting from an antigen-antibody reaction).

Another aspect provides a kit for diagnosing allergy to pollen of an oak tree plant comprising the composition or protein.

The protein, polypeptide or fragment thereof, composition, oak plant, allergy diagnosis, and the like are as described above.

The kit may be made of a variety of known kits. For example, the kit is an immunoassay kit capable of qualitatively or quantitatively analyzing the reactivity between the protein and the IgE antibody in a biological sample isolated from an individual.

The kit includes an anti-IgE antibody and other antigens (allergens)-antibodies to which the protein-coated solid substrate and a label capable of binding to IgE antibody in a biological sample isolated from an individual and generating a detectable signal are bound ( It may contain reagents necessary to detect IgE) reaction. The solid substrate may be a hydrocarbon polymer (eg, polystyrene and polypropylene, etc.), glass, metal, or gel, and may be a microtiter plate. Labels that generate the detectable signal are chemical substances (e.g., biotin, etc.), enzymes (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, cytochrome P450, etc.) , A radioactive material, a fluorescent material (eg, fluorescein, etc.), a luminescent material, a chemiluminescent material, and a fluorescence resonance energy transfer (FRET), but are limited thereto. no. Various labeling and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. The kit may be manufactured as a plurality of separate packaging or compartments including the above-described components.

Another aspect provides a method of detecting the binding of an IgE antibody to the protein in a biological sample isolated from an individual in order to provide information necessary for diagnosing allergy to pollen of an oak tree plant.

The protein, polypeptide or fragment thereof, composition, oak plant, allergy diagnosis, and the like are as described above.

The "individual" means an individual who wants to confirm or predict whether or not they have an allergy to pollen of an oak tree plant or a possibility of having an allergy to pollen of an oak tree plant. The individual may be a vertebrate, specifically mammals, amphibians, reptiles, birds, etc., may be more specifically mammals, more specifically humans ( Homo sapiens ). The human may be Korean.

The "biological sample" may include a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine isolated from an individual, but is not limited thereto.

The method can be carried out by an immunoassay (antigen-antibody reaction) method. The immunoassay methods include radioactive immunoassay, radioactive immunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, It may include immunofluorescence staining and immunoaffinity purification, but is not limited thereto. Methods of the immunoassay include Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which may be referred to.

For example, when the method is carried out by radioactive immunoassay ^{, antibodies labeled with radioactive isotopes (eg, C 14} , I1 ²⁵ , P ³² and S ^35, etc.) (antibodies that specifically bind to IgE) Is used. In addition, the method may use an antibody to which a label that generates the above-described detectable signal is bound. Or, for example, when the method is performed by ELISA, (i) coating the identified protein on the surface of a solid substrate; (Ii) adding a biological sample isolated from the subject to the coated solid substrate; (Iii) reacting the result of step (ii) with an enzyme-conjugated anti-IgE secondary antibody; And (iv) measuring the activity of the enzyme. The enzyme bound to the secondary antibody may include an enzyme that catalyzes a color development reaction, a fluorescence reaction, a light emission reaction, or an infrared reaction, but is not limited thereto, for example, alkaline phosphatase, β-galactosida Enzyme, horseradish peroxidase, luciferase or cytochrome P450. When alkaline phosphatase is used as the enzyme bound to the secondary antibody, bromochloroindolyl phosphate, nitro blue tetrazolium, naphthol-AS-B1-phosphate or enhanced chemifluorescence (ECF) as a substrate ), and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate) , Resorupine benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (p-phenylenediamine-HCl and pyrocatechol: HYR) , Tetramethylbenzidine (TMB), 2,2'-azine-di[3-ethylbenzothiazoline sulfonate] (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]: ABTS), o-phenyl Substrates such as rendiamine, naphthol/pyronine, glucose oxidase and t-nitroblue tetrazolium (t-NBT) or m-phenazine methosulfate (m-PMS) are available. Can be used.

Providing the information necessary for diagnosing an allergy to pollen of an oak tree plant may be for predicting or diagnosing the relative risk of an allergy to pollen of an oak tree plant. In the above method, the final enzyme activity measurement or signal measurement may be performed according to various methods known in the art. The detection of such a signal indicates that IgE in a biological sample isolated from an individual is bound to the protein, and this information can be used to diagnose allergies to pollen of an oak tree plant.

