Dermatophagoides farinae pure mite body allergen raw material and preparation method of leach liquor thereof
By preparing the pure allergen raw materials of dust mite body and its extract solution, the problem of rapid reproduction and culture of dust mites is solved, and efficient proliferation and high-purity dust mite cultures are achieved, and effective preparations for specific immunotherapy are provided.
Patent Information
- Application Number
- CN202411434711.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-15
- Publication Date
- 2025-03-04
AI Technical Summary
The rapid reproduction and culture of large-scale dust mites has not yet been achieved in China, resulting in the fact that dust mites-free allergen preparation products cannot be used for specific immunotherapy.
Provide a method for preparing a pure mite allergen raw material of dust mite body and its leaching solution, including mite species collection, separation and identification, mite species culture, inoculation, culture, harvesting, purification, degreasing, drying, preservation and leaching solution preparation, so as to realize efficient proliferation of dust mites and preparation of leaching solution.
The efficient proliferation of dust mites is achieved. The cultured dust mites have a high density and a purity of up to 95% to 99%. It can produce sterile dust mites cultures on a large scale and high yield basis. The prepared extract has great clinical application value.
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Figure CN119552209A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biology, and more specifically to a method for preparing a raw material of pure dust mite allergen and an extract thereof. Background Art
[0002] Dust mites are the most potent allergens for allergic reactions worldwide. Dust mites are a type of arthropod that is only the size of a speck of dust and is difficult to discern with the naked eye. They live extensively in carpets, sofas, mattresses, and thick curtains, feeding on the dandruff shed by humans and animals. Both living and dead dust mites and their secretions can cause allergic reactions in people. Dust mites prefer to breed in warm and humid environments and are the most important allergens for rhinitis, asthma, and dermatitis.
[0003] For the treatment of allergic diseases, the World Health Organization (WHO) has proposed a "four-in-one" comprehensive treatment plan. The plan recommends that patients be treated with allergen-specific immunotherapy (ASIT) combined with symptomatic drugs under the premise of avoiding contact with allergens as much as possible, and that patients be treated with comprehensive management and education in order to achieve stable short-term and long-term therapeutic effects. Among them, ASIT is currently the only treatment method that may change the natural course of allergic diseases. ASIT has been listed as the first-line treatment for AR and does not require the ineffectiveness of symptomatic drug treatment.
[0004] The key to the success of specific immunotherapy is the use of standardized, sustainably produced, high-quality allergen preparations, which is essential to the safety, effectiveness, reliability and accuracy of allergen products. At the same time, it is also crucial to be able to mass-produce allergen raw materials with controllable and stable quality.
[0005] At present, large-scale rapid reproduction and cultivation of dust mites has not been achieved in China. Products without dust mite allergen preparations can be used for specific immunotherapy.
[0006] Based on the above problems, we provide a method for preparing a raw material of pure dust mite allergen and its extract. The preparation method can prepare a raw material of pure dust mite allergen and its extract. It can not only achieve efficient proliferation of dust mites, but also be simple to operate and low in cost. The density of the cultured dust mites is high, and the purity is as high as 95% to 99%. It can be cultured on a large scale with high yield to obtain sterile dust mite cultures. The prepared extract has great clinical application value in the diagnosis and treatment of dust mite allergic diseases.
[0007] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention
[0008] In order to solve the problems raised in the above background technology, the present invention provides a method for preparing a raw material of pure dust mite allergen and an extract thereof.
[0009] The present invention provides a method for preparing a pure dust mite allergen raw material and an extract thereof using the following technical scheme:
[0010] A method for preparing a raw material of pure dust mite allergen and an extract thereof, comprising the following steps:
[0011] Step 1: Mite collection: During the peak breeding period of dust mites, which is May-June and September-October every year, collect leftover dust from the ground of grain stores and flour mills and from empty rice and flour bags;
[0012] Step 2, separation and identification: Pass the dust through a No. 2 sieve to remove impurities; then pass through a No. 4 sieve to remove fine dust particles. The middle layer is the dust containing mites. Pour the above dust containing mites into a culture dish, smooth the surface, place it in a cool and humid place away from light, and observe it regularly. When the activity of mites destroys the smooth surface of the dust and small protrusions appear, pick up a few protrusions to make slices for morphological identification, and select active dust mites under a microscope for mite species cultivation;
[0013] Step 3: Mite culture: Inoculate the selected active dust mites into a sterile cell culture bottle filled with dust mite culture medium under sterile conditions and tighten the bottle cap. Cultivate at 24-27°C and 60-80% relative humidity. Monitor the purity, contamination of other mites, and fungal contamination of dust mites under a dissecting microscope at least once a week. After two generations of purification and culture, select the dust mite species with high activity and good growth conditions;
[0014] Step 4: Inoculation: Under sterile conditions, take a dust mite species and culture medium at an inoculation ratio of about 1:25 by weight and inoculate it into a sterile cell culture bottle filled with dust mite culture medium, and tighten the bottle cap.
