Culicoides leucoides allergen as well as identification method, antibody and application of culicoides leucoides allergen

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a culicoides streptonicoides allergen as well as an identification method, an antibody and application thereof. The culicoides chromogen is maltase Cel t 1, and the amino acid sequence of the maltase Cel t 1 is as shown in SEQ NO. 1. According to the application disclosed by the invention, the maltase Curet 1 obtained by screening is used as the culicoides chromogen, so that the defect of single research object of blood-sucking midges in the early stage can be overcome, and a good foundation is laid for subsequent blood-sucking midge chromogen research. Meanwhile, one of sensitizers in the culicoides is defined on the basis of the culicoides-mouse sensitization model constructed through early-stage research, an efficient technical method is provided for research on identification of the culicoides sensitizers, and meanwhile basic data is provided for subsequent vaccine prevention and treatment research.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a Culicoides banded allergen and an identification method, an antibody and an application thereof. Background Art

[0002] Blood-sucking midges belong to the order Diptera, the suborder Longicornis, and the family Ceratidae. They are some midges that can spread insect-borne pathogens by sucking the blood of humans and animals. They are a type of holometabolous insect. Among them, Culicoides is the genus with the most species among blood-sucking midges. With the spread of midge-borne diseases, it poses a great threat to animal and human health. This is mainly reflected in the fact that during the bite of blood-sucking midges, the pathogens they carry are deposited into the host body with saliva. Their saliva is rich in salivary proteins, some of which have dual functions, which can not only digest blood meals, but also cause allergic reactions in the host.

[0003] Allergic reactions caused by blood-sucking midge bites are mainly divided into two types, namely type I and type IV hypersensitivity reactions: Type I, also known as an immediate immune response, is an immediate immune response mediated by immunoglobulin E (IgE). Allergic symptoms such as redness, swelling, and urticaria usually appear at the bite site within a few minutes to 1 hour after the bite. The allergenic components of blood-sucking midges are presented to T cells by antigens in the skin. These Th2 T cells release IL-4 and IL-13 to guide B cell activation. After activation, B cells may undergo class switch recombination (CSR), converting the isotype of their BCR from IgM / IgD to IgG, IgA or IgE. These different types of secreted antibodies have different functional effects, and the function of these antibody-mediated effectors is inseparable from binding to Fc receptors. IgE usually binds to FcεRI, which is a high-affinity receptor abundant in mast cells and basophils; the binding of IgE to cognate antigens activates FcεRI and promotes degranulation of these cells, triggering allergic symptoms such as vasodilation, increased permeability, itching, swelling, and release of leukotrienes and histamine. Type IV hypersensitivity, also known as delayed hypersensitivity, is characterized by infiltration of lymphocytes and eosinophils at the site of the bite, and the patient's peripheral mononuclear cells respond to midge extracts and secrete significantly higher levels of interferon-γ (IFN-c), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-a) than subjects who are not allergic to midges.

[0004] To date, research on blood-sucking midges at home and abroad has mainly focused on virus transmission vectors, ignoring the fact that blood-sucking midge bites can cause symptoms such as allergies; in some cases, saliva can even cause acute allergic reactions, but due to the extremely low survival rate of Culicoides in captivity, it is extremely challenging to rely on biting hosts / artificially collecting saliva to identify its allergens.

[0005] It is known that the salivary glands of blood-sucking midges contain a series of bioactive molecules, including anticoagulant factors, immunomodulatory factors and protein allergens. These allergens trigger allergic reactions in the host, leading to dermatitis, urticaria and more serious allergic diseases, such as insect bite allergy (IBH). Through humoral and cellular immune analysis, the credibility of allergens in saliva is the highest. Wilson et al. found through immunohistochemistry that IgE of allergic horses preferentially binds to salivary gland structures. Schaffartzik et al. injected systemic extracts of Culicoides into the skin of horses infected with IBH, simulating natural contact reactions and triggering type I allergic reactions. There was infiltration of T cells and eosinophils at the injection site. The subcutaneous test results were positive at 30 minutes (immediate reaction) and 4 hours (delayed reaction) after injection, and there was a significant difference with the control group. In vitro studies also found that Culicoides allergens can cause the release of thioleukotrienes and histamine.