Another aspect provides a composition for preventing or treating an allergy to pollen of an oak tree plant comprising the protein. The composition may be an immunotherapy composition for preventing or treating an allergy to pollen of an oak tree plant.

The "allergen immunotherapy" refers to a treatment for reducing sensitivity and irritability to a specific allergen by gradually increasing the amount of allergen, a substance causing allergic diseases, from a small amount to an individual for a certain period of time by injection. In other words, it means palliative therapy for the causative allergen. The immunotherapy may be administered subcutaneously and/or sublingually. This immunotherapy consists of two stages: initial therapy that starts at a low concentration and gradually increases the amount of allergen to reach a maintenance dose, and maintenance therapy that lasts for 3 to 5 years. It may be to administer a high concentration of the allergen for 1 day to 1 week, or to administer the allergen at a low concentration for several years. Through immunotherapy in which the protein or composition is administered, it is possible to obtain an effect of preventing or inhibiting the expression of symptoms due to an antigen-antibody reaction upon exposure to an allergen later. That is, since the protein is a causative agent of allergy to pollen of oak plant, it can be used as an immunotherapy agent for preventing or treating allergy to pollen of oak plant.

The composition may be a pharmaceutical composition. The pharmaceutical composition may include 0.0001 to 50% by weight of the active ingredient, the allergen, based on the total weight of the composition. The pharmaceutical composition may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and if necessary, antioxidants, Other conventional additives, such as a buffer solution and a bacteriostatic agent, may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets, and can act specifically on target organs. Thus, a target organ-specific antibody or other ligand may be used in combination with the carrier. Further, it may be preferably formulated according to each disease or component by an appropriate method in the art or by using a method described in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The pharmaceutical composition may be prepared for oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal administration, and the like. The pharmaceutical composition can be administered orally or parenterally during clinical administration, and when administered parenterally, intranasal, sublingual, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, cerebrovascular injection or chest Can be administered by intra-injection. The administered subject is a mammal, eg, a human, a pig, a cow, a horse, a dog, a cat, or an experimental animal, eg, a mouse, rat, rabbit, guinea pig, or hamster. The dosage of the composition used can be adjusted by various parameters, in particular the mode of administration or the duration of treatment. In addition, the range may be adjusted according to the individual's weight, age, sex, health status, diet, excretion rate, and severity of allergy symptoms to pollen of an oak tree plant. Daily dosage is about 0.0001 to 100 mg/kg, about 0.001 to 10 mg/kg, about 0.01 to 10 mg/kg, about 0.01 to 5 mg/kg, about 0.1 to 5 mg/kg, about 0.1 to 3 mg/kg. Kg, about 0.1 to 1 mg/kg, and about 0.5 to 1 mg/kg may be administered once or several times a day.

In one aspect, novel pathogenic related proteins can be used to diagnose allergies to pollen in oak plants. Since the protein is a major allergen of an oak tree, which is one of the causes of pollen allergy to Koreans, the result of diagnosing an allergy to pollen of an oak tree plant using the protein has high reliability and validity. In addition, the protein may be used as a composition for preventing or treating allergies to pollen of an oak tree plant.

