CN117603317A - Preparation method and application of complex specific IgY for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum - Google Patents
Preparation method and application of complex specific IgY for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum Download PDFInfo
- Publication number
- CN117603317A CN117603317A CN202311646876.9A CN202311646876A CN117603317A CN 117603317 A CN117603317 A CN 117603317A CN 202311646876 A CN202311646876 A CN 202311646876A CN 117603317 A CN117603317 A CN 117603317A
- Authority
- CN
- China
- Prior art keywords
- porphyromonas gingivalis
- intermedia
- proprietaria
- solution
- adjuvant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000605862 Porphyromonas gingivalis Species 0.000 title claims abstract description 107
- 238000002360 preparation method Methods 0.000 title claims abstract description 39
- 241000894006 Bacteria Species 0.000 claims abstract description 62
- 239000000427 antigen Substances 0.000 claims abstract description 54
- 108091007433 antigens Proteins 0.000 claims abstract description 54
- 102000036639 antigens Human genes 0.000 claims abstract description 54
- 241000605986 Fusobacterium nucleatum Species 0.000 claims abstract description 52
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 42
- 239000007788 liquid Substances 0.000 claims abstract description 40
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000002671 adjuvant Substances 0.000 claims abstract description 39
- 239000002131 composite material Substances 0.000 claims abstract description 38
- 230000001580 bacterial effect Effects 0.000 claims abstract description 27
- 239000003995 emulsifying agent Substances 0.000 claims abstract description 27
- 238000003756 stirring Methods 0.000 claims abstract description 27
- 239000008098 formaldehyde solution Substances 0.000 claims abstract description 18
- 238000002156 mixing Methods 0.000 claims abstract description 14
- 238000013329 compounding Methods 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 11
- 241000588769 Proteus <enterobacteria> Species 0.000 claims abstract description 8
- 229960005486 vaccine Drugs 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 230000000415 inactivating effect Effects 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 51
- 239000002244 precipitate Substances 0.000 claims description 43
- 235000013601 eggs Nutrition 0.000 claims description 39
- 239000006228 supernatant Substances 0.000 claims description 29
- 230000003053 immunization Effects 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 26
- 102000002322 Egg Proteins Human genes 0.000 claims description 25
- 108010000912 Egg Proteins Proteins 0.000 claims description 25
- 238000002649 immunization Methods 0.000 claims description 19
- 208000028169 periodontal disease Diseases 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 18
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 18
- 229910021641 deionized water Inorganic materials 0.000 claims description 18
- 239000000661 sodium alginate Substances 0.000 claims description 18
- 235000010413 sodium alginate Nutrition 0.000 claims description 18
- 229940005550 sodium alginate Drugs 0.000 claims description 18
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 16
- 150000001875 compounds Chemical class 0.000 claims description 16
- 235000014103 egg white Nutrition 0.000 claims description 16
- 210000000969 egg white Anatomy 0.000 claims description 16
- 239000000284 extract Substances 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 16
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 10
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 239000006781 columbia blood agar Substances 0.000 claims description 9
- 238000011033 desalting Methods 0.000 claims description 9
- 238000000502 dialysis Methods 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 238000010257 thawing Methods 0.000 claims description 9
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 238000004062 sedimentation Methods 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 239000005457 ice water Substances 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 230000001376 precipitating effect Effects 0.000 claims description 7
- 241001061264 Astragalus Species 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 235000006533 astragalus Nutrition 0.000 claims description 6
- 150000004676 glycans Chemical class 0.000 claims description 6
- 230000036541 health Effects 0.000 claims description 6
- 230000002779 inactivation Effects 0.000 claims description 6
- 229920001282 polysaccharide Polymers 0.000 claims description 6
- 239000005017 polysaccharide Substances 0.000 claims description 6
- 244000144977 poultry Species 0.000 claims description 6
- 235000015067 sauces Nutrition 0.000 claims description 6
- 210000004233 talus Anatomy 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 4
- 230000000996 additive effect Effects 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000007621 bhi medium Substances 0.000 claims description 2
- 239000006161 blood agar Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 12
- 239000002994 raw material Substances 0.000 abstract description 5
- 241000193403 Clostridium Species 0.000 abstract description 4
- 230000003239 periodontal effect Effects 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 235000013345 egg yolk Nutrition 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 230000017448 oviposition Effects 0.000 description 8
- 201000001245 periodontitis Diseases 0.000 description 7
- 206010006326 Breath odour Diseases 0.000 description 6
- 208000032139 Halitosis Diseases 0.000 description 6
- 244000052616 bacterial pathogen Species 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 241001135221 Prevotella intermedia Species 0.000 description 5
- 230000001804 emulsifying effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 239000002324 mouth wash Substances 0.000 description 3
- 229940051866 mouthwash Drugs 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 208000002064 Dental Plaque Diseases 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 241000186429 Propionibacterium Species 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 208000002925 dental caries Diseases 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 208000007565 gingivitis Diseases 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000002379 periodontal ligament Anatomy 0.000 description 2
- 229940034610 toothpaste Drugs 0.000 description 2
- 239000000606 toothpaste Substances 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010017711 Gangrene Diseases 0.000 description 1
- 206010018296 Gingivitis ulcerative Diseases 0.000 description 1
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004890 Interleukin-8 Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 208000025157 Oral disease Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 208000001143 Periodontal Abscess Diseases 0.000 description 1
- 208000005888 Periodontal Pocket Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 1
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 1
- 208000008312 Tooth Loss Diseases 0.000 description 1
- 206010044016 Tooth abscess Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- -1 amino acid compound Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 208000001277 chronic periodontitis Diseases 0.000 description 1
- 210000004081 cilia Anatomy 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- UCDVIBNDYLUWFP-UHFFFAOYSA-N glaucocalyxin A Natural products C1C(O)C2(C(C3=C)=O)C(O)C3CCC2C2(C)C1C(C)(C)C(=O)CC2 UCDVIBNDYLUWFP-UHFFFAOYSA-N 0.000 description 1
- UCDVIBNDYLUWFP-MJTHGBBVSA-N glaucocalyxin a Chemical compound C([C@@H]1[C@@H](O)[C@]2(C(C1=C)=O)[C@H](O)C1)C[C@H]2[C@@]2(C)[C@H]1C(C)(C)C(=O)CC2 UCDVIBNDYLUWFP-MJTHGBBVSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001571 immunoadjuvant effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 208000030194 mouth disease Diseases 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 208000003265 stomatitis Diseases 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and relates to an antigen and specific yolk composite antibody for resisting Proteus intermedius, carboxybacterium nucleatum and Porphyromonas gingivalis and a preparation method thereof. The antigen preparation method comprises the following steps: (1) Respectively obtaining pure Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus; (2) Mixing the Proprietas intermedia, porphyromonas gingivalis, fusobacterium nucleatum and formaldehyde solution, and inactivating; (3) Removing formaldehyde solution in the inactivated bacterial liquid, and then crushing bacteria; (4) And compounding and stirring the crushed bacterial liquid and the adjuvant to obtain the intermediate Proprietaria, the Porphyromonas gingivalis and the emulsifier of the carboxybacillus nucleatus of the complete adjuvant in the oil pocket Shui Fushi. The invention prepares the three-bacterium composite vaccine by compounding the intermediate general bacterium, the clostridium nucleatum and the porphyromonas gingivalis serving as composite antigens with the adjuvant, and the used raw materials have high biological safety, good stability and high activity.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a preparation process of a yolk antibody, and in particular relates to a specific yolk composite antigen for resisting Proteus intermedius, carboxybacterium nucleatum and Porphyromonas gingivalis, an antibody, a preparation method and application thereof.
