Preparation method of pure mite body allergen raw material of sterile tyrophagus putrescens
Through the third-level seed batch culture and standardized purification steps, high-purity sterile rotten mites allergen raw materials were prepared, solving the problems of low purity and insufficient yield in the prior art, and achieving efficient preparation for allergen preparations and scientific research.
Patent Information
- Application Number
- CN202411680569.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art is difficult to efficiently prepare high-purity sterile sterile pure mites allergenic raw materials, and it is impossible to achieve rapid reproduction and culture at a large scale and high yield.
The tertiary seed batch culture method was used to purify and cultivate the sterile mites under sterile conditions. Through standardized batch inoculation and purification steps, the purity of 95% to 99%, and sterilization and degreasing treatment were carried out to obtain high-purity sterile mites allergenic raw materials.
The preparation of high-purity sterile carrot mites is realized, which can be used for the production of allergen preparations, scientific research and specific IgE in vitro detection, solving the problems of low purity and insufficient yield in the prior art.
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Figure CN119504917A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biology, and more specifically to a method for preparing a sterile pure mite allergen raw material of Tyrophagous putrescentiae. Background Art
[0002] Tyrophagus putrescentiae belongs to Arachnida, Acari, Astigmata, Acaridae, and Tyrophagus. It is a type of storage mite that breeds in large quantities in stored grains such as rice, barley, wheat, flour, bran, rice bran, and soybeans, and foods with high fat and protein content such as egg powder, ham, jerky, cheese, melon seeds, almonds, and peanuts, and uses them as food. It can be spread by wind, mice, insects, and contact with people's clothes, shoes, and inspection tools.
[0003] Tyrophagous mites are distributed in all provinces (regions) of China. They are the most harmful food storage mites in China and one of the main sources of indoor allergens, which can cause dermatitis and respiratory allergies in humans. They are another important allergenic mite species after house dust mites and dust mites.
[0004] At present, the only way to stop the progression of allergic diseases is desensitization therapy. Desensitization therapy is to introduce allergen preparations into the body through repeated subcutaneous injections to induce immune tolerance, so that allergic individuals will no longer experience symptoms or their symptoms will be alleviated after they are exposed to the allergen again. Allergen immunotherapy is currently recognized by the World Health Organization (WHO) and the World Allergy Organization (WAO) as the only causal treatment method for allergic diseases. It can change the natural course of allergic diseases and prevent allergic rhinitis from developing into asthma. It has a dual role of prevention and treatment. Due to its long-term efficacy and the effect of reducing complications, allergen immunotherapy can significantly improve social production efficiency and save health economic expenditures. At present, the preparations that can be used for desensitization treatment in clinical practice in my country are very limited, far from meeting clinical needs.
[0005] The efficacy and safety of allergen immunotherapy rely on accurate allergen diagnosis and high-quality desensitization preparations, and diagnosis and allergen desensitization preparations are inseparable from high-yield and high-quality allergen raw materials. At present, the purity of the putrescent tyrophagous mite culture obtained by the existing mite culture methods in China is low, and large-scale high-yield rapid propagation and cultivation of sterile putrescent tyrophagous mites has not yet been achieved. Non-putrescent tyrophagous mite allergen preparations can be used for specific immunotherapy.
[0006] Based on the above problems, we provide a method for preparing a sterile putrescent tyrophagous mite pure mite allergen raw material. The preparation method can prepare a sterile putrescent tyrophagous mite pure mite allergen raw material. The method is simple to operate and low in cost for culturing putrescent tyrophagous mites, and the putrescent mites grow and reproduce quickly and have a high insect density. The method uses a standard three-level seed batch culture to purify putrescent tyrophagous mite species, and the purity of the raw material is as high as 95% to 99%. Sterile putrescent tyrophagous mite cultures can be obtained by large-scale, high-yield, and high-standard cultivation. The prepared sterile putrescent tyrophagous mite pure mite allergen raw material can be used for the production of allergen preparations, scientific research, and specific IgE in vitro detection.
