Method for establishing a subcutaneous specific immunotherapy model for Alternaria allergic asthma mice using Alt a 1

Abstract

The present invention provides a method for establishing a subcutaneous specific immunotherapy model of Alternaria allergic asthma mice using Alt a 1. The method comprises S1, using Alternaria crude extract (Alternaria) to perform nasal drops for sensitization and stimulation of mice; S2, using Alternaria, rAlt a 1 or nAlt a 1 protein to perform SCIT on the sensitized and stimulated mice respectively; S3, using Alternaria again to perform nasal drops for stimulation of the mice that have received SCIT. The method has a simple operation method, a moderate modeling time, a good model effect, and high repeatability, and is expected to be further promoted and used.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for establishing a subcutaneous specific immunotherapy model for Alternaria allergic asthma mice by using Alt a 1. Background Art

[0002] The prevalence of allergic asthma caused by Alternaria is increasing year by year. Currently, one of the main treatments for such patients is subcutaneous immunotherapy (SCIT) with crude extracts of Alternaria. However, the use of crude extracts for immunotherapy has been found to have major disadvantages, including: (1) For example, crude extracts such as birch pollen, dust mites, dog hair, and Alternaria have poor quality due to large differences between batches; (2) Studies have shown that some crude extracts lack important allergenic protein molecules, or the content of major allergenic proteins is extremely low, which directly leads to patients receiving low-titer immunotherapy injections all year round; (3) As the prevalence of patients with multiple allergies increases sharply, if the traditional treatment plan is maintained, that is, immunotherapy with crude extracts of multiple allergens for such patients, how much interference will there be? What is the long-term efficacy? More real-world data is needed to support this; (4) In terms of immune response, patients receiving allergen immunotherapy (AIT) with natural crude extract preparations cannot induce IgG antibodies against important allergens, which means that the increased IgE antibodies in the patient's body cannot be antagonized, resulting in poor clinical efficacy.

[0003] At present, there is no animal model research on immunotherapy for Alt a 1 protein molecules at home and abroad. As an allergen as common as dust mites and pollen, it is very necessary to explore the immunotherapy model of Alternaria allergic asthma mice.

[0004] The present invention is the first model in China and abroad to establish an immunotherapy model for asthma mouse models using Alt a 1 protein, breaking the bottleneck of previous crude extract treatment, and providing a strong basis for the immunotherapy of Alternaria to enter the era of precise and targeted treatment. The treatment of a single major sensitizing component effectively solves the problems of uncertain efficacy and poor safety caused by the complex composition of crude extracts, and properly avoids the practical problems of uneven content or low concentration of major allergenic proteins in different batches of crude extracts.

[0005] The operation method involved in the present invention is simple, the modeling time is moderate, the model effect is good, and the repeatability is high, and it is expected to be further promoted and used. Summary of the invention

[0006] The object of the present invention is to provide an animal model for subcutaneous specific immunotherapy of Alternaria allergic asthma mice using Alt a 1 protein.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for establishing a subcutaneous specific immunotherapy model for Alternaria allergic asthma mice using Alt a 1 protein comprises the following steps:

[0009] S1. Use Alternaria to sensitize and challenge mice by nasal drops;

[0010] S2, SCIT was performed on sensitized and challenged mice using Alternaria, rAlt a 1, or nAlt a 1 protein;

[0011] S3. Mice that had received SCIT were challenged again with Alternaria by nasal drops.

[0012] In the establishment method as described above, preferably, in step S1, the animals are sensitized by slow nasal drops on days 0, 7 and 14 respectively; and challenged by nasal drops continuously for 3 days on days 21-23.

[0013] In the establishment method as described above, preferably, in step S1, the mouse is a female BALB / c mouse.

[0014] In the method established as described above, preferably, the amount of the crude extract of Alternaria is 50 μL / head.

[0015] In the method established as described above, preferably, in step S2, subcutaneous specific immunotherapy is performed on mice every other day from day 30 to day 42.

[0016] In the method established as described above, preferably, the specific immunotherapy refers to gently pinching and fixing the skin on the neck of the mouse, and drawing 100 μL of the corresponding therapeutic drug with a 1 mL syringe.

[0017] In the method established as described above, preferably, in step S3, nasal drop stimulation is performed again for 3 consecutive days on days 49-51.