Background
Periodontitis (periodontis), also known as destructive periodontal disease, is a chronic inflammation caused by bacteria and calculus in dental plaque invading periodontal tissue, and can lead to destruction of gums, periodontal ligament, alveolar bone and cementum, periodontal pocket formation, loss of adhesion and absorption of alveolar bone, and further to atrophy of teeth, slow loosening of teeth, and finally to tooth loss. Periodontitis is a common oral disease in daily life of people, and according to the results of the fourth national epidemiological investigation, the periodontal health rate of adults in China is only 9.1%.
Periodontitis is mainly caused by microorganisms in dental plaque, and periodontal pathogenic bacteria colonize and multiply in a subgingival environment to generate toxic substances such as endotoxin, protease and the like to directly destroy periodontal tissues; at the same time, it can trigger a series of host immune reactions, and produce a large amount of inflammatory mediators, indirectly leading to periodontal connective tissue destruction and alveolar bone resorption. The intermediate Propionibacterium is a gram-negative melanin-producing obligate anaerobic bacillus, and the bacteria and acute intermediate Propionibacterium (Prevotella intermedia, pi) have lipopolysaccharide, lectin, cilia, vesicle, proteolytic enzyme and other pathogenic factors, can induce periodontal ligament cells to express and regulate metalloprotease, and participate in the degradation and destruction of periodontal connective tissues. Is closely related to the incidence of acute necrotizing gingivitis, chronic periodontitis, periodontal abscess and gestational gingivitis, and is also a traditional Chinese medicine pathogenic bacterium for gangrene stomatitis and pulmonary infection. Porphyromonas gingivalis (Porphyromonas gingivalis, pg) prevents apoptosis through P13K/Akt and JAK/STAT signaling pathways, inhibits IL-8 secretion by host cells, reduces chemotaxis of immune cells, and causes immune silencing. Meanwhile, the virulence factors can induce periodontal microenvironment disturbance and host immune inflammatory reaction, so that periodontal tissues are damaged and degraded into substances such as collagen, protein and the like, and the feed is provided for the proliferation of periodontal pathogenic bacteria. The carboxybacterium nucleatum (Fusobacterium nucleatum, fn) is also gram-negative obligate anaerobic, can copolymerize pathogenic bacteria by means of various adhesins, adhere and invade epithelial cells, destroy periodontal tissues by using virulence factors, metabolites and the like, induce hosts to generate immune response, and promote periodontal diseases and even systemic diseases.
Therefore, the key to preventing and treating periodontal disease is to start from among its key pathogenic bacteria, porphyromonas gingivalis and carboxybacterium nucleatum.
Currently, existing periodontal disease treatment drugs mainly include: chinese medicine components (Chinese patent invention: a Chinese medicinal composition for curing periodontitis and its preparation method and application, publication number: CN103784856B; chinese patent invention: a Mongolian medicine preparation for preventing and curing periodontal disease, publication number: CN 111388553B). Chemical components (Chinese invention patent: application of nicotinamide in preparation of anti-porphyromonas gingivalis medicines, publication No. CN113908157B; chinese invention patent: amino acid compound and composition and application thereof in treatment of periodontal diseases, CN112521322B; chinese invention patent: application of glaucocalyxin A in preparation of products for preventing and treating periodontal diseases, publication No. CN 111840263B). Protein hydrolysate (Chinese patent of invention: collagen hydrolysate as active material against periodontitis or gingivitis, publication No. CN115279457A; chinese patent of invention: novel drug target for preventing and treating periodontal disease, improving periodontal wound healing and promoting oral health, publication No. CN 101563100B). Although studies have demonstrated the efficacy of traditional Chinese medicines, chemical medicines and protein medicines in preventing and treating periodontitis without generating drug resistance and toxic and side effects, further verification is still needed in the specificity of the products.
The yolk antibody (Egg yolk antibody, igY) is main immunoglobulin in fowl serum and yolk, is similar to placenta transfer of IgG in mammal, has the advantages of single antibody, high yield and high immunodiagnosis affinity, does not cross react with mammal antigen or activate mammal complement system, and has good application in preventing and treating various oral pathogenic microorganism diseases. For example, the preparation method is applied to antibacterial (Chinese patent invention: a clear mouthwash containing IgY and a preparation method thereof, publication number: CN 108272649B) and caries prevention (Chinese patent invention: a yolk antibody for caries prevention and a preparation method thereof, and a yolk antibody preparation, publication number: CN 109593129B). However, the specificity of the egg yolk antibody used in the oral cavity protection market is still not strong enough for complex pathogenic bacteria, and the egg yolk antibody with strong specificity needs to be developed.