Figure 1 is a purified natural Que ac 1 by gel filtration chromatography.
Figure 2 shows the purified natural Que ac 1 and recombinant Que ac 1 SDS-PAGE.
3 shows subtypes of Que ac 1 by separating purified natural Que ac 1 by 2D gel electrophoresis.
Figure 4a shows the polymorphism of the Que ac 1 sequence according to the amino acid sequence, from the 1st amino acid to the 99th or 100th amino acid.
Figure 4b shows the polymorphism of the Que ac 1 sequence according to the amino acid sequence, from 100 or 101 th amino acid to 149 or 150 th amino acid.
5 is a result showing the amino acid sequence of Que ac 1 and the amino acid identity of homologous allergens derived from other plants (Que ac 1 ( from Quercus acutissima , sawtooth oak), Que m 1 (from Quercus mongolica , Mongolian oak), Que a 1 (white oak, Quercus alba , white oak, EU283863 origin), Que s 1 (cork oak, Quercus suber , cork oak, JQ290115 origin), Que r 1 (Lubra oak, Quercus rubra , northern red oak, JQ290112 origin), Bet v 1.0101 (birch, Betula verrucosa , European white birch, Z80099 origin), Cas s 1 (European chestnut, Castanea sativa , European chestnut, FJ390843 origin), Fag s 1 (European beech , Fagus sylvatica , European beech, FJ390847 origin), The c 1 (cacao tree, Theobroma cacao , cacao, XM_007035635 origin), Cor a 1 (Hazelnut, Corylus avellana , European hazelnut, CAA96549 origin), Mal d 1 (apple tree) , Malus domestica , apple, CAA96535 origin), Pru av 1 (sheep cherry tree, Prunus avium , sweet cherry, O24348 origin), Pru ar 1 (apricot tree, Prunus armeniaca , Apricot, O50001 origin)).
Figure 6 shows a comparison of the reactivity to the IgE antibody, (A) is an oak pollen extract and natural Que ac 1 (nQue ac 1), (B) is an oak pollen extract and recombinant Que ac 1 (rQue ac 1 ), (C) compares native Que ac 1 (nQue ac 1) and recombinant Que ac 1 (rQue ac 1).
Figure 7 shows the degree of suppression of IgE antibody respectively by the oak pollen extract, natural Que ac 1 (nQue ac 1), and recombinant Que ac 1 (rQue ac 1).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Purification and analysis of natural Que ac 1 and recombinant Que ac 1

1-1. Collection of serum samples from allergic patients

Serum samples were collected from 24 patients who visited the Allergy-Asthma Center at Severance Hospital in Seoul, Korea. Allergy diagnosis was based on patient history and skin prick test. Specific IgE responsiveness to white oak was determined using ImmunoCAP analysis (ThermaoFisher Scientific). The study was approved by the institution's deliberation committee (4-2017-1197).

1-2. Pollen Allergy Extract Collection

Pollen was collected from oak trees on the campus of Yonsei University. The collected pollen was degreased with ethyl ether, and extracted with phosphate-buffered saline (PBS) for 48 hours at 4°C. Then, the extract was centrifuged at 13,000 g for 15 minutes at 4°C. The supernatant was dialyzed with distilled water using a membrane (cutoff 3,500 Da, Spectrum), filtered through a syringe-filter (0.22 μm, Millipore, Bedford), and lyophilized. Total protein content was determined by Bradford assay.

1-3. Tablets of natural Que ac 1

After purifying the natural Que ac 1 through 3-step chromatography, the sequence was analyzed. Specifically, anion exchange chromatography using DEAE (Diethylaminoethyl cellulose: DEAE) Sepharose CL-6B (GE Healthcare Bioscience) was performed at pH 8.0 of 10 mM Tri-HCl, and the protein was eluted with a 0.5 M NaCl linear gradient. Subsequently, the protein was further purified by hydrophobic interaction chromatography using a 5 mL phenylsepharose column (GE Healthcare Biosciences), and then the protein was 100 mM Tris-HCl, pH 8.0, 1.5 M (NH4) ₂ SO _It was eluted under the conditions of 4. Then, gel filtration was performed in PBS of pH 7.4 using a Sephadex G75 16/60 column (GE Healthcare Biosciences).

As a result, as shown in FIGS. 1 and 2, when the 17 kDa protein was monitored by SDS-PAGE during the purification step, two peaks appeared in the elution pattern of gel filtration chromatography, and the second peak was Que ac 1 was detected.

1-4. Sequence analysis of natural Que ac 1

For sequencing, the purified protein was transferred to a PVDF membrane (0.45 μm, Millipore) and then Edman digested using a Procise 491 HT protein sequencer (Applied Biosystems) at Korea Basic Science Institute Edman degradation).

As a result, the Que ac 1 was a sequence consisting of GVFTHESTSVIPPARLFK, and it was confirmed that it is a multi-amino acid peptide showing polymorphism as shown in Table 1 below. "Amino acid number" in Table 1 below indicates a number according to the sequence of amino acids from the N-terminus of the peptide Que ac 1, and "variant amino acid" refers to a kind of amino acid in which the amino acid of Que ac 1 exhibits polymorphism.

