A method for preparing a sage pollen allergen and a sage pollen allergen injection vaccine

Abstract

The application provides a preparation method of Artemisia sphaerocephala pollen allergen and an Artemisia sphaerocephala pollen allergen injection, and belongs to the technical field of pollen allergen vaccine preparation. After the Artemisia sphaerocephala pollen is defatted and dried, the Artemisia sphaerocephala pollen is extracted by using an extraction solution, the supernatant is filled into a dialysis bag to be dialyzed, concentrated, enzymolyzed and desalted, and then chromatography is carried out, and polypeptides with molecular weights of 23-46KD and 67-82KD are collected to be the Artemisia sphaerocephala pollen allergen. The Artemisia sphaerocephala pollen allergen injection comprises the Artemisia sphaerocephala pollen allergen, sodium chloride, PBS buffer, phenol and thiourea. The Artemisia sphaerocephala pollen allergen injection of the application fills the blank of Artemisia sphaerocephala pollen allergen injection, can effectively reduce the allergic degree of guinea pigs to Artemisia sphaerocephala pollen, and can be stably stored at 2-8 DEG C.

Description

A method for preparing Artemisia annua pollen allergen and an injectable vaccine for Artemisia annua pollen allergen. Technical Field

[0001] This invention relates to the field of pollen allergen vaccine preparation technology, and in particular to a method for preparing Artemisia argyi pollen allergen and an injectable Artemisia argyi pollen allergen vaccine. Background Technology

[0002] Allergic diseases are a major threat to human health, and the World Health Organization has listed them as one of the key diseases for research and prevention in the 21st century. Hay fever, also known as seasonal allergic rhinitis, is an allergy-related inflammation of the nasal passages, throat, and conjunctiva caused by sensitivity to airborne pollen. In 1911, Noon and Freeman first used pollen extracts to treat hay fever, marking the beginning of the treatment of allergic diseases.

[0003] Artemisia (L., sensu sricto) is a genus of plants in the Asteraceae family. It has many species and a wide distribution, with 344 species and 69 varieties worldwide. Its main habitats are in temperate to subtropical regions of the Northern Hemisphere, with the highest concentration in Eurasia and followed by North America. Surveys show that Artemisia pollen is the most abundant pollen in the air during summer and autumn in many regions, and it is the most significant allergen. Artemisia spp., belonging to the Artemisia genus of the Asteraceae family, has excellent sand-fixing effects and is a pioneer species for windbreak and sand fixation. Seasonal allergic rhinitis and / or allergic asthma are most prevalent from mid-August to mid-September each year. Common clinical symptoms include persistent sneezing and runny nose, nasal congestion (even affecting breathing), increased eye discharge, and redness and itching. Some severe cases experience asthma, shortness of breath, hearing loss, and blurred vision. Other allergy sufferers may experience skin rashes and dermatitis symptoms such as itching and redness. In recent years, research on the epidemiology, pollen allergens and sensitization evaluation, and sensitization mechanisms of Artemisia annua pollen-induced allergic diseases has increasingly attracted widespread attention from scholars both domestically and internationally. Artemisia annua pollen allergen protein is a key basis for triggering allergic reactions; therefore, obtaining high-purity Artemisia annua pollen allergen protein can be used for the diagnosis and clinical application of autumn artemisia pollen-induced allergic diseases, for the preparation of high-purity Artemisia annua pollen prick test solutions, for the development and application of pollen vaccines for specific treatment, and can provide necessary materials for research on its structure and epitopes, biological characteristics, sensitization evaluation, and assay methods.

[0004] The invention patent application number "201110433656.9" provides an injectable vaccine for Artemisia pollen allergens, wherein the Artemisia pollen allergens are Artemisia macrocarpa pollen, Artemisia annua pollen, and Artemisia argyi pollen allergens. However, to date, no Artemisia sandy pollen allergens have been reported. Furthermore, due to the complex composition and structure of Artemisia pollen allergens, they are easily affected by physical or chemical factors, resulting in loss of activity and reduced potency. Even in low-temperature environments, pollen allergen vaccines cannot guarantee the long-term stability of their allergen activity, which is not conducive to long-term preservation. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing Artemisia argyi pollen allergen and an injectable vaccine for Artemisia argyi pollen allergen, filling the gap in the field of Artemisia argyi pollen allergen and solving the problem that pollen allergen vaccines cannot be stored for a long time.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing Artemisia annua pollen allergen, comprising the following steps:

[0008] (1) After defatting and drying the pollen of Artemisia argyi, mix it with the extract and extract it to obtain the extract.