Disclosure of Invention
Aiming at the defect of the specificity of the existing antibody, the invention provides a method for preparing vaccine by taking Protopanax intermedia, porphyromonas gingivalis and carboxybacillus nucleatum as antigens, collecting eggs after the immunization of laying hens, extracting specific yolk antibody from the eggs, and related application thereof.
The method is characterized in that the egg yolk antibody is prepared by adopting Protopanax intermedia, porphyromonas gingivalis and carboxybacillus nucleatum to prepare a three-bacterium composite antibody and immunize hen, the egg is collected, then the antibody is separated and extracted by adopting a water dilution method, and the potency is measured by adopting an ELISA method. The yolk antibody has the characteristics of stable performance, good biocompatibility, strong specificity and high purity. The obtained yolk antibody has obvious effect for preventing and treating periodontal diseases.
The invention prepares antigen liquid by crushing intermediate Proprietaria, porphyromonas gingivalis and nucleus after being inactivated by formaldehyde solution. Further, the antigen liquid is mixed with an adjuvant to prepare a vaccine, the vaccine is used for immunizing an egg laying hen, finally eggs are collected, egg yolk antibodies are separated and purified from the eggs, and the activity titers of the egg yolk antibodies against Proteus intermedius, porphyromonas gingivalis and Fusobacterium nucleatum are measured.
The invention provides a preparation method of a complex antigen of Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum, which comprises the following steps:
(1) Respectively obtaining pure Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus;
(2) Mixing the Proprietas intermedia, porphyromonas gingivalis, fusobacterium nucleatum and formaldehyde solution, and inactivating;
(3) Removing formaldehyde solution in the inactivated bacterial liquid, and then crushing bacteria;
(4) And compounding and stirring the crushed bacterial liquid and the adjuvant to obtain the intermediate Proprietaria, the Porphyromonas gingivalis and the emulsifier of the carboxybacillus nucleatus of the complete adjuvant in the oil pocket Shui Fushi.
Preferably, the formaldehyde solution has a mass concentration of 0.1-0.6%.
Preferably, the inactivation is for 12-48 hours at 0-5 ℃.
Preferably, the concentration of the bacterial liquid in the inactivated bacterial liquid for removing formaldehyde solution is 1-5 multiplied by 10 9 CFU/mL。
The adjuvant is a complete adjuvant; preferably, the adjuvant is Freund's adjuvant, white oil Ban Zuoji or Astragalus polysaccharide adjuvant.
Preferably, the Shui Fushi in oil complete adjuvant Proprietaria intermedia, porphyromonas gingivalis and Bacillus nucleatus emulsifiers contain 10-40 hundred million bacteria per milliliter of emulsifier.
Preferably, the preparation method comprises the following steps: inoculating middle Proprietas, porphyromonas gingivalis and carboxybacillus nucleatus on Columbia blood agar medium for amplification culture, hanging strains through PBS, centrifugally washing, uniformly mixing bacterial precipitate with 0.1-0.6% formaldehyde aqueous solution after washing, and inactivating at 4 ℃ for more than 24 hours; centrifuging the inactivated bacteria solution, precipitating with PBS, washing for 3 times, removing formaldehyde solution, and adjusting bacteria concentration to 3×10 9 CFU/mL; crushing bacteria of the bacterial liquid under the concentration by using an ultrasonic crusher under the ice water bath condition; will beThe crushed bacteria liquid antigen with the same volume is compounded with Freund's adjuvant, and is stirred and emulsified by a high-speed stirrer, so that the water-in-oil three-bacteria composite emulsifier is finally prepared, and each milliliter of adjuvant emulsifier contains 10-40 hundred million bacteria.
Preferably, the Columbia blood agar medium is BHI medium or blood agar medium.
The invention also provides application of the preparation method of the complex antigen of the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacter nucleatum, which is used for preparing the complex specific IgY for resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacter nucleatum.
Preferably, the three-bacterium composite vaccine is prepared by taking the middle general bacterium, the clostridium nucleatum and the porphyromonas gingivalis as composite antigens and compounding with an adjuvant.
Preferably, the anti-Proprietas intermedia, porphyromonas gingivalis and Bacillus nucleatus compound specific IgY is used as a medicine or a reagent for preventing and treating periodontal diseases, and the additive is added into oral health products.
Preferably, the application comprises:
immunization of animals, and preparation of complex specific IgY extracts against P.intermedia, porphyromonas gingivalis and F.nucleatum.
Preferably, the animal is poultry.
Preferably, immunizing the animal comprises subcutaneously and/or intramuscularly injecting the complex antigens of Proteus intermedium, porphyromonas gingivalis and Fusobacterium nucleatum into the poultry, wherein each poultry is injected with 0.5-2mL of emulsifier and immunized 1 time every 8-10 days for 3-6 times in total;
the preparation of the complex specific IgY extract against Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum comprises the following steps:
collecting fowl eggs subjected to immunization for the 2 nd time, sterilizing the surfaces of the fowl eggs, and separating egg white;
adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:4-6, and regulating the pH value to be 5.0-5.5 by using glacial acetic acid;
freezing at-20deg.C for 24-48 hr, thawing, centrifuging, and collecting supernatant;
adding sodium alginate solution with the mass concentration of 0.5-1.5% and calcium chloride solution with the mass concentration of 13-18% into the supernatant, wherein the adding ratio of the sodium alginate to the calcium chloride solution is 8-10:4, standing at room temperature, settling for 48 hours, and taking the supernatant after settling;
slowly adding an ammonium sulfate solution into the settled supernatant;
centrifuging to obtain precipitate, adding the collected precipitate into ammonium sulfate solution with volume 2-3 times of that of yolk liquid, centrifuging, and collecting precipitate;
and (3) dissolving the precipitate, filling the solution into a dialysis bag, dialyzing, and desalting to obtain the composite specific IgY antibody for resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacillus nucleatus.