Amino acid number Amino Acids of Que ac 1 Mutated amino acids One Gly Asp 2 Val Glu, Pro, Ile 3 Phe Tyr, Ile 4 Thr Glu 5 His Val 6 Glu Lys 7 Ser Asn 8 Thr Ala 15 Arg Ala

1-5. Amino acid sequence analysis of Que ac 1

It was intended to analyze the amino acid sequence of the purified natural Que ac 1. Specifically, the purified natural Que ac 1 was subjected to 2-dimensional gel electrophoresis. Isoelectric focusing was performed on 11 IPG strips (ReadyStrip IPG Strips, Protean IEF system; Bio-Rad) using a gradient immobilized from pH 4 to 7. Then, 15% SDS-polyacrylamide gel electrophoresis was performed. After excising from the gel for the identification of the spot, electrospray ionization-tandem mass spectrometry and Edman decomposition combined with liquid chromatography were performed. In-gel tryptic digestion for serial mass spectrometry was performed at ProteomeTech (Seoul), and Edman digestion was performed at the Korean Institute of Basic Science.

As a result, as shown in Table 2 and FIG. 3, the purified Que ac 1 was further separated into at least 13 spots in the 2-dimensional gel. Of these, 12 spots were identified as PR-10 molecules by serial mass spectrometry. In addition, six specific spots were identified as PR-10 molecules by Edman digestion.

Spot Diagnostic peptide Protein name Bell Sequence coverage Mascot score Edman disassembly One K.LVASPDGGSIVK.T Pru ar 1 Prunus armeniaca 54 GVITYESEDASVIXPAXLFX (SEQ ID NO: 2) K.SIEIVEGNGGPGTIKK.L Disease resistance response protein DRRG49-C Pisum sativum 10% 27 2 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 65 GVITYESEDASVIPPARLFF (SEQ ID NO: 3) K.LVASPDGGSIVK.T Pru ar 1 Prunus armeniaca 47 3 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 86 K.LVASPDGGSIVK.T Pru ar 1 Prunus armeniaca 37 4 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 9% 46 5 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 10% 324 GVYTHESQETSVIPPAXLFX (SEQ ID NO: 4) 6 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 10% 125 7 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 10% 100 K.LVASPDGGSIVK.T Pru ar 1 Prunus armeniaca 48 8 K.LTFVEDGETK.Y Disease resistance response protein DRRG49-C Pisum sativum 10% 52 GVITTESEDASVIPPAXLFX (SEQ ID NO: 5) K.LVASPDGGSIVK.T Pru ar 1 Prunus armeniaca 51 9 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 115 K.LVASPDGGSIVK.T Pru ar 1 Prunus armeniaca 41 10 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 231 GVYTXESQETXXIPXA (SEQ ID NO: 6) -.AFVLDADNLIPK.K Fra a 1 Fragaria ananassa 23 11 K.SIEIVEGNGGPGTIK.K Disease resistance response protein DRRG49-C Pisum sativum 263 GVYTHESQETSVIPPARLFF (SEQ ID NO: 7) K.LVASPDGGSIVK.T Pru ar 1 Prunus armeniaca 33 12 R.AVVVHADPDDLGK.G Superoxide dismutase Ananas comosus 65 13 K.TVVEVAGGGGGGGGGGGGGENAVR.Q Dynein alpha chain Chlamydomonas reinhardtii 17

In the Edman decomposition amino acid sequence of Table 2, X represents an arbitrary amino acid, and the species of Table 2 are respectively apricot tree ( Prunus armeniaca ), pea ( Pisum sativum), strawberry ( Fragaria ananassa) , pineapple ( Ananas comosus) . it means.

1-6. Polymorphism analysis of Que ac 1 at the RNA level

We tried to analyze the polymorphism of Que ac 1 at the RNA level using reverse transcription polymerase chain reaction (RT-PCR). Specifically, total RNA was isolated from oak pollen using TRIzol reagent (Invitrogen) for RT-PCR. The first strand cDNA was synthesized with 10 μg of total RNA. Subsequently, ExTaq DNA polymerase (Takara) was used to amplify the cDNA encoding Que ac 1 by polymerase chain reaction, and the following table designed based on the N-terminal sequence of natural Que ac 1 and identity allergens as primers The sequence of 3 was used. Among the sequences in Table 3 below, the italicized sequence was introduced for directional replication, and three independent PCRs were performed. The PCR product was cloned into a pET6xHN-N vector (Clonetech Laboratories, Inc. A Takara Bio Company), and the DNA sequence was determined in both directions in BioFact (Daejeon). A total of 242 sequences were analyzed.

