KR20200129470A - Compositions for diagnosis of allergy disease to silkworm, methods for diagnosing of allergy disease to silkworm, and composition for immunotherapy of allergy disease to silkworm - Google Patents

Abstract

The present invention relates to a composition and kit for diagnosing allergic diseases to silkworm, to a method for diagnosing allergic diseases for silkworm, and to a composition for immune treatment of allergic diseases to silkworm. A novel 30 kDa lipoprotein according to one aspect can be used to diagnose an allergy to silkworm. Since the lipoprotein is a major allergen of a silkworm allergy, a result of diagnosing the allergy to silkworm using the protein has high reliability and validity. In addition, the lipoprotein is used as the composition for immune treatment for preventing or treating the allergy to silkworm.

Description

Compositions for diagnosis of allergy disease to silkworm, methods for diagnosing of allergy disease to silkworm, and composition for immunotherapy of allergy disease to silkworm}

It relates to a composition and kit for diagnosing allergic diseases to silkworms, a method for diagnosing allergic diseases to silkworms, and a composition for immune treatment of allergic diseases to silkworms.

The silkworm ( Bombyx mori ) is known as an industrially useful insect that becomes a pupa from larva and makes silk threads from the cocoon of the pupa. However, silkworm urine, silkworm moth scales, and substances derived from silkworms such as sericin (silk gum) can cause respiratory allergies such as occupational asthma, asthma, and allergic rhinitis.

In addition, pupae are used as dietary supplements and medicines, and are also used for food and animal waste treatment. Approximately 1,500-2,000 species of insects are consumed worldwide as food. Pupa is a traditional food in Korea. Boiled and seasoned pupae are often sold as snacks. In China, grilled pupae are consumed, and dried pupae is used not only as an expectorant, but also to relieve flatulence and muscle spasms.

Allergies to edible insects as described above may seriously affect vulnerable individuals, so caution is required. Meanwhile, pupa is recognized as an important cause of food allergies. In Korea, 9.4% of allergy patients are known to be sensitized to the pupa, and skin reaction tests have been identified as the most frequently sensitized allergens among 62 major food allergens. Among them, allergic reactions to silkworm pupae are serious, and in East Asia, silkworm pupa allergy is a frequent cause of anaphylaxis.

Bomb m 1 (arginine kinase) has been reported to be a major allergen of silkworm pupa allergy (Int Arch Allergy Immunol 2009;150:8-14), and a recent study reported that 27 kDa glycoprotein and tropomyosin are major allergens of silkworm pupa allergy I did. However, no other pupa allergens other than the silkworm allergens have been reported, and the reported allergens are easily denatured with acids and the like because their recognition sites are composed of proteins, so they are not easily denatured by acids and other digestive enzymes as food allergens. Efforts to find allergens that do not exist are continuing.

Accordingly, the present inventors completed the invention by analyzing IgE reactivity in Korean silkworm allergy patients showing various clinical signs, and identifying a 30kDa lipoprotein allergen that is specific for silkworm allergic diseases and is particularly useful for diagnostic purposes for silkworm allergy.

One aspect provides a composition for diagnosing an allergic disease to a silkworm containing a 30kDa lipoprotein of silkworm or a protein region thereof.

Another aspect provides a kit for diagnosing an allergic disease to a silkworm.

Another aspect provides a method of providing information necessary for diagnosing an allergic disease to a silkworm.

Another aspect provides a composition for immune treatment of allergic diseases to silkworms containing a 30kDa lipoprotein of silkworm or a protein region thereof.

One aspect provides a composition for diagnosing an allergic disease to a silkworm containing a 30kDa lipoprotein or a protein region thereof.

Lipoprotein according to an aspect may include a protein region including an amino acid sequence represented by SEQ ID NO: 1. The lipoprotein relates to an allergen derived from silkworm pupa that can be used for diagnosis of silkworm allergy, and by confirming that it binds to IgE antibody in the blood of a patient with silkworm allergy, the 30 kDa lipoprotein is the cause of silkworm allergy (allergen). I did.

The amino acid sequence of the 30 kDa lipoprotein of the silkworm is described in NCBI as GenBank accession No. It is published as NM_001044021. The amino acid sequence corresponds to the first sequence of the sequence listing attached to the present specification.

