CN116199750A - 一种核糖体蛋白s11及制备方法和在疫苗佐剂中的应用 - Google Patents
一种核糖体蛋白s11及制备方法和在疫苗佐剂中的应用 Download PDFInfo
- Publication number
- CN116199750A CN116199750A CN202310021719.2A CN202310021719A CN116199750A CN 116199750 A CN116199750 A CN 116199750A CN 202310021719 A CN202310021719 A CN 202310021719A CN 116199750 A CN116199750 A CN 116199750A
- Authority
- CN
- China
- Prior art keywords
- ribosomal protein
- protein
- eluting
- natural immune
- immune memory
- 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
- 108090000930 ribosomal protein S11 Proteins 0.000 title claims abstract description 72
- 102000004413 ribosomal protein S11 Human genes 0.000 title claims abstract description 70
- 239000012646 vaccine adjuvant Substances 0.000 title claims abstract description 13
- 229940124931 vaccine adjuvant Drugs 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 230000006054 immunological memory Effects 0.000 claims abstract description 34
- 102000002278 Ribosomal Proteins Human genes 0.000 claims abstract description 19
- 108010000605 Ribosomal Proteins Proteins 0.000 claims abstract description 19
- 230000006698 induction Effects 0.000 claims abstract description 9
- 241000194032 Enterococcus faecalis Species 0.000 claims abstract description 8
- 229940032049 enterococcus faecalis Drugs 0.000 claims abstract description 8
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 230000001939 inductive effect Effects 0.000 claims description 21
- 239000006228 supernatant Substances 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 15
- 235000019750 Crude protein Nutrition 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 238000000855 fermentation Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 238000003259 recombinant expression Methods 0.000 claims description 11
- 210000003024 peritoneal macrophage Anatomy 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 4
- 238000004255 ion exchange chromatography Methods 0.000 claims description 4
- 229910052759 nickel Inorganic materials 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 3
- 238000001042 affinity chromatography Methods 0.000 claims description 3
- 238000005349 anion exchange Methods 0.000 claims description 3
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 claims description 3