In Table 3, Y is C or T, W is A or T, M is A or C, R is A or G, and S is G or C.

Nucleotide sequence Sequence number Forward primer 5'- TAAggCCTCTgTCgAC ATgggTgTYWWCAMTYAYg-3' 8 Reverse primer 5'- CAgAATTCgCAAgCTT TTAgTTgTAgRSATCRgRg-3' 9

As a result, as shown in FIGS. 4 to 5, the results were summarized by repeating at least twice in order to eliminate possible errors due to polymerase chain reaction and sequencing. At least three isoallergens (1.0101, 10201, 10301) exist, and Que ac 1.0101 (SEQ ID NO: 1), the most dominant isoform, was deposited with GenBank under accession number MN201198. Que ac 1.0101 showed 58.1% to 83.0% sequence identity to allergens. In addition, various subtypes showing the polymorphism of Que ac 1, shown in FIGS. 4A and 4B, showed 70% or more sequence identity with the sequence of Que ac 1.0101, and most of them showed 90% or more sequence identity. Meanwhile, 58.1% of Bet v 1 and 73.1% of Que a 1, the most commonly used molecule for diagnosing allergy to pollen of Fagars, were shared.

Example 2. Comparison of IgE reactivity of each allergen

To compare the reactivity of each allergen to the IgE antibody obtained from the patient's serum sample. Specifically, the major subtype of Que ac 1 was transduced into E. coli Rosetta-2 (DE3) (Novagen). Then, 1 mM isopropyl-1-thio-β-galactopyranoside (IPTG) was added to induce protein expression. The recombinant protein was purified from the inclusion body using Ni-NTA resin (Qiagen).

Then, in order to evaluate the IgE reactivity by enzyme-linked immunosorbent assay, Plates were coated with 2 μg/mL of recombinant or native protein in 100 μL/well at 4° C. overnight. The plate was washed, blocking non-specific binding, and incubated with the patient's serum (1:4) for 1 hour. Biotinylated goat anti-human IgE (1:1000) (Vector, Burlingame) and streptavidin (1:1000) (Sigma-Aldrich) conjugated with horseradish peroxidase. (bound) IgE was detected. Color was developed using KPL SureBlueTM Microwell Peroxidase Substrate (1-component) (Kirkegaard & Perry Laboratories, Gaithersburg). 0.5 MH ₂ SO ₄ was added to stop the reaction, and absorbance at 450 nm was measured.

As a result, as shown in Figure 6, the recombinant protein reacted with 91.7% of the IgE antibody in the serum of an oak allergy patient, and the IgE reactivity to the recombinant Que ac 1 and the natural Que ac 1 was the reactivity to the pollen extract. (Pearson correlation coefficient = 0.98 and 0.93), but the IgE reactivity to natural Que ac 1 was strongly correlated with recombinant Que ac 1 (Pearson correlation coefficient = 0.95).

Example 3. Confirmation of IgE inhibition of each allergen

An analysis of inhibition of IgE obtained from the patient's serum sample was performed. Specifically, the plate was coated with 10 μg/mL of pollen extract overnight at 4°C. At the same time, the patient's serum (1: 4) was pre-incubated at 4° C. overnight with various concentrations of allergens. IgE bound with biotinylated goat anti-human IgE (1:1000) as described above was detected. Percent inhibition was calculated as (1-absorbance with inhibitor/absorbance without inhibitor)×100.