[0006] Although the research on allergic reactions caused by blood-sucking midge bites has been continuously deepened in recent years, there are still few known allergens. Studies have found that several potential allergens of blood-sucking midges have been identified through SDS-PAGE and Western Blotting. For example, Langner et al. used serum from IBH-infected horses and control serum to incubate the allergen Culs1 in the salivary glands of Culicoides sonorensis based on protein immunoblotting experiments. Chen et al. found allergens Fort 1, Fort 2 and Fort 3 in the extract of Forcipomyia taiwana (Taiwan midge), purified Fort 2 by affinity chromatography, and constructed a Fort2DNA vaccine that can slow down the occurrence of allergic reactions by molecular biology technology. It was confirmed that the vaccine has a certain protective effect on Fort 2-sensitized mice. However, the allergens from different species of blood-sucking midges have different sensitization properties. Different allergens are used to develop vaccines for the same disease from different species of blood-sucking midges. Therefore, these known allergens cannot replace allergens from other species of blood-sucking midges for practical applications.

[0007] Since the banded midge is a type of blood-sucking midge widely distributed in southern my country, especially in Zunyi, Guizhou, this type of blood-sucking midge can have a significant harassing and blood-sucking effect on even-toed ungulate livestock. Severe allergic reactions will lead to a significant decrease in the meat and milk production of livestock. Discovering effective allergens and developing them into vaccines is of great significance for the prevention of midge-borne allergic diseases. However, these currently known allergens cannot replace allergens derived from the banded midge for actual vaccine development. Therefore, it is urgent to continuously discover allergens of widespread blood-sucking midges. Summary of the invention

[0008] In order to solve the problem that the known allergens in the prior art cannot replace the allergens from Culicoides stripes for actual vaccine development, the present invention is based on the Culicoides stripes-mouse sensitization model constructed in previous studies, clarifies one of the allergens in Culicoides stripes, and obtains a Culicoides stripes allergen. To achieve the above purpose, the present invention adopts the following technical solution.

[0009] The invention provides a Culicoides striped allergen. The Culicoides striped allergen is maltase Cul t1. The amino acid sequence of the maltase Cul t 1 is shown in SEQ NO.1:.

[0010] Different allergens have different effects as antigens, and allergens from different species of blood-sucking midges have different sensitizing properties. Therefore, the allergens used to develop vaccines for allergens from different species of blood-sucking midges for the same disease are also different. If antibodies are developed using allergens from other known species of blood-sucking midges, it is not necessarily possible to prevent the allergic reactions caused by the striped Culicoides in the present invention. Therefore, if a widely distributed species of Culicoides (inducing IBH) is found in a certain area, it is an effective solution to separate its allergens and then develop antibodies. The striped Culicoides allergen provided by the present invention is maltase Cul t 1, which is an allergen derived from the striped Culicoides, and can be used for actual vaccine development against the striped Culicoides, and has the effect of preventing allergic reactions caused by the striped Culicoides, thereby solving the problem in the prior art that the currently known allergens cannot replace the allergens derived from the striped Culicoides for actual vaccine development.

[0011]

[0012] The present invention also provides a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium, which can express the above-mentioned striped Culicoides allergen. At the same time, the present invention also provides a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium, which has the above-mentioned nucleic acid encoding the striped Culicoides allergen.

[0013] The present invention also provides a preventive or therapeutic agent with the above-mentioned Culicoides striped allergen as an effective ingredient.

[0014] The present invention also provides a use of the above-mentioned Culicoides striped allergen as an effective ingredient of a preventive or therapeutic agent.

[0015] The present invention also provides the use of the Culicoides banded allergen as an antigen, a hapten, an immunogen or an immunomodulator.

[0016] In practical applications, the allergens of Culicoides bandedi can be used as antigens for diagnosis, immunotherapy and vaccine research of allergic diseases. These applications rely on the purification of allergens, specificity verification and in-depth understanding of their immune response mechanisms. Specifically, the application is that the allergens are used to develop diagnostic reagents for allergic diseases:

[0017] (1) Skin prick test (SPT): The purified Culicoides stripe allergen (protein) can be used for allergic skin prick test to detect whether patients who are allergic to the bites of this insect have specific IgE antibodies.

[0018] (2) In vitro immunodiagnosis: Enzyme-linked immunosorbent assay (ELISA) is used to detect the IgE response level in the patient's serum to determine the sensitivity to the allergen in the salivary fluid of the striped midge. Some studies have developed standardized kits to improve the efficiency of allergen screening.