[0015] Step 5, Cultivation: The cultivation temperature is 24-27°C, and the relative humidity is 60-80%. The temperature and relative humidity are recorded every day. The purity, contamination of other mites, and fungal contamination of dust mites are monitored under a dissecting microscope at least once a week. The cultivation time is 2-3 months. During the cultivation process, the culture bottles contaminated by fungi and other mites are promptly picked out;
[0016] Step 6: Harvest: When the insect density reaches about 30-50 / mg of culture, it can be harvested. Seal the cell culture bottle with cultured dust mites and put it in the refrigerator at -18℃ for more than 24 hours;
[0017] Step 7: Purification: The dust mite culture is separated and purified layer by layer according to the different physical properties and specific gravity of the mites and the feed. This operation is repeated 4 to 5 times, and the dust mite content under microscopic examination reaches 95% to 99%;
[0018] Step 8: Degreasing: The collected raw materials of dust mites are repeatedly washed with a degreasing solvent for degreasing treatment;
[0019] Step 9, drying: evenly spread the defatted dust mites, and dry them at room temperature, in a vacuum or in a fluidized bed to obtain the dust mite allergen raw material;
[0020] Step 10: Storage: Freeze and store the raw materials of dust mite allergens;
[0021] Step 11, preparation of dust mite allergen extract: add pH7.9-8.5 buffered saline extract to the dust mite allergen raw material at a weight volume ratio of 1:50-1:10, and shake while adding to disperse the dust mite allergen raw material in the buffered saline extract to ensure that there is no agglomeration in the mixed solution; extract at 2-8°C for 48-72 hours; centrifuge the buffered saline extract with a centrifuge; collect the supernatant, and perform multiple filtration on the supernatant in sequence; then perform membrane sterilization filtration in a Class A sterile clean environment, and finally package and store.
[0022] By adopting the above technical solution, pure dust mite allergen raw materials and extracts thereof can be prepared.
[0023] Preferably, in step three and step four, the culture medium components are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% complex vitamins, and 0.2% yeast powder, and the culture medium is sterilized by cobalt 60 radiation at a dose of 10KGy-20KGy.
[0024] Preferably, in step eight, the process of repeatedly immersing the dust mite allergen raw material in a degreasing solvent for degreasing treatment includes: adding the degreasing solvent to the allergen raw material, stirring or shaking for degreasing for 1 to 3 hours, then standing for stratification, pouring out the upper layer liquid, adding new degreasing solvent, and repeating the degreasing until the upper layer liquid is clear, the weight volume ratio of the allergen raw material to the degreasing solvent is 1:5 to 1:1, and the degreasing solvent is at least one of acetone, petroleum ether, ethanol, methanol or cyclohexane.
[0025] Preferably, in step ten, the dust mite allergen raw material is frozen and stored at -40°C.
[0026] Preferably, in step eleven, the buffered saline extract is a bicarbonate extract or a hydrogen phosphate extract.
[0027] Preferably, in step eleven, the centrifugal speed of the centrifuge is 6000-12000 rpm, the centrifugal temperature is 2-8° C., and the centrifugal time is set to 10-20 minutes.
[0028] Preferably, in step eleven, the supernatant is subjected to multiple filtrations in sequence, the supernatant is first filtered through filter paper, and then filtered through 0.65 μm, 0.45 μm and 0.22 μm filter membranes in sequence, and the filtration is positive pressure filtration.
[0029] Preferably, in step eleven, in a Class A sterile clean environment, the membrane sterilization filtration is performed by sterilization filtration through a 0.22 μm filter membrane.
[0030] In summary, the present invention includes the following beneficial technical effects:
[0031] The method for preparing a raw material of pure dust mite allergen and its extract of the present invention can prepare a raw material of pure dust mite allergen and its extract. It can not only realize the efficient proliferation of dust mites, but also be simple to operate and low in cost. The density of the cultured dust mites is large, the purity is as high as 95% to 99%, and sterile dust mite cultures can be obtained by large-scale high-yield cultivation. The prepared extract has great clinical application value in the diagnosis and treatment of dust mite allergic diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 The present invention is a flow chart of a method for preparing a pure dust mite allergen raw material and an extract thereof in an embodiment of the present invention.