In a preferred embodiment of the invention, the preparation method of the complex specific IgY extract against P.intermedia, porphyromonas gingivalis and Fusobacterium nucleatum comprises the following steps:
collecting fowl eggs subjected to immunization for the 2 nd time, sterilizing the surfaces of the fowl eggs by using 75% alcohol, opening the fowl eggs, separating egg white by using an egg white separator, adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:4-6, fully stirring, adjusting the pH value to be 5.0-5.5 by using glacial acetic acid, standing at-40 ℃ for 32 hours, taking out for thawing, centrifuging to obtain supernatant, adding sodium alginate solution with the mass concentration of 0.5-1.5% and calcium chloride solution with the mass concentration of 13-18%, wherein the adding ratio of sodium alginate to calcium chloride solution is 8-10:4, standing at room temperature for 48 hours, and taking supernatant after sedimentation;
slowly adding 50% saturated ammonium sulfate solution into the settled supernatant, fully stirring, standing at 4 ℃ for 2 hours, centrifuging at high speed, taking precipitate, adding 20% ammonium sulfate solution with mass concentration which is 2-3 times of the volume of yolk liquid into the collected precipitate, stirring and mixing uniformly, standing at room temperature for 3 hours, centrifuging at high speed for 20 minutes, and collecting precipitate;
dissolving the precipitate in PBS, placing the solution into a dialysis bag, dialyzing with PBS solution, collecting the dialyzate, and desalting to obtain the composite specific IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus.
The method of the invention comprises the following steps:
1. preparation of anti-Proprietaria, porphyromonas gingivalis and composite antigen of carboxybacillus nucleatus
Inoculating the intermediate Proprietas, the Porphyromonas gingivalis and the carboxybacillus nucleatus on a Columbia blood agar culture medium for amplification culture, hanging strains through PBS, centrifugally washing, uniformly mixing bacterial precipitate with formaldehyde aqueous solution with the mass concentration of 0.1-0.6%, and inactivating at the temperature of 4 ℃ for more than 24 hours. Centrifuging the inactivated bacteria solution, precipitating with PBS, washing for 3 times, removing formaldehyde solution, and adjusting bacteria concentration to 3×10 9 CFU/mL. Bacteria are crushed by an ultrasonic crusher under the condition of ice water bath. The crushed bacteria liquid antigen with the same volume is compounded with Freund's adjuvant, and is stirred and emulsified by a high-speed stirrer, so that the water-in-oil three-bacteria composite emulsifier is finally prepared, and each milliliter of adjuvant emulsifier contains 10-40 hundred million bacteria.
2. Immunization:
three-bacteria composite immunogen was subcutaneously and intramuscularly injected into egg-laying hens using a syringe, each hen was injected with 1mL of emulsifier, and then immunized 1 time every 9 days for a total of 5 times.
3. Preparation of anti-Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum complex specific IgY extract:
collecting the eggs after the 2 nd immunization, sterilizing the surfaces of the eggs by using 75% alcohol, opening the eggs by using an egg opener, separating egg white by using an egg white separator, adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:4-6, fully stirring, adjusting the pH value to be 5.0-5.5 by using glacial acetic acid, standing at-40 ℃ for 32 hours, taking out for thawing, centrifuging, taking supernatant, adding sodium alginate solution with the mass concentration of 0.5-1.5% and calcium chloride solution with the mass concentration of 13-18%, adding sodium alginate and calcium chloride solution according to the addition ratio of 8-10:4, standing at room temperature for 48 hours, and taking supernatant after sedimentation.
Slowly adding 50% saturated ammonium sulfate solution into the supernatant after sedimentation, fully stirring, standing at 4 ℃ for 2 hours, centrifuging at high speed, taking the precipitate, adding 20% ammonium sulfate solution with mass concentration which is 2-3 times of the volume of the yolk liquid into the collected precipitate, stirring and mixing uniformly, standing at room temperature for 3 hours, centrifuging at high speed for 20 minutes, and collecting the precipitate. Dissolving the precipitate in PBS, placing the solution into a dialysis bag, dialyzing with PBS solution, collecting the dialyzate, and desalting to obtain the composite specific IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus.
The Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum used in the invention are all purchased from Shanghai preservation biotechnology center.
The adjuvant is Freund's adjuvant, white oil sauce Ban Zuoji and astragalus polysaccharide adjuvant, preferably white oil sauce Ban Zuoji.
The invention has the advantages that:
(1) The invention prepares the three-bacteria composite vaccine by taking the middle general bacteria, the clostridium nucleatum and the porphyromonas gingivalis as composite antigens and compounding with an adjuvant. The raw materials used are high in biological safety, and part of the raw materials are commercial products.
(2) The invention uses three bacteria composite antibody to immunize chicken, and extracts antibody in yolk, the antibody can react with the composite antigen prepared by three bacteria, and has good stability and high activity.
(3) The three-bacterium composite antibody prepared by the invention can be used as an additive to be added into oral health products such as toothpaste, mouthwash and the like so as to be applied to the prevention and treatment of periodontal diseases.
(4) The preparation method provided by the invention has the advantages of simple and convenient flow and strong operability, and can further meet the requirements of production and application.
Detailed Description
The invention relates to a preparation method and application of a composite specificity egg yolk IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum. The method prepares a three-bacterium composite antigen by compounding middle Proprietaria, porphyromonas gingivalis, bacterial cells and bacterial fragments of Fusobacterium nucleatum with an immunoadjuvant, extracts specific IgY antibodies in yellow after the antigen is immunized with hen, and determines the biological titer of the composite IgY antibodies against the combination of middle Proprietaria, porphyromonas gingivalis and Fusobacterium nucleatum by ELISA. The specific egg yolk antibody has low cost, high biocompatibility and high specificity. The invention prepares the three-bacteria composite vaccine by taking the middle general bacteria, the clostridium nucleatum and the porphyromonas gingivalis as composite antigens and compounding with an adjuvant. The raw materials used are high in biological safety, and part of the raw materials are commercial products. The invention uses three bacteria composite antibody to immunize chicken, and extracts antibody in yolk, the antibody can react with the composite antigen prepared by three bacteria, and has good stability and high activity. The three-bacterium composite antibody prepared by the invention can be used as an additive to be added into oral health products such as toothpaste, mouthwash and the like so as to be applied to the prevention and treatment of periodontal diseases. The preparation method provided by the invention has the advantages of simple and convenient flow and strong operability, and can further meet the requirements of production and application.