[0019] The present invention also provides the use of the Culicoides banded allergen in preparing products resistant to sensitization by the Culicoides banded allergen.

[0020] Preferably, the product is a vaccine and / or a medicine.

[0021] The application of the striped Culicoides allergen in immunotherapy is mainly manifested in the following forms:

[0022] (1) Allergen Immunotherapy (AIT): The purified allergens of Culicoides stripes are exposed to the patient's immune system multiple times at low doses, allowing the body to gradually build up immune tolerance, thereby reducing clinical allergic symptoms. Major allergens such as Cult 1 have become potential candidates for desensitization treatment of patients with IBH (insect bite allergy).

[0023] (2) Vaccine research: The striped Culicoides allergen Cul t 1 is used as an antigenic basis, and its immunogenicity is enhanced by adjuvants, laying the foundation for the development of preventive or therapeutic vaccines. Among them, the striped Culicoides allergen Cul t 1 is referred to as the striped Culicoides allergen.

[0024] The present invention also provides an antibody obtained by screening the strip Culicoides allergen as a target.

[0025] Preferably, the antibody is one of a polyclonal antibody, a monoclonal antibody or a chimeric antibody.

[0026] The present invention also provides a method for studying the immune response mechanism caused by insect bites, which is obtained by inducing the allergen of Culicoides stripes as an antigen.

[0027] (1) Host immune response assessment

[0028] By analyzing the B cell or T cell responses triggered by these antigens, we can gain insight into the sensitization process and characteristics of allergic reactions to insect bites.

[0029] (2) Genetic Engineering Expression System

[0030] The allergen gene is inserted into yeast or mammalian cells for expression and the recombinant protein is prepared for more efficient research and application development.

[0031] The present invention provides a method for identifying the allergen of the striped Culicoides, comprising the following steps:

[0032] Preparation of Culicoides thorax extract:

[0033] The thorax extract of Culicoides stripes induces an allergic reaction, collects in vitro serum samples after the allergic reaction, and screens for allergens;

[0034] Detecting the binding of the striped Culicoides thorax extract to the specific antibody to determine the presence of potential allergens in the striped Culicoides thorax extract

[0035] Preparation of the thorax extract of Culicoides stripes: Take the thorax of Culicoides stripes, add PBS buffer, grind, and centrifuge. After centrifugation, take out the supernatant and filter it to obtain the extract.

[0036] The thorax extract of Culicoides stripes induces an allergic reaction, and an in vitro serum sample is collected after the allergic reaction; and allergens are screened.

[0037] Identification of candidate allergens: The binding of the thorax extract of Culicoides stripes with specific antibodies is detected by SDS-PAGE or protein immunoblotting to determine the presence of potential allergens in the thorax extract of Culicoides stripes.

[0038] The potential allergen is identified, and the obtained potential allergen is the Culicoides banded allergen.

[0039] Preferably, mass spectrometry detection and Uniport analysis are used to identify the Culicoides allergen in the strip.

[0040] Mass spectrometry detection was performed using electrospray ionization quadrupole analysis of protein fragments time-of-flight-tandem mass spectrometry.

[0041] Preferably, the parameters of the mass spectrometry detection are as follows:

[0042] Multiple reaction monitoring mode was used for positive ion detection;

[0043] Interface voltage: 3.8kV~4.2kV; interface temperature: 298℃~302℃; nebulizing gas flow rate: 2.9L / min~3.1L / min; drying gas flow rate: 9.8L / min~10.2L / min; heating gas flow rate: 9.8L / min~10.2L / min; heating block temperature: 398℃~402℃; desolvation tube temperature: 248℃~252℃; desolvation temperature: 518℃~522℃; collision gas is high-purity argon.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. The present invention provides a Culicoides allergen. The present invention is based on the Culicoides allergen-mouse sensitization model constructed in previous studies, clarifies one of the allergens in Culicoides, and obtains a Culicoides allergen. The Culicoides allergen is maltase Cul t 1. The Culicoides allergen is maltase Cul t 1, which is an allergen derived from Culicoides bands and can be used for actual vaccine development against Culicoides bands, and has the effect of preventing allergic reactions caused by Culicoides bands, thereby solving the problem in the prior art that the currently known allergens cannot replace the allergens derived from Culicoides bands for actual vaccine development.