The lipoprotein may be produced recombinantly using animal cells or bacteria. The protein may contain an additional amino acid sequence. The protein may include, for example, 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, for example, to apply to affinity column chromatography. The bacterium may be a microorganism of the genus Escherichia , and may be, for example, E. coli . The bacterium may be Pichia . In the above composition, when the protein is recombinantly produced and expressed in bacteria, it has a nucleotide sequence encoding a polypeptide or fragment thereof comprising an amino acid sequence having at least 95% sequence homology with the amino acid sequence of SEQ ID NO. It can be obtained by introducing polynucleotides into bacteria. The polynucleotide having the nucleotide sequence may be codon-optimized for bacteria. In a specific aspect, the lipoprotein or protein region thereof may be recombinantly produced using E. coli without post-translational modification.

Introduction of the polynucleotide into bacteria may be, for example, introduction in the form of an expression cassette or introduction of the polynucleotide itself. 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 with 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, cellulose, fats such as soybean oil, sunflower oil, castor oil, 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 microbial culture media 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 appropriate 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.

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 any kind of substance including an allergen substance, an antigen-antibody reaction may occur when re-intake or re-contact of the same substance occurs.

Since the 30 kDa lipoprotein of the silkworm according to one aspect is an allergen that causes silkworm allergy, the reaction between IgE in the blood and the allergen is checked after processing the biological sample to be tested on the lipoprotein or its protein region. Can be effectively diagnosed.

The "diagnosis" means identifying the presence or characteristics of a pathological condition. Accordingly, the diagnosis of an allergic disease to a silkworm may mean confirming or predicting whether to have an allergic disease to a silkworm or a possibility of having an allergic disease to a silkworm.

The term "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 attached to receptors on the surface of mast cells or basophils. IgE antibodies may cause anaphylaxis (specific symptoms resulting from an antigen-antibody reaction).

The composition according to an aspect may include a natural 30 kDa lipoprotein of a silkworm, that is, a 30 kDa lipoprotein directly isolated from a silkworm larva, a silkworm pupa, or a silkworm moth. In a specific example, a protein region of a 30 kDa lipoprotein recombinantly prepared using E. coli was prepared.

It was confirmed that the reactivity of IgE to 30 kDa lipoprotein was not dependent on the protein part by confirming the binding of IgE of the allergic patient even when the protein region was denatured by treating the recombinantly produced 30 kDa lipoprotein. . Therefore, it can be confirmed that the Lipoprotein according to one aspect exhibits IgE reactivity even when the protein region is denatured by the treatment of urea.

When the allergen is expressed in bacterial cells, it can be expressed after codon optimization. After codon optimization of the open reading frame (GenBank accession No. NM_001044021) of 30 kDa lipoprotein of silkworm published in NCBI, it can be successfully expressed and purified in E. coli.

Another aspect provides a kit for diagnosing an allergic disease to a silkworm.

Since the kit includes the composition for diagnosing silkworm allergy, descriptions in common between the two are omitted in order to avoid excessive complexity of the specification.

The kit for diagnosing silkworm allergy may be made of various types of known kits. For example, the kit of the present invention may be prepared as an immunoassay kit capable of qualitatively or quantitatively analyzing the reactivity between a silkworm allergen identified in the present invention and an IgE antibody in a blood sample to be diagnosed.

The kit includes a solid substrate coated with a 30 kDa lipoprotein of silkworm or a protein region thereof, and an anti-IgE antibody in which a label capable of binding to an IgE antibody in a blood sample to be diagnosed and generating a detectable signal is bound. It may contain reagents necessary to detect an antigen (allergen)-antibody (IgE) reaction.

Suitable as said solid substrate are, for example, hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal, gel, or microtiter plates.

Labels that generate the detectable signal are, for example, chemical substances (e.g., biotin), enzymes (alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P450), radioactive substances, fluorescence. A substance (eg, fluorescein), a luminescent material, a chemiluminescent material, and a fluorescence resonance energy transfer (FRET) are included, but are not limited thereto. Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

The kit according to an aspect may be manufactured as a plurality of separate packaging or compartments including the one component.

Another aspect is a method for providing information necessary for diagnosis of an allergic disease to a silkworm, comprising the step of detecting binding of an IgE antibody to a 30 kDa lipoprotein or a protein region thereof in a biological sample isolated from an individual. It provides a way.