[0009] (2) Centrifuge the extract and collect the supernatant;

[0010] (3) Place the supernatant into a dialysis bag, place the dialysis bag in the extract for dialysis, change the extract every 3 to 6 hours, and dialysis for 36 to 72 hours to obtain Artemisia annua pollen protein extract;

[0011] (4) Concentrate the Artemisia annua pollen protein extract to 1 / 10 to 1 / 8 of its original volume, mix it with ammonium bicarbonate solution, adjust the pH value to 7.5 to 8.5 to obtain a diluted solution, add trypsin to the diluted solution, and enzymatically hydrolyze it at 36 to 38°C for 8 to 12 hours to obtain an enzymatic hydrolysate.

[0012] (5) After desalting the enzymatic hydrolysate, dilute it to a polypeptide concentration of 10-15 mg / mL and perform chromatography to collect polypeptides with molecular weights of 23-46 KD and 67-82 KD, which are Artemisia argyi pollen allergens.

[0013] Preferably, the defatting is performed by mixing pollen and defatting solvent at a mass-volume ratio of 1g:3-8mL, stirring or shaking for 20-40 minutes, allowing the mixture to stand and separate into layers, pouring out the upper layer, adding new defatting solvent, and repeating this process 2-3 times.

[0014] The degreasing solvent is acetone or diethyl ether.

[0015] Preferably, the extract comprises the following components at concentrations of: 4.5–5.5 g / L sodium chloride, 2–3 g / L sodium bicarbonate, and 2–4 mg / L phenol.

[0016] Preferably, the mass-to-volume ratio of defatted and dried Artemisia pollen to extract is 1g:10-40mL.

[0017] Preferably, the extraction temperature is 12-18°C, the extraction time is 20-26 hours, and the extraction is accompanied by stirring at a speed of 100-300 rpm.

[0018] Preferably, the molecular weight cutoff of the dialysis bag is 8-14 KD.

[0019] Preferably, the weight-to-volume ratio of the trypsin to the diluent is 1g:80-100mL.

[0020] The present invention also provides Artemisia annua pollen allergen prepared by the modified preparation method.

[0021] The present invention also provides an injectable vaccine for Artemisia annua pollen allergen, comprising the aforementioned Artemisia annua pollen allergen, sodium chloride, PBS buffer, phenol, and thiourea; wherein the mass concentration of the Artemisia annua pollen allergen is 2–300 μg / mL, the molar concentration of the PBS buffer is 40–100 mM, the mass concentration of the sodium chloride is 0.8–1.5%, the mass concentration of the phenol is 0.2–0.5%, and the mass concentration of the thiourea is 1–2%.

[0022] Preferably, the activity of the Artemisia annua pollen allergen is 50–7500 AU / mL.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects: the invented Artemisia pollen allergen injection vaccine fills the gap in Artemisia allergen injection vaccines; it effectively reduces the allergic reaction of guinea pigs to Artemisia pollen and can be stably stored at 2-8℃. Detailed Implementation

[0024] This invention provides a method for preparing Artemisia annua pollen allergen, comprising the following steps:

[0025] (1) After defatting and drying the pollen of Artemisia argyi, mix it with the extract and extract it to obtain the extract.

[0026] (2) Centrifuge the extract and collect the supernatant;

[0027] (3) Place the supernatant into a dialysis bag, place the dialysis bag in the extract for dialysis, change the extract every 3 to 6 hours, and dialysis for 36 to 72 hours to obtain Artemisia annua pollen protein extract;

[0028] (4) Concentrate the Artemisia annua pollen protein extract to 1 / 10 to 1 / 8 of its original volume, mix it with ammonium bicarbonate solution, adjust the pH to 7.5 to 8.5 to obtain a diluted solution, add trypsin and hydrolyze at 36 to 38°C for 8 to 12 hours to obtain a hydrolysate.