- 229910001626 barium chloride Inorganic materials 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000002953 preparative HPLC Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 230000006870 function Effects 0.000 claims description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000002671 adjuvant Substances 0.000 abstract description 22
- 230000000694 effects Effects 0.000 abstract description 21
- 229960005486 vaccine Drugs 0.000 abstract description 15
- 238000011161 development Methods 0.000 abstract description 8
- 239000013543 active substance Substances 0.000 abstract description 6
- 241000222122 Candida albicans Species 0.000 abstract description 5
- 229940095731 candida albicans Drugs 0.000 abstract description 5
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 5
- 230000003013 cytotoxicity Effects 0.000 abstract description 5
- 230000002949 hemolytic effect Effects 0.000 abstract description 4
- 230000015788 innate immune response Effects 0.000 abstract description 4
- 230000015654 memory Effects 0.000 abstract description 4
- 230000004721 adaptive immunity Effects 0.000 abstract description 2
- 239000000843 powder Substances 0.000 abstract description 2
- 239000007787 solid Substances 0.000 abstract description 2
- 108010058846 Ovalbumin Proteins 0.000 description 24
- 229940092253 ovalbumin Drugs 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 15
- 239000000427 antigen Substances 0.000 description 14
- 108091007433 antigens Proteins 0.000 description 14
- 102000036639 antigens Human genes 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 11
- 230000003053 immunization Effects 0.000 description 9
- 238000002649 immunization Methods 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 210000000952 spleen Anatomy 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 241000191967 Staphylococcus aureus Species 0.000 description 5
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 5
- 229910052782 aluminium Inorganic materials 0.000 description 5
- 230000002708 enhancing effect Effects 0.000 description 5
- 230000002757 inflammatory effect Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 241000426497 Chilo suppressalis Species 0.000 description 3
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 3
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 230000024932 T cell mediated immunity Effects 0.000 description 3
