CN106928326A - A kind of coronavirus vaccine of the receptor binding domain subunit based on dimerization - Google Patents
A kind of coronavirus vaccine of the receptor binding domain subunit based on dimerization Download PDFInfo
- Publication number
- CN106928326A CN106928326A CN201511021535.8A CN201511021535A CN106928326A CN 106928326 A CN106928326 A CN 106928326A CN 201511021535 A CN201511021535 A CN 201511021535A CN 106928326 A CN106928326 A CN 106928326A
- Authority
- CN
- China
- Prior art keywords
- rbd
- antigen
- seq
- cov
- albumen
- 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.)
- Granted
Links
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 22
- 238000006471 dimerization reaction Methods 0.000 title claims abstract description 15
- 229960005486 vaccine Drugs 0.000 title abstract description 23
- 102000005962 receptors Human genes 0.000 title abstract description 7
- 108020003175 receptors Proteins 0.000 title abstract description 7
- 239000000539 dimer Substances 0.000 claims abstract description 28
- 241000238631 Hexapoda Species 0.000 claims abstract description 12
- 239000000427 antigen Substances 0.000 claims description 37
- 102000036639 antigens Human genes 0.000 claims description 37
- 108091007433 antigens Proteins 0.000 claims description 37
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 241000711443 Bovine coronavirus Species 0.000 claims description 12
- 150000001413 amino acids Chemical class 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 230000005847 immunogenicity Effects 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- 239000002671 adjuvant Substances 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000004411 aluminium Substances 0.000 claims description 7
- 229910052782 aluminium Inorganic materials 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 239000003636 conditioned culture medium Substances 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 238000001890 transfection Methods 0.000 claims description 4
- 108020005038 Terminator Codon Proteins 0.000 claims description 3
- 238000005349 anion exchange Methods 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- 230000009261 transgenic effect Effects 0.000 claims description 3
- 238000013519 translation Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 239000003795 chemical substances by application Substances 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims 1
- 239000000178 monomer Substances 0.000 abstract description 27
- 208000025370 Middle East respiratory syndrome Diseases 0.000 abstract description 21
- 230000003472 neutralizing effect Effects 0.000 abstract description 14
- 102100031673 Corneodesmosin Human genes 0.000 abstract description 10
- 101710139375 Corneodesmosin Proteins 0.000 abstract description 10
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 abstract description 7
- 230000002163 immunogen Effects 0.000 abstract description 2
- 239000001749 Calcium fumarate Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 31
- 241000315672 SARS coronavirus Species 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 241001112090 Pseudovirus Species 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 241000700605 Viruses Species 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 7
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000006386 neutralization reaction Methods 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 2
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 2
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 2
- 229940124678 MERS-CoV vaccine Drugs 0.000 description 2
- 101710141347 Major envelope glycoprotein Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 2
- 108091005634 SARS-CoV-2 receptor-binding domains Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 235000014304 histidine Nutrition 0.000 description 2
- 150000002411 histidines Chemical class 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 206010034674 peritonitis Diseases 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000029058 respiratory gaseous exchange Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 description 1
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000283725 Bos Species 0.000 description 1
- 241000711506 Canine coronavirus Species 0.000 description 1
- 208000001528 Coronaviridae Infections Diseases 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 101000955144 Drosophila melanogaster WASH complex subunit 1 Proteins 0.000 description 1
- 241000711475 Feline infectious peritonitis virus Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 101710114810 Glycoprotein Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 101710167605 Spike glycoprotein Proteins 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 208000030303 breathing problems Diseases 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940090124 dipeptidyl peptidase 4 (dpp-4) inhibitors for blood glucose lowering Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- A61K39/12—Viral antigens
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- A61K2039/55505—Inorganic adjuvants
-
- 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
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of coronavirus vaccine of the receptor binding domain subunit based on dimerization, belong to pharmaceutical technology field.The present invention expresses RBD (E367 Y606) regions of MERS CoV albumen and the RBD (R294 F515) of ARS CoV by baculoviral body in insect cell, so that RBD by the cysteine residues of S protein itself 603 can form dimer or form dimer by the cysteine residues of S protein itself 512, Balb/c mouse are immunized using the RBD albumen dimer and monomer of purifying respectively.The RBD of dimerization of the invention overcomes the not enough shortcoming of RBD monomeric immunogenics, and the neutralizing antibody for substantially increasing mouse for MERS CoV is produced.