[0046] 2. The identification method of the strip Culicoides allergen provided by the present invention is simple to operate and convenient to obtain and collect samples, which makes up for the shortcoming of the single research object of blood-sucking midges in the early stage and lays a good foundation for subsequent research on blood-sucking midge allergens.

[0047] 3. The present invention is based on the striped Culicoides-mouse sensitization model constructed by the research team in the early stage, clarifies one of the allergens in the striped Culicoides, provides an efficient technical method for the research on the identification of Culicoides allergens, and also provides basic data for subsequent vaccine prevention and treatment research. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1This is a graph showing the behavioral evaluation of mice using the thorax extract of Culicoides stripes in the present invention.

[0049] Figure 2 is the result of immunoblotting analysis of the Culicoides strip extract in the present invention; wherein, Figure 2 Figure A shows the strips of Culicoides extract separated by 10% polyacrylamide gel and stained with Coomassie Brilliant Blue; Figure 2 Figure B shows that the protein in Figure A was transferred to a polyvinylidene fluoride membrane and then incubated with serum from the midge-sensitized mouse group; Figure 2 Figure C shows the protein in Figure A transferred to a polyvinylidene fluoride membrane and then incubated with the serum of a control group of mice; wherein M represents the position of the molecular weight standard.

[0050] Figure 3 A protocol was established for the Culicoides striped thoracic extract-mouse sensitization model. DETAILED DESCRIPTION

[0051] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0052] Example 1

[0053] A method for identifying allergens of Culicoides bandeda comprises the following steps:

[0054] 1. Preparation of the Thorax Extract of Culicoides tricolor

[0055] (1) Weigh 0.2 g of the thorax of Culicoides bandedi and place it in an EP tube. Add 1 mL of PBS buffer and 6 stainless steel grinding beads with a diameter of 5 mm. Grind it for 60 seconds at 60 Hz using a high-throughput tissue grinder. After grinding, centrifuge it at 8,000 g for 15 minutes. After centrifugation, aspirate the supernatant and filter it using a 0.22 μm filter to obtain the thorax extract of Culicoides bandedi.

[0056] Among them, the stainless steel grinding beads with a diameter of 5 mm are the supporting experimental equipment for the high-throughput tissue grinder and were purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0057] The method for obtaining the banded Culicoides was as follows: the insects were collected from the cattle shed of the Shuiyanlin group of Xinpu Town, Zunyi City, Guizhou Province. The cattle shed was mixed with water buffalo and yellow cattle. The midges were collected by ultraviolet lamps. Two collection lamps were placed in the cattle shed, 2 meters apart, and each lamp was about 2 meters from the ground. In order to reduce the entry of larger insects into the collection net, a filter sieve plate was placed in the middle of the collection net, which was opened 2 hours after sunset and closed 2-3 hours after sunrise. The collection time was about 12 hours. The collected insects were immediately brought back to the laboratory and briefly cooled in a low-temperature refrigerator (-20℃) to inactivate them. According to the classification method of "Chinese Ceratidae Insects" compiled by Yu Yixin, the unsaturated blood banded Culicoides were selected under a stereo microscope.

[0058] The above-selected unsaturated blood-banded Culicoides are the Culicoides used in the examples of the present invention.

[0059] The pH of PBS buffer is 7.6, and its formula composition is: TrisHCL (1M, pH7.5) 50mL, NaCl 8g, KCL0.2g, Tween 0.5mL, and water is the solvent.

[0060] BCA was used to detect the protein concentration in the thorax extract of Culicoides fasciatus prepared above. The specific detection method is as follows:

[0061] (1) Preparation of protein standards

[0062] A. Take 1.2mL of protein standard solution and add it to a tube of protein standard (30mg BSA). After fully dissolving, prepare a 25mg / mL protein standard solution. It can be used immediately after preparation or stored at -20℃ for a long time.

[0063] B. Take 20μL of 25mg / mL protein standard and add 980μL of diluent to PBS buffer to prepare 0.5mg / mL protein standard. Dilute the protein standard with PBS buffer. The diluted 0.5mg / mL protein standard can be stored at -20℃ for a long time.