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Preparation of complex antigen against Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum
The intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are inoculated on a Columbia blood agar medium for amplification culture, and the intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are identified. And (3) hanging strains through PBS, performing centrifugal washing, uniformly mixing bacterial precipitates with formaldehyde aqueous solution with the mass concentration of 0.4%, and placing at the temperature of 4 ℃ for inactivation for more than 24 hours. Centrifuging the inactivated bacteria solution, precipitating with PBS, washing for 3 times, removing formaldehyde solution, and adjusting bacteria concentration to 3×10 9 CFU/mL. Bacteria are crushed by an ultrasonic crusher under the condition of ice water bath. Compounding the crushed bacterial liquid antigen with Freund's complete adjuvant in equal volume, and emulsifying with a high-speed stirrer to obtain intermediate Proprietaria, porphyromonas gingivalis and Bacillus nucleatus emulsifier as complete adjuvant in oil pocket Shui FushiThe adjuvant emulsifier contains 10-40 hundred million bacteria.
(2) Immunization:
three-bacteria complex antigen was subcutaneously and intramuscularly injected into egg-laying hens using a syringe, each hen was injected with 1mL of the emulsifier, and then immunized 1 time every 9 days for a total of 5 times.
(3) Preparation of anti-Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum complex specific IgY extract:
collecting the eggs after the 2 nd immunization, sterilizing the surfaces of the eggs by using 75% alcohol, opening the eggs by using an egg opener, separating egg white by using an egg white separator, adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:5, fully stirring, adjusting the pH value to be 5.0-5.5 by using glacial acetic acid, standing at the temperature of minus 40 ℃ for 32 hours, taking out for thawing, centrifuging, taking supernatant, adding sodium alginate solution with the mass concentration of 1% and calcium chloride solution with the mass concentration of 15%, adding sodium alginate and calcium chloride solution according to the addition ratio of 9:4, standing at room temperature for 48 hours, and taking supernatant after sedimentation.
Slowly adding 50% saturated ammonium sulfate solution into the settled supernatant, stirring thoroughly, standing at 4deg.C for 2 hr, centrifuging at high speed, collecting precipitate, adding 20% ammonium sulfate solution with mass concentration of 2.5 times of yolk liquid volume into the precipitate, stirring, standing at room temperature for 3 hr, centrifuging at high speed for 20 min, and collecting precipitate. Dissolving the precipitate in PBS, placing the solution into a dialysis bag, dialyzing with PBS solution, collecting the dialyzate, and desalting to obtain the composite specific IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus.
The specific IgY antibody of the compound specificity resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacterium nucleatum is detected by ELISA method, and the activity titer of the specific yolk antibody in each milligram of protein of the specific IgY antibody of the compound specificity resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacterium nucleatum can reach 1:15000/mg of protein.
Example 2
(1) Preparation of complex antigen against Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum
The intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are inoculated on a Columbia blood agar medium for amplification culture, and the intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are identified. And (3) hanging strains through PBS, performing centrifugal washing, uniformly mixing bacterial precipitates with formaldehyde aqueous solution with the mass concentration of 0.5%, and placing at the temperature of 4 ℃ for inactivation for more than 24 hours. Centrifuging the inactivated bacteria solution, precipitating with PBS, washing for 3 times, removing formaldehyde solution, and adjusting bacteria concentration to 3×10 9 CFU/mL. Bacteria are crushed by an ultrasonic crusher under the condition of ice water bath. The method comprises the steps of compounding an equal volume of broken bacterial liquid antigen with white oil sauce Ban Zuoji, stirring and emulsifying by using a high-speed stirrer, and finally preparing a water-in-oil white oil sauce Ban Zuoji middle Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum emulsifier, wherein each milliliter of white oil sauce Ban Zuoji emulsifier contains 10-40 hundred million bacteria.
(2) Immunization:
three-bacteria complex antigen was subcutaneously and intramuscularly injected into egg-laying hens using a syringe, each hen was injected with 1mL of the emulsifier, and then immunized 1 time every 9 days for a total of 5 times.
(3) Preparation of anti-Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum complex specific IgY extract:
collecting the eggs after the 2 nd immunization, sterilizing the surfaces of the eggs by using 75% alcohol, opening the eggs by using an egg opener, separating egg white by using an egg white separator, adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:5, fully stirring, adjusting the pH value to be 5.0-5.5 by using glacial acetic acid, standing at the temperature of minus 40 ℃ for 32 hours, taking out for thawing, centrifuging, taking supernatant, adding sodium alginate solution with the mass concentration of 1% and calcium chloride solution with the mass concentration of 14%, adding sodium alginate and calcium chloride solution according to the addition ratio of 8:4, standing at room temperature for 48 hours, and taking supernatant after sedimentation.
Slowly adding 50% saturated ammonium sulfate solution into the settled supernatant, fully stirring, standing at 4 ℃ for 2 hours, centrifuging at high speed, taking precipitate, adding 20% ammonium sulfate solution with mass concentration which is 2 times of the volume of the egg yolk liquid into the collected precipitate, stirring and mixing uniformly, standing at room temperature for 3 hours, centrifuging at high speed for 20 minutes, and collecting the precipitate. Dissolving the precipitate in PBS, placing the solution into a dialysis bag, dialyzing with PBS solution, collecting the dialyzate, and desalting to obtain the composite specific IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus.
The specific IgY antibody of the compound specificity resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacterium nucleatum is detected by ELISA method, and the activity titer of the specific yolk antibody in each milligram of protein of the specific IgY antibody of the compound specificity resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacterium nucleatum can reach 1:25600/mg of protein.
Example 3
(1) Preparation of complex antigen against Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum
The intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are inoculated on a Columbia blood agar medium for amplification culture, and the intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are identified. And (3) hanging strains through PBS, performing centrifugal washing, uniformly mixing bacterial precipitates with formaldehyde aqueous solution with the mass concentration of 0.4%, and placing at the temperature of 4 ℃ for inactivation for more than 24 hours. Centrifuging the inactivated bacteria solution, precipitating with PBS, washing for 3 times, removing formaldehyde solution, and adjusting bacteria concentration to 3×10 9 CFU/mL. Bacteria are crushed by an ultrasonic crusher under the condition of ice water bath. The method comprises the steps of compounding an equal volume of broken bacterial liquid antigen with an astragalus polysaccharide adjuvant, stirring and emulsifying by using a high-speed stirrer, and finally preparing water-in-oil astragalus polysaccharide adjuvant intermediate Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum emulsifying agent, wherein each milliliter of astragalus polysaccharide adjuvant emulsifying agent contains 10-40 hundred million bacteria.
