A galectin 10 crystal and an antibody igy and a pharmaceutical preparation prepared therefrom

Abstract

The application provides a galactoside agglutinin 10 (Gal10) crystal and a preparation method and prepared antibody IgY and a pharmaceutical preparation thereof, and belongs to the technical field of medicines. The application uses the galactoside agglutinin 10 crystal as an antigen to prepare an egg yolk neutralizing antibody IgY capable of dissolving the Gal10 crystal, has the effects of improving rhinitis or asthma of a patient, preventing and treating rhinitis and asthma, and is safe, has no side effects, has a long-lasting curative effect, and has no drug resistance, thereby providing a safe, simple, hygienic, effective, economic and personalized treatment mode for prevention and treatment of rhinitis and asthma, and having a high market value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a galactolectin 10 crystal and the antibody IgY and pharmaceutical preparations prepared therefrom. Background Technology

[0002] Rhinitis, or nasal inflammation, is an inflammation of the nasal mucosa caused by viruses, bacteria, allergens, various physical and chemical factors, and certain systemic diseases. The main pathological changes in rhinitis include congestion, swelling, exudation, hyperplasia, atrophy, or necrosis of the nasal mucosa. Allergic rhinitis (commonly known as allergic rhinitis) is a chronic disease with a high incidence, long course, and frequent recurrence. Severe allergic rhinitis can affect sleep in 70% of cases and daily life and work in 94% of cases, impacting quality of life even more than major diseases such as hypertension and diabetes. Furthermore, allergic rhinitis can be complicated by otitis media, sinusitis, nasal polyps, asthma, and other diseases, posing a greater threat to health. Allergic rhinitis can also worsen and lead to nasopharyngeal carcinoma. Therefore, the prevention and treatment of rhinitis should be given sufficient attention. Currently, clinical treatment often uses hormones, antihistamines, and leukotriene inhibitors, but the side effects cannot be ignored. Repeated use easily leads to drug resistance, and long-term use of hormones can affect the development of children and adolescents.

[0003] Asthma is a heterogeneous disease characterized by chronic airway inflammation involving multiple cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components. This chronic inflammation is associated with airway hyperresponsiveness and usually presents with widespread and variable reversible expiratory airflow limitation, leading to recurrent episodes of wheezing, shortness of breath, chest tightness, and / or cough, with the intensity varying over time. It often occurs and worsens at night and / or in the early morning. If asthma is not diagnosed and treated in time, irreversible airway narrowing and airway remodeling can occur as the disease progresses. Currently, the treatment of asthma is generally divided into four stages based on the severity of the disease: (1) Long-term anti-inflammatory treatment is the basic treatment, with inhaled corticosteroids being the first choice. Commonly used inhaled drugs include beclomethasone (BDP), budesonide, fluticasone, and mometasone, etc. The latter two have stronger biological activity and longer-lasting effects. It usually takes more than a week of regular inhalation to take effect. (2) The first-line drug for emergency symptom relief is an inhaled β2 agonist. β2 agonists mainly work by stimulating β2 receptors in the respiratory tract, activating adenylate cyclase, increasing intracellular cyclic adenosine monophosphate (cAMP) levels, and decreasing free calcium, thereby relaxing bronchial smooth muscle. It is the first-line drug for controlling acute asthma attacks. (3) For patients whose condition is not well controlled after regular inhaled corticosteroids, it is advisable to add inhaled long-acting β2 agonists, sustained-release theophylline, or leukotriene modifiers (combined therapy); increasing the dosage of inhaled corticosteroids may also be considered. (4) For patients with severe asthma who still experience long-term recurrent attacks after the above treatment, intensive treatment may be considered. That is, treatment is carried out as a severe asthma attack, with high-dose corticosteroids and other treatments. After the symptoms are completely controlled, lung function recovers to the best level, and PEF fluctuation is normal, the dosage of corticosteroids is gradually reduced 2 to 4 days later. Some patients achieve ideal condition control after the intensive treatment phase.

[0004] It is evident that the most common and effective treatment for rhinitis and asthma is hormone therapy. However, hormone therapy has serious side effects and cannot meet the medication requirements of patients. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a galactolectin 10 crystal, which is prepared by in vitro recombinant expression and can produce antibodies that dissolve the galactolectin 10 crystal when immunized in animals.

[0006] The present invention also aims to provide an antibody IgY against galactolectin 10 crystals, wherein the antibody IgY completely neutralizes the inflammatory response caused by galactolectin 10 crystals by dissolving them, thereby achieving the purpose of treating asthma and rhinitis.

[0007] The present invention also aims to provide a drug for the prevention and treatment of rhinitis and asthma, which is safe, effective and economical.

[0008] The present invention provides a galactolectin 10 crystal, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] This invention provides a method for preparing the galactose lectin 10 crystals, comprising the following steps:

[0010] 1) Construct a recombinant vector containing the coding sequence of the purified tag-galactosyllectrin 10 crystals;

[0011] 2) The recombinant vector described in step 1) is transferred into the expression system, and after induction of expression, recombinant expression cells are obtained;

[0012] 3) Isolate and purify the recombinant protein from the recombinant expression cells described in step 2), remove the purification tag, adjust the concentration to not less than 2 mg / ml, and obtain galactolectin 10 crystals.

[0013] Preferably, the purification tag in step 1) is a His tag; the nucleotide sequence encoding the purification tag - galactolectin 10 crystal is shown in SEQ ID NO:2.

[0014] This invention provides an antibody IgY for dissolving galactolectin 10 crystals, the preparation method comprising the following steps:

[0015] The galactolectin 10 crystals or the galactolectin 10 crystals prepared by the above preparation method are used as antigens to immunize laying hens.

[0016] Eggs were collected 14 days after the initial immunization, and the lecithin and other proteins in the egg yolk aqueous solution were removed under acidic conditions. The supernatant was then passed through 0.45 μm and 50,000 molecular weight filter membranes to obtain antibody IgY.

[0017] Preferably, the method for immunizing laying hens is to immunize them in three separate immunizations, with an interval of 28-30 days between each immunization. Each immunization uses a three-point immunization method, and the immunization dose of antigen at each immunization site is 30-50 μg per hen.

[0018] Preferably, the acidic condition is that the pH value of the egg yolk aqueous solution is preferably 4.8 to 5.5;

[0019] The method for removing the precipitate from the egg yolk aqueous solution is centrifugation; the centrifugation speed is preferably 7500-8500 rpm, and the centrifugation time is preferably 15-25 min.