[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 a person of ordinary skill in the art. 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 sterile pure mite body allergen raw material of Tyrophagus putrescentiae.
[0009] The present invention provides a method for preparing a sterile pure mite allergen raw material of Tyrophagous putrefaciens by adopting the following technical scheme:
[0010] A method for preparing a sterile pure mite allergen raw material of Tyrophagus putrescentiae, comprising the following steps:
[0011] Step 1: Initial mite species collection: Collect storage samples from long-stored food such as flour, rice, paddy, corn, feed, and dried fruits; sieve the collected storage samples and spread them flat in a culture dish, smooth the surface, place them in a dark, cool, and humid place, and observe them regularly. When small protrusions are found, pick up a few of the protrusions to make slices for morphological identification.
[0012] Step 2: Purification of mite species: Under sterile conditions, use a dissecting microscope and an inoculation needle to select male and female putrescent tyrophagous mites with high activity and good growth conditions, and inoculate them into a sterile cell culture bottle containing putrescent tyrophagous mite culture medium. Follow this method to purify and culture for two generations in a constant temperature incubator at 25°C and 75% relative humidity, and ensure that there is no contamination from other mites and fungi before it can be used as the initial population.
[0013] Step 3, original seed batch culture: Under sterile conditions, take the purified Tyrophagous mite species and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mite culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, promptly pick out the culture bottles contaminated by fungi and mites. After 2 to 3 months of culture, the mite density reaches about 10,000 per kilogram, which can be used as the original seed batch after morphological and biological identification.
[0014] Step 4, main seed batch culture: Under sterile conditions, take the original seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time. After 2 to 3 months of culture, the mite density reaches about 100,000 per kilogram, which can be used as the main seed batch after morphological and biological identification.
[0015] Step 5, working seed batch culture: Under sterile conditions, take the main seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, promptly pick out the culture bottles contaminated by fungi and mites. After 2 to 3 months of culture, the mite density reaches about 1 million per kilogram, which can be used as a working seed batch after morphological and biological identification.
[0016] Step 6, batch inoculation culture: Under sterile conditions, take the working seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25 and inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Cultivate in a constant temperature incubator at 25°C and relative humidity of 75%, record the temperature and relative humidity every day, and monitor the purity, mite contamination, and fungal contamination of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time, and culture for 2 to 3 months. When the insect body density reaches about 30 to 50 / mg of culture, it can be harvested.
[0017] Step 7: Harvest: Seal the cell culture bottle in which the putrescentia tyrophagous mites have been cultured and place it in a refrigerator, freeze it at -18°C and keep it for more than 24 hours;
[0018] Step 8: Purification: The culture of Tyrophagoides putrescentiae 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. The content of Tyrophagoides putrescentiae under microscopic examination reaches 95% to 99%;
[0019] Step 9, sterilization and degreasing: the collected raw materials of Tyrophagous putrescentiae are cleaned and sterilized with anhydrous ethanol, and then the solvent is filtered out with multiple layers of gauze, and then a degreasing solvent is added and repeatedly immersed for degreasing treatment;
[0020] Step 10, drying: evenly spread the defatted tyrophagous mites, and dry them at room temperature, in a vacuum or in a fluidized bed to obtain the tyrophagous mites allergen raw material;
[0021] Step 11: Preservation: freezing the raw material of tyrophagous mite allergen;
[0022] By adopting the above technical solution, sterile tyrophagous mite pure mite allergen raw material can be prepared.
[0023] Preferably, in steps two, three, four, five and six, the culture medium components are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% complex vitamins, 0.2% yeast powder, and the culture medium is sterilized by cobalt 60 radiation at a dose of 10KGy-20KGy.