A method for diagnosing allergy to a silkworm may be performed by an immunoassay (antigen-antibody reaction) method. The immunoassay methods include, for example, radioactive immunoassay, radioactive immunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, immunofluorescence staining. And immunoaffinity purification, but is not limited thereto.

The biological sample refers to a sample obtained from an organism. The biological sample is, for example, blood, tissue, urine, mucus, saliva, tears, plasma, serum, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary fluid, breast milk, lymphatic fluid, semen , Cerebrospinal fluid, organ system fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof.

Methods of the immunoassay include Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA) , in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is carried out according to a radioactive immunoassay method, an antibody labeled with a radioactive isotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ^35, etc.) (which specifically binds to IgE) Antibodies) can be used. In addition, the method of the present invention can use an antibody to which a label that generates the above-described detectable signal is bound.

For example, when the method of the present invention is carried out by ELISA method, a specific embodiment of the present invention is, (i) coating a 30 kDa lipoprotein or protein region thereof of a silkworm, an allergen identified in the present invention, on the surface of a solid substrate. step; (Ii) treating a blood sample to be diagnosed on 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 "individual" refers to an individual who wants to confirm or predict whether or not to have an allergic disease to a silkworm or a possibility of having an allergic disease to a silkworm. The individual may be a vertebrate animal, specifically mammals, amphibians, reptiles, birds, etc., may be more specifically mammals, for example, humans ( Homo sapiens ). The human may be a Korean.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes that catalyze a color development reaction, a fluorescence reaction, a light emission reaction, or an infrared reaction, and examples include alkaline phosphatase, β-galactosidase, and hose. Radish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme that binds to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate -B1-phosphate) and ECF (enhanced chemifluorescence) are used, 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), HYR (p-phenylenediamine- HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD), naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS Substrates such as (phenzaine methosulfate) may be used.

In the immunoassay method, the final enzyme activity measurement or signal measurement may be performed according to various methods known in the art. The detection of this signal indicates that IgE in the blood sample is bound to the 30 kDa lipoprotein or protein region of the silkworm, and this information can be used for diagnosis of pupa allergy.

Another aspect provides a composition for immune treatment of an allergic disease to a silkworm containing a 30kDa lipoprotein or a protein region thereof.

According to one aspect, since the 30 kDa lipoprotein or protein region of the silkworm is a causative agent of silkworm allergy, it can be used as an immunotherapy agent for pupa allergy.

The "allergen immunotherapy" refers to a treatment for reducing sensitivity and irritability to a specific allergen by gradually increasing the amount of allergen, which is a causative agent of an allergic disease, 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 to the respiratory system. This immunotherapy consists of two stages: initial therapy that starts at a low concentration and gradually increases the amount of allergen to reach the 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 lipoprotein or composition is administered, it is possible to obtain an effect of preventing or suppressing the expression of symptoms due to an antigen-antibody reaction when exposed to an allergen later. That is, since the lipoprotein is a substance that causes allergies to silkworms, it can be used as an immunotherapy for preventing or treating allergic diseases to silkworms.

The composition for immunological treatment of allergic diseases 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 active ingredients described above for administration. Pharmaceutically acceptable carriers may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of 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 can 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. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or by using the method described in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The composition for immunological treatment of allergic diseases 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, intranasally, sublingually, in the respiratory tract, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, cerebrovascular It can be administered by injection or intrathoracic injection. The administered subject may be a mammal, for example, a human, a pig, a cow, a horse, a dog, a cat, or an experimental animal, for example, 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 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 silkworms. The daily dosage may be about 0.0001 to 100 mg/kg, or 0.001 to 10 mg/kg, and may be administered once to several times a day. In the course of the allergic reaction, monitoring the sensitization pattern and profile for the allergen accurately predicts and identifies the clinical symptoms of the patient, thereby enabling more accurate diagnosis and immunotherapy.

The silkworm allergic disease may be an unnecessary immune response, that is, hypersensitivity reaction by an external antigen, an allergen, of anaphylaxis, urticaria, allergic asthma, allergic rhinitis, dermatitis, or a combination thereof.

