CN114853885A - Preparation method of specific biological extraction protein-gamma protein - Google Patents
Preparation method of specific biological extraction protein-gamma protein Download PDFInfo
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- CN114853885A CN114853885A CN202210618446.5A CN202210618446A CN114853885A CN 114853885 A CN114853885 A CN 114853885A CN 202210618446 A CN202210618446 A CN 202210618446A CN 114853885 A CN114853885 A CN 114853885A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 114
- 101710179016 Protein gamma Proteins 0.000 title claims abstract description 58
- 238000000605 extraction Methods 0.000 title claims abstract description 33
- 239000000427 antigen Substances 0.000 claims abstract description 138
- 102000036639 antigens Human genes 0.000 claims abstract description 138
- 108091007433 antigens Proteins 0.000 claims abstract description 138
- 229960005486 vaccine Drugs 0.000 claims abstract description 92
- 238000002649 immunization Methods 0.000 claims abstract description 78
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- 239000006228 supernatant Substances 0.000 claims abstract description 32
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 21
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- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 15
- 238000005520 cutting process Methods 0.000 claims abstract description 15
- 239000012528 membrane Substances 0.000 claims abstract description 15
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- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims description 15
- 229940114124 ferulic acid Drugs 0.000 claims description 15
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims description 15
- 235000001785 ferulic acid Nutrition 0.000 claims description 15
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims description 15
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- WHMDKBIGKVEYHS-IYEMJOQQSA-L Zinc gluconate Chemical compound [Zn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O WHMDKBIGKVEYHS-IYEMJOQQSA-L 0.000 claims description 14
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 9
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 9
- 108010020346 Polyglutamic Acid Proteins 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 229920002643 polyglutamic acid Polymers 0.000 claims description 8
- 125000005227 alkyl sulfonate group Chemical group 0.000 claims description 7
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/284—Atractylodes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/42—Cucurbitaceae (Cucumber family)
- A61K36/428—Trichosanthes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/75—Rutaceae (Rue family)
- A61K36/752—Citrus, e.g. lime, orange or lemon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/894—Dioscoreaceae (Yam family)
- A61K36/8945—Dioscorea, e.g. yam, Chinese yam or water yam
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
- A61K36/8998—Hordeum (barley)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
- C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
- C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application relates to the technical field of biological medicines, and particularly discloses a preparation method of a specific biological extraction protein-gamma protein, which comprises the steps of pretreating rotavirus antigen by using a treatment solution to obtain a treatment antigen; adding adjuvant into the treated antigen, and fully emulsifying to obtain the immune vaccine; applying the immune vaccine to chicken immunization, and collecting immune eggs; cutting the immune egg into yolk, uniformly stirring and dispersing the yolk, phosphoric acid solution and polyethylene glycol, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, and spray drying to obtain the required specific biological extracted protein-gamma protein. The gamma protein prepared by the method has high purity, good stability, high specificity and sensitivity, can be used for preparing oral specific gamma protein antibodies, has small immune interference, simple preparation method and low preparation cost, and can be widely applied.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to a preparation method of a specific biological extraction protein-gamma protein.
Background
Rotavirus (RV for short) is a double-stranded ribonucleic acid virus, belonging to the reoviridae family. Rotavirus is the first pathogen causing severe diarrhea of children under 5 years old worldwide, severe rotavirus enteritis mainly occurs in infants between 2 months and 3 years old, dehydration, acidosis and electrolyte disturbance are frequently caused, and the health of the children is seriously threatened. The rotavirus infection route is a fecal-oral route, and mainly infects epithelial cells of the small intestine, thereby causing cell damage and causing diarrhea. There are seven rotaviruses, which are numbered A, B, C, D, E, F and G in English letters. Of these A, B, C, three groups were able to infect humans, whereas group A was the most common one, and more than 90% of cases of human rotavirus infection were also caused by group A.
Once rotavirus enteritis happens, no specific medicine treatment exists at present, and the means which is the most effective means for preventing and controlling rotavirus infection is vaccine at present. The oral rotavirus live vaccine can prevent rotavirus infection and reduce severe rotavirus enteritis, but in the related technology, the vaccine protection rate can only reach about 80 percent.