Description
Technical field
The present invention relates to a kind of coronavirus vaccine of the receptor binding domain subunit based on dimerization, belong to medical science
Field.
Background technology
Coronavirus belongs to coronaviridae coronavirus genus, is the positive chain RNA virus for having cyst membrane, in all RNA virus
In its genome it is maximum.Animals and humans are all the hosts of coronavirus.Coronavirus mainly infects mammal and birds
Respiratory tract and alimentary canal.Wherein there are 6 kinds of coronavirus infection people.That wherein threaten maximum to global public health is 2002-
2003 China's outburst serious respiration syndrome coronavirus (SARS-CoV) and break out and continue to this day within 2012
Eastern respiration syndrome coronavirus (MERS-CoV).Coronavirus also triggers many serious Animal diseases, especially to agriculture domestic animal
Poultry and Belt for pets are seriously threatened.Such as, transmissible gastro-enteritis virus (TGEV) can trigger the severe diarrhea of pig, the death rate
It is high；Its deleted virus PRCV (PRCV) can cause the serious breathing problem of pig；Cat peritonitis virus
(FIPV) cat peritonitis and ascites can be caused to assemble, fatal rate is very high；Canine coronavirus (CCoV) can then make dog that different journeys occur
The enterogastritis symptom of degree, propagates fast, hardly possible control；Porcine epidemic diarrhea virus (PEDV) causes the enteron aisle disease such as pig epidemic diarrhea
Disease, easily propagates fatal rate very high in swinery.In addition with coronavirus such as mouse, oxen.These coronavirus are to humans and animals
Health cause serious threat.Therefore, develop a kind of safe and effective for coronavirus SARS-CoV and MERS-CoV
Vaccine it is very urgent, have great significance.
Surface spikes albumen (S protein) is the main neutralization antigen of coronavirus.MERS-CoV, SARS-CoVS albumen
Receptor binding domain (Receptor Binding Domain, RBD) is considered as to induce body to produce the topmost of neutralizing antibody
Antigen target region.Body can be stimulated the neutralizing antibody for producing more to focus on the acceptor knot for virus by RBD as vaccine
Close, the immunogenicity and Immune efficiency of vaccine can be improved.MERS-CoV by the acceptor of RBD and host cell (CD26, also known as
DPP4) combine and invade cell.Additionally, SARS-CoV is combined with host cell receptor ACE2 by its RBD and is entered cell.
Currently reported prepares the vaccine for being directed to MERS-CoV and SARS-CoV by using coronavirus RBD, makes
It is the albumen of RBD albumen or fusion monomer RBD with Fc areas of monomer.Inside MERS-CoV, the RBD for reporting before is mono-
Body is only capable of evoking relatively low pseudovirus neutralizing antibody level in Mice Body.This prompting uses the RBD monomer conducts of MERS-CoV
The immunogenicity of vaccine is limited.It is therefore desirable to be improved to the vaccine based on MERS-CoVRBD.Have been reported that again before and pass through
RBD C-terminal merge IgG Fc regions, expression prepares RBD-Fc be used for for MERS-CoV vaccine.RBD-Fc can be lured
Pseudovirus neutralization activity higher is given birth in artificial delivery.But as vaccine, its security also needs to many deep grinding to protein fusion expression Fc
Study carefully evaluation, limit the progress of its Clinical practice.
Additionally, the research of SARS-CoV vaccines also uses RBD monomers before, with certain neutralization immunogenicity.
In a word, the vaccine that the RBD monomers of coronavirus S protein are developed is currently based on, immunogenicity is limited.And it is based on RBD
Fusion protein (Fc sections of such as IgG) then there is the not enough defect of safety evaluation.Therefore, develop that a kind of immunogenicity is high, peace
The more approved SARS-CoV or MERS-CoV vaccines of full property, are the problems of current urgent need to resolve.
The content of the invention
In order to solve the above problems, the present invention develops a kind of new method that vaccine is designed based on coronavirus RBD, utilizes
The cysteine residues of MERS-CoV, SARS-CoV itself form homodimer to prepare vaccine, and vaccine of the invention can show
The level of raising host's neutralizing antibody of work, preparation method is simple, albumen is without label and is easy to purifying, can be applied to face faster
The use of bed.