[0020] The present invention provides a medicament for the prevention and treatment of rhinitis and / or asthma, the medicament comprising the antibody IgY and pharmaceutically acceptable excipients.

[0021] Preferably, the dosage form of the drug is a spray, and the spray comprises the following components in weight percentage:

[0022] Antibody IgY 0.1-0.4%, lysozyme 0.1-0.9%, glycerol 1-10%, chlorhexidine gluconate 0.04-0.1%, and distilled water 88.6-98.76%.

[0023] Preferably, the dosage form of the drug is an atomizing agent;

[0024] The atomizing agent preferably comprises the following components in weight percentage:

[0025] 0.1–0.4%, lysozyme 0.1–0.9%, nisin 0.005–0.02%, glycerol 1–10%, chlorhexidine gluconate 0.04–0.1%, and distilled water 88.58–98.755%.

[0026] This invention provides the use of the antibody IgY in the preparation of drugs for the prevention and treatment of rhinitis and / or asthma.

[0027] The galactolectin 10 crystals provided by this invention have the amino acid sequence shown in SEQ ID NO:1. This invention utilizes an in vitro recombinant expression method to prepare recombinant galactolectin 10 protein. After purification and removal of the purification tag, the concentration is adjusted, and galactolectin 10 crystals are precipitated. The galactolectin 10 crystals prepared by this invention are similar to Charcot-Leyden crystals (CLCs) obtained from patients, providing an effective antigen for the preparation of corresponding antibodies and laying the foundation for the subsequent application of these antibodies in pharmaceuticals.

[0028] This invention provides an antibody IgY that dissolves galactolectin 10 crystals. Using Gal10 protein crystals as an antigen, this invention immunizes laying hens and extracts egg yolk antibody IgY from the egg yolk. Functionally equivalent to mammalian blood antibody IgG, it lacks the Fc fragment of IgG, is safe, and contains no pathogens that may remain in the plasma. The antibody IgY provided by this invention takes only a little over two months from antigen protein expression to antibody production in immunized chickens. Furthermore, it is easily mass-produced to meet the needs of treating rhinitis and asthma in the general population. Since the IgY antibody is non-immunogenic when taken orally or topically, it will not cause immune rejection. Using egg yolk antibody IgY without Gal10 crystals extracted from eggs laid by hens before immunization as a negative control, the Gal10 crystal egg yolk antibody IgY of this invention, at a concentration of 3 mg / ml, can completely eliminate the symptoms of rhinitis and asthma, while the control group showed no significant neutralizing activity.

[0029] This invention provides a medicament for the prevention and treatment of rhinitis and / or asthma, comprising the antibody IgY and pharmaceutically acceptable excipients. Existing rhinitis medications mainly consist of hormones and antihistamines, which only treat the symptoms and not the root cause. Repeated use easily leads to drug resistance, resulting in treatment failure. Hormones can also affect children's development. The medicament prepared in this invention is extracted from eggs, is natural, safe, non-toxic, and has no side effects. It contains no hormones, antihistamines, antibiotics, or chemical drugs, thus treating both the symptoms and the root cause. It fundamentally eliminates the Gal10 protein crystals that cause rhinitis, thereby fundamentally eliminating the symptoms of rhinitis without inducing drug resistance. Attached Figure Description

[0030] Figure 1 The results are for the small-scale identification of FL389-TEV, where 1 is the result of recombinant TEV enzyme expression before induction culture, and 2 is the result of recombinant TEV enzyme expression before induction culture.

[0031] Figure 2 The results show the expression form of FL3891-TEV(B), where 1 is the supernatant and 2 is the precipitate.

[0032] Figure 3 The purification effect of FL3891-TEV(B) is evaluated. 1 is the elution, and 2-4 are elution buffers of 50mM, 200mM, 500mM, and imidazole, respectively.

[0033] Figure 4 The results of the small-scale identification of FL3892-GAL10 are as follows: 1 is the expression result of recombinant Gal10 before induction culture, and 2 is the expression result of recombinant Gal10 before induction culture.

[0034] Figure 5 The results show the expression form of FL3892-GAL10(B), where 1 is the supernatant and 2 is the precipitate.

[0035] Figure 6 The purification effect of FL3892-GAL10(B) is evaluated. Among them, 1-3 are 50mM, 200mM, 500mM, and imidazole elution buffers, and 4 is the eluent.

[0036] Figure 7 Image of the finished FL3892-GAL10(B) protein;

[0037] Figure 8 A diagram showing the crystal morphology of the recombinantly expressed Gal10 protein;

[0038] Figure 9 The results show the molecular weights of the heavy and light chains of the egg yolk neutralizing antibody IgY prepared in this invention. Detailed Implementation

[0039] The present invention provides a galactolectin 10 crystal, the amino acid sequence of which is shown in SEQ ID NO:1.

[0040] This invention provides a method for preparing the galactose lectin 10 crystals, comprising the following steps:

[0041] 1) Construct a recombinant vector containing the coding sequence of the purified tag-galactosyllectrin 10 crystals;

[0042] 2) The recombinant vector described in step 1) is transferred into the expression system, and after induction of expression, recombinant expression cells are obtained;

[0043] 3) Isolate and purify the recombinant protein from the recombinant expression cells described in step 2), remove the purification tag, adjust the concentration to not less than 2 mg / ml, and obtain galactolectin 10 crystals.

[0044] This invention first constructs a recombinant vector containing the coding sequence of the purified tag-galactose lectin 10 crystal.

[0045] In this invention, the method for constructing a recombinant vector containing the coding sequence of the purified tag-galactolectin 10 crystals preferably includes the following steps:

[0046] The coding sequence of the purified tag-galactosyllectrin 10 crystal was inserted into the backbone vector through enzyme digestion and ligation cloning or homologous recombination. After screening and verification, the recombinant vector was obtained.

[0047] In this invention, the purification tag is preferably a His tag; the nucleotide sequence encoding the purification tag - galactolectin 10 crystal - is preferably as shown in SEQ ID NO:2. This invention does not impose any particular limitation on the enzyme digestion and ligation methods; any enzyme digestion and ligation methods well known in the art can be used. This invention also does not impose any particular limitation on the type of backbone vector; any type of backbone vector well known in the art can be used, such as pRK793.

[0048] Having obtained the recombinant vector, the present invention transfers the recombinant vector into an expression system, and after induction of expression, obtains recombinant expression bacteria.