Chicken egg Yolk antibodies (IgY), also known as gamma protein, are polyclonal antibodies produced by avian B lymphocytes and transferred into the Yolk under specific receptor-mediated stimulation. As the relative relationship between the poultry and the human is far and the species line difference is large, the IgY has strong reactivity to the high-conservative protein of the mammal, and a large amount of specific IgY which can be continuously expressed can be obtained in the immune egg yolk by very little antigen stimulation; meanwhile, the IgY has the advantages of good stability, high specificity and sensitivity, small immune interference, low background and the like in detection and diagnosis. IgY is combined with antigen, can effectively eliminate pathogen invading body, neutralizes toxin produced by pathogen, and is an important component of immune environment.
With the continuous and deep research and application of the IgY field, the application prospect of the IgY is wider and wider, and the research proves that the oral specific IgY antibody has the resistance effect on bovine and human rotavirus, but the existing oral specific IgY antibody has limited resistance effect on bovine and human rotavirus, and the effective prevention and treatment on rotavirus enteritis can not be realized.
Disclosure of Invention
In order to realize effective prevention and treatment of rotavirus enteritis, the application provides a preparation method of a specific biological extraction protein-gamma protein.
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme: a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating rotavirus antigen by using a treatment solution to obtain a treatment antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with an adjuvant to prepare an immune vaccine;
s3, obtaining immune eggs: applying the immune vaccine obtained in the step S2 to chicken immunization, and collecting immune eggs;
s4, yolk protein extraction: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol uniformly according to the mass ratio of 1:8-12:2-3, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, and then performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
By adopting the technical scheme, the gamma protein prepared by the method has high purity, good stability, high specificity and sensitivity, small immune interference, simple preparation method and low preparation cost, can be used for preparing oral specific gamma protein products, and can be widely applied.
Preferably, the rotavirus antigen in the step S1 comprises a group a rotavirus antigen, a group B rotavirus antigen and a group C rotavirus antigen; the mass ratio of the group A rotavirus antigen to the group B rotavirus antigen to the group C rotavirus antigen is 89-95:1-5: 1-3.
By adopting the technical scheme, the mass ratio of three groups of different rotavirus antigens is controlled, the immunogen is prepared by selecting the combined rotavirus antigen and the adjuvant, and the finally prepared gamma protein has high specificity and sensitivity, can be used for treating rotavirus enteritis caused by single-type or multi-type rotavirus, and has obvious treatment effect.
Preferably, the treating fluid in step S1 includes the following raw materials in parts by weight: 3-8 parts of sodium secondary alkyl sulfonate, 40-60 parts of 5% hydrogen peroxide solution by mass fraction and 12-18 parts of patchouli oil.
By adopting the technical scheme, the treatment solution can promote the denaturation of virus protein and accelerate the decomposition of virus nucleic acid, so that the rotavirus antigen obtained by treatment has antigenicity and fusion activity and simultaneously reduces the infection activity.
Preferably, the preprocessing in step S1 specifically includes the following operations: firstly, under the condition of 47-51 ℃, the treatment time is 20-40min, then the temperature is raised to 53-57 ℃, the treatment is continued for 10-18min, finally, the temperature is lowered to 17-22 ℃, and the heat preservation is carried out for 10-20min, thus obtaining the treated antigen.
By adopting the technical scheme, the pretreatment temperature and time are controlled, and the mode of heating and cooling is adopted, so that the protein structure of the virus is damaged, the virus loses the infection, pathogenicity and reproductive capacity, and meanwhile, the useful antigen in the virus body is not damaged.
Preferably, the vaccines immunized in step S2 include prime vaccine, secondary vaccine and tertiary vaccine.
Preferably, the preparation of the immune vaccine is specifically performed as follows:
fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; and (3) fully emulsifying the treated antigen obtained in the step S1 with a compound adjuvant according to the mass ratio of 1:1 to obtain the three-immunity vaccine.
By adopting the technical scheme, in each immunization process, the antigen is selected and matched with different adjuvants, so that the side effect of the vaccine can be effectively reduced, the antigen-antibody reaction and the immunogen sustained release capacity can be enhanced, the antigen can be effectively delivered to a target site, and the local immune reaction can be stimulated.
Preferably, the compound adjuvant is prepared by compounding normal saline, lipopolysaccharide, polyglutamic acid and sodium citrate in a mass ratio of 7-11:3-5:1-2: 1-2.