The formulation of the composition for immunotherapy of one aspect is not largely limited, for example, in the form of a solution, suspension or emulsion in an aqueous or oil medium, or may be in the form of granules, tablets, or capsules, and additionally includes a dispersant or a stabilizer. can do.

A novel 30kDa lipoprotein according to one aspect can be used to diagnose allergy to silkworms. Since the lipoprotein is a major allergen of silkworm allergy, the result of diagnosing the allergy to silkworm using the protein has high reliability and validity. In addition, the lipoprotein may be used as an immunotherapy composition for preventing or treating allergies to silkworms.

1 is a diagram showing the results of performing SDS-PAGE experiments of silkworm pupa extract (FIG. 1A) and results of performing IgE immunoblotting for 13 serum samples (FIG. 1B).
Figure 2 is a result of performing 2D gel electrophoresis by performing the identification of a 30 kDa lipoprotein by LC-MS / MS analysis (Figure 2A) and the result of confirming the IgE-reactive molecule from the pooled serum sample (Figure 2B). Is also.
Figure 3 is a result of performing SDS-PAGE for the glycoprotein of 27kDa, 30kDa lipoprotein, Tropomyosin, Bomb m1, which are four recombinant protein allergens (Figure 3A), and immunoblotting confirming IgE reactivity for a comparative experiment for IgE reactivity It is a figure showing the result (FIG. 3B).
Figure 4 is a diagram showing the results of confirming the IgE reactivity when treated with the serum of a patient allergic to 30kDa lipoprotein treated with 4M urea.
5 is a 30kDa lipoprotein allergen and IgE inhibition immunoblotting analysis of silkworm extract, confirming the IgE response inhibitory effect of silkworm extract (Figure 5A), confirming the IgE response inhibitory effect of urea-treated silkworm extract The results (Fig. 5B) and the results of confirming the inhibitory effect of the 30kDa lipoprotein allergen on the IgE response (Fig. 5C) are shown.

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. Preparation of allergens for diagnosis of pupa allergy in silkworm and preparation of serum samples for confirmation of IgE reactivity

1.1 Preparation of allergen allergen antigen

Frozen silkworms were purchased from Anysilk (www.anysilk.com, Boeun, Chungbuk, Korea). After the purchased frozen silkworm was pulverized in liquid nitrogen, the pulverized product was degreased in 5 volumes with respect to a 1:1 mixture of ethyl ether and ethyl acetate. Then, 6 mM 2-mercaptoethanol, 1:1000 dilution ratio of protease inhibitor cocktail set III (Calbiochem, San Diego, CA, USA) and 1 mg/mL of 1-phenyl-3-(2-thiazyl)-2 Protein was extracted at 4° C. using phosphate buffered saline (PBS) of pH 7.4 containing urea (Sigma-Aldrich, St. Louis, MO, USA). The supernatant was collected after centrifuging at 10,000 g at 4° C. for 30 minutes, filtered through a syringe (0.22 μm, Millipore, Bedford, MS, USA), and maintained at -70° C. until use. Protein concentration was determined by Bradford analysis (Bio-rad, Hercules, CA, USA).

1.2 Subject and serum sample

Serum samples were collected from 17 silkworm allergy patients (ages 24-50 years old, average 35 years old) who visited the Severance Hospital Allergy Asthma Clinic, 11 false-positive patients with allergen sensitization but no symptoms, and 13 normal subjects. Based on the temporal relationship between the intake of silkworm pupae and the onset of skin allergy symptoms and the positive skin prick test for the total extract of silkworm pupa, it was confirmed whether or not the subjects exhibited silkworm pupa allergy, which are shown in Table 1. . The allergic symptoms include urticaria and anaphylaxis, and occurred within 30 minutes after ingestion. Total serum samples of 13 normal subjects (ages 22-72 years old, average 39 years old) who did not have allergic reactions to the silkworm pupa and showed negative reactions to allergens using the skin rash test ImmunoCAP (Phadia, Uppsala, Sweden). Serum samples of kU _A / L or less were used as a negative control and cutoff values were set. In addition, in Table 1 below, the silkworm allergy patient group is represented by group S (S01 to S17), and the group of false-positive patients who were sensitized to the allergen but without symptoms was represented by group A (groups A01 to A11). The study was conducted under the approval of the relevant institutional review committee (4-2013-0397).