[0049] This invention does not impose any particular limitation on the method of transforming the recombinant vector into the expression system; any transformation method well-known in the art, such as the CaCl2 method, can be used. The expression system is preferably a prokaryotic expression system. The prokaryotic expression system is preferably *E. coli* BL21 competent cells. This invention does not impose any particular limitation on the method of inducing expression; any induction method well-known in the art can be used, such as adding 1.0 mM IPTG to the culture medium and inducing expression for 3 hours.

[0050] After obtaining the recombinant expression bacterial cells, the present invention isolates and purifies the recombinant protein from the recombinant expression bacterial cells, removes the purification tag, adjusts the concentration to not less than 2 mg / ml, and obtains galactolectin 10 crystals.

[0051] In this invention, the method for isolating recombinant proteins from the recombinant expressed bacterial cells preferably involves lysing the bacterial cells, centrifuging, and obtaining a supernatant. The lysis method is preferably autoclaving or ultrasonic lysis. During autoclaving, the bacterial solution needs to be kept on ice. The bacterial precipitate is removed from a -20°C freezer, and 5-6 times the volume of Buffer NiA is added to resuspend the bacterial cells. The cells are then placed in a beaker containing water to thaw, thoroughly shaken, and the pressure in the autoclave is increased to 1100-1300 MPa (this pressure is used to break E. coli cells). The bacterial solution is repeated four times until the E. coli are observed to be completely ruptured, and the bacterial solution drips like water without being viscous. The broken bacterial solution is aliquoted into 50 mL high-speed centrifuge tubes. The centrifugation speed is preferably 15000-20000 rpm, more preferably 17000 rpm, and the centrifugation temperature is preferably 4°C. The centrifugation time is preferably 1-1.5 h, more preferably 1 h. The recombinant protein is mainly expressed in the form of supernatant and inclusion bodies. Slowly pour the supernatant into a brand new 50mL centrifuge tube for subsequent sample loading and purification. The preferred purification method is NI column purification. The preferred conditions for NI column purification are: first, rinse the entire peristaltic pump tubing with 50mL of ultrapure water; then, rinse with Buffer NiB for 5 column volumes (approximately 5mL per column volume); next, equilibrate with Buffer NiA for 5-6 column volumes, ensuring the entire tubing is immersed in buffer. After column equilibration, transfer the supernatant from the high-speed centrifuged protein sample into a 50mL centrifuge tube for sample loading, using a peristaltic pump at a loading rate of 3mL / min. After loading, rinse the column with 50mL Buffer NiA, then with 50mL of 10% Buffer NiB. Finally, elute the target protein with 50mL of 100% Buffer NiB. The sonication lysis conditions are: ice bath, 60% power, 2s sonication, 2s interval, 35min. The centrifugation speed is preferably 11500–12500 rpm, more preferably 12000 rpm. The centrifugation time is preferably 12–18 min, more preferably 15 min. The centrifugation temperature is preferably 4℃. The recombinant protein is mainly expressed in the form of supernatant and inclusion bodies. The purification method is preferably NI column purification. The NI column purification conditions are preferably as follows: first, rinse the entire peristaltic pump tubing with 50 mL of ultrapure water, then rinse with Buffer NiB for 5 column volumes (one column volume is approximately 5 mL), then equilibrate with Buffer NiA for 5-6 column volumes and rinse the peristaltic pump tubing, ensuring the entire tubing is immersed in buffer. After equilibration, the supernatant of the protein sample after high-speed centrifugation is taken out and placed into a 50 mL centrifuge tube for loading, with the peristaltic pump loading speed at 3 mL / min. After loading, rinse the column with 50 mL of Buffer NiA, and then rinse the column with 50 mL of 10% Buffer NiB.Finally, the target protein was eluted with 50 mL of 100% Buffer NiB.

[0052] In this invention, the method for removing the purification tag is preferably achieved by enzymatic digestion with TEV enzyme. This invention does not have any particular limitation on the source of the TEV enzyme; any TEV enzyme well-known in the art can be used, such as commercially available or recombinant expression. In this embodiment, the recombinant expression method of the TEV enzyme preferably involves cloning the nucleotide sequence as shown in SEQ ID NO:3 into a backbone vector to form a recombinant vector, introducing the recombinant vector into a prokaryotic expression system for expression, inducing culture, and collecting the recombinant protein. Verification of the recombinant protein yields the expected product, indicating successful preparation of the recombinant TEV enzyme. During the enzymatic digestion, the mass ratio of TEV enzyme to galactolectin 10 recombinant protein is preferably 1:100, and the concentration of the galactolectin 10 recombinant protein is 2 mg / ml or higher, preferably 10 mg / ml. The digestion time is preferably 10–14 h, more preferably 12 h. The digestion temperature is preferably 20–28 °C, most preferably 25 °C.

[0053] In this invention, after enzymatic digestion, the digestion product is preferably centrifuged, the precipitate is washed, redissolved, and the concentration is adjusted to an aqueous solution with a concentration greater than 2 mg / ml. The solution is then placed at room temperature, and crystals are observed. The centrifugation conditions are preferably 600 × g for 5 min. The washing solution is preferably a PBS solution with pH 7.4. The redissolved solvent is preferably a PBS solution with pH 7.4.

[0054] In this invention, the prepared galactolectin 10 crystals are highly similar to CLCs isolated from patients. Furthermore, it was verified that injecting the prepared Gal10 crystals into the airway of mice induced the secretion of inflammatory factors; however, injecting a mutant Gal protein that cannot form crystals into the airway of mice did not induce the secretion of inflammatory factors. Therefore, it is evident that Gal10 crystals induce an inflammatory response, and dissolving the Gal10 crystals may inhibit this inflammatory response. Based on this, this invention develops an antibody for dissolving Gal10 crystals.

[0055] This invention provides an antibody IgY for dissolving galactolectin 10 crystals, the preparation method comprising the following steps:

[0056] The galactolectin 10 crystals or the galactolectin 10 crystals prepared by the above preparation method are used as antigens to immunize laying hens.

[0057] After three initial immunizations, and 14 days after the third immunization, eggs were collected. The lecithin and other proteins in the egg yolk aqueous solution were removed under acidic conditions. The resulting supernatant was then passed through 0.45 μm and 50,000 molecular weight filter membranes to obtain antibody IgY.

[0058] In this invention, the concentration of galactolectin 10 crystals in the antigen is preferably 50–100 μg / ml, more preferably 100 μg / ml. The solvent for the antigen is preferably PBS solution, more preferably PBS.