[0029] (5) After desalting the enzymatic hydrolysate, dilute it to a polypeptide concentration of 10-15 mg / mL and perform chromatography to collect polypeptides with molecular weights of 23-46 KD and 67-82 KD, which are Artemisia argyi pollen allergens.

[0030] This invention preferably involves collecting branches of *Artemisia arenaria* with flower buds during its flowering season (mid-August to late September). The branches are placed on a clean glass plate indoors, and after 24–48 hours, the pollen naturally falls off. The pollen is collected and sieved, with a mesh size of 100–150 mesh, more preferably 110–140 mesh, and even more preferably 120 mesh. The sieved pollen is then collected and dried, using vacuum drying, a dryer, or a fluidized bed drying method, until a constant weight is achieved.

[0031] In this invention, after obtaining Artemisia annua pollen, the pollen is mixed with acetone or ether for defatting. The mass-to-volume ratio of Artemisia annua pollen to acetone / ether is 1g:3-8mL, more preferably 1g:4-7mL, and even more preferably 1g:6mL. After mixing the pollen with acetone / ether, the mixture is stirred or shaken, allowed to stand to separate into layers, the upper layer is poured off, and fresh acetone or ether is added. This process is repeated 2-3 times until the upper layer is clear. This invention does not have specific limitations on the stirring or shaking parameters, as long as sufficient mixing of the pollen with acetone / ether is achieved. The stirring or shaking time is 20-40 minutes, more preferably 25-35 minutes, and even more preferably 30 minutes. After defatting, the pollen is dried.

[0032] The present invention involves defatting and drying Artemisia annua pollen, and then preparing an extract containing the following components at concentrations: 4.5–5.5 g / L sodium chloride, 2–3 g / L sodium bicarbonate, and 2–4 mg / L phenol.

[0033] In this invention, the mass concentration of sodium chloride in the extract is 4.5–5.5 g / L, more preferably 4.8–5.2 g / L, and even more preferably 5 g / L;

[0034] The mass concentration of sodium bicarbonate in the extract is 2-3 g / L, more preferably 2.3-2.7 g / L, and even more preferably 2.5 g / L;

[0035] The mass concentration of phenol in the extract is 2 to 4 mg / L, more preferably 2.5 to 3.5 mg / L, and even more preferably 3 mg / L.

[0036] This invention involves mixing defatted and dried Artemisia annua pollen with an extract, followed by extraction to obtain an extract. The mass-to-volume ratio of Artemisia annua pollen to the extract is 1g:10-40mL, more preferably 1g:20-30mL, and even more preferably 1g:25mL. The extraction temperature is 12-18℃, more preferably 14-16℃, and even more preferably 15℃. The extraction time is 20-26h, more preferably 22-24h, and even more preferably 23h. The extraction is accompanied by stirring at a speed of 100-300rpm, more preferably 150-250rpm, and even more preferably 200rpm. After obtaining the extract, the mixture is centrifuged, and the supernatant is collected. The centrifugal force is 6000-9000g, more preferably 7000-8000g, and even more preferably 7500g. The centrifugation time is 10-15min, more preferably 11-14min, and even more preferably 12min.

[0037] In this invention, the supernatant is placed in a dialysis bag, and the dialysis bag is placed in the extract for dialysis to obtain Artemisia annua pollen protein extract; the dialysis time is 36-72 hours, more preferably 40-60 hours, and more preferably 50 hours; during the dialysis process, the extract is replaced every 3-6 hours, more preferably 4-5 hours, and more preferably 4.5 hours; the molecular weight cutoff of the dialysis bag is 8-14 KD, more preferably 10-12 KD, and more preferably 11 KD; after dialysis, Artemisia annua pollen protein extract is obtained.

[0038] In this invention, after obtaining the Artemisia annua pollen protein extract, it is concentrated. The concentration is preferably dialysis concentration. Specifically, polyethylene glycol 20000 is placed around the dialysis bag containing the Artemisia annua pollen protein extract, and the extract is concentrated to 1 / 10 to 1 / 8 of the original volume of the Artemisia annua pollen protein extract, more preferably 1 / 9. The role of polyethylene glycol 20000 is to act as an absorbent, absorbing and removing the solution to concentrate the protein, thereby obtaining the Artemisia annua pollen protein concentrate.