[0064] (2) Preparation of BCA working solution

[0065] According to the number of samples, prepare BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly. For example, add 5 mL of BCA reagent A to 100 μL of BCA reagent B, mix well, and prepare 5.1 mL of BCA working solution. BCA working solution is stable at room temperature for 24 hours.

[0066] (3) Protein concentration detection

[0067] A. Add 0, 1μL, 2μL, 4μL, 8μL, 12μL, 16μL and 20μL of the protein standard solution to the standard wells of a 96-well plate, and add standard diluent to make up to 20μL, which is equivalent to the concentration of the protein standard solution being 0, 0.025mg / mL, 0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL and 0.5mg / mL, respectively.

[0068] B. Add the sample to the sample well of the 96-well plate. If the sample is less than 20 μL, add the standard diluent to make it up to 20 μL. Please remember to record the sample volume. The sample is the Culicoides thorax extract of the above strip.

[0069] C. Add 200 μL of BCA working solution to each well and incubate at 37°C for 25 minutes.

[0070] D. Use an ELISA reader to measure the absorbance of the wavelength of A562.

[0071] E. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0072] 2. Animal model construction

[0073] The thorax extract of Culicoides fasciatus prepared above was intraperitoneally injected into mice to prepare an allergic mouse model. The specific method for constructing the allergic mouse model is as follows:

[0074] According to the method of constructing a Culicoides extract-mouse sensitization model in the early stage by the research group of the inventor of the present invention, a Culicoides thorax extract-mouse sensitization model was constructed. The method for constructing a Culicoides extract-mouse sensitization model can be found in "Bao, S., Lu, X., Xiang, S., & Hou, X. (2024). Establishment of a mouse allergy model for Culicoides (Diptera: Ceratopogonidae). Veterinary Medicine and Science, 10, e1462.". The specific operation is as follows:

[0075] 80 mice were selected and divided into two groups, 40 in each group. One group was the midge sensitization group (experimental group) and the other was the control group. The mice were sensitized by a two-step method. The midge sensitization group was sensitized by intraperitoneal injection of 20 μg of Culicoides stripes chest extract and 2 mg of alum adjuvant with a syringe on days 0, 7, 14 and 21, while the control group was injected with 2 mg of alum adjuvant and PBS buffer with a syringe on days 0, 7, 14 and 21. On days 28, 31 and 35, 10 μg of Culicoides stripes chest extract was injected subcutaneously with a syringe for sensitization, and the control group was injected with PBS buffer in the same way. On the 35th day of the last sensitization, the scratching behavior of the mice was recorded. And on the 40th day, after stimulation with 10 μg of Culicoides stripes chest extract, the skin of the stimulated site was taken for subsequent experiments.

[0076] Among them, mice were purchased from the Experimental Animal Center of Zunyi Medical University. They were all 6-week-old C57 female mice with a body weight of 20±2 g.

[0077] Dosage regimen Figure 3 shown.

[0078] 3. Collect mouse serum samples

[0079] After the mice were anesthetized with ether, 0.5 mL of blood was collected from the retro-orbital venous plexus of the anesthetized mice using capillaries and collected in a sterilized and labeled 1.5 mL EP tube prepared in advance. Serum samples were collected from the two groups of mice on days 0, 14, 28, and 42, and 0.5 mL of blood was collected from the retro-orbital venous plexus to obtain isolated mouse venous blood.

[0080] The isolated mouse venous blood was allowed to stand at room temperature for 20 minutes, and then centrifuged at 3,000 rpm for 10 minutes; after centrifugation, the serum and red blood cells were quickly and carefully separated, and the upper serum was collected to obtain the sensitized mouse serum, which was stored at -20°C for future use. The sensitized mouse serum is the isolated mouse serum sample.

[0081] On the 42nd day, the spleen of the mice was collected and placed in an EP tube and stored at -80°C for later use.

[0082] 4. Screening for allergens

[0083] 4.1. Coomassie Brilliant Blue staining was used to detect the protein expression of the Culicoides thoracis extract prepared above. The specific method is as follows:

[0084] (1) Sample preparation

[0085] A mixed solution was prepared in the ratio of 4 volumes of sample to 1 volume of 5× loading buffer, boiled at 100°C for 5 min, cooled to room temperature, centrifuged to collect the liquid on the tube wall to the bottom, and stored at -20°C for later use.