[0033] Figure 2 This is a picture of the live mount of cultured mites of the present invention.
[0034] Figure 3 The purity test result of the PCR of the cultured mites of the present invention (1: the gene product of the mites in the bottle by PCR with Der f 1 specific primers; 2: the result of PCR with Der f 2 specific primers; M: DNA marker)
[0035] Figure 4 It is the gene sequence of the PCR product after sequencing.
[0036] Figure 5 It is the gene sequence peak diagram after sequencing of the PCR product of the present invention.
[0037] Figure 6 This is the result of Blastx of the PCR product of the present invention. DETAILED DESCRIPTION
[0038] The following is combined with Figure 1 The present invention is described in further detail.
[0039] The embodiment of the present invention discloses a method for preparing a raw material of pure dust mite allergen and its extract. Figure 1 A method for preparing a raw material of pure dust mite allergen and its extract comprises the following steps:
[0040] Step 1: Mite collection: During the peak breeding period of dust mites, which is May-June and September-October every year, collect leftover dust from the ground of grain stores and flour mills and from empty rice and flour bags;
[0041] Step 2, separation and identification: Pass the dust through a No. 2 sieve to remove impurities; then pass through a No. 4 sieve to remove fine dust particles. The middle layer is the dust containing mites. Pour the above dust containing mites into a culture dish, smooth the surface, place it in a cool and humid place away from light, and observe it regularly. When the activity of mites destroys the smooth surface of the dust and small protrusions appear, pick up a few protrusions to make slices for morphological identification, and select active dust mites under a microscope for mite species cultivation;
[0042] Step 3: Mite culture: Inoculate the selected active dust mites into a sterile cell culture bottle filled with dust mite culture medium under sterile conditions and tighten the bottle cap. Cultivate at 24-27°C and 60-80% relative humidity. Monitor the purity, contamination of other mites, and fungal contamination of dust mites under a dissecting microscope at least once a week. After two generations of purification and culture, select the dust mite species with high activity and good growth conditions;
[0043] Step 4: Inoculation: Under sterile conditions, take a dust mite species and culture medium at an inoculation ratio of about 1:25 by weight and inoculate it into a sterile cell culture bottle filled with dust mite culture medium, and tighten the bottle cap.
[0044] Step 5, Cultivation: The cultivation temperature is 24-27°C, and the relative humidity is 60-80%. The temperature and relative humidity are recorded every day. The purity, contamination of other mites, and fungal contamination of dust mites are monitored under a dissecting microscope at least once a week. The cultivation time is 2-3 months. During the cultivation process, the culture bottles contaminated by fungi and other mites are promptly picked out;
[0045] Step 6: Harvest: When the insect density reaches about 30-50 / mg of culture, it can be harvested. Seal the cell culture bottle with cultured dust mites and put it in the refrigerator at -18℃ for more than 24 hours;
[0046] Step 7: Purification: The dust mite culture is separated and purified layer by layer according to the different physical properties and specific gravity of the mites and the feed. This operation is repeated 4 to 5 times, and the dust mite content under microscopic examination reaches 95% to 99%;
[0047] Step 8: Degreasing: The collected raw materials of dust mites are repeatedly washed with a degreasing solvent for degreasing treatment;
[0048] Step 9, drying: evenly spread the defatted dust mites, and dry them at room temperature, in a vacuum or in a fluidized bed to obtain the dust mite allergen raw material;
[0049] Step 10: Storage: Freeze and store the raw materials of dust mite allergens;
[0050] Step 11, preparation of dust mite allergen extract: add pH7.9-8.5 buffered saline extract to the dust mite allergen raw material at a weight volume ratio of 1:50-1:10, and shake while adding to disperse the dust mite allergen raw material in the buffered saline extract to ensure that there is no agglomeration in the mixed solution; extract at 2-8°C for 48-72 hours; centrifuge the buffered saline extract with a centrifuge; collect the supernatant, and perform multiple filtration on the supernatant in sequence; then perform membrane sterilization filtration in a Class A sterile clean environment, and finally package and store.
[0051] Specifically, in step three and step four, the culture medium components are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% complex vitamins, and 0.2% yeast powder. The culture medium is sterilized by cobalt 60 radiation at a dose of 10KGy-20KGy.