[0039] In this invention, the concentrated pollen protein solution of Artemisia annua is mixed with an ammonium bicarbonate solution at a volume ratio of 1:3 to 5, more preferably 1:3.5 to 4.5, and more preferably 1:4; the pH value is adjusted to 7.5 to 8.5, more preferably 7.8 to 8.2, and more preferably 8; the concentration of the ammonium bicarbonate solution is 23 to 28 mM, more preferably 24 to 26 mM, and more preferably 25 mM. A diluted solution is obtained after mixing the concentrated pollen protein solution of Artemisia annua with the ammonium bicarbonate solution.

[0040] In this invention, trypsin is added to a diluent for enzymatic hydrolysis. The mass-to-volume ratio of trypsin to the diluent is 1g:40-60mL, more preferably 1g:45-55mL, and even more preferably 1g:50mL. The hydrolysis temperature is 36-38℃, more preferably 36.5-37.5℃, and even more preferably 37℃. The hydrolysis time is 8-12h, more preferably 9-11h, and even more preferably 10h. Formic acid is added to adjust the pH to acidic to terminate the hydrolysis, yielding the hydrolysate.

[0041] After desalting and purifying the enzymatic hydrolysate, it is diluted and subjected to chromatography to collect peptides with molecular weights of 30–40 KD and 60–70 KD, which are identified as Artemisia annua pollen allergens. After dilution, the peptide concentration is 10–15 mg / mL, more preferably 11–14 mg / mL, and even more preferably 12 mg / mL, at which concentration good separation effect is observed. The chromatography is gel filtration chromatography, preferably using a dextran gel, more preferably Sephadex G-100 dextran gel, with a particle size of 40–120 μm, more preferably 60–100 μm, and even more preferably 80 μm. The present invention preferably uses 0.04–0.06 mol / L PBS buffer for elution, with a PBS buffer concentration more preferably 0.05 mol / L, and a flow rate of 4–6 mL / h, more preferably 4.5–5.5 mL / h, and even more preferably 5 mL / h.

[0042] The present invention also provides an injectable vaccine for Artemisia annua pollen allergen, comprising the aforementioned Artemisia annua pollen allergen, sodium chloride, PBS buffer, phenol, and thiourea; wherein the mass concentration of the Artemisia annua pollen allergen is 2–300 μg / mL, the molar concentration of the PBS buffer is 40–100 mM, the mass concentration of the sodium chloride is 0.8–1.5%, the mass concentration of the phenol is 0.2–0.5%, and the mass concentration of the thiourea is 1–2%.

[0043] In the Artemisia argyi pollen allergen injection vaccine of the present invention, the mass concentration of Artemisia argyi pollen allergen is 2-300 μg / mL, more preferably 50-200 μg / mL, and more preferably 100 μg / mL;

[0044] The molar concentration of the PBS buffer is 40–100 mM, more preferably 50–80 mM, and even more preferably 60 mM;

[0045] The mass concentration of sodium chloride is 0.8-1.5%, more preferably 1.0-1.3%, and even more preferably 1.1%.

[0046] The phenol has a mass concentration of 0.2-0.5%, more preferably 0.3-0.4%, and even more preferably 0.35%; the function of phenol is to inhibit bacteria.

[0047] The mass concentration of thiourea is 1-2%, more preferably 1.2-1.8%, and even more preferably 1.5%, and the function of thiourea is to resist oxidation.

[0048] In the Artemisia argyi pollen allergen injection vaccine of the present invention, the activity range of the Artemisia argyi pollen allergen is 50-7500 AU / mL, more preferably 2000-6000 AU / mL, and even more preferably 4000 AU / mL.

[0049] The Artemisia annua pollen allergen injectable vaccine of the present invention may further include solvents and adjuvants.

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] In early September, branches of Artemisia argyi with flower buds were collected and placed on a clean glass plate indoors. After 24 hours, the pollen was naturally shed. The collected pollen was passed through a 100-mesh sieve and vacuum dried at 10°C until the pollen reached a constant weight.