[0059] In this invention, the preferred method for immunizing laying hens is to administer three immunizations, with an interval of 28-30 days between each immunization. Each immunization uses a three-point immunization method, with an antigen dose of 30-50 μg per hen at each immunization site. The hens are normal young hens, specifically hens aged 16-20 weeks and weighing 1.2-2.0 kg.

[0060] In this invention, since the effectiveness of antibody production decreases over time, it is preferable to perform booster immunizations every two months after the third immunization. The booster immunizations preferably employ a three-point immunization method, with an antigen dose of 30–50 μg per hen at each immunization site.

[0061] In this invention, the acidic condition is preferably a pH value of 4.8 to 5.5 for the egg yolk aqueous solution. The method for removing the precipitate from the egg yolk aqueous solution is preferably centrifugation. The centrifugation speed is preferably 7500 to 8500 rpm, more preferably 8000 rpm. The centrifugation time is preferably 15 to 25 minutes, more preferably 20 minutes. The antibody IgY content is 2 to 4 mg / ml, with a purity of 40 to 50%.

[0062] In this invention, the obtained antibody IgY is preferably verified by electrophoresis. Electrophoresis results show that the heavy chain of the yolk-neutralizing antibody IgY is 67–70 KD, and the light chain is 22–30 KD. The titer of the antibody IgY was detected using ELISA, and the results showed that the IgY ELISA titer was 2. 16 That is, 65536.

[0063] In this invention, experimental verification showed that antibody IgY can dissolve Gal10 crystals within 2 hours, and can also dissolve CLCs crystals in the mucus of patients with chronic sinusitis. In a mouse model of house dust mite-induced asthma, the CLCs dissolution treatment with 1D11 completely neutralized the pro-inflammatory effect of CLCs, thereby weakening or eliminating excessive lung inflammation or mucus accumulation in rhinitis or asthma, thus achieving the purpose of preventing and treating rhinitis and asthma.

[0064] The present invention provides a medicament for the prevention and treatment of rhinitis and / or asthma, the medicament comprising the antibody IgY and pharmaceutically acceptable excipients.

[0065] In this invention, when the drug is used to prevent and treat rhinitis, the dosage form of the drug is preferably a spray, and the spray preferably comprises the following components in the following mass percentages: antibody IgY 0.1-0.4%, lysozyme 0.1-0.9%, glycerol 1-10%, chlorhexidine gluconate 0.04-0.1%, and distilled water 88.6-98.76%.

[0066] In this invention, the preferred dosage form of the drug is an nebulizer. The nebulizer comprises the following components in the following mass percentages: 0.1–0.4%, lysozyme 0.1–0.9%, nisin 0.005–0.02%, glycerol 1–10%, chlorhexidine gluconate 0.04–0.1%, and distilled water 88.58–98.755%.

[0067] The present invention does not impose any special restrictions on the preparation method of the drug; any preparation method of antibody-containing drugs well known in the art can be used.

[0068] This invention provides the use of the antibody IgY in the preparation of drugs for the prevention and treatment of rhinitis and / or asthma.

[0069] The following detailed description, in conjunction with embodiments, illustrates a galactoglobulin 10 crystal, its preparation method, and the prepared antibody IgY and pharmaceutical formulation provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0070] Example 1

[0071] A method for preparing galactose lectin 10 crystals

[0072] I. Expression and purification of TEV enzymes

[0073] 1. Method

[0074] 1.1 Small-scale expression

[0075] Plasmid transformation:

[0076] For the ice-based operation, add 1 μL of addgene plasmid#8827 (backbone vector pRK793) to 50 μL of BL21 competent cells, incubate on ice for 30 min, heat shock at 42°C for 1 min, and then incubate on ice for 5 min. Add 1 mL of LB medium (without antibiotics), incubate at 37°C on a shaker for 45 min. Stir the bacterial culture at 6000 rpm for 3 min, aspirate the supernatant, leaving approximately 50 μL, mix well, and drop a few drops onto an AP-resistant LB agar plate, then streak evenly and incubate overnight at 37°C.

[0077] Single colonies were picked and cultured in the corresponding liquid culture medium for 15 hours, and the glycerol bacteria were stored at -80℃.

[0078] Induction of expression: The next day, the bacterial strain was diluted 1:50 and cultured in 5 mL at 37°C until OD was reached. 600 =0.5, collect 2.5 mL of bacterial culture and process it as a pre-induction control. Add 1.0 mM IPTG to the remaining 2.5 mL to a final concentration of 0.4 mM / mL, and induce expression at 37℃ for 3 h. Centrifuge and collect the bacterial cells, process them as post-induction samples, and use SDS-PAGE to identify protein expression.

[0079] 1.2 Protein Expression and Purification

[0080] 1.2.1 Activation of microbial strains

[0081] The bacterial strain was revived and incubated overnight at 37°C for activation.

[0082] 1.2.2 Induced Expression

[0083] Large-scale in vitro culture of target protein

[0084] The transformed single colonies grown on the plate were inoculated into 100 mL of LB medium containing AP resistance and cultured overnight at 37°C with shaking at 220 rpm. From the 100 mL overnight culture, 15 mL of the culture was inoculated into 1 LLB medium containing AP resistance and cultured at 37°C with shaking at 220 rpm for expansion. When the bacterial OD... 600 When the OD value reaches 0.6, cool the shaker down to 18°C ​​and continue shaking incubation at 200 rpm. 600 When the culture medium reaches approximately 0.8, add 200 μL of 1M IPTG to each 1L of medium and continue incubation overnight at 18°C ​​with shaking at 200 rpm for 16–18 hours. 1.2.3 Cell collection.

[0085] The cultured bacterial solution was divided into batches and transferred to 1L centrifuge bottles. The bottles were then placed in a BECKMAN COULTRAvanti J-26XPI centrifuge and centrifuged at 4°C and 5000 rpm for 15 min, collecting the precipitate. 20 mL of 1×PBS (pH 7.4) was added, and the precipitate was resuspended by pipetting. The resuspended solution was transferred to 50 mL centrifuge tubes and centrifuged at 4°C and 4000 rpm for 30 min in a floor-standing centrifuge, collecting the precipitate. For short-term storage, the precipitate was stored at -20°C; for long-term storage, it was stored at -80°C.