[0018] According to the establishment method as described above, preferably, the amino acid sequence of the rAlt a 1 protein is as shown in SEQ ID NO.1.

[0019] In the establishment method as described above, preferably, the protein expression of rAlt a 1 is carried out using the gene sequence shown in SEQ ID NO.2.

[0020] Through multiple studies, it was found that when the number of stimulations exceeded 3 times, the inflammation of the mouse lung tissue was very serious, causing great damage to the mice, so it was preferred to perform nasal drops again for 3 consecutive days. The amount of crude extract in the model was 50 μL / mouse and the amount of SCIT was 100 μL / mouse, both of which were based on the administration volume of crude extracts abroad, and there was no reference to the administration volume of Alt a 1. In order to achieve comparability between different experimental groups, the treatment volume of Alt a 1 must also be consistent with the volume of the crude extract.

[0021] The beneficial effects of the present invention are:

[0022] The present invention is the first animal model in China that uses Alt a 1 protein to perform subcutaneous specific immunotherapy on mice with Alternaria allergic asthma, filling the gap in Alt a 1 in mouse immunotherapy models.

[0023] The method for establishing a subcutaneous specific immunotherapy model of Alternaria allergic asthma mice using Alt a 1 provided by the present invention has a simple operation method, a moderate modeling time, a good model effect, and high repeatability, and is expected to be further promoted and used. The model established by the method of the present invention can be used for subsequent immunotherapy research on Alternaria, providing a repeatable research model for researchers who need to conduct Alternaria immunity research, and on the other hand, provides an animal model for a novel Alternaria immunotherapy preparation-Alt a 1. Various tissue and cytological experiments can be carried out on this model in the future, so as to achieve the experimental purpose desired by the researchers.

[0024] The present invention proves that 5 μg of Alt a 1 has a good immunotherapeutic effect on the Alternaria asthma mouse model, becoming the first model in China to verify the therapeutic concentration and therapeutic effect of Alt a 1.

[0025] The model provided by the present invention utilizes Alt a 1, the main sensitizing component, for immunotherapy, thereby reducing the toxic and side effects of the crude extract of Alternaria alternata on the asthma mouse model, and has application space in future clinical treatment; the use of rAlta 1 has the characteristics of easy availability, good stability between product batches, and low cost, which greatly improves the economic and safety issues brought about by the use of crude extracts for treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the SCIT scheme.

[0027] Figure 2 Alt a 1 improves airway resistance in mice.

[0028] Figure 3 This is the result of total cell count in the supernatant of bronchoalveolar lavage fluid.

[0029] Figure 4 It is the IL-4 content level in the supernatant of bronchoalveolar lavage fluid.

[0030] Figure 5 It is the IL-10 content level in the supernatant of bronchoalveolar lavage fluid.

[0031] Figure 6 It is the IL-13 content level in the supernatant of bronchoalveolar lavage fluid.

[0032] Figure 7 is the concentration of MMCP-1 in the supernatant of bronchoalveolar lavage fluid.

[0033] Figure 8 is the antibody concentration of IgE and sIgG in serum; where (A) total IgE level in serum. (B) Alternaria-sIgE and Alt a 1-sIgE levels in serum. (C) Alternaria and Alt a 1-sIgG1, sIgG2a, sIgG2b levels in serum. (n = 8 / group). Values ​​are mean ± SD. *P < .05, **P < .01 and ***P < .01 compared with PC group. NC: healthy control group, PC: positive control group.

[0034] Fig. 9 The results are the ratios of sIgG to sIgE in serum after SCIT; (A) Alternaria-related neutralizing antibody levels in serum. (B) Alt a 1-related neutralizing antibody levels in serum. (n = 8 / group). Values ​​are mean ± SD. *P < .05, **P < .01 compared with the PC group. NC: healthy control group, PC: positive control group.

[0035] Fig.10 Alt a 1 can inhibit the inflammatory response of asthmatic mice induced by Alternaria alternata; (A) H&E staining of lung tissues of mice in 5 groups. Scale bar = 250 μm. (B) Semi-quantitative scoring of inflammatory pathology of lung tissues. (n = 8 / group). Values ​​are mean ± SD. *P < .05 compared with PC group. NC: healthy control group, PC: positive control group.