number age gender SPT wheal Symptom CAP (kU _A /L) SPT+ S01 40 M ND hives ND S02 25 M ND hives ND S03 39* F ND Anaphylaxis f23 (1.28), f24 (1.48) ND S04 35 F 3.5 hives f23 (0.68), f24 (0.78) ND S05 36 F ND hives f23 (0.06), f24 (0.04) ND S06 37 F 4.5 hives f23 (6.55), f24 (6.84) f4 S07 40 M ND hives f23 (0.00), f24 (0.05) ND S08 25 M ND hives f23 (0.02), F24 (0.05) ND S09 43 M ND Anaphylaxis f23 (11.0), f24 (8.41) ND S10 28 F 8.5 hives f23 (3.18), f24 (3.64), f4 (0.02) Pupa, f23, f24 S11 27 F 4 hives d2 (1.35), i6 (0.64), f23 (0.87), f24 (1.11) d1, i206, pupa S12 39 M 5.5 hives f23 (3.27), f24 (3.27) pupa S13 50 M ND hives d2 (1.79), i6 (2.54) ND S14 25 M ND hives m6 (4.98), d2 (21.9), f23 (6.02), f24 (10.4) ND S15 49 F ND hives d2 (0.58) f24 (2*2), crab (2*2) S16 24 F 4 Anaphylaxis d2 (47.0), e5 (1.17), f23 (22.1), f24 (23.2), f416 (0.06), f423 (0.07) f23, f24, f25, f80, f84, f85, pupa S17 35 F ND hives c2 (0.04), c7 (0.03) ND A01 48 M 3*3 Pupa false positive ND pupa A02 23 M 3*3 Pupa stomach positive f4 (0.00), f416 (0.00), a-gal (0.00) pupa A03 57 F 3*3 Pupa stomach positive i1 (12.0), i2 (0.28), i3 (0.08), i4 (0.01), i5 (0.12), d1 (0.84), d2 (1.03) pupa A04 22 M 3*3 Pupa stomach positive f3 (0.25), f24 (0.13) f3, pollock, stingray, f313, early, eel, f290, f20, f11, sunflower seed, pupa A05 53 F 2*2 Pupa stomach positive t2 (44.6), t3 (69.4), t7 (40.7), w1 (9.39), w6 (0.36), f22 (0.44) f11, f17, f20, f14, f12, f256, sunflower seed, f35, f95, f85, f270, f204), pupa A06 36 M 9*5 Pupa stomach positive f49 (0.66) f17, f20, f85, f214, f59, f24, f80, f26, f23, pupa A07 28 M ND Pupa stomach positive m6 (2.15), d2 (1.57) ND A08 28 M 3*3 Pupa stomach positive f4 (0.10), f416 (1.82) Stingray, trout, f313, f258, octopus, f24, f23, f290, pupa A09 72 F 2*2 Pupa stomach positive d2 (0.50), f4 (0.23), f24 (0.27), f27 (0.03) f25, pupa A10 34 F 2*2 Pupa stomach positive ND octopus, f24, f23, f20, f85, pupa A11 30 F 2*2 Pupa stomach positive f245 (0.04), f83 (0.02), d2 (0.65) pupa

c1, peniciloyl G; c5, ampiciloyl; c7, Sepakler; d1, vertical pattern dust mite ( Dermatophagoides pteronyssinus ); d2, large leg dust mite ; f4, wheat; f23, crab; f24, shrimp; f206, mackerel; f290, oyster; i6, cockroach;

1.3 Identification of 30 kDa lipoprotein

Allergens extracted from silkworm pupa were separated by 12% SDS-PAGE and electroblotting on a nitrocellulose membrane. After blocking with 5% skim milk in PBST (containing 0.05% Tween 20 in PBS), the membrane was incubated overnight at 4°C with pooled serum (diluted at a ratio of 1:10). The IgE reaction component was reacted with biotinylated anti-human IgE (Vector Laboratories Inc., Burglingame, CA) and streptavidin-horseradish peroxidase (BioLegend, San Diego, CA) for 2 hours at 37°C. Subsequently, the blot was detected with an enhanced chemiluminescence (ECL) kit (Pierce ^TM ECL Western Blotting Substrate; Pierce Biotechnology, Rockford, IL). Subsequently, two-dimensional gel electrophoresis and IgE immunoblotting were performed to identify the 30 kDa allergen, followed by LC-coupled ESI MS/MS analysis.