By adopting the technical scheme, the physiological saline, the lipopolysaccharide, the polyglutamic acid and the sodium citrate are compounded to be used as the compounded adjuvant for the three-immunity vaccine, so that the antigen can be effectively transmitted to a target position, the local immune response is stimulated, the biological activity of the antigen is kept, the action time of the antigen is prolonged, and the immune effect is enhanced.
Preferably, the chicken immunization scheme in the step S3 is as follows: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.5-1 mg/chicken; the immune dose of the three-immunization is 0.1-0.3 mg/mouse, 2 weeks after the three-immunization, immune eggs are collected, marked and detected in a laboratory.
By adopting the technical scheme, the antigen is selected and matched with different adjuvants for three times of immunization, the required high-titer immune eggs can be obtained by controlling the immunization dose and the immunization interval time and immunizing for 3 times, the vaccine dosage is small, the immune cycle is short, and the immune effect is obvious.
Preferably, the step S4 further includes a sterilization process before the spray drying, and the specific operations are as follows: sterilizing the supernatant after dialysis and purification at 67-71 deg.C for 12-18min, and sterilizing at 75-78 deg.C for 3-10 min.
By adopting the technical scheme, a two-stage low-temperature sterilization method is adopted, and the sterilization temperature and the sterilization time are controlled, so that microbial cells in the prepared gamma protein can be largely destroyed and die; the effective sterilization is realized, and simultaneously, the nutrient substances and the biological activity of the gamma protein are ensured not to be damaged.
Preferably, the prepared gamma protein can be used for preparing protein products.
Preferably, the protein product comprises the following raw materials in percentage by weight: 0.1-2% of gamma protein, 8-15% of yam powder, 0.1-0.5% of lactulose, 0.3-0.8% of ferulic acid, 1-5% of zinc gluconate, 0.5-1.5% of active extract and the balance of water.
Preferably, the protein product is prepared by the following method: sequentially adding gamma protein, rhizoma Dioscoreae powder and active extract into water under stirring, adding lactulose, ferulic acid and zinc gluconate, mixing, and packaging to obtain the desired protein product.
By adopting the technical scheme, the proportion of the selected raw materials is controlled, the prepared gamma protein is matched with the raw materials to be mixed to prepare the required protein product, the preparation method is simple and suitable for industrial production, the obtained protein product has strong pertinence, high safety and good stability, has obvious treatment effect on rotavirus enteritis caused by single-type or multi-type rotavirus, and can quickly achieve the effects of relieving illness state, shortening course of disease and finally curing after oral administration; the health-care food also has excellent effects of resisting bacteria and viruses, tonifying spleen and qi, conditioning intestines and stomach, enhancing human immunity and the like, and is beneficial to repairing gastrointestinal functions and improving physical quality of patients.
Preferably, the active extract comprises the following raw materials: 1-3 parts of snakegourd fruit extract, 2-7 parts of dried orange peel extract, 5-15 parts of malt extract and 1-3 parts of bighead atractylodes rhizome extract.
By adopting the technical scheme, the snakegourd fruit extract has the effects of clearing heat and removing phlegm, relieving chest stuffiness and eliminating stagnation, moistening dryness and lubricating intestines and the like; the dried orange peel extract can promote humoral immunity and cellular immunity, and has the obvious functions of removing oxygen free radicals, regulating gastrointestinal functions, benefiting gallbladder and the like; the malt extract has the effects of promoting the circulation of qi, strengthening the spleen, invigorating the stomach, promoting digestion, aiding digestion, resisting bacteria and the like; the Atractylodis rhizoma extract has effects of invigorating spleen and replenishing qi, and invigorating spleen and regulating stomach function; the raw materials are mixed to prepare the active extract for adding and preparing the protein product, the active extract can play a good synergistic effect with gamma protein, the resistance effect of the prepared protein product on rotavirus is further enhanced, and the final cure rate is further improved.
In summary, the present application has the following beneficial effects:
the application provides a preparation method of a specific biological extraction protein-gamma protein, the prepared gamma protein has high purity, good stability, high specificity and sensitivity, and an oral specific gamma protein product prepared by matching yam powder, lactulose, ferulic acid, zinc gluconate and an active extract has strong pertinence, high safety and good stability, has obvious treatment effect on rotavirus enteritis caused by single-type or multi-type rotavirus, and can quickly achieve the effects of relieving the state of illness, shortening the course of disease and finally curing after oral administration; the obtained protein product also has excellent effects of resisting bacteria and viruses, tonifying spleen and qi, regulating intestines and stomach, enhancing human immunity and the like, and is beneficial to the repair of gastrointestinal functions and the improvement of physical quality of patients.