[0024] Preferably, in step nine, the process of washing and sterilizing the putrescent tyrophagous mite allergen raw material with anhydrous ethanol and then repeatedly immersing it in a degreasing solvent for degreasing treatment includes: adding anhydrous ethanol to the putrescent tyrophagous mite raw material, stirring and immersing it for 30 minutes, filtering it with multiple layers of gauze to remove the solvent, and the weight volume ratio of the allergen raw material to the anhydrous ethanol is 1:3 to 1:1; placing the filtered putrescent tyrophagous mite raw material in a container, adding a degreasing solvent, and stirring or shaking to degrease for 1 to 3 hours, then standing to separate, pouring out the upper layer liquid, adding new degreasing solvent, and repeating the degreasing until the upper layer liquid is clear, and 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 eleven, the sterile tyrophagous mite allergen raw material is frozen and stored at -40°C.
[0026] In summary, the present invention includes the following beneficial technical effects:
[0027] The method for preparing a sterile putrescent tyrophagous mite pure mite allergen raw material of the present invention can prepare a sterile putrescent tyrophagous mite pure mite allergen raw material. The method is simple to operate and low in cost in culturing putrescent tyrophagous mites, and the putrescent tyrophagous mites grow and reproduce quickly and have a high insect density. The method adopts a standard three-level seed batch culture to purify putrescent tyrophagous mite species, and the purity of the raw material is as high as 95% to 99%. Sterile putrescent tyrophagous mite cultures can be obtained by large-scale, high-yield, and high-standard cultivation. The prepared sterile putrescent tyrophagous mite pure mite allergen raw material can be used for the production of allergen preparations, can also be used for scientific research, and can also be used for specific IgE in vitro detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 The present invention is a flow chart of a method for preparing a sterile pure mite allergen raw material of Tyrophagous putrescentiae in an embodiment of the present invention.
[0029] Figure 2 This is a picture of the live mount of cultured mites of the present invention.
[0030] Figure 3 This is the result of Blastx of the PCR product of the present invention. DETAILED DESCRIPTION
[0031] The following is combined with Figure 1 The present invention is described in further detail.
[0032] The embodiment of the present invention discloses a method for preparing a sterile pure mite allergen raw material of Tyrophagus putrescentiae. Figure 1 , a method for preparing a sterile pure mite allergen raw material of Tyrophagus putrescentiae, comprising the following steps:
[0033] Step 1: Initial mite species collection: Collect storage samples from long-stored food such as flour, rice, paddy, corn, feed, and dried fruits; sieve the collected storage samples and spread them flat in a culture dish, smooth the surface, place them in a dark, cool, and humid place, and observe them regularly. When small protrusions are found, pick up a few of the protrusions to make slices for morphological identification.
[0034] Step 2: Purification of mite species: Under sterile conditions, use a dissecting microscope and an inoculation needle to select male and female putrescent tyrophagous mites with high activity and good growth conditions, and inoculate them into a sterile cell culture bottle containing putrescent tyrophagous mite culture medium. Follow this method to purify and culture for two generations in a constant temperature incubator at 25°C and 75% relative humidity, and ensure that there is no contamination from other mites and fungi before it can be used as the initial population.
[0035] Step 3, original seed batch culture: Under sterile conditions, take the purified Tyrophagous mite species and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mite culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, promptly pick out the culture bottles contaminated by fungi and mites. After 2 to 3 months of culture, the mite density reaches about 10,000 per kilogram, which can be used as the original seed batch after morphological and biological identification.
[0036] Step 4, main seed batch culture: Under sterile conditions, take the original seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time. After 2 to 3 months of culture, the mite density reaches about 100,000 per kilogram, which can be used as the main seed batch after morphological and biological identification.
[0037] Step 5, working seed batch culture: Under sterile conditions, take the main seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, promptly pick out the culture bottles contaminated by fungi and mites. After 2 to 3 months of culture, the mite density reaches about 1 million per kilogram, which can be used as a working seed batch after morphological and biological identification.
[0038] Step 6, batch inoculation culture: Under sterile conditions, take the working seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25 and inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Cultivate in a constant temperature incubator at 25°C and relative humidity of 75%, record the temperature and relative humidity every day, and monitor the purity, mite contamination, and fungal contamination of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time, and culture for 2 to 3 months. When the insect body density reaches about 30 to 50 / mg of culture, it can be harvested.