(2) Immunization:
three-bacteria complex antigen was subcutaneously and intramuscularly injected into egg-laying hens using a syringe, each hen was injected with 1mL of the emulsifier, and then immunized 1 time every 9 days for a total of 5 times.
(3) Preparation of anti-Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum complex specific IgY extract:
collecting the eggs after the 2 nd immunization, sterilizing the surfaces of the eggs by using 75% alcohol, opening the eggs by using an egg opener, separating egg white by using an egg white separator, adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:4, fully stirring, adjusting the pH value to 5.0-5.5 by using glacial acetic acid, standing at-40 ℃ for freezing for 32 hours, taking out for thawing, centrifuging, taking supernatant, adding sodium alginate solution with the mass concentration of 1.5% and calcium chloride solution with the mass concentration of 16%, adding sodium alginate and calcium chloride solution according to the addition ratio of 8:4, standing at room temperature for 48 hours, and taking supernatant after sedimentation.
Slowly adding 50% saturated ammonium sulfate solution into the settled supernatant, stirring thoroughly, standing at 4deg.C for 2 hr, centrifuging at high speed, collecting precipitate, adding 20% ammonium sulfate solution with mass concentration of 2.5 times of yolk liquid volume into the precipitate, stirring, standing at room temperature for 3 hr, centrifuging at high speed for 20 min, and collecting precipitate. Dissolving the precipitate in PBS, placing the solution into a dialysis bag, dialyzing with PBS solution, collecting the dialyzate, and desalting to obtain the composite specific IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus.
The specific IgY antibody of the anti-middle Proprietas, the Porphyromonas gingivalis and the carboxybacillus nucleatum is detected by ELISA method, and the activity titer of the specific yolk antibody in each milligram of protein of the specific IgY antibody of the anti-middle Proprietas, the Porphyromonas gingivalis and the carboxybacillus nucleatum can reach 1:3700/mg of protein.
Example 4
(1) Preparation of complex antigen against Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum
The intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are inoculated on a Columbia blood agar medium for amplification culture, and the intermediate Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are identified. And (3) hanging strains through PBS, performing centrifugal washing, uniformly mixing bacterial precipitates with formaldehyde aqueous solution with the mass concentration of 0.4%, and placing at the temperature of 4 ℃ for inactivation for more than 24 hours. Centrifugally collecting the inactivated bacterial liquid, precipitating with PBS, washing for 3 times, and removing formaldehydeSolution, and the concentration of the bacterial solution is adjusted to 3 multiplied by 10 9 CFU/mL. Bacteria are crushed by an ultrasonic crusher under the condition of ice water bath. Compounding the crushed bacterial liquid antigen with Phosphate Buffer (PBS) in equal volume, and stirring and emulsifying by using a high-speed stirrer to finally prepare the water-in-oil adjuvant-free emulsifying agent of the Proprietas intermedia, the Porphyromonas gingivalis and the Fusobacterium nucleatum, wherein each milliliter of the emulsifying agent contains 10-40 hundred million bacteria.
(2) Immunization:
three-bacteria complex antigen was subcutaneously and intramuscularly injected into egg-laying hens using a syringe, each hen was injected with 1mL of the emulsifier, and then immunized 1 time every 9 days for a total of 5 times.
(3) Preparation of anti-Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum complex specific IgY extract:
collecting the eggs after the 2 nd immunization, sterilizing the surfaces of the eggs by using 75% alcohol, opening the eggs by using an egg opener, separating egg white by using an egg white separator, adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:5, fully stirring, adjusting the pH value to be 5.0-5.5 by using glacial acetic acid, standing at the temperature of minus 40 ℃ for 32 hours, taking out for thawing, centrifuging, taking supernatant, adding sodium alginate solution with the mass concentration of 1% and calcium chloride solution with the mass concentration of 15%, adding sodium alginate and calcium chloride solution according to the addition ratio of 9:4, standing at room temperature for 48 hours, and taking supernatant after sedimentation.
Slowly adding 50% saturated ammonium sulfate solution into the settled supernatant, stirring thoroughly, standing at 4deg.C for 2 hr, centrifuging at high speed, collecting precipitate, adding 20% ammonium sulfate solution with mass concentration of 2.5 times of yolk liquid volume into the precipitate, stirring, standing at room temperature for 3 hr, centrifuging at high speed for 20 min, and collecting precipitate. Dissolving the precipitate in PBS, placing the solution into a dialysis bag, dialyzing with PBS solution, collecting the dialyzate, and desalting to obtain the composite specific IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus.
The specific IgY antibody of the compound specificity resisting the intermediate Proprietas, the Porphyromonas gingivalis and the carboxybacillus nucleatum is detected by ELISA method, and the activity titer of the specific yolk antibody in each milligram of protein of the specific IgY antibody of the compound specificity resisting the intermediate Proprietas, the Porphyromonas gingivalis and the carboxybacillus nucleatum can reach 1:6400/mg of protein.
Example 5
The three bacteria composite antigens of the middle Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are immunized to egg-laying hens according to the invention, eggs are immunized 30 days after the first immunization, the composite specificity IgY antibody extract for resisting the middle Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum is prepared according to the method of the invention, and ELISA is used for measuring the biological titers of the different antigens combined by the middle Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum (table 1):
from the above results, it can be seen that: after the mixed antigen of the Proprietas intermedia, the Porphyromonas gingivalis and the Fusobacterium nucleatum is used for immunizing hens, specific IgY antibodies against three bacteria can be generated; the titers of specific IgY antibodies raised against the three bacteria do not vary with the amount of each bacterium used when immunizing eggs, which correlates with the immunogenicity of the antigen; the compound specific antibody can react with the compound antigens of three bacteria, and the titer of the total specific IgY antibody is increased, so that the titer has a superposition effect.