[0086] The sample refers to the thorax extract of Culicoides stripedensis prepared as above.

[0087] (2) SDS-PAGE gel configuration

[0088] 1) Preparation of separation gel: Fix a glass plate (long inside and short outside) on a gel preparation rack, prepare separation gel according to the separation gel preparation ingredients (Table 1), and stop injecting the gel 1.5 cm from the top of the thin glass plate. Add anhydrous ethanol to make the gel surface flat, and let it stand at room temperature for 30 minutes to allow the gel to solidify.

[0089] Table 1 SDS-PAGE gel preparation ingredients

[0090] Element 10% separation gel 5% stacking gel Total volume 10mL 5mL <![CDATA[ddH2O]]> 4.0mL 3.42mL 30% Acr / Bis (29:1) 3.3mL 0.83mL 1MTris-Hcl(PH6.8) 0.625mL 1.5MTris-Hcl(PH8.8) 2.5mL 10% SDS 100μL 50μL 10% PAGE gel coagulant 100μL 75μL PAGE gel coagulant 10μL 7.5μL

[0091] 2) Preparation of concentrated gel: Remove the glass plate, absorb anhydrous ethanol with absorbent paper, and then fix it on the gel rack. Prepare concentrated gel according to Table 6. Fill the gap between the two glass plates with concentrated gel. Insert the corresponding type of comb vertically into the concentrated gel and place it at room temperature for 28 minutes to allow the gel to solidify.

[0092] 3) Sample loading: Pull the comb upwards with both hands at the same time. If the sample holes are not neat, you can use a needle to correct them. Add 4μL protein marker to the first hole, add the corresponding samples to the remaining holes, and make corresponding records.

[0093] 4) Electrophoresis: Set the voltage to 80 V to migrate the sample out of the stacking gel, then adjust the voltage to 120 V and stop the electrophoresis when bromophenol blue migrates to 1 cm from the bottom of the separation gel.

[0094] 5) Transfer: Prepare a PVDF membrane corresponding to the size of the target band, and place it in a container filled with methanol to activate it for 15-30 seconds. According to the principle of "black gel and white membrane", use a gel cutting board to place the gel on the filter paper on the black scissors side, then place the activated PVDF membrane on the gel, use a gel cutting board to drive away bubbles, cover with another layer of filter paper, and clamp the electrotransfer clamp. Add appropriate electrotransfer solution, cover with a lid, and transfer the membrane at 200mA for 1 hour.

[0095] 6) Blocking: Wash 4 times with TBST and then block in a 37°C shaker for 1 h.

[0096] 7) Primary antibody incubation: Dilute the primary antibody with TBST according to the target band size. The dilution volume is generally 3 mL and incubate at 4°C overnight. Overnight means ≥ 12 hours.

[0097] 8) Washing the membrane: After the primary antibody incubation is completed, use TBST to wash the PDVF membrane 4 times, 5 minutes each time (phosphorylated antibodies can be washed 3 times, 5 minutes each time).

[0098] 9) Secondary antibody incubation: dilute the secondary antibody with 5%wt milk blocking solution, the dilution multiple depends on the situation, and incubate the membrane in the secondary antibody at room temperature for 2h.

[0099] 10) Membrane washing: same as above.

[0100] 11) Mix solution A and solution B according to the instructions of the ultrasensitive ECL chemiluminescence kit, add a certain amount of exposure solution on the PVDF membrane, incubate in the dark for 1 min, place the membrane in the chemiluminescence imaging system for exposure, save the image and analyze the results.

[0101] (3) SDS-PAGE gel staining

[0102] Place the SDS-PAGE gel containing the target band into a container filled with Coomassie Brilliant Blue staining solution, place it on a horizontal shaker, and stain for more than 2.5 hours.

[0103] (4) SDS-PAGE gel destaining

[0104] Transfer the stained SDS-PAGE gel to the elution buffer, place it on a horizontal shaker, and decolorize it overnight. During this period, change the elution buffer twice until the blue color of the gel is basically removed. Observe the protein expression and take pictures for record.