- 230000000240 adjuvant effect Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 206010057249 Phagocytosis Diseases 0.000 description 2
- 108010026552 Proteome Proteins 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229940037003 alum Drugs 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000001280 germinal center Anatomy 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 230000028996 humoral immune response Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
- 239000000902 placebo Substances 0.000 description 2
- 229940068196 placebo Drugs 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
- 229940022962 COVID-19 vaccine Drugs 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 206010018691 Granuloma Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 229940124615 TLR 7 agonist Drugs 0.000 description 1
- 229940124614 TLR 8 agonist Drugs 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000001609 comparable effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000000074 matrix-assisted laser desorption--ionisation tandem time-of-flight detection Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229940023146 nucleic acid vaccine Drugs 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000002516 postimmunization Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000001973 thioglycolate broth Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000029069 type 2 immune response Effects 0.000 description 1
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- 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
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
Abstract
本发明公开了一种核糖体蛋白S11及制备方法和在疫苗佐剂中的应用,提供了一种新的核糖体蛋白S11,是一种粪肠球菌产生的诱导先天免疫记忆活性物质核糖体蛋白;与灭活的白色念珠菌相比,其显示出较强的诱导天然免疫记忆作用;该核糖体蛋白具有良好的稳定性,低细胞毒性和溶血活性,为实际应用奠定理论基础,确定了核糖体蛋白作为疫苗佐剂的作用,为未来开发为新型佐剂、同时具备先天免疫记忆和适应性免疫的双记忆疫苗奠定坚实的基础。
Description
技术领域
本发明公开一种诱导天然免疫记忆的核糖体蛋白及其制备方法,同时还提供其作为疫苗佐剂的用途,属于生物医药技术领域。
背景技术
疫苗是预防和控制疾病感染的有效措施。疫苗佐剂是指加入疫苗中能够非特异地增强疫苗抗原免疫原性的物质,可以促进、延长或增强疫苗抗原特异性免疫应答。在疫苗中使用佐剂能够提高弱免疫原性抗原的免疫效果;改善现有疫苗的免疫效果;减少抗原用量从而降低抗原产能压力或提高疫苗的产量;降低疫苗的价格。疫苗佐剂的生物学作用包括如下几个方面:使抗原缓慢释放、延长抗原在体内停留时间;扩大抗原的表面积,增强吞噬细胞的吞噬和抗原提呈;增强抗原的免疫原性,使无免疫原性或仅有微弱免疫原性的物质变成有效的免疫原;引起或增强迟发型超敏反应,促进局部的炎症反应;增强机体对抗原刺激的反应性,可以提高初次应答和再次应答所产生抗体的滴度;改变抗体类型,使由产生IgM转变为产生IgG。