[0052] Specifically, in step eight, the process of repeatedly immersing the dust mite allergen raw material in a degreasing solvent for degreasing treatment includes: adding the degreasing solvent to the allergen raw material, stirring or shaking for degreasing for 1 to 3 hours, then standing for stratification, pouring out the upper layer liquid, adding new degreasing solvent, and repeating the degreasing until the upper layer liquid is clear, the weight volume ratio of the allergen raw material to the degreasing solvent is 1:5 to 1:1, and the degreasing solvent is at least one of acetone, petroleum ether, ethanol, methanol or cyclohexane.
[0053] Specifically, in step ten, the dust mite allergen raw material is frozen and stored at -40°C.
[0054] Specifically, in step eleven, the buffered saline extract is a bicarbonate extract or a hydrogen phosphate extract.
[0055] Specifically, in step eleven, the centrifugal speed of the centrifuge is 6000-12000 rpm, the centrifugal temperature is 2-8° C., and the centrifugal time is set to 10-20 minutes.
[0056] Specifically, in step eleven, the supernatant is subjected to multiple filtrations in sequence. The supernatant is first filtered through filter paper, and then filtered through 0.65 μm, 0.45 μm and 0.22 μm filter membranes in sequence, and the filtration is positive pressure filtration.
[0057] Specifically, in step eleven, in a Class A sterile clean environment, membrane sterilization filtration is performed by using a 0.22 μm filter membrane for sterilization filtration.
[0058] Example 1. A method for preparing a raw material of pure dust mite allergen
[0059] Step 1: Mite collection: During the peak breeding period of dust mites in May and June, collect leftover dust from empty flour bags in flour mills;
[0060] Step 2, separation and identification: Pass the dust through a No. 2 sieve to remove impurities; then pass through a No. 4 sieve to remove fine dust particles. The middle layer is the dust containing mites. Pour the above dust containing mites into a culture dish, smooth the surface, place it in a cool and humid place away from light, and observe it regularly. When the activity of mites destroys the smooth surface of the dust and small protrusions appear, pick up a few protrusions to make slices for morphological identification, and select active dust mites under a microscope for mite species cultivation;
[0061] Step three, culture of mite species: inoculate the selected active dust mites into a sterile cell culture bottle containing dust mite culture medium under sterile conditions, and tighten the bottle cap. The components of the culture medium are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% multivitamins, and 0.2% yeast powder. The culture medium has been sterilized by cobalt 60 radiation. Culture at 25°C and relative humidity of 60-80%, and monitor the purity, miscellaneous mite contamination, and fungal contamination of dust mites under a dissecting microscope at least once a week. After two generations of purification and culture, select the dust mite species with high activity and good growth condition;
[0062] Step 4: Inoculation: Under sterile conditions, take the dust mite species and the culture medium at a weight ratio of about 1:25 and inoculate it into a sterile cell culture bottle containing the dust mite culture medium, and tighten the bottle cap. The culture medium components are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% multivitamins, and 0.2% yeast powder. The culture medium is sterilized by cobalt 60 radiation at a dose of 10KGy-20KGy.
[0063] Step 5, Cultivation: Cultivation temperature is 25°C, relative humidity is 60-80%, record the temperature and relative humidity every day, and monitor the purity, contamination of dust mites, and fungal contamination under a dissecting microscope at least once a week. Cultivation time is 3 months. During the cultivation process, timely pick out the culture bottles contaminated by fungi and mites;
[0064] Step 6: Harvest: When the insect density reaches about 30-50 / mg of culture, it can be harvested. Seal the cell culture bottle with cultured dust mites and put it in the refrigerator at -18℃ for more than 24 hours;
[0065] Step 7: Purification: The dust mite culture is separated and purified layer by layer according to the different physical properties and specific gravity of the mites and the feed. This operation is repeated 4 to 5 times, and the dust mite content under microscopic examination reaches 95% to 99%;
[0066] Step 8: Degreasing: Add acetone to the allergen raw material at a mass volume ratio of 1:3, and stir to degrease for 2 hours, then let it stand for stratification, pour out the upper layer, add new acetone, and repeat the degreasing until the upper layer is clear;
[0067] Step 9, drying: evenly spread the defatted dust mites and dry them at room temperature to obtain the dust mite allergen raw material;
[0068] Step 10: Storage: freeze the raw materials of dust mite allergens at -40°C;
[0069] After 3 months of cultivation, an appropriate amount of dust mite culture was weighed and placed on a glass slide, and a sealing agent was added. The mite species density was calculated by counting under an optical microscope. The mite species density was about 46 / mg of culture. At the same time, the whole culture was collected and tested according to the sterility test method 1101 of the third general rule of the "Chinese Pharmacopoeia" (2020 edition). After the culture was completed, the sterility test result was sterile growth.