[0105] The results are as follows Figure 2 As shown in Figure A. Figure 2 Figure A shows the protein expression of C. tainanus chest extracts detected by SDS-PAGE (10%) and Coomassie Brilliant Blue staining, and then the binding of C. tainanus chest extracts to IgE was detected by Western blotting (B and C). The Western blotting method is as follows: After electrotransfer, block in 5% skim milk for 2 hours, use sensitized mouse serum diluted 1:10 as the primary antibody, and incubate overnight; the control group uses unsensitized mice and horseradish peroxidase (HRP) goat anti-mouse IgE antibody as the secondary antibody, incubate at room temperature for two hours, and finally detect the binding with IgE using ECL working solution.

[0106] Depend on Figure 2 As shown in Figure A, the bands in the thorax extract of Culicoides are expressed at 0-180 kd, but there are more obvious bands near 48 KD and 66 KD, indicating that proteins of this size are more abundant in the extract.

[0107] The above results showed that the proteins contained in the thorax extract of Culicoides stripes were expressed in the molecular weight range of 0-180kD.

[0108] 4.2. The binding of IgE in the thoracic extract of Culicoides midge and mouse serum samples was detected by Western blotting.

[0109] The specific steps and methods are the same as the above-mentioned "Coomassie Brilliant Blue staining to detect the protein expression of the above-prepared Culicoides thoracic extract", and the protein immunoblotting method is used to detect the bands of the two groups of mouse sera on the gel for comparison with the thoracic extract.

[0110] After electroporation, the cells were blocked in 5% wt skim milk for 2 h, and sensitized mouse serum was diluted 1:10 as the primary antibody and incubated overnight. The control group used unsensitized mice and horseradish peroxidase (HRP) goat anti-mouse IgE antibody as the secondary antibody, incubated at room temperature for two hours, and finally the binding with IgE was detected by ECL working solution.

[0111] The results are as follows Figure 2 Figure B and Figure 2 As shown in Figure C. Figure 2 Figure B and Figure 2 Figure C shows the bands of Culicoides separated by 10% polyacrylamide gel and used for Coomassie brilliant blue staining (A) or transferred to polyvinylidene fluoride membrane. Among them, lanes 1 and 2 in Figure B were incubated with serum from mice sensitized with midges, and lanes 1 and 2 in Figure C were incubated with serum from control mice. After incubation with mouse IgE-specific monoclonal antibody and detection antibody labeled with horseradish peroxidase, IgE binding was determined by chemiluminescence. M indicates the position of the molecular mass standard.

[0112] Depend on Figure 2 Figure B and Figure 2 As shown in Figure C, proteins that can bind to IgE are distributed in the range of about 0-100 kd. Comparison of the two figures (midge-sensitized group and control group) revealed that proteins at 48 KD and 66 KD are unique bands in the midge-sensitized group of mice, which are the target bands for subsequent experiments.

[0113] Western blotting showed that proteins that can bind to IgE were expressed between 25kD and 100kD. Combining SDS-PAGE, Western blotting and the size of the allergenic proteins of Culicoides reported previously, proteins with differences between the midge sensitization group and the control group were selected for subsequent experiments (66KD).

[0114] 4. Mass spectrometry detection of allergens

[0115] (1) According to the results of protein immunoblotting, stain with Coomassie Brilliant Blue and cut out the corresponding band of SDS-PAGE gel that binds to IgE.

[0116] According to the results of protein immunoblotting, the corresponding bands of SDS-PAGE gel and IgE binding were cut out by Coomassie Brilliant Blue staining and sent to Shanghai Biotechnology Company for detection by electrospray ionization quadrupole analysis of protein fragments time of flight (QtoF)-tandem mass spectrometry. Figure 2 The bands pointed by arrows in Figure C are target proteins of approximately 66 kd and 48 kd in size.

[0117] The data exported from the final test were compared and analyzed with the protein database Uniprot, and the results are shown in Table 2.

[0118] Table 2 Bands of Culicoides extracts immunoblot analysis

[0119]

[0120] The results in Table 2 show that a total of 55 amino acids were obtained, accounting for 9.2% of the entire protein sequence, and it was identified as one of the allergens of Culicoides bandedus.

[0121] According to the regulations of the World Health Organization (WHO) / International Union of Immunology (IUIS), the above allergen is a maltase named Cul t 1, also known as maltase Cul t 1.