[0086] High-pressure sterilization

[0087] During the lysis process, the bacterial solution needs to be kept on ice. Since the first step in Gal10 protein purification is nickel column affinity chromatography, Buffer NiA is used for bacterial resuspension. The bacterial pellet is removed from the -20°C freezer, and 5-6 times its volume of Buffer NiA is added to resuspend the bacteria. The pellet is then placed in a beaker containing water to dissolve, and thoroughly shaken. To prevent large bacterial aggregates, it is best to filter the solution once with a 2mm needle tip to avoid damaging the instrument. The laboratory uses a JN-3000plus lysis instrument. Before lysis, the compressor needs to be turned on for pre-cooling. Under low pressure, the 20% ethanol stored in the tubing is rinsed off with ultrapure water. Then, the entire tubing is rinsed with Buffer NiA, and the lysis instrument is pressurized to 1100-1300 MPa (this pressure is used to lyse E. coli cells). The bacterial solution is lysed four times consecutively until the E. coli are observed to be completely ruptured, and the bacterial solution drips like water without being viscous. After sterilization, rinse the pipeline with deionized water 2-3 times, and finally fill the pipeline with 20% ethanol.

[0088] 1.2.5 Centrifugation of bacterial culture

[0089] The disrupted bacterial culture was aliquoted into 50mL high-speed centrifuge tubes. After pairwise balancing on a balance (with an error not exceeding 0.02g), the tubes were placed in a BECKMAN COULTRAvanti J-26XPI centrifuge. Centrifugation was performed at 17000rpm and 4°C for 1 hour using a high-speed rotor. The supernatant was then slowly poured into new 50mL centrifuge tubes for subsequent sample loading. This step requires care to prevent the bacterial precipitate from being accidentally resuspended and poured into new tubes. SDS-PAGE analysis was performed to determine the expression pattern of the target protein.

[0090] 1.2.6 Primary Ni-NTA (nickel) column affinity chromatography

[0091] Nickel ion affinity chromatography columns utilize the specificity of nickel ions in the column medium to generate an affinity interaction with the histidine tag on proteins, while other proteins do not have this interaction, thereby achieving the purification of the target protein.

[0092] First, rinse the entire peristaltic pump tubing with 50 mL of ultrapure water. Then, rinse with Buffer NiB (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM imidazole) for 5 column volumes (approximately 5 mL per column volume). Next, equilibrate the peristaltic pump tubing with Buffer NiA (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 25 mM imidazole) for 6 column volumes, ensuring the entire tubing is immersed in buffer. After equilibration, transfer the supernatant from the high-speed centrifuged protein sample into a 50 mL centrifuge tube for loading. Load the sample using the peristaltic pump at a rate of 3 mL / min. After loading, rinse the column with 50 mL of Buffer NiA, followed by 50 mL of 10% Buffer NiB. Finally, elute the target protein with 50 mL of 100% Buffer NiB.

[0093] 1.2.7 Dialysis

[0094] Since the eluted target protein in the miconazole buffer might affect crystallization, the target protein was dialyzed into PBS. A section of the dialysis bag was cut and placed in ultrapure water for 5 minutes to remove impurities. The dialysis bag was then rinsed several times in dialysis buffer (prepared PBS and refrigerated at 4°C). Once the temperature had cooled to a comfortable level, the clamp was secured, and the eluted target protein was added to the dialysis bag. Finally, the bag was sealed with the clamp. Dialysis was performed at 4°C for approximately 4 hours.

[0095] The target protein was mainly expressed in the form of supernatant and inclusion bodies. The supernatant was purified by NI column and the purification effect was detected by SDS-PAGE.

[0096] 2. Results

[0097] 2.1 Small-scale expression

[0098] SDS-PAGE analysis showed that in the small-scale trial, pRK793(B) was expressed. Figure 1 ).

[0099] 2.2 Preparation method of recombinant protein

[0100] pRK793(B) protein was induced to express in large quantities at 18℃. The bacterial cells were then sonicated and identified by SDS-PAGE. The results showed that pRK793(B) protein was mainly expressed in supernatant and inclusion bodies. Figure 2 ).

[0101] The supernatant protein was purified by NI column chromatography, and the purification effect was detected by SDS-PAGE. Figure 3 ).

[0102] II. Methods for Recombinant Expression of Gal10

[0103] 1. Cloning Gal 10

[0104] 1.1 Gal 10 gene synthesis

[0105] 1.2 Wuxi Youlong Biotechnology Co., Ltd. was commissioned to synthesize the Gal 10 gene.

[0106] 1.3Gal 10 gene cloning

[0107] The Gal 10 gene was cloned into the expression vector pET28a, following the method described in Molecular Cloning.

[0108] 1.4 Small-scale expression

[0109] Plasmid transformation: Transform the required plasmid into BL21(B), spread it on the corresponding solid culture medium, and incubate at 37℃ for 12-16h.

[0110] Single colonies were picked and cultured in LB liquid medium for 15 hours, and the glycerol bacteria were stored at -80℃.

[0111] Strain activation: Resuscitate the strain and incubate overnight at 37°C for activation.

[0112] Induction of expression: The next day, the bacterial strain was diluted 1:50 and cultured in 5 mL at 37°C until OD was reached. 600 =0.5, collect 2.5 mL of bacterial culture and process it as a pre-induction control. Add 1.0 mM IPTG to the remaining 2.5 mL, induce expression at 37℃ for 3 h, centrifuge and collect the bacterial cells and process them as the post-induction sample, and use SDS-PAGE to identify the protein expression.

[0113] 1.5 Protein Expression and Purification

[0114] Large-scale in vitro culture of target protein

[0115] The transformed single colonies grown on the plate were inoculated into 100 mL of LB medium containing kanamycin resistance and cultured overnight at 37°C with shaking at 220 rpm. From the 100 mL overnight culture, 15 mL of the culture was inoculated into 1 L of LB medium containing kanamycin resistance and cultured at 37°C with shaking at 220 rpm for expansion. When the OD of the bacterial culture reached... 600 When the OD value reaches 0.6, cool the shaker down to 18°C ​​and continue shaking incubation at 200 rpm. 600 When the concentration reaches approximately 0.8, add 200 μL of 1M IPTG to each 1L of culture medium and continue to incubate overnight at 18°C ​​with shaking at 200 rpm for 18 hours.

[0116] 1.6 Bacterial cell collection

[0117] Divide the cultured bacterial suspension into 1L centrifuge bottles and centrifuge in a BECKMAN COULTRAvanti J-26XPI centrifuge at 4°C and 5000 rpm for 15 min, collecting the precipitate. Add 20 mL of 1×PBS (pH 7.4) and pipette to resuspend the bacterial precipitate. Transfer the resuspended bacterial suspension to 50 mL centrifuge tubes and centrifuge in a floor-standing centrifuge at 4°C and 4000 rpm for 30 min, collecting the bacterial precipitate. For short-term storage, store at -20°C; for long-term storage, store at -80°C.