[0053] Mix 20g of Artemisia annua pollen with 60mL of acetone, stir for 20min, defatted, and dried to constant weight. Mix 10g of defatted and dried Artemisia annua pollen with 100mL of extract, and extract at 12℃ and 100rpm for 20h. After extraction, centrifuge at 6000g for 10min and collect the supernatant. The extract contains 4.5g / L sodium chloride, 2g / L sodium bicarbonate, and 2mg / L phenol.

[0054] Take 100 mL of the above supernatant and place it into a dialysis bag with a molecular weight cutoff of 8 KD. Place the dialysis bag in the extract and dialyze for 36 hours, changing the extract every 3 hours. After dialysis, obtain the Artemisia annua pollen protein extract. Place polyethylene glycol 20000 around the dialysis bag containing the Artemisia annua pollen protein extract and concentrate it to 10 mL to obtain the Artemisia annua pollen protein concentrate.

[0055] The concentrated protein solution of Artemisia annua pollen was mixed with 30 mL of 23 mM ammonium bicarbonate solution, the pH was adjusted to 7.5, 1 g of trypsin was added, and the mixture was enzymatically hydrolyzed at 36 °C for 8 h. Formic acid was added to adjust the pH to acidic, and the enzymatic hydrolysis was terminated to obtain the hydrolysate.

[0056] After desalting and purifying the enzymatic hydrolysate, the solution was diluted to a peptide concentration of 10 mg / mL and subjected to dextran gel chromatography. A Sephadex G-100 dextran gel with a particle size of 40 μm was packed into a column after a 6-hour boiling water bath, and chromatography was performed using the following parameters:

[0057] Diameter: 1.6 cm; Column length: 95 cm; Volume: 190 mL; Flow rate: 4 mL / h; Buffer: 0.04 mol / L PBS.

[0058] Polypeptides with molecular weights of 23–46 KD and 67–82 KD were collected and identified as Artemisia argyi pollen allergens.

[0059] Example 2

[0060] In mid-September, branches of Artemisia argyi with flower buds were collected and placed on a clean glass plate indoors. After 48 hours, the pollen naturally fell off. The collected pollen was passed through a 120-mesh sieve and vacuum dried at 15°C until the Artemisia argyi pollen reached a constant weight.

[0061] Mix 20g of Artemisia annua pollen with 100mL of ether, shake for 30min to defatt the mixture, and dry to constant weight. Mix 10g of defatted and dried Artemisia annua pollen with 200mL of extract, and extract at 15℃ and 200rpm for 24h. After extraction, centrifuge at 8000g for 13min and collect the supernatant. The extract contains 5g / L sodium chloride, 2.5g / L sodium bicarbonate, and 3mg / L phenol.

[0062] Take 200 mL of the supernatant and place it into a dialysis bag with a molecular weight cutoff of 10 KD. Place the dialysis bag in the extract and dialyze for 48 hours, changing the extract every 4 hours. After dialysis, obtain the Artemisia annua pollen protein extract. Place polyethylene glycol 20000 around the dialysis bag containing the Artemisia annua pollen protein extract and concentrate to 23 mL to obtain the Artemisia annua pollen protein concentrate.

[0063] The concentrated pollen protein solution of Artemisia annua was mixed with 92 mL of 25 mM ammonium bicarbonate solution, the pH was adjusted to 8, 1.8 g of trypsin was added, and the mixture was enzymatically hydrolyzed at 37 °C for 10 h. Formic acid was added to adjust the pH to acidic, and the enzymatic hydrolysis was terminated to obtain the hydrolysate.

[0064] After desalting and purifying the enzymatic hydrolysate, the solution was diluted to a peptide concentration of 12 mg / mL and subjected to dextran gel chromatography. A Sephadex G-100 dextran gel with a particle size of 80 μm was packed into a column after a 6-hour boiling water bath, and chromatography was performed using the following parameters:

[0065] Diameter: 1.6 cm; Column length: 95 cm; Volume: 190 mL; Flow rate: 5 mL / h; Buffer: 0.05 mol / L PBS.

[0066] Polypeptides with molecular weights of 23–46 KD and 67–82 KD were collected and identified as Artemisia argyi pollen allergens.

[0067] Example 3

[0068] In late September, branches of Artemisia argyi with flower buds were collected and placed on a clean glass plate indoors. After 72 hours, the pollen was naturally shed. The collected pollen was passed through a 150-mesh sieve and vacuum dried at 20°C until the Artemisia argyi pollen reached a constant weight.