[0036] Fig.11 Alt a 1 alleviates the destruction of goblet cells and mucus secretion in the lung tissue of asthmatic mice. (A) PAS staining of lung tissue of 5 groups of mice. Scale bar = 250 μm. (B) Semi-quantitative score of goblet cell destruction. (n = 8 / group). Values ​​are mean ± SD. NC: healthy control group, PC: positive control group. DETAILED DESCRIPTION

[0037] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the present invention all fall within the scope of the present invention.

[0038] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are all of analytical purity or above. Among them, the Alternaria crude extract was purchased from Greer Company (Lot: 362093), the nAlta1 protein can be purchased commercially, and rAlta1 was obtained by preparation and purification. The amino acid sequence corresponding to the purified rAlta1 protein is shown in SEQ ID NO.1: APLESRQDTASCPVTTEGDYVWKISEFYGRKPEGTYYNSLGFNIKATNGGTLDFTCSAQADKLEDHKWYSCGENSFMDFSFD SDRSGLLLKQKVSDDITYVATATLPNYCRAGGNGPKDFVCQGVADAYITLVTLPKSS. The gene sequence encoding it was transferred into the pCZN1 plasmid, and the recombinant plasmid was introduced into Escherichia coli, and IPTG was used to induce synthesis. In the following examples, female BALB / c mice can be purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the certificate number of the mice used is No. 110011201109895154, and the license number is SCXK (Beijing) 2016-0006; New Zealand rabbits can be purchased from the Experimental Animal Farm in Fengdong New City, Xixian New Area, Xi'an. The license number of the New Zealand rabbits used in the following examples is: SCXK (Shaanxi)-2017_002).

[0039] Example 1

[0040] Preparation method and purification method of rAlta1:

[0041] 1. First, synthesize the gene Alta1, and the sequence is shown in SEQ ID NO.2

[0042] catatgGCACCTCTGGAAAGCCGTCAGGATACCGCAAGCTGTCCGGTTAC

[0043] CACCGAAGGTGATTATGTTTGGAAAATTAGTGAATTTTATGGTCGTAA

[0044] ACCGGAAGGTACCTATTATAATAGTCTGGGTTTTAATATCAAAGCCAC

[0045] CAATGGTGGTACTCTGGATTTTACCTGTAGTGCACAGGCAGATAAACT

[0046] GGAAGATCATAAATGGTATAGCTGTGGTGAAAACAGTTTTATGGATTT

[0047] TAGTTTTGATAGCGATCGTTCCGGTCTGCTGCTGAAACAGAAAGTTAG

[0048] TGATGATATCACCTATGTTGCAACGGCAACGCTGCCGAATTATTGTCG

[0049] TGCGGGTGGTAATGGTCCGAAAGATTTTGTTTGTCAGGGTGTTGCGGATGCTTATATTACCCTGGTTACCCTGCCGAAAAGCAGCtaatctaga), where lowercase letters are protective bases, which make the entire sequence more stable. The sequence shown in SEQ ID NO.2 was connected between the NdeI and XbaI sites of the vector pCZN1 (Biotech Biotechnology Co., Ltd.); the obtained recombinant plasmid pCZN1-Alt a 1 was transferred into the Top10 cloning strain, and the positive clones were selected for sequencing. The sequencing results were compared with the expected sequence, and the plasmid was identified by enzyme digestion. The plasmid enzyme digestion identification method was double enzyme digestion, taking plasmid: 3μL, endonuclease 1 (NdeI): 0.25μL, endonuclease 2 (XbaI): 0.25μL, 10×buffer: 1.0μL, DDW: up to 10μL for identification. The enzyme digestion was double enzyme digestion, the electrophoresis morphology was linear, and the correctness of the synthetic plasmid was detected by enzyme digestion. There were two fragments after enzyme digestion, one was 4500bp (bright band), and the other was between 200-500bp. The positive clone was recorded as pCZN1-Alt a 1 plasmid.

[0050] 2. Add the test plasmid to 100 μL of competent cell Top10 bacteria (available from Biotech Biotech), place on ice for 20 minutes, activate for 90 seconds at 42°C, quickly place on ice, maintain for 5 minutes, and then add 0.6 mL of LB culture solution. Shake for 1 hour at 37°C, 220 r / min, centrifuge, and spread all on LB transformation plates containing 50% PBS, invert and culture overnight at 37°C.