[0039] Step 7: Harvest: Seal the cell culture bottle in which the putrescentia tyrophagous mites have been cultured and place it in a refrigerator, freeze it at -18°C and keep it for more than 24 hours;
[0040] Step 8: Purification: The culture of Tyrophagoides putrescentiae 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. The content of Tyrophagoides putrescentiae under microscopic examination reaches 95% to 99%;
[0041] Step 9, sterilization and degreasing: the collected raw materials of Tyrophagous putrescentiae are cleaned and sterilized with anhydrous ethanol, and then the solvent is filtered out with multiple layers of gauze, and then a degreasing solvent is added and repeatedly immersed for degreasing treatment;
[0042] Step 10, drying: evenly spread the defatted tyrophagous mites, and dry them at room temperature, in a vacuum or in a fluidized bed to obtain the tyrophagous mites allergen raw material;
[0043] Step 11: Preservation: freezing the raw material of tyrophagous mite allergen;
[0044] Specifically, in steps two, three, four, five and six, the culture medium components are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% complex vitamins, 0.2% yeast powder, and the culture medium is sterilized by cobalt 60 radiation at a dose of 10KGy-20KGy.
[0045] Specifically, in step nine, the process of washing and sterilizing the putrescent tyrophagous mite allergen raw material with anhydrous ethanol and then repeatedly immersing it in a degreasing solvent for degreasing treatment includes: adding anhydrous ethanol to the putrescent tyrophagous mite raw material, stirring and immersing it for 30 minutes, filtering it with multiple layers of gauze to remove the solvent, and the weight volume ratio of the allergen raw material to the anhydrous ethanol is 1:3 to 1:1; placing the filtered putrescent tyrophagous mite raw material in a container, adding a degreasing solvent, and stirring or shaking to degrease for 1 to 3 hours, then standing to separate the layers, pouring out the upper layer liquid, adding new degreasing solvent, and repeating the degreasing until the upper layer liquid is clear, and 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.
[0046] Specifically, in step eleven, the raw material of tyrophagous mite allergen is frozen and stored at -40°C.
[0047] Example 1. A method for preparing a sterile pure mite allergen raw material of Tyrophagus putrescentiae
[0048] Step 1: Initial mite species collection: Collect storage samples from long-stored food such as flour, rice, paddy, corn, feed, and dried fruits; sieve the collected storage samples and spread them flat in a culture dish, smooth the surface, place them in a dark, cool, and humid place, and observe them regularly. When small protrusions are found, pick up a few of the protrusions to make slices for morphological identification.
[0049] Step 2: Purification of mite species: Under sterile conditions, use a dissecting microscope and an inoculation needle to select male and female putrescent tyrophagous mites with high activity and good growth conditions, and inoculate them into a sterile cell culture bottle containing putrescent tyrophagous mite culture medium. Follow this method to purify and culture for two generations in a constant temperature incubator at 25°C and 75% relative humidity, and ensure that there is no contamination from other mites and fungi before it can be used as the initial population.
[0050] Step 3, original seed batch culture: Under sterile conditions, take the purified Tyrophagous mite species and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mite culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time. After 3 months of culture, the mite density reaches about 10,000 per kilogram, which can be used as the original seed batch after morphological and biological identification.
[0051] Step 4, main seed batch culture: Under sterile conditions, take the original seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time. After 3 months of culture, the mite density reaches about 100,000 per kilogram, which can be used as the main seed batch after morphological and biological identification.
[0052] Step 5, working seed batch culture: Under sterile conditions, take the main seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, promptly pick out the culture bottles contaminated by fungi and mites. After 3 months of culture, the mite density reaches about 1 million per kilogram, which can be used as a working seed batch after morphological and biological identification.
[0053] Step 6, batch inoculation culture: Under sterile conditions, take the working seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25 and inoculate into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Cultivate in a constant temperature incubator at 25°C and 75% relative humidity, record the temperature and relative humidity every day, and monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time, and harvest when the insect body density reaches about 30 to 50 / mg of culture for 3 months.