[0069] Mix 20g of Artemisia annua pollen with 160mL of acetone, stir for 40min, defatted, and dried to constant weight. Mix 10g of defatted and dried Artemisia annua pollen with 400mL of extract, and extract at 18℃ and 300rpm for 26h. After extraction, centrifuge at 9000g for 15min and collect the supernatant. The extract contains 5.5g / L sodium chloride, 3g / L sodium bicarbonate, and 4mg / L phenol.

[0070] Take 400 mL of the supernatant and place it into a dialysis bag with a molecular weight cutoff of 14 KD. Place the dialysis bag in the extract and dialyze for 72 hours, changing the extract every 6 hours. After dialysis, obtain the Artemisia annua pollen protein extract. Place polyethylene glycol 20000 around the dialysis bag containing the Artemisia annua pollen protein extract and concentrate to 40 mL to obtain the Artemisia annua pollen protein concentrate.

[0071] The concentrated pollen protein solution of Artemisia annua was mixed with 200 mL of 28 mM ammonium bicarbonate solution, the pH was adjusted to 8.5, 4 g of trypsin was added, and the mixture was enzymatically hydrolyzed at 38 °C for 12 h. Formic acid was added to adjust the pH to acidic, and the enzymatic hydrolysis was terminated to obtain the hydrolysate.

[0072] After desalting and purifying the enzymatic hydrolysate, the solution was diluted to a peptide concentration of 15 mg / mL and subjected to dextran gel chromatography. A Sephadex G-100 dextran gel with a particle size of 120 μm was packed into a column after a 6-hour boiling water bath, and chromatography was performed using the following parameters:

[0073] Diameter: 1.6 cm; Column length: 95 cm; Volume: 190 mL; Flow rate: 6 mL / h; Buffer: 0.06 mol / L, PBS.

[0074] Polypeptides with molecular weights of 23–46 KD and 67–82 KD were collected and identified as Artemisia argyi pollen allergens.

[0075] Example 4

[0076] An injectable vaccine for Artemisia annua pollen allergen is composed of the following components: 2 μg / mL Artemisia annua pollen allergen prepared in Example 1, 40 mM PBS buffer, sodium chloride at a mass concentration of 0.8%, phenol at a mass concentration of 0.2%, and thiourea at a mass concentration of 1%.

[0077] Example 5

[0078] An injectable vaccine for Artemisia annua pollen allergen is composed of the following components: 100 μg / mL Artemisia annua pollen allergen prepared in Example 2, 60 mM PBS buffer, sodium chloride at a mass concentration of 1.2%, phenol at a mass concentration of 0.4%, and thiourea at a mass concentration of 1.5%.

[0079] Example 6

[0080] An injectable vaccine for Artemisia annua pollen allergen is composed of the following components: 300 μg / mL Artemisia annua pollen allergen prepared in Example 3, 100 mM PBS buffer, sodium chloride at a mass concentration of 1.5%, phenol at a mass concentration of 0.5%, and thiourea at a mass concentration of 2%.

[0081] Experimental Example 1: Acute Toxicity Test

[0082] Acute toxicity tests were conducted on the Artemisia argyi pollen allergen injection vaccine prepared in Example 2. Twenty-four SPF-grade mice (weighing 18–22 g), half male and half female, were used as experimental animals and divided into two groups. Each mouse in the experimental group was injected with the Artemisia argyi pollen allergen injection vaccine prepared in Example 2, while each mouse in the control group was injected with an equal volume of physiological saline. The mice were observed for 7 days, and their weight, mental state, behavior, and stool were recorded daily. After the 7th day of observation, no mice died, and no significant abnormalities were observed in the mice's mental state, behavior, or stool. The weight gain of the mice was the same as that of the normal physiological saline control group, demonstrating that the Artemisia argyi pollen allergen injection vaccine of this invention is safe and feasible.