[0118] 1.7 High-pressure sterilization

[0119] During the lysis process, the bacterial suspension needs to be kept on ice. Since the first step in Gal10 protein purification is nickel column affinity chromatography, Buffer NiA is used for bacterial resuspension. The bacterial pellet is removed from the -20°C freezer, and 6 volumes of Buffer NiA are added to resuspend the bacteria. The pellet is then placed in a beaker containing water to dissolve and thoroughly shaken. To prevent large bacterial aggregates, it is best to filter the suspension once with a 2mm needle tip to avoid damaging the instrument. The laboratory uses a JN-3000plus lysis instrument. Before lysis, the compressor needs to be turned on for pre-cooling. Under low pressure, the 20% ethanol stored in the tubing is rinsed off with ultrapure water. Then, the entire tubing is rinsed with Buffer NiA, and the lysis instrument is pressurized to 1100-1300 MPa (this pressure is used to lyse E. coli cells). The bacterial suspension is lysed four times consecutively until the E. coli are observed to be completely ruptured, and the bacterial suspension drips like water without being viscous. After sterilization, rinse the pipeline three times with deionized water, and finally fill the pipeline with 20% ethanol.

[0120] 1,8 bacterial culture centrifugation

[0121] The disrupted bacterial culture was aliquoted into 50mL high-speed centrifuge tubes. After pairwise balancing on a balance (with an error not exceeding 0.02g), the tubes were placed in a BECKMAN COULTRAvanti J-26XPI centrifuge. Centrifugation was performed at 17000rpm and 4°C for 1 hour using a high-speed rotor. The supernatant was then slowly poured into new 50mL centrifuge tubes for subsequent sample loading. This step requires care to prevent the bacterial precipitate from being accidentally resuspended and poured into new tubes. SDS-PAGE analysis was performed to determine the expression pattern of the target protein.

[0122] 1.9 Primary Ni-NTA (nickel) column affinity chromatography

[0123] Nickel ion affinity chromatography columns utilize the specificity of nickel ions in the column medium to generate an affinity interaction with the histidine tag on proteins, while other proteins do not have this interaction, thereby achieving the purification of the target protein.

[0124] First, rinse the entire peristaltic pump tubing with 50 mL of ultrapure water. Then, rinse with 5 column volumes (approximately 5 mL per column volume) of Buffer NiB. Next, equilibrate the peristaltic pump tubing with 5-6 column volumes of Buffer NiA, ensuring the entire tubing is immersed in buffer. After column equilibration, transfer the supernatant from the high-speed centrifuged protein sample into a 50 mL centrifuge tube for loading. Load the sample using the peristaltic pump at a rate of 3 mL / min. After loading, rinse the column with 50 mL of Buffer NiA, followed by 50 mL of 10% Buffer NiB. Finally, elute the target protein with 50 mL of 100% Buffer NiB. Buffer NiA: 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 25 mM imidazole; Buffer NiB: 20 ​​mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM imidazole.

[0125] 2.0 Dialysis

[0126] Since the eluted target protein in the miconazole buffer might affect crystallization, the target protein was dialyzed into PBS. A section of the dialysis bag was cut and placed in ultrapure water for 5 minutes to remove impurities. The dialysis bag was then rinsed several times in dialysis buffer (prepared PBS and refrigerated at 4°C). Once the temperature had cooled to a comfortable level, the clamp was secured, and the eluted target protein was added to the dialysis bag. Finally, the bag was sealed with the clamp. Dialysis was performed at 4°C for approximately 4 hours.

[0127] The target protein was mainly expressed in the form of supernatant and inclusion bodies. The supernatant was purified by NI column and the purification effect was detected by SDS-PAGE.

[0128] 2. Results

[0129] 2.1 Carrier Synthesis

[0130] Nucleic acid sequence:

[0131]

[0132] Note: The unbold bases at the beginning are HIS and TEV restriction sites, and the bolded bases at the end are the GAL10 sequence.

[0133] The amino acid sequence is shown in SEQ ID NO:1:

[0134] MASTTHHHHHHDTDIPTTGGGSRPDDDDKENLYFQGHMSLLPVPYTEAASLSTGSTVTIKGRPLACFLNEPYLQVDFHTEMKEESDIVFHFQVCFGRRVVMNSREYGAWKQQVESKNMPFQDGQEFELSISVLPDKYQVMVNGQSSYTFDHRIKPEAVKMVQVWRDISLTKFNVSYLKR*

[0135] * is the TAG termination codon.

[0136] 3.4 Small-scale expression

[0137] SDS-PAGE analysis showed that in the small-scale trial, pET28a-GAL10(B) was expressed. Figure 4 ).

[0138] 3.5 Preparation of Recombinant Protein

[0139] pET28a-GAL10(B) was induced to express in large quantities at 18℃. The cells were then subjected to high-pressure disruption and SDS-PAGE analysis. The results showed that pET28a-GAL10(B) protein was mainly expressed in the supernatant and inclusion bodies. Figure 5 );

[0140] The supernatant protein was purified by NI column chromatography, and the purification effect was detected by SDS-PAGE. Figure 6 );

[0141] SDS-PAGE protein identification showed that the final target protein had a purity of 95%, a protein concentration of 6 mg / ml, and a protein content of 60 mg. Figure 7 ).

[0142] III. Preparation method of Gal 10 crystal

[0143] 1. Enzyme digestion

[0144] Pour the dialyzed protein into a new 50mL centrifuge tube and measure the concentration using a spectrophotometer.

[0145] Gal10 protein concentration (mg / mL) = A280 uptake peak / 1.2

[0146] Then, concentrate the target protein using a concentration tube until the concentration is basically above 2 mg / mL.

[0147] Two different Gal10 protein concentrations were set: 10 mg / mL and 5 mg / mL, with a total volume of 200 μL. The system details are shown in Tables 1 and 2.

[0148] Table 1. Enzyme digestion system of 10 mg / mL Gal10 protein

[0149]

[0150] Table 2. Enzyme digestion system for 5 mg / mL Gal10 protein

[0151]

[0152] Prepare the enzyme digestion system according to the table above and incubate at room temperature overnight.

[0153] 2. Crystallization: On the second day, gently mix the protein solution in the EP tube five times, then incubate at room temperature for 30 minutes. The solution turns milky white, indicating crystal formation. Centrifuge at 600×g for 5 minutes, gently aspirate the supernatant, wash the precipitate twice with PBS buffer, and finally resuspend the protein precipitate in 5 mL of PBS. Incubate at 4°C. Crystals were observed (see...). Figure 8 ).