[0054] Step 7: Harvest: Seal the cell culture bottle in which the putrescentia tyrophagous mites have been cultured and place it in a refrigerator, freeze it at -18°C and keep it for more than 24 hours;
[0055] Step 8: Purification: The culture of Tyrophagoides putrescentiae 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. The content of Tyrophagoides putrescentiae under microscopic examination reaches 95% to 99%;
[0056] Step 9, sterilization and degreasing: add anhydrous ethanol to the collected Tyrophagoides putrescentiae allergen raw materials at a mass volume ratio of 1:2 to clean and sterilize, stir and soak for 30 minutes, filter with multiple layers of gauze to remove the solvent, then add acetone to the allergen raw materials at a mass volume ratio of 1:3, stir and degrease for 2 hours, then stand and separate, pour out the upper layer liquid, add new acetone, and repeat the degreasing until the upper layer liquid is clear;
[0057] Step 10, drying: evenly spread the defatted tyrophagous mites, and dry them at room temperature to obtain the tyrophagous mites allergen raw material;
[0058] Step 11: Storage: The raw material of tyrophagous mite allergen is frozen and stored at -40°C;
[0059] For insect density check, weigh an appropriate amount of Tyrophagous putrescentia mites culture and place it on a glass slide, add sealing agent, and calculate the mite density by counting under an optical microscope. The original seed batch mite density is about 10 / g culture, the main seed batch mite density is about 100 / g, the working seed batch mite density is about 1000 / g, and the mite raw material culture density is about 40 / mg.
[0060] Sterility test: Collect the whole culture and test it according to the sterility test method in 1101 of Part III of the Chinese Pharmacopoeia (2020 edition). After the culture is completed, the sterility test result is sterile growth.
[0061] Morphological identification of live mites: live mites were selected from the culture bottle, and the mite slide specimens were observed under a high-power microscope. The mite body was oval, about 300-440 μm long, with four pairs of legs, small chelae, and long hairs at the end of the body. Based on the morphology, it was identified as Tyrophagus putrescentiae. Figure 2 .
[0062] Molecular biological identification: Genomic DNA samples of cultured mites were extracted, and the products obtained by PCR amplification were sequenced after detection by 1% agarose gel electrophoresis. The obtained sequences were compared for similarity using BLAST software on the website of the National Center for Biotechnology Information of the United States. Combined with the gene sequence of tyrophagous mites in GenBank, multiple sequence alignment was performed using SeqMan software, and data analysis was performed using sequence analysis software. The results showed that the COⅠ gene sequence of the mite obtained after PCR amplification was 300-400bp. After BLAST comparison analysis of the NCBI database, the measured COⅠ gene sequence of tyrophagous mites was 100% homologous to the COⅠ gene sequence of tyrophagous mites included in GenBank. The result after PCR product Blastx is shown in the figure. Figure 3 .
[0063] Mite purity test: Take a small amount of raw material of putrescent tyrophagous mites to make slices, use the counting method under a microscope to calculate the ratio of mites to feed, and the mite content reaches 98%.
[0064] The total protein content of the extract was detected by Bradford protein concentration assay kit and was 17.60 mg / g.