近些年,随着DNA重组技术的快速发展,重组亚单位疫苗、合成肽疫苗和核酸疫苗等新型疫苗的研发取得了快速的发展。但是与传统疫苗相比,这些疫苗抗原的相对分子重量较小,通常免疫原性较弱,所以需要佐剂增强其作用。铝佐剂和油水乳剂的使用由来已久,此外微生物也是佐剂的重要来源,如卡介苗、病毒样颗粒、CPG-DNA、MPLA等。微生物及其代谢产物具有易获取、成本低,易刺激机体的免疫反应等优点,微生物佐剂在疫苗佐剂中均发挥着不可忽视的作用。
尽管已有多种佐剂被许可使用,然而这些现有的佐剂仍存在诸多问题或不足,体现在:佐剂活性仍然较弱;诱生Th2类免疫应答和体液免疫,不能诱生Th1类和CTL细胞免疫应答,在应对胞内感染或肿瘤等时乏力;存在抗原特异性,通用性不强;毒性大,导致注射部位的溃疡、肉芽肿和疼痛及以及更频繁的全身症状等毒副作用。现有佐剂类别难以达到或满足实际疫苗生产的需求,新型疫苗佐剂的研发仍十分必要。近来一些SARS-CoV-2疫苗在保护效力和时长方面体现的不足,不仅促使人们积极开发更新换代疫苗,也突出了对新型佐剂的急迫需求。免疫学理论和技术最新发展,对新型佐剂的开发具有重要的指导意义。本发明从粪肠球菌的次级代谢产物中分离纯化出诱导天然免疫记忆的活性物质核糖体蛋白S11,佐剂活性分析其具有优良的疫苗佐剂活性,能作为疫苗佐剂开发应用,并为研制能同时诱导天然免疫记忆和适应性免疫记忆的双记忆疫苗提供了原材料。
发明内容
本发明的目的之一在于提供一种诱导天然免疫记忆的核糖体蛋白S11,是一种粪肠球菌产生的诱导先天免疫记忆活性物质核糖体蛋白,其氨基酸序列如SEQ ID NO.1所示,编码基因如SEQ ID NO.2所示。
本发明的目的之二在于提供一种诱导天然免疫记忆的核糖体蛋白S11的纯化制备方法,包括如下步骤:
(1)发酵上清制备:将保存于-80℃的粪肠球菌于TSB平皿划线,37℃恒温培养至出现单菌落,然后挑取单菌落至5 mL TSB液体培养基中,37℃、180 rpm振荡培养3 h,然后以1%(v/v)比例转接至10 L TSB液体培养基中,37℃、180 rpm振荡培养6 h,10000g、4℃下离心10 min,0.22μm滤器过滤得发酵上清;
(2)粗蛋白捕获:将发酵上清以80%饱和度硫酸铵溶液在4℃沉淀16 h,然后12000g、4℃下离心20 min,收集沉淀,使用蒸馏水复溶,然后用1000 Da的透析袋透析除去硫酸铵,直至硫酸铵被完全去除,采用1%氯化钡检测硫酸铵是否除干净,最后通过真空冷冻干燥获得粗蛋白,保存于-80℃;
(3)纯化:将粗蛋白应用离子交换层析和制备型HPLC进行纯化。将粗蛋白通过Q-Tanrose 6FF阴离子交换层析柱,采用阶梯固定梯度洗脱方式,以0 M NaCl-1.0 M NaCl固定时间程序进行洗脱,每个梯度洗脱3个柱体积,流速为1mL/min,以280nm为检测波长,收集各洗脱峰,利用小鼠腹腔巨噬细胞天然免疫记忆模型,确定诱导天然免疫记忆的目标活性峰。进一步将目标峰利用Ultimate XB-C18液相色谱柱经HPLC进行纯化,流动相A为蒸馏水;B为乙腈,线性梯度洗脱,洗脱程序为:0-5min,20%乙腈洗脱;5-25 min,20%-100%乙腈洗脱;25-30 min,100%乙腈洗脱。多次重复进样后,将检测具有诱导天然免疫记忆的活性峰合并,冷冻干燥获得核糖体蛋白S11。
本发明的目的之三在于提供一种诱导天然免疫记忆的核糖体蛋白S11的重组表达方法,包括如下步骤:
(1)构建表达载体:设计引物,扩增核糖体蛋白S11基因,经酶切和连接,并且通过转化构建表达菌株大肠杆菌BL21(DE3);
(2)诱导表达:得到的重组表达菌株在37℃,180 rpm振荡培养至OD600=0.6-0.8时,再加入异丙基-β-D-硫代半乳糖苷至终浓度为500 μmol/L,16℃,160 rpm诱导24 h。
(3)纯化:得到的发酵液在8000g、4℃离心10 min,收集菌体。超声破碎菌体后的上清10000g、4℃离心20 min,应用镍柱进行亲和层析纯化,分别用20 mM、100 mM咪唑洗脱杂蛋白,收集500 mM咪唑洗脱部分,应用超滤除去咪唑、甘油等杂质,然后进行真空冷冻干燥纯化得到重组表达核糖体蛋白。
本发明的目的之四在于提供核糖体蛋白S11的佐剂效应应用分析。该方法包括如下步骤:
为研究核糖体蛋白S11对模式抗原卵清白蛋白(OVA)免疫小鼠的佐剂作用,本研究将50只雌性C57/BL6小鼠随机分为空白对照组(Saline)、单独免疫OVA组(OVA)、OVA+铝佐剂组(OVA+Alum)、OVA+核糖体蛋白组低、高(4mg/kg、20 mg/kg)剂量组。在实验的第1 d和第15d进行免疫,共免疫两次,在第29 d、36 d、43 d、50 d时时,利用ELISA法检测血清中OVA特异性抗体IgG水平。结果显示,与铝佐剂相比,OVA+核糖体蛋白S11组具有显著增强体液免疫应答效果,诱导更强烈的抗体反应,产生更高水平的特异性IgG,维持了更长时间的血清抗体水平;与OVA组相比,OVA+核糖体蛋白S11免疫后小鼠脾组织体积明显增大,脾组织重量增加明显;组织病理学染色结果表示OVA+核糖体蛋白S11免疫后小鼠脾组织的生发中心的大小和数量显著增加。CCK8法检测OVA+核糖体蛋白S11免疫后脾淋巴细胞增殖能力显著增强,表明核糖体蛋白S11可以诱导细胞免疫反应;以上结果表明核糖体蛋白S11具有成为新型佐剂的巨大潜力。
本发明的积极效果在于:提供了一种新的核糖体蛋白S11,是一种粪肠球菌产生的诱导先天免疫记忆活性物质核糖体蛋白;与灭活的白色念珠菌相比,其显示出较强的诱导天然免疫记忆作用;该核糖体蛋白具有良好的稳定性,低细胞毒性和溶血活性,为实际应用奠定理论基础,进一步的,确定了核糖体蛋白作为疫苗佐剂的作用,为未来开发为新型佐剂、同时具备先天免疫记忆和适应性免疫的双记忆疫苗奠定坚实的基础。
附图说明
图1. 核糖体蛋白S11的HPLC分析及SDS-PAGE检测;
图2. 核糖体蛋白S11的重组表达;
图3. 核糖体蛋白S11的诱导天然免疫记忆活性分析;
图4. 核糖体蛋白S11的细胞安全性评价;
图5. 核糖体蛋白S11的佐剂活性评价。
具体实施方式
下面由一些特定的具体实施例进一步说明本发明。应当明确,下面所描述的实施例是本发明的一部分实施例,而不是全部实施例,但并不因此将本发明限制在所述的实施例范围之中。下述实施例中未注明具体条件的实验方法,按照常规方法进行,或按照商品说明书进行。
实施例1、核糖体蛋白S11的纯化及鉴定
(1)发酵上清制备:
取-80℃冻存的粪肠球菌于TSB平板划线,37℃恒温培养至形成单菌落,然后挑取单菌落至5mL TSB液体培养基中,37℃、180rpm振荡培养3 h,调整菌液OD600=1.00,然后以1%(v/v)比例转接至10 L TSB液体培养基中,37℃、180 rpm振荡培养6 h,10000g、4℃下离心10 min,0.22μm滤器过滤得发酵上清;
(2)粗蛋白捕获:
将发酵上清以80%饱和度的硫酸铵溶液在4℃沉淀16 h,然后12000g、4℃下离心20min,收集沉淀,使用蒸馏水复溶,然后用1000 Da的透析袋透析除去硫酸铵,直至硫酸铵被完全去除,采用1%氯化钡检测硫酸铵是否除干净,以溶液中未出现白色沉淀为判断标准,最后通过真空冷冻干燥获得粗蛋白;
(3)纯化:
将粗蛋白应用离子交换层析和制备型HPLC进行纯化。将粗蛋白通过Q-Tanrose6FF阴离子交换层析柱,采用阶梯固定梯度洗脱方式,以0 M NaCl-1.0 M NaCl固定时间程序进行洗脱,以280 nm为检测波长,收集各洗脱峰,如图1的a所示,离子交换层析后得到6个组分。利用小鼠腹腔巨噬细胞天然免疫记忆模型,测定诱导天然免疫记忆的目标活性峰为F4组分;
将目标峰利用Ultimate XB-C18液相色谱柱经HPLC进行纯化,流动相A为蒸馏水;B为乙腈,0-30 min线性梯度洗脱,如图1的b所示,得到四个洗脱组分。经过天然免疫记忆模型确定活性组分为F4-2组分。多次重复进样后,将检测具有诱导天然免疫记忆的活性峰合并,冷冻干燥获得核糖体蛋白S11;
进一步用分析型HPLC检测纯度和SDS-PAGE检测分子量大小,结果如图1的c和1的d所示,HPLC结果显示在3.468 min出现纯度较高的单一峰,且SDS-PAGE检测在接近14 kDa处有单一条带;然后将目的条带切下进行MALDI-TOF/TOF质谱鉴定,鉴定结果显示为核糖体蛋白S11,氨基酸序列如SEQ ID NO.1所示。
实施例2、核糖体蛋白S11的重组表达
根据鉴定的核糖体蛋白S11序列及比对分析获得的相应编码基因,设计引物如下:
上游引物F:CTGGGATCCATGGCAGCAAAAAAAG;
下游引物R:CTGAAGCTTTTAAACACGACGGCG;
以粪肠球菌基因组为模板,PCR扩增核糖体蛋白S11目的基因;然后将核糖体蛋白S11目的基因片段与表达载体pET-28a质粒,经酶切、连接、转化后,应用菌液PCR对阳性克隆进行检测,结果显示表达载体构建成功;进一步,将测序正确的重组质粒转化至表达菌株(大肠杆菌BL21(DE3))中,应用菌液PCR筛选获得阳性克隆表达菌株。将得到的重组表达菌株在37℃,180 rpm振荡培养至OD600=0.6-0.8时,再加入异丙基-β-D-硫代半乳糖苷至终浓度为500 μmol/L,16℃,160 rpm诱导24 h;
将得到的发酵液8000g、4℃离心10 min,收集菌体。超声破碎菌体后的上清10000g、4℃离心20 min,应用镍柱进行亲和层析纯化,分别用20 mM、100 mM咪唑洗脱杂蛋白,收集500 mM咪唑洗脱部分,应用超滤除去咪唑、甘油等杂质,然后进行真空冷冻干燥纯化得到重组表达核糖体蛋白,利用SDS-PAGE检测核糖体蛋白S11的表达和纯度,如图2的a所示,诱导后菌体中核糖体蛋白S11的表达量增加,利用镍柱纯化后500 mM咪唑洗脱后得到纯度较高的核糖体蛋白S11;
小鼠腹腔巨噬细胞天然免疫记忆模型结果显示重组后的核糖体蛋白S11具有诱导天然免疫记忆的活性,如图2的b所示,重组核糖体蛋白S11预刺激小鼠后,腹腔巨噬细胞经过R848(TLR7/TLR8激动剂)刺激后,与对照组相比,TNF-α的分泌水平显著增加,表明核糖体蛋白预刺激可以增强接受第二次刺激时炎性细胞因子的分泌,以上结果说明核糖体蛋白S11可被重组表达成功。
实施例3、核糖体蛋白S11的诱导天然免疫记忆活性分析
利用前期研究建立的大蜡螟天然免疫记忆模型和小鼠腹腔巨噬细胞天然免疫记忆模型分析了纯化和重组表达的核糖体蛋白S11的诱导天然免疫记忆活性。
小鼠于-7 d注射等剂量的纯化的核糖体蛋白S11(F4-2)和重组的核糖体蛋白S11,-4d注射3%的巯基乙酸盐肉汤募集腹腔巨噬细胞,第0 d分离细胞,待细胞贴壁,以10ng/mL 的LPS进行第二次刺激,24 h后离心收集细胞培养上清,检测炎性介质一氧化氮的释放和炎性细胞因子TNF-α的分泌水平,如图3的a和3的b所示,纯化和重组的核糖体蛋白S11均可以显著增强炎性介质NO的释放和炎性细胞因子TNF-α的分泌水平。大蜡螟初次刺激注射等剂量的纯化的核糖体蛋白S11(F4-2)和重组的核糖体蛋白S11,注射等体积的PBS作为对照,经过3d的间隔期,第二次刺激注射致死剂量的白色念珠菌,评价初次刺激的保护作用。如图3的c所示,纯化和重组表达的核糖体蛋白S11在保护大蜡螟免受致死剂量的白色念珠菌感染时作用相当。腹腔巨噬细胞吞噬和杀伤金黄色葡萄球菌的结果表明,如图3的d所示,核糖体蛋白S11诱导的天然免疫记忆增强了巨噬细胞对金黄色葡萄球菌的吞噬能力;如图3的e和3的f所示,金黄色葡萄球菌感染细胞6 h和24 h后,成功诱导天然免疫记忆的巨噬细胞对胞内金黄色葡萄球菌的杀伤能力增强,显著降低了胞内金黄色葡萄球菌的数量。以上结果表明,纯化和重组的核糖体蛋白S11具有相当的诱导先天免疫记忆的作用。
实施例4、核糖体蛋白S11的细胞安全性评价
核糖体蛋白S11的溶血活性测定方法为采用抗凝管收集鼠和鸡的血液,将血液转移到离心管中,1500g室温离心5min后,将上层液体及白细胞层吸出,加入适量PBS重悬,再次离心后吸出上层液体及白细胞层,2000g室温离心10 min后,吸出上层清液,加入适量PBS,配置成2%的红细胞悬液。然后将红细胞悬液加入96孔板中,每孔200 μL,向孔中加入核糖体蛋白S11,使终浓度分别为0.03125、0.0625、0.125、0.25、0.5、1.0 mg/mL,加入1%的Triton X-100作为阳性对照,混合均匀,置于37℃恒温培养箱中孵育1h,将细胞以1000g室温离心5min,收集上清液,测定OD450值,计算溶血率。如图4的a和4的b所示,在核糖体蛋白S11浓度低于0.5 mg/mL,对小鼠红细胞和鸡的红细胞均没有溶血作用。利用小鼠腹腔巨噬细胞和RAW 264.7细胞测定核糖体蛋白S11的细胞毒性,将细胞接种至96孔板中孵育,待细胞贴壁,加入终浓度为0.03125、0.0625、0.125、0.25、0.5、1.0 mg/mL的核糖体蛋白S11,同时设置不含核糖体蛋白S11及不含细胞的孔作为对照,加入0.5%的Triton X-100作为阳性对照,37℃恒温培养箱孵育48 h后,PBS洗两次,加入新鲜培养基和CCK8试剂,孵育1 h,测定OD450值,计算细胞的存活率,以此评价核糖体蛋白S11的细胞毒性作用。如图4的c和4的d所示,核糖体蛋白S11浓度低于0.5 mg/mL,对小鼠腹腔巨噬细胞、RAW264.7细胞均没有明显的细胞毒性。这些结果也证明,在有效使用浓度下,核糖体蛋白S11不具有细胞毒性和溶血活性,为进一步的临床应用奠定基础。
实施例5、核糖体蛋白S11的佐剂活性评价
为研究核糖体蛋白S11对模式抗原卵清白蛋白(OVA)免疫小鼠的佐剂作用,本研究将50只雌性C57/BL6小鼠随机分为空白对照组(Saline)、单独免疫OVA组(OVA)、OVA+铝佐剂组(OVA+Alum)、OVA+核糖体蛋白组低、高(4 mg/kg、20 mg/kg)剂量组。如图5的a所示,在实验的第1 d和第15 d进行免疫,共免疫两次,分别于第29 d、36 d、43 d、50 d时,利用ELISA法检测血清中OVA特异性抗体IgG水平;我们检测了免疫前后小鼠的体重,如图5的b所示,结果表明免疫前后各组小鼠体重均增加无显著性差异,表明核糖体蛋白免疫后对小鼠体重没有明显影响;免疫后第50天处死小鼠,分离脾组织,如图5的c所示,OVA+20 mg/kg核糖体蛋白免疫组小鼠脾组织与OVA组相比较脾组织重量显著增加;免疫后第29 d分析了血清中的IgG抗体水平,如图5的d所示,结果表明,与OVA组相比,OVA+4 mg/kg、20 mg/kg核糖体蛋白组血清中的IgG抗体水平均高于OVA组,与铝佐剂相比,OVA+核糖体蛋白组具有显著增强体液免疫应答效果,诱导更强烈的抗体反应,产生更高水平的特异性IgG;此外,我们检测了血清中IgG抗体的维持时间,如图5的f所示,在第二次加强免疫后的四周内,核糖体蛋白免疫组血清中OVA特异性抗体水平均维持了较长时间;如图5的g所示,组织病理学染色结果表示OVA+核糖体蛋白免疫后小鼠脾组织的生发中心的大小和数量显著增加;淋巴细胞增殖反映了细胞免疫应答的水平,如图5的e所示,CCK8法检测OVA+核糖体蛋白免疫后脾淋巴细胞增殖能力显著增强,表明核糖体蛋白S11可以诱导细胞免疫反应。
由上述实施例可以看出,本发明提供了一种诱导天然免疫记忆活性物质核糖体蛋白S11,并阐明了其纯化制备方法,同时,提供了一种核糖体蛋白S11的重组表达方法。进一步,评估了核糖体蛋白S11诱导天然免疫记忆活性及佐剂活性分析,但也可以看出,在本发明基础上,可以对之做出一些修改和拓展,因此,在不偏离本发明精神和原则的基础上所做的修改或改进,均属于本发明要求保护的范围。
Claims (5)
1.一种核糖体蛋白S11,其特征在于:
其氨基酸序列如SEQ ID NO.1所示,编码基因如SEQ ID NO.2所示。
2.如权利要求1所述的一种核糖体蛋白S11的纯化制备方法,包括如下步骤:
(1)发酵上清制备:
将保存于-80℃的粪肠球菌于TSB平皿划线,37℃恒温培养至出现单菌落,然后挑取单菌落至5 mL TSB液体培养基中,37℃、180 rpm振荡培养3 h,然后以1%(v/v)比例转接至10L TSB液体培养基中,37℃、180 rpm振荡培养6 h,10000g、4℃下离心10 min,0.22μm滤器过滤得发酵上清;
(2)粗蛋白捕获:
将发酵上清以80%饱和度硫酸铵溶液在4℃沉淀16 h,然后12000g、4℃下离心20 min,收集沉淀,使用蒸馏水复溶,然后用1000 Da的透析袋透析除去硫酸铵,直至硫酸铵被完全去除,采用1%氯化钡检测硫酸铵是否除干净,最后通过真空冷冻干燥获得粗蛋白,保存于-80℃;
(3)纯化:将粗蛋白应用离子交换层析和制备型HPLC进行纯化;
将粗蛋白通过Q-Tanrose 6FF阴离子交换层析柱,采用阶梯固定梯度洗脱方式,以0 MNaCl-1.0 M NaCl固定时间程序进行洗脱,每个梯度洗脱3个柱体积,流速为1mL/min,以280nm为检测波长,收集各洗脱峰,利用小鼠腹腔巨噬细胞天然免疫记忆模型,确定诱导天然免疫记忆的目标活性峰;
将目标峰利用Ultimate XB-C18液相色谱柱经HPLC进行纯化,流动相A为蒸馏水;B为乙腈,线性梯度洗脱,洗脱程序为:0-5min,20%乙腈洗脱;5-25 min,20%-100%乙腈洗脱;25-30min,100%乙腈洗脱;
多次重复进样后,将检测具有诱导天然免疫记忆的活性峰合并,冷冻干燥获得核糖体蛋白S11。
3.如权利要求1所述的核糖体蛋白S11的重组表达方法,包括如下步骤:
(1)构建表达载体:设计引物,扩增核糖体蛋白S11基因,经酶切和连接,并且通过转化构建表达菌株大肠杆菌BL21(DE3);
(2)诱导表达:得到的重组表达菌株在37℃,180 rpm振荡培养至OD600=0.6-0.8时,再加入异丙基-β-D-硫代半乳糖苷至终浓度为500 μmol/L,16℃,160 rpm诱导24 h;
(3)纯化:得到的发酵液在8000g、4℃离心10 min,收集菌体;超声破碎菌体后的上清10000g、4℃离心20 min,应用镍柱进行亲和层析纯化,分别用20 mM、100 mM咪唑洗脱杂蛋白,收集500 mM咪唑洗脱部分,应用超滤除去咪唑、甘油等杂质,然后进行真空冷冻干燥纯化得到重组表达核糖体蛋白。
4.如权利要求1所述的一种核糖体蛋白S11在制备诱导天然免疫记忆制剂中的用途。
5.如权利要求1所述的一种核糖体蛋白S11在制备疫苗佐剂中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310021719.2A CN116199750A (zh) | 2023-01-06 | 2023-01-06 | 一种核糖体蛋白s11及制备方法和在疫苗佐剂中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310021719.2A CN116199750A (zh) | 2023-01-06 | 2023-01-06 | 一种核糖体蛋白s11及制备方法和在疫苗佐剂中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116199750A true CN116199750A (zh) | 2023-06-02 |
Family
ID=86507047
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310021719.2A Pending CN116199750A (zh) | 2023-01-06 | 2023-01-06 | 一种核糖体蛋白s11及制备方法和在疫苗佐剂中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116199750A (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1268178A (zh) * | 1997-05-06 | 2000-09-27 | 人体基因组科学有限公司 | 粪肠球菌多核苷酸和多肽 |
CN105188748A (zh) * | 2013-04-08 | 2015-12-23 | 米迪缪尼有限公司 | 疫苗组合物及使用方法 |
WO2016030872A1 (en) * | 2014-08-31 | 2016-03-03 | Instytut Biochemii I Biofizyki Polskiej Akademii Nauk | Enterococcus faecalis strains for the production of bacteriophage preparations |
CN113721022A (zh) * | 2021-09-07 | 2021-11-30 | 中国农业科学院油料作物研究所 | 农田黄曲霉毒素产毒菌相对丰度快速鉴别方法及其应用 |
EP4067373A1 (en) * | 2019-11-27 | 2022-10-05 | Onegene Biotechnology Inc. | Multifunctional multispecific multimeric biomolecule polymer having prolonged in-vivo duration |
-
2023
- 2023-01-06 CN CN202310021719.2A patent/CN116199750A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1268178A (zh) * | 1997-05-06 | 2000-09-27 | 人体基因组科学有限公司 | 粪肠球菌多核苷酸和多肽 |