[0070] Identification of the morphology of living mites: Pick the living mites in the culture bottle and observe the morphology of the living mites in the bottle under a microscope. The body is relatively round and full, about 0.3 to 0.4 mm long, the dorsal hook is not obvious, with front and back dorsal plates, the back dorsal plate is wide and short, the end of the foot is sucker-shaped, foot I is the thickest, and foot III is longer and thicker than foot IV, which is consistent with the morphological characteristics of dust mite, and it is dust mite. The picture of the live mite is shown in Figure 2 .
[0071] Mite purity test: Extract the genomic DNA of the insects in the culture bottle, sieve the insects in the bottle through a 200-mesh sieve, pick about 500 insects for liquid nitrogen grinding, and extract the mite genomic DNA using the kit. Use the genomic DNA as a template, add Der f 1 and Derp 1 specific primers and perform PCR. The results show that the first band of the extracted genomic DNA shows the gene product of PCR with Der f1 specific primers, and the second band does not show the gene product of PCR with Der f2 specific primers. See the figure for the mite PCR purity test results. Figure 3 .
[0072] Mite gene sequence detection: The gene sequence of the test sample of the mite in the bottle after PCR with Der f1 specific primers is 429bp long; after Blastx, it can be observed that it is similar to the protein sequence of Dermatophagoides farinae group I, and the gene of the test sample is dust Der f1. The gene sequence of the mite PCR product after sequencing is shown in Figure 4 The gene sequence peak diagram of the mite PCR product after sequencing is shown in Figure 5 .
[0073] The total protein content of the extract was detected by Bradford protein concentration assay kit and was 14.72 mg / g.
[0074] The indoorbiotechnology EILSA detection kit (EPC-DP1-1, EPC-DF1-1) was used to detect the content and activity of the main allergens Derf1 and Derp1. The test results showed that the contents of the main allergens Der f1 and Derp1 were both high, with the Derf1 content being 47.23μg / mg and the Derp1 content being 10.10μg / mg.
[0075] Example 2. A method for preparing a pure dust mite allergen extract
[0076] The pH 8.2 bicarbonate saline extract is added to the dust mite allergen raw material at a weight-to-volume ratio of 1:50, and the mixture is shaken while being added to disperse the dust mite allergen raw material in the buffered saline extract to ensure that there is no agglomeration in the mixed solution; the mixture is extracted at 2-8°C for 72 hours; the buffered saline extract is centrifuged in a centrifuge, the centrifugal speed of the centrifuge is 8000 rpm, the centrifugal temperature is 2-8°C, and the centrifugal time is set to 15 minutes; the supernatant is collected, and the supernatant is filtered multiple times in sequence, the supernatant is first filtered through filter paper, and then filtered through 0.65μm, 0.45μm and 0.22μm filter membranes in sequence, and the filtration is positive pressure; then, in a Class A sterile clean environment, the supernatant is sterilized and filtered through a 0.22μm filter membrane, and finally packaged and stored.
[0077] The total protein content of the dust mite allergen extract prepared was detected by Bradford protein concentration determination kit and was 0.11 mg / ml, wherein the total protein content standard was 0.05-0.12 mg / ml.