[0065] 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 sterile pure mite allergen raw material of Tyrophagus putrescentiae, characterized in that: The following steps are involved: Step 1: Initial mite species collection: Collect storage samples from long-stored food such as flour, rice, paddy, corn, feed, and dried fruits; sieve the collected storage samples and spread them flat in a culture dish, smooth the surface, place them in a dark, cool, and humid place, and observe them regularly. When small protrusions are found, pick up a few of the protrusions to make slices for morphological identification. Step 2: Purification of mite species: Under sterile conditions, use a dissecting microscope and an inoculation needle to select male and female putrescent tyrophagous mites with high activity and good growth conditions, and inoculate them into a sterile cell culture bottle containing putrescent tyrophagous mite culture medium. Follow this method to purify and culture for two generations in a constant temperature incubator at 25°C and 75% relative humidity, and ensure that there is no contamination from other mites and fungi before it can be used as the initial population. Step 3, original seed batch culture: Under sterile conditions, take the purified Tyrophagous mite species and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mite culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, promptly pick out the culture bottles contaminated by fungi and mites. After 2 to 3 months of culture, the mite density reaches about 10,000 per kilogram, which can be used as the original seed batch after morphological and biological identification. Step 4, main seed batch culture: Under sterile conditions, take the original seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time. After 2 to 3 months of culture, the mite density reaches about 100,000 per kilogram, which can be used as the main seed batch after morphological and biological identification. Step 5, working seed batch culture: Under sterile conditions, take the main seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25, inoculate into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Culture in a constant temperature incubator at 25°C and 75% relative humidity. Record the temperature and relative humidity every day. Monitor the purity, contamination of mites and fungi of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, promptly pick out the culture bottles contaminated by fungi and mites. After 2 to 3 months of culture, the mite density reaches about 1 million per kilogram, which can be used as a working seed batch after morphological and biological identification. Step 6, batch inoculation culture: Under sterile conditions, take the working seed batch of Tyrophagous mites and the culture medium at a weight ratio of about 1:25 and inoculate it into a sterile cell culture bottle filled with Tyrophagous mites culture medium, and tighten the bottle cap. Cultivate in a constant temperature incubator at 25°C and relative humidity of 75%, record the temperature and relative humidity every day, and monitor the purity, mite contamination, and fungal contamination of Tyrophagous mites under a dissecting microscope at least once a week. During the culture process, pick out the culture bottles contaminated by fungi and mites in time, and culture for 2 to 3 months. When the insect body density reaches about 30 to 50 / mg of culture, it can be harvested. Step 7: Harvest: Seal the cell culture bottle in which the putrescentia tyrophagous mites have been cultured and place it in a refrigerator, freeze it at -18°C and keep it for more than 24 hours; Step 8: Purification: The culture of Tyrophagoides putrescentiae 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. The content of Tyrophagoides putrescentiae under microscopic examination reaches 95% to 99%; Step 9, sterilization and degreasing: the collected raw materials of Tyrophagous putrescentiae are cleaned and sterilized with anhydrous ethanol, and then the solvent is filtered out with multiple layers of gauze, and then a degreasing solvent is added and repeatedly immersed for degreasing treatment; Step 10, drying: evenly spread the defatted tyrophagous mites, and dry them at room temperature, in a vacuum or in a fluidized bed to obtain the tyrophagous mites allergen raw material; Step 11: Storage: The raw materials of tyrophagous mite allergen are frozen for storage.
2. The method for preparing a sterile pure mite allergen raw material of Tyrophagus putrescentiae according to claim 1, characterized in that: In steps two, three, four, five and six, the culture medium components are 31% whole wheat flour, 40% corn flour, 23.7% protein powder, 4.8% mineral mixture, 0.3% complex vitamins, 0.2% yeast powder, and the culture medium is sterilized by cobalt 60 radiation at a dose of 10KGy-20KGy.
3. The method for preparing a sterile pure mite allergen raw material of Tyrophagus putrescentiae according to claim 1, characterized in that: In step nine, the process of washing and sterilizing the putrescent tyrophagous mite allergen raw material with anhydrous ethanol and then repeatedly immersing it in a degreasing solvent for degreasing treatment includes: adding anhydrous ethanol to the putrescent tyrophagous mite raw material, stirring and immersing it for 30 minutes, filtering it with multiple layers of gauze to remove the solvent, and the weight volume ratio of the allergen raw material to the anhydrous ethanol is 1:3 to 1:1; placing the filtered putrescent tyrophagous mite raw material in a container, adding a degreasing solvent, and stirring or shaking to degrease for 1 to 3 hours, then standing to separate, pouring out the upper layer liquid, adding new degreasing solvent, and repeating the degreasing until the upper layer liquid is clear, and 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 sterile pure mite allergen raw material of Tyrophagus putrescentiae according to claim 1, characterized in that: In step 11, the sterile tyrophagous mite allergen raw material is frozen and stored at -40°C.