CN105188748A (zh) * | 2013-04-08 | 2015-12-23 | 米迪缪尼有限公司 | 疫苗组合物及使用方法 |
WO2016030872A1 (en) * | 2014-08-31 | 2016-03-03 | Instytut Biochemii I Biofizyki Polskiej Akademii Nauk | Enterococcus faecalis strains for the production of bacteriophage preparations |
EP4067373A1 (en) * | 2019-11-27 | 2022-10-05 | Onegene Biotechnology Inc. | Multifunctional multispecific multimeric biomolecule polymer having prolonged in-vivo duration |
CN113721022A (zh) * | 2021-09-07 | 2021-11-30 | 中国农业科学院油料作物研究所 | 农田黄曲霉毒素产毒菌相对丰度快速鉴别方法及其应用 |
Non-Patent Citations (2)
Title |
---|
LIU, S.等: ""Enterococcus faecalis strain T30 chromosome, complete genome"", 《GENBANK》, pages 113831 * |
王涛 等: ""长春地区鸡源奇异变形杆菌的分离鉴定及毒力分析"", 《微生物学通报》, vol. 49, no. 12, pages 4987 - 4998 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20160008454A1 (en) | Compositions and methods for treatment of microbial infections | |
CN106176609A (zh) | 一种nk细胞膜仿生脂质体药物载体、制作方法及其应用 | |
Martell et al. | Host defense peptides as immunomodulators: The other side of the coin | |
CN116102640A (zh) | 重组乳铁蛋白衍生肽及其在提高免疫力方面的应用 | |
JPH10500128A (ja) | マイコバクテリア感染症に対するワクチン | |
CN101514229B (zh) | 人干扰素α衍生物及其聚乙二醇化修饰物 | |
CN111548395A (zh) | 一种口蹄疫病毒二价多表位重组病毒样颗粒及其应用 | |
CN112662695B (zh) | 一种细菌生物膜囊泡bbv作为疫苗载体的构建方法和应用 | |
CN101094686A (zh) | 鞭毛蛋白在肿瘤免疫疗法中的用途 | |
CN108948163B (zh) | 澳洲坚果植物防御素及其应用 | |
CN103948910A (zh) | 免疫调节多肽zl-1在制备抗肿瘤药物中的用途 | |
JP5227028B2 (ja) | インターロイキン−2の中和能を有する免疫治療用製剤 | |
CN116199750A (zh) | 一种核糖体蛋白s11及制备方法和在疫苗佐剂中的应用 | |
CN101544693B (zh) | 重组胸腺素α1二串体蛋白及其制备方法 | |
KR102061735B1 (ko) | 황색포도알균 약독화 장독소 및 세포독소 재조합 단백질을 포함하는 백신 조성물 | |
CN110714000A (zh) | 一种Toll样受体配体蛋白在抗细菌感染中的应用 | |
CN104667272A (zh) | 一种动物免疫佐剂及其制备方法和使用方法 | |
RU2311197C1 (ru) | Способ получения протективной белоксодержащей фракции бактерий | |
CN102266551B (zh) | 可用于治疗人或动物免疫相关性疾病的白介素-4治疗性疫苗 | |
CN109022522B (zh) | 一种gfp-2小肽及其制备方法和应用 | |
CN113603793A (zh) | 一种新型冠状病毒的重组s蛋白、重组质粒、重组菌及制备外泌体药物或外泌体疫苗的应用 | |
JPH08500846A (ja) | 抗エイズ免疫調節剤複合体 | |
CN111088269B (zh) | OprJ-N-M融合基因及其融合蛋白、融合蛋白制备方法以及用途 | |
US11564993B2 (en) | Intranasal vaccine that induces cellular immunity | |
RU2786222C1 (ru) | Поликомпонентная вакцина для иммунопрофилактики и иммунотерапии заболеваний, вызываемых условно патогенными микроорганизмами |
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 |