1.4 Production of recombinant 30 kDa lipoprotein and Bomb m 1

RT-PCR for cloning of 30 kDa lipoprotein was performed using oligonucleotide primers designed based on the sequence of GenBank (Accession No. NM_001044021); Forward primer, 5'-GCCACACTTGCACCAAGAAC-3', reverse primer, 5'-TTAGTAGGGGACGATGTACCATGC-3'. Arginine kinase (Bomb m1) was also cloned using an oligonucleotide primer designed based on GenBank sequence (Accession No. NM_001044021, forward primer: 5'-ATGGTCGACGCCGCAACC-3', reverse primer, 5'-CTACAGAGACTTCTCGATCTTGAT-3'). . The DNA fragment amplified by PCR was expressed in Escherichia coli using pEXP-5NT / TOPO vector (Invitrogen, Carlsbad, CA, USA), and the recombinant protein was purified using Ni-affinity column chromatography and then purified. The resulting protein was analyzed under reducing conditions with an SDS-polyacrylamide gel, and the protein concentration was determined by Bradford analysis. In addition, in order to express the open reading frame (NM_001043413) of the 27 kDa glycoprotein of silkworm in E. coli, the codon was optimized and synthesized. Subsequently, PCR amplification was performed using oligonucleotide primers (forward 5'-ATgAtgTggAAgACTgTCTTg-3', reverse 5'-TTAACggAAAgAAgTCCgACgg-3',), and then ligated to pEXP5NT/TOPO vector (Invitrogen). After transformation into E. coli BL21 (DE3), expression of the recombinant protein was induced by adding 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG). The recombinant protein was purified using Ni-NTA agarose (Qiagen) under denaturing conditions using 6 M urea. Recombinant silkworm tropomyosin was also expressed using the same expression system, and then purified from the soluble fraction using Ni-resin.

Example 2. Confirmation of the properties of purified allergens for diagnosis of silkworm allergy

The results of confirming the allergen for diagnosis of silkworm allergy selected in Example 1 through SDS-PAGE and IgE immunoblotting analysis from silkworm pupa extract are shown in FIGS. 1A and 1B, respectively. As confirmed in FIG. 1, it was confirmed that the protein having a mass of about 30 kDa appeared in all 13 serum samples isolated from allergic patients.

An experiment was additionally performed to confirm the properties of the 30 kDa lipoprotein by LC-MS/MS analysis, and the protein was separated by 2D gel electrophoresis, and the result of staining the protein with Coomassie blue is shown in FIG. 2A. , The results of confirming the IgE reactive molecule from the pooled serum sample isolated from the patient are shown in FIG. 2A and 2B, the protein was confirmed to be a low molecular weight 30 kDa lipoprotein through 2D gel electrophoresis and LC-binding ESI MS/MS.

Example 3. Confirmation of diagnostic ability through IgE reactivity experiment of 30 kDa lipoprotein

An experiment was performed to compare the IgE reactivity between the 30 kDa lipoprotein and the silkworm allergen, and the result of this was confirmed by SDS-PAGE is shown in FIG. 3A and the result of IgE immunoblotting is shown in FIG. 3B.

As confirmed in FIGS. 3A and 3B, it was confirmed that only 30 kDa lipoproteins among the four allergens tested showed remarkable IgE reactivity in IgE immunoblotting after SDS-PAGE under reducing conditions.