[0051] 3. Pick the single colony on the transformation plate and inoculate it into a test tube containing 3 mL LB culture medium of the single colony picked from the 50 plates, shake at 37 °C, 220 rpm and incubate overnight.

[0052] Shake the culture medium: inoculate at a ratio of 1:200, shake at 37°C, 220 rpm for 5 h until the OD600 value of the culture medium reaches 0.6-0.8.

[0053] IPTG induction: At the same time, an uninduced group was designed. The induced group was added with prepared IPTG at a ratio of 1:200, and shaken at 37°C and 220r / min for 4-5h to induce the expression of fusion protein; the uninduced group was not added with IPTG.

[0054] Take 1mL of culture, wash with PBS, resuspend the bacterial precipitate with PBS, ultrasonically disrupt the resuspended liquid in an ultrasonic disruptor, and then add 5× loading buffer to the supernatant and precipitate after disruption and mix them well; detect with 12% SDS-PAGE, stain with Coomassie Brilliant Blue and develop the bands. The results show that the target protein exists in the precipitate after induced disruption.

[0055] 4. The induced E. coli cell pellet was resuspended in 20 mL of lysis buffer and broken under ultrasonic conditions (power: 400 W, working 4 s, rest 8 s, total 20 min). Then, at 4°C, 10000 r / min, 20 min, the pellet was collected.

[0056] The precipitate was washed three times with an aqueous solution having a final concentration of 20 mM Tris, 1 mM EDTA, 2 M urea, 1 M sodium chloride, 1% Triton X-100, pH 8.0.

[0057] The washed precipitate was dissolved with an aqueous solution having a final concentration of 20 mM Tris, 5 mM DTT, 8 M urea, pH 8.0, and taken out after overnight at 4°C. The precipitate was centrifuged at room temperature at 10,000 r / min for 15 min, and the supernatant was collected.

[0058] Dialysis after dilution: Add 20mM Tris, 0.15M sodium chloride, pH8.0 buffer solution to the supernatant after centrifugation, dilute with stirring while adding, put the solution into a boiled dialysis bag, fix both ends with dovetail clips, and carefully place it in 20mM Tris-HCl, 0.15M sodium chloride, pH=8.0 solution for dialyzing overnight.

[0059] 5. The collected protein solution was placed in a dialysis bag after boiling for 10 minutes (boiling is to remove harmful substances in the dialysis bag that rupture and release proteins) and cooled, and then dialyzed in a beaker filled with PBS overnight. After electrophoresis using 12% SDS-PAGE, protein purity analysis was performed using Coomassie Brilliant Blue dye, and the purity was >98%. The results showed that rAlt a1 was a homodimer with a molecular weight of about 30 kDa.

[0060] Example 2

[0061] A total of 50 healthy female BALB / c mice were randomly divided into 5 groups after one week of adaptive feeding, namely NC (healthy group), PC (positive control group), rAlt a 1, nAlt a 1 and Alternaria group, as shown in Table 1:

[0062] Table 1 Alt a 1 grouping and intervention methods in asthmatic mice undergoing SCIT (n = 8 mice / group)

[0063]

[0064] SCIT program, as follows Figure 1 As shown: On days 0, 7, and 14, mice were sensitized with slow nasal drops (50 μL / mouse), and nasal drops were continuously challenged for 3 days from day 21 to 23 (50 μL / mouse). On days 30 to 42, mice were given subcutaneous specific immunotherapy every other day, that is, the skin of the mouse neck was gently pinched and fixed, and 100 μL of the corresponding therapeutic drug (including: rAlt a 1, nAlt a 1, Alternaria, PBS) was drawn with a 1mL syringe and injected along the pinched skin. At this time, a slightly bulging skin papule could be touched, indicating that the subcutaneous administration was successful. After an interval of 1 week, nasal drops were challenged again for 3 consecutive days on days 49 to 51. The respiratory status, weight changes, and skin status of the subcutaneous injection site of the mice were closely observed throughout the SCIT period.

[0065] Evaluation of the effect of subcutaneous specific immunotherapy (the evaluation order should be pathology first → airway resistance → other indicators)

[0066] (1) Lung tissue pathology for inflammation assessment

[0067] 1) HE staining and inflammation grading:

[0068] Lung tissue sampling: After cutting the sternum to expose the chest cavity, flush both lungs with normal saline through the right ventricle until they turn white, then separate and place in a sample tube containing 4% paraformaldehyde for fixation.