Detailed Description
The present application will be described in further detail with reference to examples.
Preparation examples 1-5 provide methods for the preparation of rotavirus antigens.
Preparation example 1
Mixing the group A rotavirus antigen, the group B rotavirus antigen and the group C rotavirus antigen according to the mass ratio of 89:1:1 to obtain the required rotavirus antigen.
Preparation example 2
Mixing the group A rotavirus antigen, the group B rotavirus antigen and the group C rotavirus antigen according to the mass ratio of 89:5:3 to obtain the required rotavirus antigen.
Preparation example 3
Mixing the group A rotavirus antigen, the group B rotavirus antigen and the group C rotavirus antigen according to the mass ratio of 92:3:2 to obtain the required rotavirus antigen.
Preparation example 4
Mixing the group A rotavirus antigen, the group B rotavirus antigen and the group C rotavirus antigen according to the mass ratio of 89:1:3 to obtain the required rotavirus antigen.
Preparation example 5
Mixing the group A rotavirus antigen, the group B rotavirus antigen and the group C rotavirus antigen according to the mass ratio of 95:5:1 to obtain the required rotavirus antigen.
Preparation examples 6 to 10 provide methods for preparing the treatment liquids.
Preparation example 6
3kg of sodium secondary alkyl sulfonate, 40kg of hydrogen peroxide solution with the mass fraction of 5 percent and 12kg of patchouli oil are ground and uniformly dispersed to obtain the required treatment solution.
Preparation example 7
5kg of sodium secondary alkyl sulfonate, 45kg of hydrogen peroxide solution with the mass fraction of 5 percent and 14kg of patchouli oil are ground and uniformly dispersed to obtain the required treatment solution.
Preparation example 8
5kg of sodium secondary alkyl sulfonate, 50kg of hydrogen peroxide solution with the mass fraction of 5 percent and 15kg of patchouli oil are ground and uniformly dispersed to obtain the required treatment solution.
Preparation example 9
Grinding and uniformly dispersing 7kg of sodium secondary alkyl sulfonate, 55kg of hydrogen peroxide solution with the mass fraction of 5% and 16kg of patchouli oil to obtain the required treatment solution.
Preparation example 10
8kg of sodium secondary alkyl sulfonate, 60kg of hydrogen peroxide solution with the mass fraction of 5 percent and 18kg of patchouli oil are ground and uniformly dispersed to obtain the required treatment solution.
Preparation examples 11-15 provide methods for preparing the compounded adjuvants.
Preparation example 11
And grinding and uniformly dispersing the normal saline, the lipopolysaccharide, the polyglutamic acid and the sodium citrate according to the mass ratio of 7:3:1:1 to obtain the required compound adjuvant.
Preparation example 12
And grinding and uniformly dispersing the normal saline, the lipopolysaccharide, the polyglutamic acid and the sodium citrate according to the mass ratio of 8:4:1:2 to obtain the required compound adjuvant.
Preparation example 13
And grinding and uniformly dispersing the normal saline, the lipopolysaccharide, the polyglutamic acid and the sodium citrate according to the mass ratio of 9:4:2:1 to obtain the required compound adjuvant.
Preparation example 14
And grinding and uniformly dispersing the normal saline, the lipopolysaccharide, the polyglutamic acid and the sodium citrate according to the mass ratio of 10:5:1:1 to obtain the required compound adjuvant.
Preparation example 15
Grinding and uniformly dispersing the normal saline, the lipopolysaccharide, the polyglutamic acid and the sodium citrate according to the mass ratio of 11:3:2:1 to obtain the required compound adjuvant.
Preparation examples 16-20 provide methods for preparing active extracts.
Preparation example 16
1kg of snakegourd fruit extract, 2kg of tangerine peel extract, 5kg of malt extract and 1kg of bighead atractylodes rhizome extract are uniformly mixed to obtain the active extract.
Preparation example 17
Mixing fructus Trichosanthis extract 1.5kg, pericarpium Citri Tangerinae extract 3kg, fructus Hordei Germinatus extract 8kg, and Atractylodis rhizoma extract 1.5kg to obtain active extract.
Preparation example 18
Mixing fructus Trichosanthis extract 2kg, pericarpium Citri Tangerinae extract 5kg, fructus Hordei Germinatus extract 10kg, and Atractylodis rhizoma extract 2kg to obtain active extract.
Preparation example 19
Mixing fructus Trichosanthis extract 2.5kg, pericarpium Citri Tangerinae extract 6kg, fructus Hordei Germinatus extract 12kg, and Atractylodis rhizoma extract 2.5kg to obtain active extract.
Preparation example 20
Mixing fructus Trichosanthis extract 3kg, pericarpium Citri Tangerinae extract 7kg, fructus Hordei Germinatus extract 15kg, and Atractylodis rhizoma extract 3kg to obtain active extract.
Examples 1-5 provide a method for the preparation of specific bio-extractable protein-gamma.
Example 1
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 1 by the treatment liquid in preparation example 6, firstly treating at 47 ℃ for 40min, then heating to 53 ℃, continuing to treat for 18min, finally cooling to 17 ℃, and preserving heat for 20min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 11 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immune dose of the primary immunity and the secondary immunity is 0.5 mg/chicken; the immune dose of the three-immunity is 0.1 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk, stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol uniformly according to the mass ratio of 1:8:2, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant through a semipermeable membrane, sterilizing at the temperature of 67 ℃ for 18min, sterilizing at the temperature of 75 ℃ for 10min, and finally performing spray drying to prepare the required specific biological extract protein-gamma protein.
Example 2
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 2 by the treatment solution in preparation example 7, firstly treating at 48 ℃ for 35min, then heating to 54 ℃, continuing to treat for 16min, finally cooling to 18 ℃, and preserving heat for 18min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 12 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a subcutaneous multipoint injection mode, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.6 mg/chicken; the immune dose of the three-immunity is 0.15 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol uniformly according to the mass ratio of 1:9:3, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing at 68 ℃ for 16min, sterilizing at 76 ℃ for 8min, and finally performing spray drying to obtain the required specific biological extract protein-gamma protein.
Example 3
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 3 by the treatment liquid in preparation example 8, firstly treating for 30min at 49 ℃, then heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 13 according to the mass ratio of 1:1 to obtain the three-immune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Example 4
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 4 by the treatment liquid in preparation example 9, firstly treating at 50 ℃ for 35min, then heating to 56 ℃, continuing to treat for 12min, finally cooling to 18 ℃, and preserving heat for 12min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 14 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.25 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol uniformly according to the mass ratio of 1:11:2, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing at the temperature of 70 ℃ for 14min, sterilizing at the temperature of 77 ℃ for 4min, and finally performing spray drying to prepare the required specific biological extract protein-gamma protein.
Example 5
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 5 by the treatment liquid in preparation example 10, firstly treating at 51 ℃ for 20min, then heating to 57 ℃, continuing to treat for 10min, finally cooling to 22 ℃, and preserving heat for 10min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 15 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a subcutaneous multipoint injection mode, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 1 mg/chicken; the immune dose of the three-immunity is 0.3 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into egg yolks, stirring and dispersing the egg yolks, 0.1mol/L phosphoric acid solution and polyethylene glycol uniformly according to the mass ratio of 1:12:3, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant through a semipermeable membrane, sterilizing at the temperature of 71 ℃ for 12min, sterilizing at the temperature of 78 ℃ for 3min, and finally performing spray drying to prepare the required specific biological extract protein-gamma protein.
Comparative example 1
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the group A rotavirus antigen by the treatment solution in the preparation example 8, firstly treating for 30min at 49 ℃, then heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 13 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a subcutaneous multipoint injection mode, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Comparative example 2
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 3 by using a treatment solution (obtained by grinding and uniformly dispersing 3kg of secondary alkyl sodium sulfonate and 52kg of hydrogen peroxide solution with the mass fraction of 5%) at 49 ℃ for 30min, heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 13 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Comparative example 3
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 3 by using a treatment solution (obtained by uniformly grinding and dispersing 3kg of secondary alkyl sodium sulfonate and 52kg of patchouli oil), firstly treating at 49 ℃ for 30min, then heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 13 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Comparative example 4
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 3 by using a treatment solution (obtained by uniformly grinding and dispersing 42kg of hydrogen peroxide solution with the mass fraction of 5% and 13kg of patchouli oil), firstly treating at 49 ℃ for 30min, then heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen obtained in the step S1 and the compound adjuvant obtained in the preparation example 13 according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Comparative example 5
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 3 by the treatment liquid in preparation example 8, firstly treating for 30min at 49 ℃, then heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Comparative example 6
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 3 by the treatment liquid in preparation example 8, firstly treating for 30min at 49 ℃, then heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the hyperimmune vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Comparative example 7
The application provides a preparation method of specific biological extraction protein-gamma protein, which adopts the following technical scheme:
a preparation method of specific biological extraction protein-gamma protein specifically comprises the following steps:
s1, preparation of antigen: pretreating the rotavirus antigen in preparation example 3 by the treatment liquid in preparation example 8, firstly treating for 30min at 49 ℃, then heating to 55 ℃, continuing to treat for 14min, finally cooling to 19 ℃, and preserving heat for 15min to obtain a treated antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine; fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine; fully emulsifying the treated antigen in the step S1 with a compound adjuvant (obtained by grinding and dispersing normal saline, lipopolysaccharide and sodium citrate uniformly according to the mass ratio of 7:3: 1) according to the mass ratio of 1:1 to obtain the three-immunity vaccine;
s3, obtaining immune eggs: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.8 mg/chicken; the immune dose of the three-immunity is 0.2 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, uniformly stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol according to the mass ratio of 1:10:2.5, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, sterilizing for 15min at the temperature of 69 ℃, sterilizing for 7min at the temperature of 76 ℃, and finally performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
Examples 6-10 provide a method of preparing a protein product.
Example 6
The application provides a preparation method of a protein product, which adopts the following technical scheme:
weighing 1.2kg of gamma protein, 15kg of yam powder, 0.5kg of lactulose, 0.8kg of ferulic acid, 5kg of zinc gluconate, 1.3kg of active extract and 76.2kg of water in the embodiment 1 for later use;
sequentially adding gamma protein, rhizoma Dioscoreae powder and active extract into water under stirring, adding lactulose, ferulic acid and zinc gluconate, mixing, and packaging to obtain the desired protein product.
Example 7
The application provides a preparation method of a protein product, which adopts the following technical scheme:
weighing 0.5kg of gamma protein, 10kg of yam powder, 0.1kg of lactulose, 0.3kg of ferulic acid, 1kg of zinc gluconate, 1.1kg of active extract and 87kg of water in the embodiment 2 for later use;
sequentially adding gamma protein, rhizoma Dioscoreae powder and active extract into water under stirring, adding lactulose, ferulic acid and zinc gluconate, mixing, and packaging to obtain the desired protein product.
Example 8
The application provides a preparation method of a protein product, which adopts the following technical scheme:
weighing 0.8kg of gamma protein, 8kg of yam powder, 0.2kg of lactulose, 0.5kg of ferulic acid, 2kg of zinc gluconate, 1kg of active extract and 87.5kg of water in the embodiment 3 for later use;
sequentially adding gamma protein, rhizoma Dioscoreae powder and active extract into water under stirring, adding lactulose, ferulic acid and zinc gluconate, mixing, and packaging to obtain the desired protein product.
Example 9
The application provides a preparation method of a protein product, which adopts the following technical scheme:
weighing 1kg of gamma protein, 12kg of yam powder, 0.3kg of lactulose, 0.5kg of ferulic acid, 3kg of zinc gluconate, 1.2kg of active extract and 82kg of water in the embodiment 4 for later use;
sequentially adding gamma protein, rhizoma Dioscoreae powder and active extract into water under stirring, adding lactulose, ferulic acid and zinc gluconate, mixing, and packaging to obtain the desired protein product.
Example 10
The application provides a preparation method of a protein product, which adopts the following technical scheme:
weighing 1.5kg of gamma protein, 10kg of yam powder, 0.4kg of lactulose, 0.7kg of ferulic acid, 4kg of zinc gluconate, 0.9kg of active extract and 82.5kg of water in the embodiment 5 for later use;
sequentially adding gamma protein, rhizoma Dioscoreae powder and active extract into water under stirring, adding lactulose, ferulic acid and zinc gluconate, mixing, and packaging to obtain the desired protein product.
To verify the performance of the protein products provided herein, the applicant set comparative examples 1-9, in which:
comparative example 1, like example 9, differs only in that: the gamma protein of example 4 was compared to the gamma protein prepared in example 1.
Comparative example 2, like example 9, differs only in that: the gamma protein of example 4 was compared to the gamma protein prepared in example 2.
Comparative example 3, like example 9, differs only in that: the gamma protein of example 4 was compared to the gamma protein prepared in example 3.
Comparative example 4, like example 9, differs only in that: the gamma protein of example 4 was compared to the gamma protein prepared in example 4.
Comparative example 5, like example 9, differs only in that: the gamma protein of example 4 was compared to the gamma protein prepared in example 5.
Comparative example 6, like example 9, differs only in that: the gamma protein of example 4 was compared to the gamma protein prepared in example 6.
Comparative example 7, like example 9, differs only in that: the gamma protein of example 4 was compared to the gamma protein prepared in example 7.
Comparative example 8, like example 9, differs only in that: replacing the active extract with water.
Comparative example 9, like example 9, differs only in that: the ferulic acid is replaced by water.
The protein products prepared in examples 6 to 10 of the present application and comparative examples 1 to 9 were analyzed for stability and virus inhibitory activity:
1. stability testing of protein products prepared in examples 6-10 and comparative examples 1-9
The protein product is subjected to stability test by an accelerated test method, the protein product is placed in a constant temperature box at 37 ℃ for 45 days, the titer of the gamma protein antibody of the effective component in the preparation is respectively measured before and after the placement, and the reduction rate of the effective component is calculated, and the result is shown in table 1.
2. Test of Virus inhibitory Activity of protein products prepared in examples 6 to 10 and comparative examples 1 to 9 about 2X 10 addition of 150. mu.L of vero cells per well in 96-well plate 5 Wherein 50 mu of LPBS buffer solution is added to the control group 1, 25 mu of L A mixed virus solution of group rotavirus, group B rotavirus and group C rotavirus (the mass ratio of the group A rotavirus, the group B rotavirus and the group C rotavirus is 92:3:2) and 25 mu of LPBS buffer solution are added to the control group 2, 25 mu of LA mixed virus solution of group rotavirus, the group B rotavirus and the group C rotavirus (the mass ratio of the group A rotavirus, the group B rotavirus and the group C rotavirus is 92:3:2) and 25 mu of corresponding protein product are added to the examples 6 to 10 and the comparative examples 1 to 9 respectively, and CO is added at 37 ℃ under the condition that CO is mixed with the group A rotavirus, the group B rotavirus and the group C rotavirus 2 Culturing in an incubator, observing cell suspension, rounding and aggregation after 48 hours, and calculating the cell death rate of each group, wherein the results are shown in table 1.
Table 1:
the results shown in Table 1 above show that: the protein products prepared in the embodiments 6-10 of the application have good stability and strong activity of resisting rotavirus, and the comprehensive performance is far superior to that of the comparative examples 1-9.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A preparation method of specific biological extraction protein-gamma protein is characterized by comprising the following steps:
s1, preparation of antigen: pretreating rotavirus antigen by using a treatment solution to obtain a treatment antigen;
s2, preparing an immune vaccine: fully emulsifying the treated antigen in the step S1 with an adjuvant to prepare an immune vaccine;
s3, obtaining immune eggs: applying the immune vaccine obtained in the step S2 to chicken immunization, and collecting immune eggs;
s4, extracting gamma protein: and (2) cutting the immune eggs in the step (S3) into yolk by the yolk, stirring and dispersing the yolk, 0.1mol/L phosphoric acid solution and polyethylene glycol uniformly according to the mass ratio of 1:8-12:2-3, standing and centrifuging, collecting supernatant, dialyzing and purifying the supernatant by a semipermeable membrane, and then performing spray drying to prepare the required specific bio-extracted protein-gamma protein.
2. The method for preparing specific bio-extracted protein-gamma according to claim 1, wherein the rotavirus antigen in step S1 comprises group a rotavirus antigen, group B rotavirus antigen and group C rotavirus antigen; the mass ratio of the A group rotavirus antigen, the B group rotavirus antigen and the C group rotavirus antigen is 89-95:1-5: 1-3.
3. The method for preparing specific bio-extracted protein-gamma according to claim 1, wherein the treatment solution in step S1 comprises the following raw materials in parts by weight: 3-8 parts of sodium secondary alkyl sulfonate, 40-60 parts of 5% hydrogen peroxide solution by mass fraction and 12-18 parts of patchouli oil.
4. The method for preparing specific bio-extracted protein-gamma according to claim 1, wherein the pre-treatment in step S1 is performed as follows: firstly, under the condition of 47-51 ℃, the treatment time is 20-40min, then the temperature is raised to 53-57 ℃, the treatment is continued for 10-18min, finally, the temperature is lowered to 17-22 ℃, and the heat preservation is carried out for 10-20min, thus obtaining the treated antigen.
5. The method for preparing specific bio-extracted protein-gamma protein according to claim 1, wherein the immune vaccine in step S2 includes prime vaccine, di-immune vaccine and tri-immune vaccine.
6. The method for preparing specific bio-extracted protein-gamma protein according to claim 5, wherein the immune vaccine is prepared by the following steps:
fully emulsifying the treated antigen in the step S1 with Freund' S complete adjuvant according to the mass ratio of 1:1 to obtain a prime vaccine;
fully emulsifying the treated antigen in the step S1 with Freund' S incomplete adjuvant according to the mass ratio of 1:1 to obtain the vaccine;
and (3) fully emulsifying the treated antigen obtained in the step S1 with a compound adjuvant according to the mass ratio of 1:1 to obtain the three-immunity vaccine.
7. The method for preparing specific bio-extracted protein-gamma protein according to claim 6, wherein the compounded adjuvant is prepared by compounding normal saline, lipopolysaccharide, polyglutamic acid and sodium citrate in a mass ratio of 7-11:3-5:1-2: 1-2.
8. The method for preparing specific bio-extracted protein-gamma protein according to claim 1, wherein the chicken immunization scheme in step S3 is as follows: the immune vaccine obtained in the step S2 is used for chicken immunization by adopting a mode of subcutaneous multipoint injection, 3 times of immunization are carried out, 2 weeks are separated each time, and the immunization dose of the primary immunization and the secondary immunization is 0.5-1 mg/chicken; the immune dose of the three-immunity is 0.1-0.3 mg/mouse, 2 weeks after the three-immunity, immune eggs are collected, marked and detected in a laboratory.
9. The method for preparing specific bio-extracted protein-gamma according to claim 1, wherein the step S4 further comprises a sterilization treatment before the spray drying, and the specific operations are as follows: sterilizing the supernatant after dialysis and purification at 67-71 deg.C for 12-18min, and sterilizing at 75-78 deg.C for 3-10 min.
10. The method for preparing specific bio-extracted protein-gamma protein according to any one of claims 1 to 9, wherein the prepared gamma protein can be used for preparing protein products;
the protein product comprises the following raw materials in percentage by weight: 0.1-2% of gamma protein, 8-15% of yam powder, 0.1-0.5% of lactulose, 0.3-0.8% of ferulic acid, 1-5% of zinc gluconate, 0.5-1.5% of active extract and the balance of water;
the active extract comprises the following raw materials: 1-3 parts of snakegourd fruit extract, 2-7 parts of dried orange peel extract, 5-15 parts of malt extract and 1-3 parts of bighead atractylodes rhizome extract.
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| CN1201693A (en) * | 1998-04-01 | 1998-12-16 | 中德联合研究所 | Chick lecithal immune globulin products against rolavirus and its use and its testing method of activity |
| CN103554254A (en) * | 2013-11-05 | 2014-02-05 | 四川理工学院 | Chicken egg yolk antibody resisting human A rotavirus as well as preparation method and application thereof |
| CN106617068A (en) * | 2017-01-10 | 2017-05-10 | 威海百合生物技术股份有限公司 | Bitter-free health care product capable of promoting digestion |
| CN107441419A (en) * | 2017-08-17 | 2017-12-08 | 皖南医学院 | A kind of syrup of clearing heat and moistening lung preventing phlegm from forming and stopping coughing and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1201693A (en) * | 1998-04-01 | 1998-12-16 | 中德联合研究所 | Chick lecithal immune globulin products against rolavirus and its use and its testing method of activity |
| CN103554254A (en) * | 2013-11-05 | 2014-02-05 | 四川理工学院 | Chicken egg yolk antibody resisting human A rotavirus as well as preparation method and application thereof |
| CN106617068A (en) * | 2017-01-10 | 2017-05-10 | 威海百合生物技术股份有限公司 | Bitter-free health care product capable of promoting digestion |
| CN107441419A (en) * | 2017-08-17 | 2017-12-08 | 皖南医学院 | A kind of syrup of clearing heat and moistening lung preventing phlegm from forming and stopping coughing and preparation method thereof |
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