An experiment to confirm whether the IgE reactivity of the recombinant protein appears even when an element causing protein denaturation is added was performed by ELISA. Recombinant protein (2 μg/mL) was coated on microplates overnight in 0.05 M carbonate buffer (pH 9.6). After blocking with 3% skim milk, a serum sample (diluted at a ratio of 1:4) was added and the plate was incubated for 1 hour. IgE antibody was incubated with biotinylated goat anti-human IgE (diluted at 1:1000 ratio) (Vector, Burlingame, CA, USA) for 1 hour and then diluted at 1:1000 ratio of streptavidin-peroxidase. It was detected as a conjugate (Sigma-Aldrich). Color development was confirmed by adding the substrate 3,3'5,5'-tetramethyl-benzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). 0.5MH ₂ SO ₄ was added to stop the enzyme reaction, and then the absorbance at 450 nm was measured. A value twice the standard deviation of the mean absorbance + 2 of the serum obtained from the healthy control group was used as the cutoff value. For ELISA inhibition, silkworm pupa extract (10 μg/mL) was put into a coated microtiter plate and incubated overnight at 4°C. After blocking with 3% skim milk, the wells to which serum samples (1: 4) obtained from 2 patients positive for the recombinant protein previously incubated with various concentrations of extract or a solution containing the recombinant protein were added were incubated for 2 hours. . Then, IgE antibodies were detected as described, and recombinant 30 kDa lipoprotein was used as an inhibitor for comparison, and weak IgE reactivity was confirmed by ELISA. 4 M urea was added to the serum sample to promote antibody interaction, and the experimental results are shown in FIG. 4.

As confirmed in FIG. 4, 30 kDa lipoprotein was recognized by all (17 of 17) serum samples when 4 M urea was added, but the remaining 3 allergens (Bomb m1, 27 kDa glycoprotein) were among 17 serum samples. One sample did not show reactivity, and tropomyosin also showed very low IgE reactivity, below 50%.

Example 4. Identification of the role of 30kDa lipoprotein through inhibition analysis

In order to investigate the role of 30kDa lipoprotein in silkworm allergy, inhibition analysis was performed. For IgE immunoblotting, 5 μg of the recombinant protein was performed on a 12% SDS-PAGE gel under reducing conditions. The separated protein was electroblotting onto a PVDF membrane. After blocking with 3% skim milk in PBST, the membrane was incubated overnight with serum samples (1: 4) obtained from 2 patients positive for the recombinant protein. Full serum samples (1: 4 dilution) were examined for reactivity to IgE. IgE antibodies were detected for 1 hour with alkaline phosphatase-conjugated goat anti-human IgE (1:1000) (Sigma-Aldrich). Color development was confirmed by adding nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Promega, Madison, WI, USA), which are shown in Figs. 5A to C.

As confirmed in Figure 5A, it was confirmed that the silkworm extract did not inhibit the IgE reaction. As confirmed in Figure 5B, when 4 M urea was included in the serum, it was confirmed that the silkworm extract showed minimal inhibition (maximum 16.1%). However, as can be seen in Figure 5C, it was confirmed that self-inhibition of 93% or more of 30 kDa lipoproteins appeared, and the inhibition of about 30% of the extracts appearing in the extract through the above results It was confirmed that it appears by the recombinant 30 kDa lipoprotein contained within. Therefore, it was confirmed that 30kDa lipoprotein is a major cause of allergy to silkworm pupae, and is more significant in diagnosing allergy to silkworm pupae.