[0083] Experimental Example 2: Pharmacodynamic Evaluation of Artemisia annua Pollen Allergen Injectable Vaccine

[0084] The experiment was divided into four groups, with 10 clean-grade sensitized guinea pigs in each group. The control group, allergen-treated group 1, allergen-treated group 2, and allergen-treated group 3 were administered vaccines. The control group received physiological saline, while allergen-treated groups 1, 2, and 3 received the vaccines prepared in Examples 4, 5, and 6, respectively, via subcutaneous injection (0.5 mL). Sensitization was repeated every 10 days for a total of three times. After the administration of the vaccines, each group of guinea pigs received a subcutaneous injection of 0.5 mL of a 100 AU / mL Artemisia annua pollen allergen solution at different injection sites. The diameters (average of the major and minor axis diameters) of wheals and erythema generated at different injection sites were measured for each guinea pig. The arithmetic mean of the diameters of wheals and erythema for each pollen allergen in each group was calculated. The results are shown in Table 1.

[0085] Table 1. Pharmacodynamic evaluation of Artemisia pollen allergen injection vaccine

[0086] Group wheal and erythema diameter (mm): Control group 38.5, Allergen-treated group 1 30.7, Allergen-treated group 2 22.6, Allergen-treated group 3 13.9 surface

[0087] As shown in Table 1, the Artemisia argyi pollen allergen injection vaccine of the present invention can effectively reduce the allergy level of guinea pigs to Artemisia argyi pollen, and to a certain extent, the anti-allergy effect is enhanced as the concentration of Artemisia argyi pollen allergen in the Artemisia argyi pollen allergen increases.

[0088] Experiment Example 3: Stability Test of Artemisia pollen allergen injectable vaccine

[0089] After testing the physicochemical properties of the Artemisia argyi pollen allergen injection vaccines in Examples 4-6, the quality standards shown in Table 2 were established.

[0090] Table 2 Physicochemical Properties and Quality Standards of Artemisia pollen allergen injectable vaccines

[0091] Test Items and Quality Standards: pH value 7.0–8.0, Sodium chloride content 0.8–1.5% (mass percentage), Phenol content 0.2–0.5% (mass percentage), Thiourea content 1–2% (mass percentage), Artemisia annua pollen allergen content 2–300 μg / mL, PBS buffer molar concentration 40–100 mM, Total allergen activity 45–7500 AU / mL surface

[0092] Long-term stability studies were conducted on the Artemisia argyi pollen allergen injection vaccines of Examples 4-6 at 2-8°C. The pH value, Artemisia argyi pollen allergen content, and total allergen activity of the three batches of samples were measured at 0 hours, 3 months, 3 months, 9 months, and 12 months. The results are shown in Table 3.

[0093] Table 3. Results of stability test for Artemisia argyi pollen allergen vaccine

[0094]

[0095] The stability results above show that, during the 12-month long-term trial, although the pH value, pollen allergen content, and total allergen activity of the Artemisia argyi pollen allergen injection vaccine fluctuated, they were all within the quality control range of the quality standard. Moreover, the trends of change between different batches were basically consistent. This indicates that the Artemisia argyi pollen allergen injection vaccine described in this invention can be stably stored for 12 months under conditions of 2–8°C.

[0096] As can be seen from the above embodiments, the present invention provides a method for preparing Artemisia argyi pollen allergen and an injectable vaccine for Artemisia argyi pollen allergen. The injectable vaccine for Artemisia argyi pollen allergen of the present invention fills the gap in injectable vaccines for Artemisia argyi allergens. The injectable vaccine for Artemisia argyi pollen allergen of the present invention can effectively reduce the degree of allergy to Artemisia argyi pollen in mice and can be stably stored for 12 months at 2-8℃.