The specific IgY for resisting Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum can prevent periodontal disease, halitosis and the like caused by the bacteria; meanwhile, the compound specificity IgY for resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the Fusobacterium nucleatum can be used for preventing periodontal diseases, halitosis and the like caused by any one of the bacteria; thus, it is also possible to expand the range of the prevention of periodontal disease, halitosis and the like caused by bacteria having common antigenic determinants with the above bacteria, by using the complex specific IgY against P.intermedia, porphyromonas gingivalis and F.nucleatum of the present invention.
Example 6
The three bacteria composite antigens of the middle Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum are immunized to egg-laying hens according to the invention, eggs are immunized 60 days after the first immunization, the composite specificity IgY antibody extract for resisting the middle Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum is prepared according to the method of the invention, and ELISA is used for measuring the biological titers of the different antigens combined by the middle Proprietas, the Porphyromonas gingivalis and the Fusobacterium nucleatum (Table 2):
from the above results, it can be seen that: after the mixed antigen of the Proprietas intermedia, the Porphyromonas gingivalis and the Fusobacterium nucleatum is used for immunizing hens, specific IgY antibodies against three bacteria can be generated; the titers of specific IgY antibodies raised against the three bacteria do not vary with the amount of each bacterium used when immunizing eggs, which correlates with the immunogenicity of the antigen; the compound specific antibody can react with the compound antigens of three bacteria, and the titer of the total specific IgY antibody is increased, so that the titer has a superposition effect.
The specific IgY of the combination of the anti-Proprietaria intermedia, the Porphyromonas gingivalis and the Fusobacterium nucleatum can prevent periodontal disease, halitosis and the like caused by the bacteria; meanwhile, the compound specificity IgY for resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the Fusobacterium nucleatum can prevent periodontal disease, halitosis and the like caused by any one of the bacteria; thus, it is also possible to expand the range of the prevention of periodontal disease, halitosis and the like caused by bacteria having common antigenic determinants with the above bacteria, by using the complex specific IgY against P.intermedia, porphyromonas gingivalis and F.nucleatum of the present invention.
In the description of the present invention, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element. It should be noted that, in this document, relational terms such as "first," "second," or steps (1), (2), and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
The above-described embodiments are merely specific embodiments of the present application, but the scope of protection of the present application is not limited thereto, and any changes or substitutions that can be suggested by one skilled in the art without creative efforts are intended to be included in the scope of protection of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims in the present application.
Claims (10)
1. A preparation method of a complex antigen of Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum is characterized by comprising the following steps:
(1) Respectively obtaining pure Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus;
(2) Mixing the Proprietas intermedia, porphyromonas gingivalis, fusobacterium nucleatum and formaldehyde solution, and inactivating;
(3) Removing formaldehyde solution in the inactivated bacterial liquid, and then crushing bacteria;
(4) And compounding and stirring the crushed bacterial liquid and the adjuvant to obtain the intermediate Proprietaria, the Porphyromonas gingivalis and the emulsifier of the carboxybacillus nucleatus of the complete adjuvant in the oil pocket Shui Fushi.
2. The method for preparing the complex antigen of Proteus intermedium, porphyromonas gingivalis and carboxybacterium nucleatum according to claim 1, wherein the method for preparing the complex antigen comprises the following steps:
the mass concentration of the formaldehyde solution is 0.1-0.6%;
the inactivation is carried out for 12-48h at 0-5 ℃;
the concentration of the bacterial liquid in the inactivated bacterial liquid for removing formaldehyde solution is 1-5 multiplied by 10 9 CFU/mL；
The adjuvant is a complete adjuvant;
the oil bag Shui Fushi contains 10-40 hundred million bacteria per milliliter of emulsifier in complete adjuvant of Proprietaria intermedia, porphyromonas gingivalis and Bacillus nucleatus.
3. The method for preparing a complex antigen of Proprietaria intermedia, porphyromonas gingivalis and Bacillus nucleatus according to claim 1, wherein the adjuvant is Freund's adjuvant, white oil sauce Ban Zuoji or Astragalus polysaccharide adjuvant.
4. The method for preparing the complex antigen of Proteus intermedium, porphyromonas gingivalis and carboxybacterium nucleatum according to claim 1, wherein the method for preparing the complex antigen comprises the following steps: inoculating middle Proprietas, porphyromonas gingivalis and carboxybacillus nucleatus on Columbia blood agar medium for amplification culture, hanging strains through PBS, centrifugally washing, uniformly mixing bacterial precipitate with 0.1-0.6% formaldehyde aqueous solution after washing, and inactivating at 4 ℃ for more than 24 hours; centrifuging the inactivated bacteria solution, precipitating with PBS, washing for 3 times, removing formaldehyde solution, and adjusting bacteria concentration to 3×10 9 CFU/mL; crushing bacteria of the bacterial liquid under the concentration by using an ultrasonic crusher under the ice water bath condition; the crushed bacteria liquid antigen with the same volume is compounded with Freund's adjuvant, and is stirred and emulsified by a high-speed stirrer, so that the water-in-oil three-bacteria composite emulsifier is finally prepared, and each milliliter of adjuvant emulsifier contains 10-40 hundred million bacteria.
5. The method for preparing the complex antigen of Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus according to claim 1, wherein the Columbia blood agar medium is BHI medium or blood agar medium.
6. The use of a method for the preparation of a complex antigen of Proteus intermedia, porphyromonas gingivalis and Bacillus nucleatus as claimed in any one of claims 1 to 5 for the preparation of complex specific IgY against Proteus intermedia, porphyromonas gingivalis and Bacillus nucleatus.
7. The use according to claim 6, wherein the three-bacterium compound vaccine is prepared by compounding a compound antigen of the group consisting of a middleprovium, fusobacterium nucleatum and porphyromonas gingivalis with an adjuvant; or,
the anti-Proprietas, porphyromonas gingivalis and carboxybacillus nucleatus compound specific IgY are used as medicines or reagents for preventing and treating periodontal diseases, and the additive is added into oral health products.
8. The application of claim 6, wherein the application comprises:
immunizing an animal, and
preparation of anti-Proprietas, porphyromonas gingivalis and Fusobacterium nucleatum complex specific IgY extract.