As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Industry-Academic Cooperation Foundation, Yonsei University ProteomeTech Inc. <120> Compositions for diagnosis of allergy disease to silkworm, methods for diagnosing of allergy disease to silkworm, and composition for immunotherapy of allergy disease to silkworm <130> PN126528 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 240 <212> PRT <213> Unknown <220> <223> Recombinant 30 kDa lipoprotein <400> 1 Ala Thr Leu Ala Pro Arg Thr Asn Asp Val Leu Ala Glu Gln Leu Tyr 1 5 10 15 Met Ser Val Val Ile Gly Glu Tyr Glu Thr Ala Ile Ala Lys Cys Ser 20 25 30 Glu Tyr Leu Lys Glu Lys Lys Gly Glu Val Ile Lys Glu Ala Val Lys 35 40 45 Arg Leu Ile Glu Asn Gly Lys Arg Asn Thr Met Asp Phe Ala Tyr Gln 50 55 60 Leu Trp Thr Lys Asp Gly Lys Glu Ile Val Lys Ser Tyr Phe Pro Ile 65 70 75 80 Gln Phe Arg Val Ile Phe Thr Glu Gln Thr Val Lys Leu Ile Asn Lys 85 90 95 Arg Asp His His Ala Leu Lys Leu Ile Asp Gln Gln Asn His Asn Lys 100 105 110 Ile Ala Phe Gly Asp Ser Lys Asp Lys Thr Ser Lys Lys Val Ser Trp 115 120 125 Lys Phe Thr Pro Val Leu Glu Asn Asn Arg Val Tyr Phe Lys Ile Met 130 135 140 Ser Thr Glu Asp Lys Gln Tyr Leu Lys Leu Asp Asn Thr Lys Gly Ser 145 150 155 160 Ser Asp Asp Arg Ile Ile Tyr Gly Asp Ser Thr Ala Asp Thr Phe Lys 165 170 175 His His Trp Tyr Leu Glu Pro Ser Met Tyr Glu Ser Asp Val Met Phe 180 185 190 Phe Val Tyr Asn Arg Glu Tyr Asn Ser Val Met Thr Leu Asp Glu Asp 195 200 205 Met Ala Ala Asn Glu Asp Arg Glu Ala Leu Gly His Ser Gly Glu Val 210 215 220 Ser Gly Tyr Pro Gln Leu Phe Ala Trp Tyr Ile Val Pro Tyr *** Ala 225 230 235 240 <210> 2 <211> 722 <212> DNA <213> Unknown <220> <223> Nucleotide sequence of recombinant 30 kDa lipoprotein <400> 2 gccacacttg caccaagaac taatgacgta ctggcggagc agctgtatat gagtgtcgtc 60 attggtgaat acgagaccgc tatcgccaaa tgctctgaat atctgaagga aaagaaggga 120 gaggttatca aggaagccgt gaagcgtctg atcgaaaacg gcaagaggaa caccatggac 180 ttcgcctacc agttatggac aaaggatgga aaggaaatcg tcaaatctta cttccccatc 240 cagtttagag tgatcttcac cgagcagact gtcaagctca taaacaaaag ggaccatcac 300 gccctcaagt tgatcgacca acaaaaccac aacaaaattg cattcggtga ctccaaagac 360 aaaaccagca agaaagtctc ctggaagttt acccccgtgt tggaaaacaa cagagtttac 420 ttcaagatca tgtccaccga ggacaaacag tacctgaagc tcgataacac gaaaggttct 480 agtgatgacc gtatcatcta cggtgatagc accgctgaca ccttcaaaca ccactggtac 540 cttgagccct ccatgtacga aagcgacgtc atgttcttcg tctacaaccg agagtacaac 600 agtgttatga cacttgatga agatatggcc gccaacgaag accgtgaagc cttggggcac 660 agcggagaag tttccggtta tccccaactt tttgcatggt acatcgtccc ctactaagct 720 tg 722 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of SW30kLp <400> 3 gccacacttg caccaagaac 20 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of SW30kLp <400> 4 ttagtagggg acgatgtacc atgc 24 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of Bomb m1 <400> 5 atggtcgacg ccgcaacc 18 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of Bomb m1 <400> 6 ctacagagac ttctcgatct tgat 24 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of 27kDa glycoprotein <400> 7 atgatgtgga agactgtctt g 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of 27kDa glycoprotein <400> 8 ttaacggaaa gaagtccgac gg 22

Claims (11)

A composition for diagnosing an allergic disease to a silkworm containing a 30kDa lipoprotein of silkworm or a protein region thereof. The composition of claim 1, wherein the protein region of the lipoprotein comprises an amino acid sequence represented by SEQ ID NO: 1. The composition of claim 1, wherein the lipoprotein is isolated from silkworm larvae, silkworm pupae, or silkworm moth. The composition according to claim 1, wherein the lipoprotein or protein region thereof is produced recombinantly. The composition of claim 1, wherein the lipoprotein maintains IgE reactivity even in the denaturation of the protein region. The composition of claim 5, wherein the denaturation of the protein region is caused by urea. A kit for diagnosing an allergic disease to silkworms comprising the composition of any one of claims 1 to 6. The kit of claim 7 is a kit, characterized in that for an immunoassay. A method for providing information necessary for diagnosis of an allergic disease to a silkworm, comprising the step of detecting binding of an IgE antibody to a lipoprotein or a protein region thereof in a biological sample isolated from an individual. The method of claim 9, wherein the biological sample is blood, tissue, urine, mucus, saliva, tears, plasma, serum, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, urogenital fluid, breast milk, lymphatic body fluid , Semen, cerebrospinal fluid, organ body fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof. A composition for immunological treatment of allergic diseases for silkworms containing a 30kDa lipoprotein of silkworm or a protein region thereof.

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