[0069] Paraffin embedding and sectioning were commissioned to Beijing Biosun Technology Co., Ltd.

[0070] Baking: Bake the slices in an oven at 60°C for 2 h.

[0071] Dewaxing: Dewax the baked slides with dewaxing liquid.

[0072] Hematoxylin staining: The dewaxed sections were placed in hematoxylin staining solution and stained for 3 minutes.

[0073] Rinse: Rinse the slices in running water for 3-5 minutes.

[0074] Hydrochloric acid alcohol differentiation: Immerse the sections in 1% hydrochloric acid alcohol for 5-15 seconds and repeat the washing step.

[0075] Eosin staining: Place the sections in 0.5% eosin alcohol solution, immerse for 2 minutes, and repeat the washing steps.

[0076] Sealing: After drying, seal the slide with neutral resin and observe under a microscope.

[0077] Inflammation grading: Grading is based on the degree of inflammatory cell infiltration around the bronchus (the thickness of 3 lymphocytes is 1 week): Grade 1 - lack of inflammatory cells around the bronchus; Grade 2 - inflammatory cells ≤ 25% of the bronchial circumference; Grade 3 - inflammatory cells around 25%-75% of the bronchial circumference; Grade 4 - inflammatory cells around the bronchus for 1 week; Grade 5 - inflammatory cells around the bronchus for 2 weeks; Grade 6 - inflammatory cells around the bronchus for ≥ 3 weeks;

[0078] 2) PAS staining and scoring:

[0079] The dewaxing treatment is the same as the HE stained part.

[0080] Oxidation: Oxidize with 0.5% high-point acid solution for 10 minutes, rinse under tap water for 5 minutes, and wipe dry.

[0081] Staining: Stain with Schiff stain for 30 minutes and repeat the washing step.

[0082] Nuclear staining: Use an appropriate amount of hematoxylin staining solution to stain the nucleus for 2-5 minutes.

[0083] The subsequent steps are basically the same as the HE staining part.

[0084] Scoring: 0 points, PAS-positive goblet cells account for ≤5% of the total airway epithelial cells; 1 point, positive goblet cells account for 5%-25%; 2 points, positive goblet cells account for 25%-50%; 3 points, positive goblet cells account for 50%-75%; 4 points, positive goblet cells ≥75%; 5 points, almost all airways have a large number of PAS-positive goblet cells.

[0085] The results are as follows Figure 2 and Figure 3 As shown, from Figure 2 A's lung tissue HE staining (200 times) showed that compared with the healthy control (NC group), the mice in the positive control group (PC group) were infiltrated by a large number of inflammatory cells around the bronchi and lung interstitium, with thickening of the bronchial wall, narrowing of the lumen, interruption of mucosal continuity, and damage to epithelial cells, which were consistent with the inflammatory conditions of the asthma model. SCIT was performed on the model using Alt a 1 and Alternaria alternata crude extracts, and it was found that all three could alleviate the inflammation of the lung tissue of asthmatic mice, mainly reflected in the reduction of inflammatory cells around the bronchi and blood vessels. Among them, the therapeutic effect of rAlt a 1 was relatively significant, and the effect of the crude extract was slightly less effective. Objectively evaluate the inflammation from the perspective of semi-quantitative scoring ( Figure 2 B), it can also be seen that the three proteins (rAlt a 1, nAlt a 1 and Alternaria crude extract) improve the inflammation of mouse lung tissue, and the rAlt a 1 treatment group is statistically significant compared with the PC group (P = 0.0143).

[0086] In addition to evaluating the therapeutic effect of Alt a 1 from the perspective of lung tissue inflammation, the present invention also evaluated the goblet cells in the mouse lungs using PAS staining ( Figure 3 A). Similar to the results of HE staining, the goblet cell destruction in the PC group was more obvious than that in the NC group. After treatment with the crude extracts of Alt a 1 and Alternaria alternata, the goblet cell destruction in the lung tissues of the three groups of mice was significantly improved. Figure 3 B), no statistical difference was found between the PC group and each treatment group, but the relief in the rAlt a 1 group was relatively obvious, which further illustrates the therapeutic effect of rAlt a 1 on asthma inflammation.