[0122] Sequencing revealed that the amino acid sequence of the maltase Cult1 is as shown in SEQ NO.1: MIIFKTLTILISSACLVLTVPQGAREKDWWETGNFYQVYPRSFMDSDGDGIGDLKGVQQKVAYLKEIGMDGVWLSPIFDSPMADFGYDISNFTKVYPKFGDMQSIQELIDECNKNDIKLILDFVPNHSSDECEWFKKSVKRDPYYSDFYIWHPGKPNPNGGRNLPPTNWVSAFRSSAWEWNDERQEYYLHQFLAAQPDLNYRNPEVVKTMKNVLKFWLNKGINGFRIDAVPYLFEVGPDASGNYPDEPLTYKCADPLSQCYYDHIYTQMRSETVNMVTQWRTVMDDWQKENGGPTRVLLVEAYAPLAKVVEIYGENGKLNGAEFPFNFEFLNYLGPKSDAADFKAIIDNYLATIPQGSTPNWVQGNHDQHRSASRLGPQKADAVNMLLQVLPGAAITYYGEELAMEDVFIPWYRTVDPQACTTDPVTFHKYSRDPARTPMIWDDQRNAGFSNANSTWLPVGPDFRSNNVELQRSERSSHLNIFKKLMQLRKQDILMYGDYDSVLLNDDVLAVKRELSNNRTLIAVLNLGFTEQMVKLSTISQWQIPQVMEVYTASVGSGMFERQPVYTDEVYVAAGIGVVLDYQVGKRLPQPRGDDPGQYEQ。

[0123]

[0124] From the above experimental results, it can be seen that the present invention obtains an allergen of Culicoides for the first time, and the effectiveness of the allergen is verified by basic experimental detection, and subsequent construction of a recombinant expression vector, induced expression, purification and in vivo experiments. The allergen of Culicoides banded provided by the present invention is maltase Cul t 1, which is an allergen derived from Culicoides banded, and can be used for actual vaccine development against Culicoides banded, and has the effect of preventing allergic reactions caused by Culicoides banded, thereby solving the problem in the prior art that the currently known allergens cannot replace the allergens derived from Culicoides banded for actual vaccine development.

[0125] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0126] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts are known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A Culicoides banded allergen, characterized in that: The allergen of Culicoides stripe is maltase Cult 1, and the amino acid sequence of the maltase Cult 1 is shown in SEQ NO.

1.

2. The Culicoides banded allergen according to claim 1, characterized in that The base sequence of the gene encoding the maltase Cult 1 is shown in SEQ NO.

2.

3. Use of the Culicoides banded allergen according to claim 1 or 2 as an antigen, hapten, immunogen or immunomodulator.

4. Use of the Culicoides stripes allergen according to claim 1 or 2 in the preparation of products resistant to sensitization to the Culicoides stripes allergen.

5. The use according to claim 4, characterized in that: The product is a vaccine and / or a medicine.

6. An antibody, characterized in that Obtained by screening using the striped Culicoides allergen described in claim 1 or 2 as a target.

7. The antibody according to claim 6, characterized in that The antibody is any one of a polyclonal antibody, a monoclonal antibody and a chimeric antibody.

8. A method for identifying the allergen of Culicoides striped midge according to claim 1, characterized in that: The following steps are involved: Strip Preparation of Culicoides thorax extract: The thorax extract of Culicoides stripes induces an allergic reaction, collects in vitro serum samples after the allergic reaction, and screens for allergens; Detecting the binding between the thorax extract of Culicoides stripes and the specific antibody to determine the presence of potential allergens in the thorax extract of Culicoides stripes; The potential allergen is identified, and the obtained potential allergen is the Culicoides banded allergen.

9. The method according to claim 8, characterized in that The allergen of Culicoides band was identified by mass spectrometry detection and Uniport analysis method; The mass spectrometry detection adopts electrospray ionization quadrupole analysis protein fragment time-of-flight-tandem mass spectrometry.

10. The method according to claim 9, characterized in that The parameters of the mass spectrometry detection are as follows: Multiple reaction monitoring mode was used for positive ion detection; Interface voltage: 3.8kV~4.2kV; interface temperature: 298℃~302℃; nebulizing gas flow rate: 2.9L / min~3.1L / min; drying gas flow rate: 9.8L / min~10.2L / min; heating gas flow rate: 9.8L / min~10.2L / min; heating block temperature: 398℃~402℃; desolvation tube temperature: 248℃~252℃; desolvation temperature: 518℃~522℃; collision gas is high-purity argon.

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