As a result, as shown in FIG. 7, when the concentration is 10 μg/mL, recombinant Que ac 1 was able to inhibit 82.6% of the IgE response to pollen extract, whereas natural Que ac 1 could inhibit 92.8%. There were, and pollen extract was able to inhibit 95.5%. Therefore, it was found that the recombinant Que ac 1 can be used as an excellent diagnostic tool for oak allergy, and can also be used for suppression of the allergy.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> Compositions for diagnosis of allergy to Fagales plant pollen, diagnosis methods using the same, and composition for prevention or treatment of allergy to Fagales plant pollen <130> PN128275 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 159 <212> PRT <213> Quercus acutissima <400> 1 Met Gly Val Tyr Asn Tyr Glu Ser Gln Glu Thr Ser Val Ile Pro Pro 1 5 10 15 Ala Arg Leu Phe Lys Ala Phe Val Leu Asp Ser Asp Asn Leu Ile Ser 20 25 30 Lys Val Leu Pro His Ala Val Lys Ser Thr Glu Ile Ile Glu Gly Asn 35 40 45 Gly Gly Pro Gly Thr Ile Lys Lys Phe Thr Phe Gly Glu Ala Ser Lys 50 55 60 Val Lys Tyr Ala Lys His Arg Ile Asp Thr Leu Asp Pro Glu Asn Cys 65 70 75 80 Ser Tyr Ser Phe Ser Val Ile Glu Gly Glu Ala Leu Thr Asp Ile Ala 85 90 95 Ser Ile Ser Thr Glu Val Lys Phe Val Ala Ser Pro Asp Gly Gly Ser 100 105 110 Ile Met Lys Ser Thr Thr Lys Tyr Gln Thr Lys Gly Gly Phe Gln Leu 115 120 125 Lys Glu Glu Gln Ile Gln Ala Ala Val Glu Lys Gly Thr Gly Leu Phe 130 135 140 Lys Ala Val Glu Ala Tyr Leu Leu Ala His Pro Asp Leu Tyr Asn 145 150 155 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Prunus armeniaca <400> 2 Gly Val Ile Thr Tyr Glu Ser Glu Asp Ala Ser Val Ile Xaa Pro Ala 1 5 10 15 Xaa Leu Phe Xaa 20 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pisum sativum <400> 3 Gly Val Ile Thr Tyr Glu Ser Glu Asp Ala Ser Val Ile Pro Pro Ala 1 5 10 15 Arg Leu Phe Phe 20 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pisum sativum <400> 4 Gly Val Tyr Thr His Glu Ser Gln Glu Thr Ser Val Ile Pro Pro Ala 1 5 10 15 Xaa Leu Phe Xaa 20 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pisum sativum <400> 5 Gly Val Ile Thr Thr Glu Ser Glu Asp Ala Ser Val Ile Pro Pro Ala 1 5 10 15 Xaa Leu Phe Xaa 20 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Pisum sativum <400> 6 Gly Val Tyr Thr Xaa Glu Ser Gln Glu Thr Xaa Xaa Ile Pro Xaa Ala 1 5 10 15 <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Pisum sativum <400> 7 Gly Val Tyr Thr His Glu Ser Gln Glu Thr Ser Val Ile Pro Pro Ala 1 5 10 15 Arg Leu Phe Phe 20 <210> 8 <211> 35 <212> RNA <213> Artificial Sequence <220> <223> forward primer <400> 8 taaggcctct gtcgacatgg gtgtywwcam tyayg 35 <210> 9 <211> 35 <212> RNA <213> Artificial Sequence <220> <223> reverse primer <400> 9 cagaattcgc aagcttttag ttgtagrsat crgrg 35

Claims (12)

A polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. The polypeptide according to claim 1 or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 1. The method according to claim 1, wherein the polypeptide is a polypeptide or a fragment thereof derived from a plant of the oak tree (Fagales). The method according to claim 1, wherein the polypeptide is derived from the pollen of an oak tree plant or a fragment thereof. The polypeptide or fragment thereof according to claim 3, wherein the oak tree plant is Sawtooth oak. A composition for diagnosing allergy to pollen of an oak tree plant comprising a polypeptide or a fragment thereof comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. The composition according to claim 6, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. The composition of claim 6, wherein the oak tree plant is an oak tree. A kit for diagnosing allergy to pollen comprising a polypeptide comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. The kit for diagnosing allergy to pollen according to claim 9, wherein the polypeptide or fragment thereof binds to IgE isolated from an allergic patient. To provide information necessary for diagnosing allergy to pollen of an oak tree plant, comprising the step of combining an IgE antibody of a biological sample isolated from an individual with an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 A method of detecting a polypeptide or fragment thereof. A composition for preventing or treating allergy to pollen of an oak tree plant, comprising a polypeptide or a fragment thereof comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1.

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