[0087] (2) Assessment of mouse airway responsiveness

[0088] Anesthesia: Within 24 hours after the last stimulation, the mice were anesthetized by intraperitoneal injection of 0.1 mL / 20 g of pentobarbital. The next step can be performed after the mouse's limbs are gently clamped with forceps and there is no response.

[0089] Endotracheal intubation: Use forceps to separate the cervical fascia, use ophthalmic scissors to make a small cut at the tracheal ring, and then insert the tracheal tube ring into the trachea to fix it. Do not induce bleeding during this process to avoid blocking the trachea and causing suffocation.

[0090] Nebulization: After placing the mice in the detector and adapting for 5 minutes, they were exposed to PBS by nebulization, and the baseline was set. Then, 1.5, 3, 6, 12, and 24 mg / mL of methacholine were added for 1 minute. During this process, the breathing and anesthesia status of the mice were closely observed.

[0091] Measuring Resistance: The Buxco Fine Pointe system automatically generates airway resistance values.

[0092] The airway resistance values ​​obtained in each group were plotted with the concentration of methacholine as the horizontal axis and the airway resistance value as the vertical axis. The values ​​are mean ± standard deviation. Figure 4 shown.

[0093] Since the main manifestation of asthma in clinical practice includes airway hyperresponsiveness, and lung function is an objective means of diagnosing asthma, it is particularly important to evaluate the airway resistance of asthma mouse models and after SCIT. The present invention uses methacholine in a concentration range of 1.5-24 mg / mL to test the airway resistance of mice ( Figure 4 ), the results showed that the airway resistance of mice in the PC group gradually increased from 3 mg / mL concentration to that of the other groups, but there was no statistical significance. There was no significant difference in airway resistance among the three treatment groups. At 6 mg / mL concentration, the difference with the PC group was the largest. This part of the results showed that Alt a 1 treatment can reduce the airway resistance of asthmatic mice, which illustrates the therapeutic effect of Alt a 1 from another perspective.

[0094] (4) Total cell count of bronchoalveolar lavage fluid (BALF)

[0095] 1) BALF collection:

[0096] Sacrifice: On the 52nd day of the experiment (within 24 hours of the last stimulation), the mice were killed by overdose of anesthesia, and the eyeballs were removed and blood was collected.

[0097] Expose the trachea: fix the limbs, fully separate the cervical fascia and blood vessels, and expose the trachea.

[0098] Endotracheal intubation: After cutting a small hole in the tracheal ring with ophthalmic scissors, insert a 24G indwelling needle into the trachea and fix it with sutures.

[0099] Lavage: pre-cooled PBS at a volume of 0.5 mL / mouse was slowly injected into the mouse lung tissue using a 1 mL syringe connected to a retention needle.

[0100] Collection: After gently pressing both lungs of the mouse, slowly draw back the milky white liquid into the syringe, and repeat this step 3 times. The recovery rate is >80%.

[0101] 2) Total cell count:

[0102] Centrifugation: Centrifuge the collected BALF (4°C, 2000 rpm, 10 min), collect the supernatant and aliquot it for freezing and storage.

[0103] Resuspend: Resuspend the pellet in 100 μL of PBS, pipette 20 μL and count on a cell counter.

[0104] The inflammation of bronchoalveolar lavage fluid is also an important indicator for evaluating asthma models. Therefore, the present invention continues to evaluate the therapeutic effect of Alt a 1 from multiple perspectives. By collecting BALF from each group of mice, the total cell count was performed first. The results are as follows Figure 5 As shown, obviously, the total cell number of PC group was significantly higher than that of NC group. After treatment with Alt a 1 and Alternaria crude extract, the cell count showed a significant decrease, among which the reduction levels of nAlt a 1 and rAlt a 1 groups were comparable (P=0.0043).

[0105] (3) Detection of the expression levels of IL-4, IL-10 and IL-13 in BALF supernatant.

[0106] Sample processing: Concentrate the supernatant to about 400 μL using an ultrafiltration tube.

[0107] Antibody coating concentration: IL-4 and IL-10: 2 μg / mL, IL-13 is pre-coated by Wuhan Huamei Company.

[0108] Rewarming: Take out the 96-well plate and rewarm it at room temperature for 30 minutes.

[0109] Wash the plate: Wash the plate three times with 300 μL of TBST.

[0110] Blocking: Block with 200 μL of TBST blocking solution containing 1% BSA at room temperature for 1 h.

[0111] Repeat the plate wash step.

[0112] Add the prepared standard and the sample to be tested and dilute according to the instructions, and incubate overnight at 4°C.

[0113] After rewarming, repeat the plate washing step.

[0114] Add 100 μL of Biotin-labeled antibody to each well, incubate at room temperature for 1 h, and then repeat the plate washing step.

[0115] Then add 100 μL of streptavidin-HRP antibody, incubate at room temperature for 30 min, and repeat the plate washing step.

[0116] Color development: Add 100 μL of TMB color development solution to each well, and a blue precipitate will be visible in about 5 minutes.

[0117] Termination reaction: Add 100 μL of concentrated sulfuric acid to each well to terminate the reaction.

[0118] Read and calculate protein concentration: Read the sample at a wavelength of OD450 nm on the microplate reader and calculate the protein concentration based on the standard curve.

[0119] The present invention detected the expression level of cytokines in BALF supernatant. The quantitative detection results of ELISA on IL-4, IL-10 and IL-13 in BALF were as follows: Figure 6-Figure 8 As shown in the figure, *P<.05, **P<.01 compared with the PC group. IL-4 and IL-13 are mainly secreted by type 2 inflammatory cells, and IL-10 is mainly produced by Breg cells, which is generally considered to have a negative regulatory effect on immunity. In view of this, the present invention found that the expression levels of IL-4 and IL-13 in the PC group were significantly higher than those in the NC group. After immunotherapy, the expression of both cytokines decreased. The degree of decrease of IL-4 in the nAlt a 1 group was the highest, which was statistically significant (P=0.0022), and the expression of IL-13 in the Alternaria crude extract group was the lowest (P=0.0411). It shows that both the Alt a 1 component and the Alternaria crude extract treatment can alleviate the type 2 inflammatory cytokines induced by asthmatic mice. As for IL-10, we observed that its level increased after rAlt a 1 and Alternaria crude extract SCIT treatment, and the increase in the nAlt a 1 group was not obvious.

[0120] (4) Detection of mouse mast cell protein-1 (MMCP-1) in serum

[0121] Rewarming: Rewarm the kit at room temperature for 30 minutes before use in the next step.

[0122] Washing: Wash the plate 3 times with TBST by shaking.

[0123] Blocking: Block with diluted assay buffer at room temperature for 1 hour, then repeat the plate washing operation.

[0124] Prepare standard curves: 4000pg / mL, 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL and blank wells.

[0125] Incubation with primary antibody: serum was diluted at a ratio of 1:10 and incubated at 37°C for 3 h, then the plate was washed repeatedly.

[0126] Add 100 μL of detection antibody to each well and incubate at room temperature for 1 hour. Repeat the plate washing operation.

[0127] Add 100 μL of diluted avidin-HRP antibody to each well, react at room temperature for 30 min, and then wash the plate three times.

[0128] Color development: Add 100 μL of TMB to each well and develop color at room temperature for 15 min.

[0129] Stop the reaction and read the results: add 100 μL of stop solution to each well, read the results at OD450 nm, and calculate the value of MMCP-1 based on the standard curve.

[0130] The results are as follows Fig. 9 As shown in the figure, the values ​​are mean ± standard deviation, MMCP-1: mast cell protease-1. The expression of MMCP-1 in asthma models is unknown. Therefore, this section also aims to provide auxiliary evidence for the therapeutic effect of the above protein in this regard. Fig. 9 It can be seen that the MMCP-1 level in the PC group was slightly higher than that in the other groups. Although it decreased in the treatment groups, there was no statistical difference, and the expression levels of MMCP-1 among the three treatment groups were also comparable. This result may suggest that although the asthma model established in this part had obvious inflammation, it did not reach the level of severe allergic reaction.

[0131] (5) Detection of T-IgE, Alternaria and rAlt a 1-sIgE, sIgG1, sIgG2a, and sIgG2b in serum

[0132] Detection of T-IgE, Alternaria and rAlt a 1-sIgE: See the table below for relevant antibody information.

[0133] Table 2 SCIT stage serum IgE antibody detection information

[0134]

[0135] The detection information of Alternaria and rAlt a 1-sIgG1, sIgG2a, sIgG2b is shown in the table below:

[0136] Table 3 SCIT stage serum IgG antibody detection information

[0137]

[0138]

[0139] The detection method is basically the same as the "IL-4 and IL-10 detection part in BALF supernatant".

[0140] Results Fig.10 and Fig.11 , from the changes in serum T-IgE ( Fig.10A), the T-IgE levels of the three treatment groups were significantly lower than those of the PC group, all of which were statistically significant. The detection of antigen-specific IgE can further verify the success of the allergic asthma model, so this part continued to monitor the sIgE levels of Alternaria alternata and Alt a 1 ( Fig.10 B) found that the expression of Alternaria-sIgE was similar to that of T-IgE, i.e., it was significantly reduced in the treatment group, but in Alt a 1-sIgE, there was no trend of decreased expression in the treatment group, but it was increased compared with the PC group, especially in the nAlt a 1 group (P=0.0022), followed by the rAlt a 1 group. This may be related to the fact that these two groups were treated with only the Alt a 1 component.

[0141] IgG antibodies are considered to have protective significance in clinical studies of AIT and are one of the hallmark antibodies for the therapeutic effect of AIT. The present invention also detected the expression of Alternaria alternata and Alt a 1-specific IgG antibodies in a mouse model ( Fig.10 C). As can be seen from the figure, only sIgG1, sIgG2a and sIgG2b of Alt a 1 showed increased levels in the two component treatment groups, with the most obvious increase in the nAlt a 1 group. The expression of various sIgG subtypes of Alternaria alternata in the treatment groups was not consistent. Alternaria alternata-sIgG1 and sIgG2b in the rAlt a 1 group were lower than those in the PC group, and only a slight increase was seen in sIgG2a. The reason may be related to the complexity of the crude extract components.

[0142] In addition, the present invention also analyzed the neutralizing antibody ratios (sIgG1 / sIgE, sIgG2a / sIgE, sIgG2b / sIgE) related to Alternaria alternata and Alt a 1. In general, the neutralizing antibody ratio of Alt a 1 showed an increased expression in the Alt a 1 immunotherapy group ( Fig.11 A), while the neutralizing antibody ratios associated with Alternaria alternata remained inconsistent ( Fig.11 B), only the increase in the expression of sIgG1 / sIgE was statistically significant (P=0.0411), and the increase in the other two ratios was not obvious.

[0143] The results of this part revealed that immunotherapy of asthmatic mice with Alt a 1 could induce an increase in the expression of IgG and its subtypes and the ratio of neutralizing antibodies compared with treatment with crude extract of Alternaria.

[0144] The animal model established by the present invention can be used for subsequent immunotherapy research on Alternaria alternata, that is, it provides a reproducible research model for researchers who need to conduct immunotherapy research on Alternaria alternata.

Claims

1. A method for establishing a subcutaneous specific immunotherapy model of Alternaria allergic asthma mice using Alt a 1 protein, characterized in that: It includes the following steps: S1. Use crude extract of Alternaria alternata to sensitize and challenge mice by nasal drops; S2, SCIT was performed on sensitized and challenged mice using crude Alternaria extract, rAlt a 1, or nAlt a 1 protein, respectively; S3, the mice that had received SCIT were challenged again with crude extract of Alternaria by nasal drops; The amino acid sequence of the rAlt a 1 protein is shown in SEQ ID NO.

1.

2. The establishment method according to claim 1, characterized in that: In step S1, animals were sensitized by slow nasal drops on days 0, 7, and 14, respectively; and challenged by nasal drops for 3 consecutive days on days 21-23.

3. The establishment method according to claim 1, characterized in that: In step S1, the mice are female BALB / c mice.

4. The establishment method according to claim 1, characterized in that: The dosage of the crude extract of Alternaria was 50 μL per individual.

5. The establishment method according to claim 1, characterized in that: In step S2, mice were given subcutaneous specific immunotherapy every other day from day 30 to day 42.

6. The establishment method according to claim 1, characterized in that: Specific immunotherapy refers to gently pinching and fixing the mouse neck skin, and using a 1mL syringe to draw 100μL of the corresponding therapeutic drug.

7. The establishment method according to claim 1, characterized in that: In step S3, on days 49-51, nasal drop challenge was performed again for 3 consecutive days.

8. The establishment method according to claim 1, characterized in that: The protein expression of rAlt a 1 is carried out using the gene sequence shown in SEQ ID NO.2.

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