[0097] 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. A method for preparing Artemisia annua pollen allergen, characterized in that, Includes the following steps: (1) After defatting and drying the pollen of Artemisia argyi, it is mixed with the extract and extracted to obtain the extract; the defatting is performed by mixing the pollen with the defatting solvent at a mass-volume ratio of 1g:5mL, stirring or shaking for 30min, allowing it to stand and separate into layers, pouring off the upper layer, adding new defatting solvent, and repeating 2-3 times; the defatting solvent is ether; the mass-volume ratio of the defatted and dried Artemisia argyi pollen to the extract is 1g:20mL; the extract uses water as a solvent and includes the following components at the following concentrations: 5g / L sodium chloride, 2.5g / L sodium bicarbonate and 3mg / L phenol; the extraction temperature is 15℃; the extraction is accompanied by stirring, the stirring speed is 200rpm; the extraction time is 24h; (2) the extract is centrifuged and the supernatant is taken; (3) the supernatant is put into a dialysis bag, the dialysis bag is placed in the extract for dialysis, the extract is changed every 4h, and dialysis is performed for 48h to obtain Artemisia annua pollen protein extract; the molecular weight cutoff of the dialysis bag is 10 KD; (4) Concentrate the Artemisia annua pollen protein extract to 1 / 10 to 1 / 8 of its original volume, mix it with ammonium bicarbonate solution, adjust the pH to 8, add 1.8g of trypsin, and enzymatically hydrolyze it at 37℃ for 10h. Add formic acid to adjust the pH to acidic, terminate the enzymatic hydrolysis, and obtain the enzymatic hydrolysate; (5) After desalting and purifying the enzymatic hydrolysate, dilute it to a polypeptide concentration of 12mg / mL, pack it into a Sephadex G-100 dextran gel with a particle size of 80μm after boiling water bath for 6h, and perform chromatography using the following parameters: diameter: 1.6cm; column length: 95cm; volume: 190mL; flow rate: 5mL / h; Buffer: 0.05 mol / L PBS; collect peptides with molecular weights of 23–46 KD and 67–82 KD, which are Artemisia annua pollen allergens.

2. A method for preparing Artemisia annua pollen allergen, characterized in that, Includes the following steps: (1) After defatting and drying the pollen of Artemisia argyi, it is mixed with the extract and extracted to obtain the extract; the defatting is performed by mixing the pollen with the defatting solvent at a mass-volume ratio of 1g:8mL, stirring or shaking for 40min, allowing it to stand and separate into layers, pouring out the upper layer, adding new defatting solvent, and repeating 2-3 times; the defatting solvent is acetone; the mass-volume ratio of the defatted and dried Artemisia argyi pollen to the extract is 1g:40mL; the extract uses water as a solvent and includes the following components at the following concentrations:

5. 5 g / L sodium chloride, 3 g / L sodium bicarbonate and 4 mg / L phenol; the extraction temperature is 18°C; the extraction is accompanied by stirring at a speed of 300 rpm; the extraction time is 26 h; (2) the extract is centrifuged and the supernatant is collected; (3) the supernatant is placed in a dialysis bag and the dialysis bag is placed in the extract for dialysis. The extract is replaced every 6 h and dialysis is performed for 72 h to obtain Artemisia annua pollen protein extract; the molecular weight cutoff of the dialysis bag is 14. KD; (4) Concentrate the Artemisia annua pollen protein extract to 1 / 10 to 1 / 8 of its original volume, mix it with ammonium bicarbonate solution, adjust the pH to 8.5, add 4g of trypsin, and enzymatically hydrolyze it at 38℃ for 12h. Add formic acid to adjust the pH to acidic, terminate the enzymatic hydrolysis, and obtain the enzymatic hydrolysate; (5) After desalting and purifying the enzymatic hydrolysate, dilute it to a polypeptide concentration of 15mg / mL, pack it into a Sephadex G-100 dextran gel with a particle size of 120μm after boiling water bath for 6h, and perform chromatography using the following parameters: diameter: 1.6cm; column length: 95cm; volume: 190mL; flow rate: 6mL / h; Buffer: 0.06 mol / L PBS; collect peptides with molecular weights of 23–46 KD and 67–82 KD, which are Artemisia annua pollen allergens.

3. The Artemisia annua pollen allergen prepared by any one of claims 1 to 2.

4. A *Artemisia annua* pollen allergen injectable vaccine, characterized in that, The mixture comprises the Artemisia annua pollen allergen, sodium chloride, PBS buffer, phenol, and thiourea as described in claim 3; wherein the mass concentration of the Artemisia annua pollen allergen is 100-300 μg / mL, the molar concentration of the PBS buffer is 40-100 mM, the mass concentration of the sodium chloride is 0.8-1.5%, the mass concentration of the phenol is 0.2-0.5%, and the mass concentration of the thiourea is 1-2%.

5. The Artemisia annua pollen allergen injectable vaccine according to claim 4, characterized in that, The activity of the Artemisia annua pollen allergen was 50~7500 AU / mL.

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