9. The use according to claim 8, wherein the animal is poultry;
the immunization of animals comprises subcutaneously and/or intramuscularly injecting the complex antigens of Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum into poultry, and injecting 0.5-2mL of emulsifier into each poultry, and immunizing 1 time every 8-10 days for 3-6 times in total;
the preparation of the complex specific IgY extract against Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum comprises the following steps:
collecting fowl eggs subjected to immunization for the 2 nd time, sterilizing the surfaces of the fowl eggs, and separating egg white;
adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:4-6, and regulating the pH value to be 5.0-5.5 by using glacial acetic acid;
freezing at-20deg.C for 24-48 hr, thawing, centrifuging, and collecting supernatant;
adding sodium alginate solution with the mass concentration of 0.5-1.5% and calcium chloride solution with the mass concentration of 13-18% into the supernatant, wherein the adding ratio of the sodium alginate to the calcium chloride solution is 8-10:4, standing at room temperature, settling for 48 hours, and taking the supernatant after settling;
slowly adding an ammonium sulfate solution into the settled supernatant;
centrifuging to obtain precipitate, adding the collected precipitate into ammonium sulfate solution with volume 2-3 times of that of yolk liquid, centrifuging, and collecting precipitate;
and (3) dissolving the precipitate, filling the solution into a dialysis bag, dialyzing, and desalting to obtain the composite specific IgY antibody for resisting the Proprietaria intermedia, the Porphyromonas gingivalis and the carboxybacillus nucleatus.
10. The use according to claim 8, wherein the preparation of the complex specific IgY extract against praecox intermedia, porphyromonas gingivalis and fusobacterium nucleatum comprises:
collecting fowl eggs subjected to immunization for the 2 nd time, sterilizing the surfaces of the fowl eggs by using 75% alcohol, opening the fowl eggs, separating egg white by using an egg white separator, adding deionized water into yolk liquid and deionized water according to the volume ratio of 1:4-6, fully stirring, adjusting the pH value to be 5.0-5.5 by using glacial acetic acid, standing at-40 ℃ for 32 hours, taking out for thawing, centrifuging to obtain supernatant, adding sodium alginate solution with the mass concentration of 0.5-1.5% and calcium chloride solution with the mass concentration of 13-18%, wherein the adding ratio of sodium alginate to calcium chloride solution is 8-10:4, standing at room temperature for 48 hours, and taking supernatant after sedimentation;
slowly adding 50% saturated ammonium sulfate solution into the settled supernatant, fully stirring, standing at 4 ℃ for 2 hours, centrifuging at high speed, taking precipitate, adding 20% ammonium sulfate solution with mass concentration which is 2-3 times of the volume of yolk liquid into the collected precipitate, stirring and mixing uniformly, standing at room temperature for 3 hours, centrifuging at high speed for 20 minutes, and collecting precipitate;
dissolving the precipitate in PBS, placing the solution into a dialysis bag, dialyzing with PBS solution, collecting the dialyzate, and desalting to obtain the composite specific IgY antibody for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacillus nucleatus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311646876.9A CN117603317A (en) | 2023-12-04 | 2023-12-04 | Preparation method and application of complex specific IgY for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311646876.9A CN117603317A (en) | 2023-12-04 | 2023-12-04 | Preparation method and application of complex specific IgY for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117603317A true CN117603317A (en) | 2024-02-27 |
Family
ID=89954526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311646876.9A Pending CN117603317A (en) | 2023-12-04 | 2023-12-04 | Preparation method and application of complex specific IgY for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117603317A (en) |
-
2023
- 2023-12-04 CN CN202311646876.9A patent/CN117603317A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4748018A (en) | Method of passive immunization of mammals using avian antibody | |
| MXPA06013471A (en) | Vaccine for periodontal disease. | |
| MXPA03006038A (en) | Immunizing compositions and methods of use. | |
| JPH0313205B2 (en) | ||
| CS244906B2 (en) | Production method of antigen agent for limitation or prevention of tooth defects | |
| EP0669971B1 (en) | Method of producing gram-negative bacterial vaccines | |
| JPH10500102A (en) | Antigen preparation for treatment or prevention of Helicobacter infection | |
| WO2023231250A1 (en) | Bovine pasteurella multocida capsular type a capsular polysaccharide vaccine and preparation method therefor | |
| JPS6176420A (en) | Membrane polysaccharide and manufacture | |
| JPS6211090A (en) | Toreponema hyodicenteriae bacterin and its method | |
| CN117603317A (en) | Preparation method and application of complex specific IgY for resisting Proprietaria intermedia, porphyromonas gingivalis and carboxybacterium nucleatum | |
| IE45145B1 (en) | Immunostimulating agent | |
| US5176910A (en) | Treponema hyodysenteriae hemolysin and uses therefor | |
| IE60324B1 (en) | Vaccine against e. coli septicaemia in poultry | |
| JP3072353B2 (en) | Food for preventing gastritis, stomach or duodenal ulcer | |
| Bosch et al. | Bacterial immunostimulant (Broncho-Vaxom) versus levamisole on the humoral immune response in mice | |
| CN110237251B (en) | Composite specificity egg yolk antibody oral spray and preparation method thereof | |
| JPS60146834A (en) | Production of antibody for inhibiting periodontitis and noncarious composition containing said antibody | |
| US4386066A (en) | Immunogenic complex from N. gonorrhoeae | |
| KR100267746B1 (en) | Oral immunological preparation containing egg-yolk antibodies, for prevention and treatment of porcine diarrhea caused by enterotoxigenic escherichia coli | |
| UA126438C2 (en) | Polyvalent combined inactivated liquid vaccine against bacterial and fungal pathogens of the nasopharynx, mouth and eyes, a method of its manufacture and a method of treatment and prevention thereof | |
| JPS61140527A (en) | Oral vaccin for preventing pheridontosis | |
| CN110357955B (en) | Composite specificity egg yolk antibody and preparation method and application thereof | |
| CN117599167A (en) | Yolk composite antibody freeze-dried powder for resisting Proprietaria intermedia, porphyromonas gingivalis and Fusobacterium nucleatum and preparation method thereof | |
| KR840000905B1 (en) | Process for preparing inactivated canine parro vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |