CN116098124B - Method for establishing a subcutaneous specific immunotherapy model for Alternaria allergic asthma mice using Alt a 1 - Google Patents

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

Allergic asthma mice to Alt a 1 method for establishing subcutaneous specific immunotherapy model

Technical Field

The invention belongs to the field of biotechnology, in particular to allergic asthma to Alt a 1a method for establishing a subcutaneous specific immune therapeutic model of mice.

Background

The prevalence of allergic asthma caused by alternaria alternata increases year by year, and one of the main therapeutic modalities currently aimed at such patients is subcutaneous specific immunotherapy of crude alternaria alternata extract (ALLERGEN SPECIFIC subcutaneous immunotherapy, SCIT). However, the significant drawbacks of using crude extracts for immunotherapy include (1) crude extracts such as birch pollen, dust mites, dog hair and alternaria leaf curl, etc., which are poor in quality due to large differences between batches, (2) studies to confirm that some crude extracts lack important protein molecules or have very low levels of major protein, which directly lead to patients receiving daily low-titer immune injection therapy, (3) as the prevalence of multiple sensitized patients increases dramatically, how much of the mutual interference is required to maintain conventional treatment regimens for such patients with multiple allergen crude extracts.

Currently, no animal model study is made for immunotherapy against Alt a 1 protein molecules at home and abroad. As an allergen common to dust mites and pollen, it is necessary to develop a model for the immunotherapy of Alternaria alternata allergic asthma mice.

The invention establishes a model for carrying out immunotherapy on an asthma mouse model by utilizing Alt a 1 protein for the first time at home and abroad, breaks the bottleneck of the prior crude extract treatment, and provides a powerful basis for the accurate and targeted treatment times of the immunity treatment of the alternaria alternata. The treatment of single main sensitization component effectively solves the problems of inaccurate curative effect, poor safety and the like caused by complex components of the crude extract, and properly avoids the practical problems of uneven content or lower concentration of main sensitization protein in the crude extracts of different batches.

The operation method related by the invention is simple, the modeling time is moderate, the model effect is good, the repeatability is high, and the method is expected to be further popularized and used.

Disclosure of Invention

The invention aims to provide an animal model for carrying out subcutaneous specific immunotherapy on Alt a1 protein-based Alt 1 allergic asthma mice.

In order to achieve the above purpose, the invention adopts the following technical scheme:

A method for establishing a subcutaneous specific immunotherapy model of Alt a1 protein in Alt a1 protein allergic asthma mice, the method comprises the following steps:

S1, nasal drip sensitization and excitation are carried out on a mouse by utilizing Alternaria;

s2, using Alternaria, rAlt a, or nAlt a proteins to sensitize and excite mice SCIT, respectively;

S3, nasal drip excitation is carried out on the mice which have received SCIT by using Alternaria again.

The method of establishment as described above is preferably performed in step S1 by slow nasal drip priming of the animals on days 0, 7, 14, respectively, and continuous nasal drip priming on days 21-23 for 3 days, respectively.

The method of establishment as described above, preferably in step S1, the mice are female BALB/c mice.

The above-mentioned method of establishment is preferably carried out in an amount of 50. Mu.L/g of crude Alternaria alternata extract.

The method of establishment as described above is preferably performed on mice subcutaneously for specific immunotherapy every alternate day on days 30-42 in step S2.

The method of establishment as described above, preferably, the specific immunotherapy means that the neck skin of the mouse is gently pinched and fixed, and 100. Mu.L of the corresponding therapeutic agent is sucked by a 1mL syringe.

The method of establishment as described above is preferably performed in step S3, at days 49-51, for 3 consecutive days, for re-nasal drip excitation.

The method for establishing the polypeptide is preferably that the amino acid sequence of rAlt a1 is shown as SEQ ID NO. 1.

The method for establishing the protein is preferably that the rAlt a protein expression is carried out by adopting a gene sequence with a sequence shown as SEQ ID NO. 2.

It was found from many studies that when the number of shots exceeds 3, the lung tissue of the mouse is severely inflamed and the mice are greatly damaged, so that the nasal drop is preferably performed again for 3 consecutive days. The amount of crude extract in the model is 50 mu L/dose and SCIT mu L/dose, and 100 mu L/dose is only the administration volume of the foreign crude extract, and the administration volume of Alt a1 is not referenced, so that the treatment volume of Alt a1 is consistent with the volume of the crude extract in order to achieve the comparability among different groups of experiments.

The invention has the beneficial effects that:

the invention is an animal model for subcutaneous specific immunotherapy of Alt a1 protein on Alt a1 allergic asthma mice in China, and fills the blank of Alt a1 on the mouse immunotherapy model.

The method for establishing the subcutaneous specific immunotherapy model of the Alt a 1-based Alt allergic asthma mice has the advantages of simple operation method, moderate modeling time, better model effect and high repeatability, is hopeful to be further popularized and used, can be used for the subsequent immunotherapy research of Alt a on one hand, the method provides a repeatable research model for researchers needing to carry out the immunity research of the alternaria alternata, and provides an animal model for the novel immunity therapeutic preparation of the alternaria alternata-Alt a1, and various tissue and cytology experiments can be carried out on the model, so that the aim of the experiments intended by the researchers is fulfilled.

The invention proves that 5 mug of Alt a1 has better immunotherapy effect on Alt a1 asthma mice model, and becomes a model for verifying the therapeutic concentration and the therapeutic effect of Alt a1 for the first time in China.

The model provided by the invention utilizes Alt a1 as a main sensitization component for immunotherapy, reduces toxic and side effects of the crude extract of Alt a1 on an asthma mouse model, has application space in future clinical treatment, has the characteristics of easy acquisition, good stability among product batches, low cost and the like, and greatly improves economic and safety problems caused by the treatment of the crude extract.

Drawings

FIG. 1 is a schematic diagram of SCIT schemes.

Figure 2 is Alt a 1 improving airway resistance in mice.

FIG. 3 is a graph showing the total cell count in the supernatant of alveolar lavage fluid.

FIG. 4 shows IL-4 levels in alveolar lavage fluid supernatant.

FIG. 5 shows IL-10 levels in alveolar lavage fluid supernatants.

FIG. 6 shows IL-13 levels in alveolar lavage fluid supernatant.

FIG. 7 shows MMCP-1 concentration in alveolar lavage fluid supernatant.

FIG. 8 shows the antibody concentrations of IgE and sIgG in serum, wherein (A) the total IgE level in serum. (B) Alternaria-sIgE and Alt a 1-sIgE levels in serum. (C) Serum Alternaria and Alt a 1-sIgG1, sIgG2a, sIgG2b levels. (n=8/group). The values are mean ± standard deviation. * P <.05, P <.01 and P <.01 were compared to the PC group. NC is healthy control group, PC is positive control group.

FIG. 9 shows the results of the ratio of sIgG to sIgE in serum after SCIT, wherein (A) the level of Alternaria-related neutralizing antibodies in serum. (B) Alt a 1-related neutralizing antibody levels in serum. (n=8/group). The values are mean ± standard deviation. * P <.05P <.01 was compared to the PC group. NC is healthy control group, PC is positive control group.

FIG. 10 shows Alt a1 inhibition caused by Alternaria alternata inflammatory response HE staining results in asthmatic mice; (A) H & E staining of lung tissue in 5 groups of mice. Scale bar = 250 μm. (B) semi-quantitative scoring of lung tissue inflammatory pathology. (n=8/group). The values are mean ± standard deviation. * P <.05 was compared to the PC group. NC is healthy control group, PC is positive control group.

FIG. 11 is a graph showing the results of Alt a1 relieving destruction of goblet cells and mucus secretion in lung tissue of asthmatic mice. (A) PAS staining of lung tissue of group 5 mice. Scale bar = 250 μm. (B) semi-quantitative scoring of goblet cell disruption. (n=8/group). The values are mean ± standard deviation. NC is healthy control group, PC is positive control group.

Detailed Description

The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.

Unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, all reagents used in the present invention are of analytical purity or above. Wherein ALTE RNARIA crude extract is purchased from Greer company (Lot: 362093), and nAlt a protein can be obtained by preparing and purifying rAlt a protein by using commercial products. The amino acid sequence corresponding to rAlt a protein prepared by purification is shown as SEQ ID NO.1：APLESRQDTASCPVTTEGDYVWKISEFYGRKPE GTYYNSLGFNIKATNGGTLDFTCSAQADKLEDHKWYSCGENSFMDFSFD SDRSGLLLKQKVSDDITYVATATLPNYCRAGGNGPKDFVCQGVADAY IT LVTLPKSS, the coded gene sequence is transferred into pCZN plasmid, the recombinant plasmid is introduced into escherichia coli, and IPTG is used for induction synthesis. In the following examples, female BALB/c mice were purchased from Vetolihua laboratory animal technologies Inc. in Beijing, the mice used were approved as No.110011201109895154, the license number SCXK (Beijing) 2016-0006, new Zealand rabbits were purchased from laboratory animal farms in New east New City, west salty, new West Ann, and the New Zealand rabbits used in the following examples were approved as No. SCXK (Shaanxi) -2017_002.

Example 1

RAlt a1 preparation method and purification method:

1. Firstly, synthesizing a gene Alt a 1, the sequence of which is shown as SEQ ID NO.2

catatgGCACCTCTGGAAAGCCGTCAGGATACCGCAAGCTGTCCGGTTAC

CACCGAAGGTGATTATGTTTGGAAAATTAGTGAATTTTATGGTCGTAA

ACCGGAAGGTACCTATTATAATAGTCTGGGTTTTAATATCAAAGCCAC

CAATGGTGGTACTCTGGATTTTACCTGTAGTGCACAGGCAGATAAACT

GGAAGATCATAAATGGTATAGCTGTGGTGAAAACAGTTTTATGGATTT

TAGTTTTGATAGCGATCGTTCCGGTCTGCTGCTGAAACAGAAAGTTAG

TGATGATATCACCTATGTTGCAACGGCAACGCTGCCGAATTATTGTCG

TGCGGGTGGTAATGGTCCGAAAGATTTTGTTTGTCAGGGTGTTGCGGATGCTTATATTACCCTGGTTACCCTGCCGAAAAGCAGCTAATCTAGA), wherein the lower case letters are protective bases, which make the whole sequence more stable. The sequence shown in SEQ ID No.2 was ligated between NdeI and XbaI sites of vector pCZN (BAOHOTAI Bio Inc.), and the obtained recombinant plasmid pCZN-Alt a1 was transferred into Top10 clone strain, and positive clones were picked up for sequencing. And comparing the sequencing result with the expected sequence, and carrying out plasmid enzyme digestion identification. The plasmid restriction enzyme identification method is double restriction enzyme, wherein 3 mu L of plasmid, 0.25 mu L of endonuclease 1 (NdeI), 0.25 mu L of endonuclease 2 (XbaI), 10 Xbuffer 1.0 mu L of plasmid and DDW up to 10 mu L of plasmid are taken for identification. The enzyme digestion is double enzyme digestion, the electrophoresis form is linear, two enzyme digestion fragments are used for detecting the correctness of the synthesized plasmid, one is 4500bp (bright band), the other is 200-500bp, and the positive clone is pCZN-Alt a1 plasmid.

2. 100. Mu.L of competent cell Top10 bacteria (available from Bai Ou Tai Bio Inc.) was added with 1 (available from Bai Ou Tai Bio Inc.), and after 20min on ice, the plasmid was activated for 90s at 42℃and rapidly placed on ice for 5min, and then 0.6mL of LB medium was added. Shaking for 1h at 37 ℃ and 220r/min, centrifuging, coating all on LB conversion plates containing 50 parts and all on a containing object, and culturing overnight in an inverted mode.

3. After picking up the monoclonal on the transformation plate, inoculating the transformation plate into a test tube containing 3mL of LB culture solution picked up by the monoclonal on a 50 plate, shaking the transformation plate uniformly at 37 liquid and 220r/min and overnight.

Shaking, namely inoculating according to the ratio of bacteria to culture medium=1:200, and shaking for 5 hours at 37 ℃ and 220r/min until the value of the OD600 of the bacteria is 0.6-0.8.

IPTG induction, namely simultaneously designing an uninduced group, adding the prepared IPTG into the induction group according to the proportion of 1:200, shaking uniformly at 37 ℃ and 220r/min for 4-5h, and inducing the expression of fusion proteins, wherein the uninduced group is not added with the IPTG.

1ML of the culture is taken, washed by PBS, then bacterial precipitate is resuspended by PBS, the resuspension is subjected to ultrasonic disruption in an ultrasonic disruption instrument, 5 x loading buffer is added into the disrupted supernatant and the sediment respectively for uniform mixing, detection is carried out by 12% SDS-PAGE, and coomassie brilliant blue is used for staining and band development. The results indicate that the target protein exists in the sediment after induced disruption.

4. The bacterial pellet of the induced escherichia coli is resuspended by 20mL of lysate and broken under ultrasonic conditions (power: 400W, work 4s, intermittent 8s, total 20 min). After 4 ℃,10000r/min,20min, the precipitate was collected.

The precipitate was washed 3 times with a final concentration of 20mM Tris,1mM EDTA,2M urea, 1M sodium chloride, 1% Triton X-100, pH 8.0 in water.

The washed precipitate was dissolved in an aqueous solution having a final concentration of 20mM Tris,5mM DTT,8M urea, pH 8.0, taken out after overnight at 4℃and centrifuged at 10000r/min for 15min at room temperature, and the supernatant after centrifugation was collected.

And (3) after dilution, dialyzing, namely dripping 20mM Tris,0.15M sodium chloride and pH8.0 buffer solution into the supernatant after centrifugation, stirring and diluting the solution while adding, putting the solution into a boiled dialysis bag, fixing two ends by using a dovetail clamp, and carefully placing the solution into a solution of 20mM Tris-HCl,0.15M sodium chloride and pH=8.0 for dialyzing overnight.

5. Placing the above collected protein solution into a dialysis bag which is boiled for 10min (boiling is to remove harmful substances in the dialysis bag to release protein) and cooled, and dialyzing overnight in a beaker containing PBS. After electrophoresis by 12% SDS-PAGE, protein purity analysis was performed by color development with Coomassie blue dye, and the purity was >98%, which indicated that rAlt a was a homodimer with a molecular weight of about 30 kDa.

Example 2

Healthy female BALB/c mice were randomly divided into 5 groups after one week of adaptive rearing, NC (healthy group), PC (positive control group), rAlt a1, nAlt a and Alternaria groups as shown in table 1 below:

Table 1alt a1 grouping and intervention pattern in asthma mouse row SCIT (n=8/group)

SCIT protocol, specifically as shown in FIG. 1, mice were subjected to slow nasal drop sensitization (50. Mu.L/dose) on days 0, 7, and 14, respectively, and continuous nasal drop challenge for 3 days 21-23 (50. Mu.L/dose). On days 30-42, subcutaneous specific immunotherapy was performed on mice every alternate day, i.e. the neck skin of mice was gently pinched and fixed, 100 μl of the corresponding therapeutic drugs (including: rAlt a1, nAlt a, ALTERNARIA, PBS) were aspirated with a 1mL syringe, and injected along the pinched skin, where slightly bulge hills were accessible, representing successful subcutaneous administration. After 1 week interval, re-nasal drip challenge was performed on days 49-51 for 3 consecutive days. The mice were closely observed for respiratory status, weight changes, and skin status at cervical subcutaneous injections throughout SCIT hours.

Subcutaneous specific immunotherapy effect evaluation (the evaluation sequence should be pathology first → airway resistance → other index)

(1) Evaluation of inflammation in lung tissue pathology

1) HE staining and inflammation fractionation:

Lung tissue is obtained by cutting sternum to expose chest, washing two lungs with normal saline via right ventricle to whiten, separating, and fixing in sample tube containing 4% paraformaldehyde.

Paraffin embedding and dicing were done by beijing baiaosen company.

Baking, namely placing the slices into a 60 ℃ oven for baking for 2 hours.

Dewaxing, namely dewaxing the baked glass slide by using dewaxing liquid.

Hematoxylin staining, namely placing the dewaxed slice into hematoxylin staining solution for 3min.

Washing, namely placing the slices into running water to wash for 3-5min.

Hydrochloric acid alcohol differentiation, namely immersing the slice into 1% hydrochloric acid alcohol for differentiation for 5-15s, and repeating the washing step.

Eosin staining, namely placing the slice into 0.5% eosin alcohol solution for dip-dyeing for 2min, and repeating the rinsing step.

Sealing the sheet, namely, sealing the sheet by neutral resin after drying, and observing the sheet under a mirror.

Grading according to the infiltration degree of inflammatory cells around the trachea (the thickness of 3 lymphocytes is 1 week), wherein the grade 1 is that inflammatory cells are lacked around the bronchus, the grade 2 is that inflammatory cells with the circumference of less than or equal to 25 percent of the bronchus, the grade 3 is that inflammatory cells with the circumference of 25 to 75 percent of the bronchus, the grade 4 is that inflammatory cells with the circumference of 1 week around the bronchus, the grade 5 is that inflammatory cells with the circumference of 2 weeks around the bronchus, and the grade 6 is that inflammatory cells with the circumference of more than or equal to 3 weeks around the bronchus;

2) PAS staining and scoring:

dewaxing treatment is the same as HE dyeing part.

Oxidizing, namely oxidizing for 10min by using 0.5% high-point acid liquor, washing for 5min under tap water, and wiping off water.

Dyeing, namely dyeing for 30min by using Schiff dye liquor, and repeating the rinsing step.

The nuclear dyeing step comprises the step of dyeing the nuclear with a proper amount of hematoxylin dye solution for 2-5min.

The subsequent steps are essentially identical to the HE staining section.

The score is 0, PAS positive goblet cells account for less than or equal to 5% of the number of epithelial cells of the whole airway, 1, 5-25% of positive goblet cells, 2, 25-50% of positive goblet cells, 3, 50-75% of positive goblet cells, 4, more than or equal to 75% of positive goblet cells, and 5, almost all airways have a large number of PAS positive goblet cells.

As shown in fig. 2 and 3, from the HE staining of lung tissue (200-fold) of fig. 2A, it can be seen that the positive control group (PC group) mice had a large number of inflammatory cells infiltrated around the bronchi and in the lung interstitium, and the bronchi wall thickened, lumen narrowing, mucosal continuity interruption, epithelial cell damage, and met the inflammatory conditions of the asthma model, as compared to the healthy control group (NC group). The model line SCIT is prepared from Alt a 1 and Alternaria alternata crude extract, and the three are found to relieve lung tissue inflammation conditions of asthmatic mice, and are mainly characterized by reduction of inflammatory cells around bronchi and blood vessels, wherein the treatment effect of rAlt a is relatively remarkable, and the effect of the crude extract is slightly poor. From the perspective of semi-quantitative scoring, the inflammatory status was objectively evaluated (fig. 2B), and it was also seen that the three proteins (rAlt a a, nAlt a a and crude extract of alternaria alternata) had a statistically significant effect on the improvement of pulmonary inflammation in mice compared to the PC group in the rAlt a treatment group (p=0.0143).

In addition to assessing the therapeutic effect of Alt a1 from lung tissue inflammation, the present invention also uses PAS staining to assess the goblet cell status of the mouse lungs (fig. 3A). Similar to HE staining results, goblet cell destruction was evident in the PC group compared to the NC group, and goblet cell destruction of lung tissue was significantly improved in the three groups of mice following Alt a1 and crude extract treatment, while no statistical difference was found between the PC group and each treatment group in semi-quantitative scoring (fig. 3B), but the relief was relatively evident in the rAlt a group. Further illustrating the therapeutic effect of rAlt a1 on asthmatic inflammation.

(2) Mouse airway responsiveness assessment

Anesthesia, in the last 24 hours of excitation, according to the pentobarbital dosage of 0.1mL/20g, the mice are subjected to intraperitoneal injection anesthesia, and after the forceps gently clamp the limbs of the mice and do not react, the next operation can be performed.

The tracheal cannula is inserted into the trachea for fixation after a small opening is cut at the tracheal ring by using forceps to separate the fascia at the neck. This procedure does not induce bleeding, so as not to occlude the trachea and cause asphyxia.

Nebulization after mice were placed in the tester for 5min, each nebulized and exposed to PBS, baseline was set, and 1.5, 3, 6, 12 and 24mg/mL methacholine nebulization was added for 1min. This procedure closely observes the respiration and anesthesia status of the mice.

Measured resistance Buxco Fine Pointe the system automatically generates airway resistance values.

The airway resistance values obtained for each group are plotted on the ordinate of concentration of methacholine, the airway resistance values are plotted on the ordinate, and the values are mean ± standard deviation, and the results are shown in fig. 4.

Since asthma is mainly manifested in clinic as airway hyperreactivity, and pulmonary function is an objective examination means for diagnosing asthma, it is particularly important to evaluate the mouse model of asthma and airway resistance after SCIT. The invention uses methacholine with concentration range of 1.5-24mg/mL to detect the airway resistance of mice (figure 4), and the result shows that the PC group mice start from the concentration of 3mg/mL, the airway resistance is gradually higher than the rest groups, but the invention has no statistical significance, the airway resistance among the three treatment groups has no obvious difference, and the difference value with the PC group is the largest at the concentration of 6 mg/mL. This part of the results shows that Alt a1 treatment can reduce airway resistance in asthmatic mice, and from another perspective demonstrates the therapeutic effect of Alt a 1.

(4) Bronchoalveolar lavage fluid (bronchoalveolar lavage fluid, BALF) total cell count

1) BALF collection:

mice were sacrificed on day 52 of the experiment (within 24h of the last challenge) and were anesthetized and bled after eye removal.

Exposing trachea, fixing limbs, fully separating fascia and blood vessel of neck, and exposing trachea.

Trachea cannula, after cutting a small opening on the trachea ring by using ophthalmic scissors, a 24G indwelling needle is inserted into the trachea and fixed by using a suture.

Lavage, pre-chilled PBS was slowly injected into the lung tissue of the mice after the indwelling needle was connected with a 1mL syringe in an amount of 0.5 mL/min.

Collecting, slightly pressing the lungs on both sides of the mouse, slowly pumping back the visible milky white liquid into the syringe, and repeating the steps for 3 times, wherein the recovery rate is more than 80%.

2) Total cell count:

centrifuging, namely centrifuging the collected BALF (at 4 ℃ C., 2000rpm,10 min), collecting the supernatant, sub-packaging and freezing for later use.

Resuspension the pellet was resuspended in 100 μl PBS and 20 μl was pipetted on a cytometer.

The inflammatory status of bronchoalveolar lavage fluid is also an important index for evaluating asthma models, and thus the present invention continues to evaluate the therapeutic efficacy of Alt a1 from a number of angles. By collecting BALF from each group of mice, total cell counts were performed first, and the results are shown in fig. 5, it is evident that the total cell count was significantly higher in the PC group than in the NC group, and that cell counts were significantly reduced after the treatment with Alt a1 and the crude extract of alternaria alternata, where nAlt a and rAlt a groups were comparable at reduced levels (p=0.0043).

(3) Detection of IL-4, IL-10 and IL-13 expression levels in BALF supernatants.

Sample treatment, the supernatant was concentrated to about 400. Mu.L with an ultrafiltration tube.

Antibody coating concentration IL-4 and IL-10:2. Mu.g/mL, IL-13 was a pre-coated plate from Wuhan Huamei corporation.

Rewarming, namely taking out the 96-well plate and rewarming for 30min at room temperature.

Wash plate 3 times with 300. Mu.L TBST.

Blocking with 200. Mu.L of TBST blocking solution containing 1% BSA for 1h at room temperature.

The plate washing step was repeated.

The prepared standard and the sample to be detected are added for dilution according to the instruction, and the mixture is incubated at 4 ℃ overnight.

And taking out the plate and repeating the plate washing step after rewarming.

100. Mu.L of Biotin-labeled antibody was added to each well, and after incubation at room temperature for 1 hour, the plate washing step was repeated.

Then, 100. Mu.L of streptavidin-HRP antibody was added, and the plate washing step was repeated after incubation at room temperature for 30 min.

Color development-100. Mu.L of TMB color development solution was added to each well, and blue precipitate was visible about 5min.

Termination of the reaction 100. Mu.L of concentrated sulfuric acid was added to each well to terminate the reaction.

And reading and calculating the protein concentration, namely reading at the OD450 nm of the enzyme label instrument wavelength, and calculating the protein concentration according to the label curve.

The invention detects the expression level of cytokines in BALF supernatant, and the quantitative detection results of ELISA on IL-4, IL-10 and IL-13 in BALF are shown in figures 6-8, wherein P is 05 and P is 01 compared with a PC group. IL-4 and IL-13 are secreted mainly by type 2 inflammatory cells, and IL-10 is produced mainly by Breg cells, and is generally thought to have negative regulatory effects on immunity. In view of this, the present invention found that IL-4 and IL-13 were expressed at significantly higher levels in the PC group than in the NC group, and that both cytokines were reduced after immunotherapy, IL-4 was most reduced in nAlt a groups to the highest degree, statistically significant (P=0.0022), and IL-13 was expressed at the lowest level in the crude Alternaria alternata extract group (P= 0.0411). It shows that the component Alt a 1 and the crude extract of Alternaria alternata can relieve the type 2 inflammatory cytokines caused by asthma mice. Whereas for IL-10, we observed an increase in levels after rAlt a a and crude Alternaria alternata extract SCIT treatment, no significant increase in group nAlt a 1.

(4) Detection of mouse mast cell protease-1 (mouse mast cell protein-1, MMCP-1) in serum

Rewarming, namely rewarming the kit at room temperature for 30min and then waiting for the next step to use.

Washing the plate, namely oscillating the plate for 3 times by using TBST.

Sealing, namely sealing for 1h at room temperature by using diluted assay buffer, and then repeating the plate washing operation.

The standard curve is prepared by preparing 4000pg/mL, 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL and blank holes.

Incubation of primary antibody serum was diluted in a 1:10 ratio and incubated at 37 ℃ for 3h before plate washing.

Mu.L of detection antibody was added to each well and incubated for 1h at room temperature. The plate washing operation is repeated again.

Mu.L of diluted avidin-HRP antibody was added to each well, and the plate was washed 3 times after 30min reaction at room temperature.

Color development, 100. Mu.L of TMB was added to each well and developed for 15min at room temperature.

The reaction was stopped and read by adding 100. Mu.L of stop solution per well, reading at OD450 nm and calculating the MMCP-1 value from the standard curve.

The results are shown in FIG. 9, where the values are mean.+ -. Standard deviation, MMCP-1: mast cell protease-1. There is no known about the expression of MMCP-1 in an asthma model. Therefore, this section is also intended to provide assistance in this regard to the therapeutic effects of the proteins described above. As can be seen from FIG. 9, MMCP-1 levels in the PC group were slightly higher than those in the other groups, although there was a decrease in the treatment groups, there was no statistical difference, and the expression levels of MMCP-1 were also comparable among the three treatment groups. This result may suggest that the asthma model established in this section, although inflammation is significant, does not reach the level of severe allergic reactions.

(5) Detection of T-IgE, alternaria and rAlt a 1-sIgE, sIgG1, sIgG2a, sIgG2b in serum

Detection of T-IgE, alternaria and rAlt a 1-sIgE relevant antibody information is shown in the following Table.

TABLE 2SCIT serum IgE antibody detection information table

The detection information for Alternaria and rAlt a 1-sIgG1, sIgG2a, sIgG2b is shown in the following table:

TABLE 3SCIT serum IgG antibody detection information table

The detection method is essentially the same as the "IL-4 and IL-10 detection portions in BALF supernatant".

The results are shown in FIGS. 10 and 11, where the T-IgE levels were significantly lower in the three treatment groups than in the PC group, as seen by the change in T-IgE in serum (FIG. 10A), all of which were statistically significant. The success of the allergic asthma model was further confirmed by the detection of antigen-specific IgE, so this section continued to monitor the level of scge of Alternaria and Alt a1 (fig. 10B), and the expression of Alternaria-scge was found to be similar to the change of T-IgE, i.e. significantly reduced in the treatment group, but in Alt a 1-scge, no trend of reduced expression was found in the treatment group, which was elevated compared to the PC group, particularly apparent in nAlt a (p=0.0022), rAlt a times. This may be relevant for both groups of treatments using only the fraction Alt a 1.

The IgG class antibodies are considered to have protective significance in the clinical study of AIT, are one of the marker antibodies for the therapeutic effect of AIT, and the expression of Alt a1 specific IgG class antibodies and Alt A mould are correspondingly detected in the mouse model (figure 10C). It can be seen from the figure that only Alt a1 of sIgG1, sIgG2a and sIgG2b exhibited elevated levels in the two-component treatment group, with the most significant elevation in the nAlt a group, that the expression of various sIgG subtypes of Alternaria alternata was not consistent in the treatment group, that the Alternaria alternata-sIgG 1 and sIgG2b of rAlt a group were reduced compared to the PC group, and that only slight elevations were also seen in sIgG2a, possibly also in relation to the complexity of the crude extract fraction.

In addition, the invention also analyzes the neutralizing antibody ratio (sIgG 1/sIgE, sIgG2a/sIgE, sIgG2 b/sIgE) related to Alt a1 and Alt. In general, the neutralizing antibody ratio of Alt a1 appeared to be elevated in the immunotherapeutic group for Alt a1 (fig. 11A), while the neutralizing antibody ratio related to alternaria alternata remained inconsistent (fig. 11B), with only an elevation in the expression of sgg 1/ige being statistically significant (p= 0.0411), with the remaining two classes of ratio elevation not evident.

The results in this section reveal that immunotherapy of asthmatic mice with Alt a1 more leads to an increase in the expression of IgG and its subtypes and the neutralizing antibody ratio than the crude extract of alternaria alternata.

The animal model established by the invention can be used for the subsequent immunotherapy research of the alternaria alternata, namely a research model with repeatability is provided for researchers needing to carry out the immunity research of the alternaria alternata.

Claims (8)

1. The method for establishing the subcutaneous specific immunotherapy model of the Alt a 1 protein-based Alt-a 1 allergic asthma mice is characterized by comprising the following steps of:

s1, nasal drip sensitization and excitation are carried out on a mouse by utilizing a crude extract of Alternaria alternata;

S2, using Alternaria crude extract, rAlt a and nAlt a protein to sensitize and excite mice SCIT;

S3, nasal drip excitation is carried out on the mice which have received SCIT by using the Alternaria crude extract again;

the amino acid sequence of rAlt a's 1 protein is shown in SEQ ID NO. 1.

2. The method according to claim 1, wherein in step S1, the animals are subjected to slow nasal drop sensitization on days 0, 7 and 14, respectively, and continuous nasal drop challenge on days 21-23 is carried out for 3 days.

3. The method of claim 1, wherein in step S1, the mice are female BALB/c mice.

4. The method according to claim 1, wherein the crude extract of Alternaria alternata is used in an amount of 50. Mu.L/min.

5. The method of claim 1, wherein in step S2, the mice are subjected to subcutaneous specific immunotherapy every alternate day on days 30-42.

6. The method according to claim 1, wherein the specific immunotherapy is to gently pinch and fix the neck skin of the mouse, and aspirate 100 μl of the corresponding therapeutic agent with a 1mL syringe.

7. The method of claim 1, wherein in step S3, re-nasal drip firing is performed on days 49-51 for 3 consecutive days.

8. The method according to claim 1, wherein the rAlt a1 protein expression is performed using a gene sequence as shown in SEQ ID NO. 2.