[0078] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A method for preparing a raw material of pure dust mite allergen and its extract, characterized in that: The following steps are involved: Step 1: Mite collection: During the peak breeding period of dust mites, which is May-June and September-October every year, collect leftover dust from the ground of grain stores and flour mills and from empty rice and flour bags; Step 2, separation and identification: Pass the dust through a No. 2 sieve to remove impurities; then pass through a No. 4 sieve to remove fine dust particles. The middle layer is the dust containing mites. Pour the above dust containing mites into a culture dish, smooth the surface, place it in a cool and humid place away from light, and observe it regularly. When the activity of mites destroys the smooth surface of the dust and small protrusions appear, pick up a few protrusions to make slices for morphological identification, and select active dust mites under a microscope for mite species cultivation; Step 3: Mite culture: Inoculate the selected active dust mites into a sterile cell culture bottle filled with dust mite culture medium under sterile conditions and tighten the bottle cap. Cultivate at 24-27°C and 60-80% relative humidity. Monitor the purity, contamination of other mites, and fungal contamination of dust mites under a dissecting microscope at least once a week. After two generations of purification and culture, select the dust mite species with high activity and good growth conditions; Step 4: Inoculation: Under sterile conditions, take a dust mite species and culture medium at an inoculation ratio of about 1:25 by weight and inoculate it into a sterile cell culture bottle filled with dust mite culture medium, and tighten the bottle cap. Step 5, Cultivation: The cultivation temperature is 24-27°C, and the relative humidity is 60-80%. The temperature and relative humidity are recorded every day. The purity, contamination of other mites, and fungal contamination of dust mites are monitored under a dissecting microscope at least once a week. The cultivation time is 2-3 months. During the cultivation process, the culture bottles contaminated by fungi and other mites are promptly picked out; Step 6: Harvest: When the insect density reaches about 30-50 / mg of culture, it can be harvested. Seal the cell culture bottle with cultured dust mites and put it in the refrigerator at -18℃ for more than 24 hours; Step 7: Purification: The dust mite culture is separated and purified layer by layer according to the different physical properties and specific gravity of the mites and the feed. This operation is repeated 4 to 5 times, and the dust mite content under microscopic examination reaches 95% to 99%; Step 8: Degreasing: The collected raw materials of dust mites are repeatedly washed with a degreasing solvent for degreasing treatment; Step 9, drying: evenly spread the defatted dust mites, and dry them at room temperature, in a vacuum or in a fluidized bed to obtain the dust mite allergen raw material; Step 10: Storage: Freeze and store the raw materials of dust mite allergens; Step 11, preparation of dust mite allergen extract: add pH7.9-8.5 buffered saline extract to the dust mite allergen raw material at a weight volume ratio of 1:50-1:10, and shake while adding to disperse the dust mite allergen raw material in the buffered saline extract to ensure that there is no agglomeration in the mixed solution; extract at 2-8°C for 48-72 hours; centrifuge the buffered saline extract with a centrifuge; collect the supernatant, and perform multiple filtration on the supernatant in sequence; then perform membrane sterilization filtration in a Class A sterile clean environment, and finally package and store.
2. The method for preparing a pure dust mite allergen raw material and an extract thereof according to claim 1, characterized in that: In step three and step four, the culture medium components are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% complex vitamins, and 0.2% yeast powder. The culture medium is sterilized by cobalt 60 radiation at a dose of 10KGy-20KGy.
3. The method for preparing a pure dust mite allergen raw material and an extract thereof according to claim 1, characterized in that: In step eight, the process of repeatedly immersing the dust mite allergen raw material in a degreasing solvent for degreasing treatment includes: adding the degreasing solvent to the allergen raw material, stirring or shaking for degreasing for 1 to 3 hours, then standing for stratification, pouring out the upper layer liquid, adding new degreasing solvent, and repeating degreasing until the upper layer liquid is clear, the weight volume ratio of the allergen raw material to the degreasing solvent is 1:5 to 1:1, and the degreasing solvent is at least one of acetone, petroleum ether, ethanol, methanol or cyclohexane.
4. The method for preparing a pure dust mite allergen raw material and an extract thereof according to claim 1, characterized in that: In step ten, the dust mite allergen raw material is frozen and stored at -40°C.
5. The method for preparing a pure dust mite allergen raw material and an extract thereof according to claim 1, characterized in that: In step 11, the buffered saline extract is a bicarbonate extract or a hydrogen phosphate extract.
6. The method for preparing a pure dust mite allergen raw material and an extract thereof according to claim 1, characterized in that: In step 11, the centrifugal speed of the centrifuge is 6000-12000 rpm, the centrifugal temperature is 2-8° C., and the centrifugal time is set to 10-20 minutes.
7. The method for preparing a pure dust mite allergen raw material and an extract thereof according to claim 1, characterized in that: In step eleven, the supernatant is subjected to multiple filtration processes in sequence, including filtering the supernatant through filter paper first, and then filtering through 0.65 μm, 0.45 μm and 0.22 μm filter membranes in sequence, and the filtration is positive pressure filtration.
8. The method for preparing a pure dust mite allergen raw material and an extract thereof according to claim 1, characterized in that: In step 11, in a Class A sterile clean environment, membrane sterilization filtration is performed by using a 0.22 μm filter membrane.