[0154] 3. Crystal Concentration Determination

[0155] Take the above crystallization treatment solution and mix it with 6M guanidine hydrochloride at a volume ratio of 1:1, with a volume of 20 μL. Then measure the concentration with a spectrophotometer and calculate the crystal concentration of each treatment group according to the following formula.

[0156] Gal10 crystal concentration (mg / ml) = A280 absorption peak value / 1.2.

[0157] The results are shown in Table 2.

[0158] Table 2. Enzyme digestion system for 5 mg / mL Gal10 protein

[0159] Gal10 protein concentration 1:25 processing group 1:50 processing group 1:100 processing group 5mg / mLGal10 protein Unable to form crystals 0.3mg / ml 1mg / ml 10 mg / mL Al10 protein Unable to form crystals 0.6mg / ml 2mg / ml

[0160] The results above show that a higher concentration of Gal10 protein in the digested product makes crystallization easier. Furthermore, the 1:25 digestion system failed to form crystals. Compared to the 1:50 digestion system, the 1:100 digestion system is more conducive to Gal10 crystal formation and significantly improves the yield of Gal10 crystals.

[0161] Example 2

[0162] Gal 10 crystal egg yolk neutralizing antibody IgY

[0163] 1. Immunize laying hens with Gal 10 crystal protein

[0164] Gal 10 crystals (the nucleotide sequence of which is shown in SEQ ID NO.1) were used as the antigen. 100 μg of the antigen was injected intramuscularly into laying hens (16-20 weeks old) with 1 ml of the antigen each time. The immunization was carried out 3 times at 3 points, with an interval of 28 days between each immunization.

[0165] 2. Extraction and purification of IgY antibody

[0166] Egg yolks from immunized hens were separated using an egg yolk sieve. Nine times the volume of distilled water was added and stirred to prepare an egg yolk solution. The pH was adjusted to 5.2 with 1 mol HCl, and the solution was allowed to stand at 4°C for 5 hours to precipitate. After centrifugation at 8000 rpm for 20 minutes, the precipitate was discarded, and the supernatant was collected. Vacuum filtration (0.09 MPa) was performed using a 0.45 μm mixed cellulose ester membrane to obtain the filtrate. An ultrafiltration membrane with a molecular weight cutoff of 50,000 was used for ultrafiltration concentration at room temperature in a single-stage intermittent operation mode to obtain concentrated IgY. This concentrated IgY was then sterilized by pasteurization and filtered through a 0.22 μm membrane to obtain the purified SARS-CoV-2 egg yolk neutralizing antibody IgY, with a content of 3 mg / ml and a purity of 50%.

[0167] 3. SDS-PAGE electrophoresis analysis

[0168] The isolated and purified IgY was analyzed by SDS-PAGE electrophoresis. Sample preparation: 3% stacking gel, 8% separating gel, 8 μl sample, voltage 80V, electrophoresis for 60 min, stop electrophoresis when the indicator reaches 1-2 cm from the front of the separating gel, Coomassie brilliant blue staining, destaining, and photographing (e.g.) Figure 9 ).

[0169] Depend on Figure 9 It can be seen that the heavy chain of the egg yolk neutralizing antibody IgY is 67-70KD, and the light chain is 22-30KD.

[0170] 4. ELISA valence analysis

[0171] The titer of Gal10 IgY antibody was determined using ELISA:

[0172] Recommended dosage for ELASA experiments:

[0173] Recommended coating amount for His-tagged Gal10 antigen: 1 μg / ml

[0174] The recommended starting dilution for primary antibody Gal 10 IgY is: dilute IgY 1:10000 with 1% BSA / TBST. The recommended dilution for control IgY is: the same concentration as the primary antibody.

[0175] Secondary antibody HRP-conjugated Affinipure Rabbit Anti-Chicken IgY (IgG) (H+L) (Shanghai Tuoran Biotechnology Co., Ltd.) Recommended dilution: 1% BSA / TBST 1:4000.

[0176] ELASA method:

[0177] 1) Dilute the antigen to 1 μg / ml with ELISA Coating Buffer. Add 100 μl to each well and coat overnight at 4°C.

[0178] 2) Block with 3% BSA / TBS, add 200 μl to each well, and incubate at room temperature for 1 hour.

[0179] 3) Wash three times with TBST.

[0180] 4) Except for the H well, add 100 μl of 1% BSA / TBST to each well to dilute the IgY sample to the recommended concentration, then dilute it 2 / 3 times. Add 200 μl of sample diluent to each well in the H well, then take 100 μl of diluent from each well in the H well and transfer it to the G well. Mix well and then dilute sequentially to the B well. After mixing wells in the B well, discard 100 μl. The A well serves as a negative control. Incubate at room temperature for two hours.

[0181] 5) Wash TBST three times

[0182] 6) Dilute the secondary antibody with 1% BSA / TBST and add 100 μl to each well. Incubate at room temperature for one hour.

[0183] 7) Wash TBST five times.

[0184] 8) Add 50 μl of substrate solution to each well and mix well.

[0185] 9) Once the color changes, add 50 μl of stop solution (1M HCl) to each well. The color change should occur within 5 minutes.

[0186] 9) Measure and record the OD value at a wavelength of 450nm.

[0187] 10) Positive result determination: A sample OD value / average OD value of negative control > 2.1 is considered positive.

[0188] Results: The ELISA titer of anti-Gal10 antibody IgY was 2. 16 That is, 65536.

[0189] Example 5

[0190] Preparation method of Gal 10 crystal egg yolk neutralizing antibody IgY spray for treating rhinitis

[0191] The components were prepared in the following weight percentages: 0.3% Gal10 crystal egg yolk neutralizing antibody IgY (prepared in Example 4 for the prevention and treatment of rhinitis), 0.5% lysozyme, 2% glycerol, and 0.04% chlorhexidine gluconate. Deionized water was added to make up to 100 ml. The solution was sterilized by pasteurization, filtered through a 0.22 μm membrane, and then sprayed into a small portable spray can for the treatment of rhinitis.

[0192] Example 6

[0193] Preparation method of Gal 10 crystal egg yolk neutralizing antibody IgY nebulizer for treating asthma

[0194] The components were formulated in the following weight percentages: Gal10 crystal egg yolk neutralizing antibody IgY 0.4%, lysozyme 0.6%, nisin (NISIN) 0.02%, glycerol 5%, and chlorhexidine gluconate 0.1%. Deionized water was added to make up the balance.

[0195] The virus was inactivated using the Pasteurization method and then sterilized by filtration through a 0.22 μm membrane. The solution was then placed in an atomizing bottle for use.

[0196] Example 7

[0197] A clinical trial of Gal10 crystal egg yolk neutralizing antibody IgY nasal spray for the treatment of rhinitis was conducted at Xinyi People's Hospital in Jiangsu Province. Patients were divided into three groups: the experimental group (Gal10IgY), the positive control group (1) topical nasal spray corticosteroids (Rhinocort nasal spray), the antihistamine nasal spray (Azelastine hydrochloride nasal spray), and the nasal mucosal decongestant (Ephedrine nasal drops), and the negative control group (PBS). Each group consisted of 5000 patients. Clinical assessment indicators were: disappearance of nasal congestion, disappearance of nasal dryness, disappearance of nasal itching, improvement of clear nasal discharge, and improvement of nasal voice. Since symptoms vary depending on the type of rhinitis, the treatment was considered effective if any one of the above assessment indicators disappeared in a patient. Dosage and administration: Spray 3-5 times per nostril each time, the number of times depending on the severity of symptoms; more sprays may be given if there is thick nasal discharge or nasal congestion. Spray again when rhinitis symptoms recur. Other procedures should follow the instructions for each nasal spray. The treatment course is one year.

[0198] Results: Experimental group: Total effective rate of polyclonal antibody (Gal10 IgY) was 98%; Positive control group: 1) Topical nasal spray corticosteroids: Rhinocort nasal spray total effective rate 50%; 2) Antihistamine nasal spray: Azelastine hydrochloride nasal spray total effective rate 55%; 3) Nasal mucosal decongestant: Ephedrine nasal drops total effective rate 49%; Negative control group: Total effective rate of PBS 5%. Compared with the positive control group, polyclonal antibody (Gal10 IgY) significantly improved nasal congestion, dryness, itching, clear nasal discharge, and nasal voice, and its efficacy was long-lasting with long-term use, indicating no drug resistance. In contrast, the efficacy of the positive control group gradually decreased over time, indicating the development of drug resistance.

[0199] Example 8

[0200] Human trial of Gal 10 crystal egg yolk neutralizing antibody IgY nebulizer for the treatment of asthma

[0201] A clinical trial was conducted at Xinyi People's Hospital in Jiangsu Province. Patients were divided into three groups: the experimental group received Gal10 IgY (a nasal polyclonal antibody); the positive control group received: 1) salbutamol inhaler (a bronchodilator for relieving asthma); 2) ipratropium bromide inhalation (an anticholinergic); 3) theophylline injection; and 4) budesonide (a corticosteroid for controlling asthma attacks). The negative control group received PBS. Each group had 2000 patients. Clinical assessment indicators: disappearance of wheezing symptoms and disappearance of lung wheezing sounds. Dosage and administration: Intermittent use: repeated four times daily. The adult dose is 0.5–1.0 ml, diluted with normal saline to 2.0–2.5 ml. The inhalation lasts for approximately ten minutes. Some adults may require a higher dose of 10 mg. In this case, 2.0 ml of the product can be placed directly into the inhaler for nebulization until the bronchi are dilated, usually taking three to five minutes. For children aged 18 months to under 12 years, the usual dose is 0.5 mg, diluted with normal saline to 2.0–2.5 ml. Some patients may require an increase to 5 mg. Other procedures should be followed according to the instructions for each nasal spray. The treatment course is one year.

[0202] Results: In the experimental group, the overall effective rate of Gal10 IgY (a polyclonal antibody for rhinitis) was 97%. In the positive control group: 1) Bronchodilators (salbutamol aerosol) had an overall effective rate of 60%; 2) Inhaled ipratropium bromide (an anticholinergic drug) had an overall effective rate of 70%; 3) Theophylline (theophylline injection) had an overall effective rate of 80%; 4) Budesonide (a glucocorticoid for controlling asthma attacks) had an overall effective rate of 40%. In the negative control group, PBS had an overall effective rate of 2%. Compared with the positive control group, Gal10 IgY significantly improved wheezing symptoms and lung sounds, and its efficacy was long-lasting with long-term use, indicating no drug resistance. In contrast, the efficacy of the positive control group gradually decreased over time, indicating the development of drug resistance.

[0203] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody IgY for dissolving galactolectin 10 crystals, characterized in that, The preparation method includes the following steps: Galactoglobulin 10 crystals were used as an antigen to immunize laying hens. The amino acid sequence of the galactoglobulin 10 crystals is shown in SEQ ID NO:

1. The concentration of galactoglobulin 10 crystals in the antigen was 50-100 μg / ml. The method of immunizing laying hens was to immunize them in three separate sessions, with an interval of 28-30 days between the two immunizations. Each immunization was performed using a three-point immunization method, and the immunization dose of antigen at each immunization site was 30-50 μg / hen. Eggs were collected after three initial immunizations and 14 days after the third immunization. The lecithin and other proteins in the egg yolk aqueous solution were removed under acidic conditions. The supernatant was then passed through 0.45 μm and 50,000 molecular weight filter membranes to obtain antibody IgY. The acidic condition is that the pH value of the egg yolk aqueous solution is 4.8~5.5; The method for removing the precipitate from the egg yolk aqueous solution is centrifugation; the centrifugation speed is 7500~8500 rpm, and the centrifugation time is 15~25 min.

2. A medicine for the prevention and treatment of rhinitis and / or asthma, characterized in that, The drug comprises the antibody IgY of claim 1 and pharmaceutically acceptable excipients.

3. The drug according to claim 2, characterized in that, The drug is in the form of a spray, and the spray comprises the following components by weight percentage: Antibody IgY 0.1~0.4%, lysozyme 0.1~0.9%, glycerol 1~10%, chlorhexidine gluconate 0.04~0.1% and distilled water 88.6~98.76%.

4. The drug according to claim 2, characterized in that, The drug is in the form of an aerosol; The atomizing agent comprises the following components in weight percentage: Antibody IgY 0.1~0.4%, lysozyme 0.1~0.9%, nisin 0.005~0.02%, glycerol 1~10%, chlorhexidine gluconate 0.04~0.1% and distilled water 88.58~98.755%.

5. The use of the antibody IgY according to claim 1 in the preparation of a medicament for the prevention and treatment of rhinitis and / or asthma.

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