First purpose of the invention is to provide the Bovine Coronavirus Antigen that a kind of immunogenicity is improved, and the antigen is dimerization
The MERS-CoV albumen of change or the RBD of SARS-CoV albumen.
The amino acid sequence of the RBD is such as shown in (1) or (2)：
(1) such as SEQ ID NO:1 or SEQ ID NO:Amino acid sequence shown in 2；
(2) amino acid sequence in (1) is substituted, lacks or adds an amino acid or several amino acid and with phase
Antigenic polypeptide as derived from (1) or its analog, and the albumen itself of its coding can form dimer.
The SEQ ID NO:1 sequence, the S protein from MERS-CoV EMC/2012 plants is (on NCBI
GenBank:AFS88936.1 a part), is the E367-Y606 regions of the RBD of MERS-CoV albumen.
The SEQ ID NO:2 sequence, from SARS-CoV BJ01 plants of S protein (spike glycoprotein
S, NCBI are GenBank:AAP30030.1 a part), is the R294-F515 regions of the RBD of SARS-CoV.
In one embodiment of the invention, the nucleotide sequence of the RBD of the dimerization such as SEQ ID NO:3 or
SEQ ID NO:Shown in 4.
Second object of the present invention is to provide the preparation method of the Bovine Coronavirus Antigen, is in SEQ ID NO:3 or
Person SEQ ID NO:3 ' ends of nucleotide sequence shown in 4 add translation termination codon, carry out clonal expression, the correct weight of screening
Group, then transfection insect cell is expressed, and cell conditioned medium is collected after expression, and purifying obtains Bovine Coronavirus Antigen.
In one embodiment of the invention, the insect cell is Insect cells Sf9 or insect cell line Hi5.
In one embodiment of the invention, the purification process of the Bovine Coronavirus Antigen, is that will express the antigen
Cell supernatant be filtered to remove cell fragment, and the capture of destination protein is carried out by HiTrap QHP anion-exchange columns,
Thick pure, the component loading drainage column containing destination protein afterwards is carried out by the buffer solution elution containing 1M NaCl immediately
HiTrap Phenyl HP, then carry out gradient elution, and the eluting peak containing target protein that will be obtained merges, then 10K retentions
Concentration, then carries out sieve chromatography and is purified using the pillars of Superdex200 Hiload 16/60 (GE).
In one embodiment of the invention, the gradient elution is to use 20mM Tris-HCl, pH 8.0.
In one embodiment of the invention, the sieve chromatography buffer solution is 20mM Tris, 150mM NaCl,
PH8.0。
In one embodiment of the invention, the destination protein is the MERS-RBD albumen or SARS- of dimerization
RBD albumen；Size is about 60Kd in the case that the MERS-RBD albumen of the dimerization (is not added with DTT) under non reducing conditions,
Size is about 30Kd (to add DTT) under the reducing conditions；The SARS-RBD albumen of dimerization size under non reducing conditions
About 55kd, size about 28Kd under reducing condition.
Third object of the present invention is to provide the DNA for encoding the antigen, and carrier, the transgenosis for expressing the antigen are thin
Born of the same parents system or recombinant bacterium, the expression cassette for carrying the antigen.
The present invention also provides the application of the antigen, is the medicine for preparing anti-coronavirus.
In one embodiment of the invention, the application is by amino acid sequence such as SEQ ID NO:1 antigen with
MF59 or aluminium adjuvant are shared.
In one embodiment of the invention, the application is by amino acid sequence such as SEQ ID NO:2 antigen with
MF59 or aluminium adjuvant are shared.
In one embodiment of the invention, the application, is for reagent preparation box；Containing in the kit
State antigen, or encode the DNA molecular of the antigen, or the recombinant vector/expression cassette/transgenic cell for expressing the antigen
System/recombinant bacterium.
Fourth object of the present invention is to provide a kind of method for improving antigen immunogenicity, and methods described is expression dimerization
The MERS-CoV albumen of change or the RBD of SARS-CoV albumen；The amino acid sequence of the RBD of the dimerization such as SEQ ID NO:
1 or SEQ ID NO:Shown in 2.
Beneficial effects of the present invention：
The present invention expresses RBD (E367-Y606) region of MERS-CoV albumen by baculoviral body in insect cell,
So that RBD can form dimer by the cysteine residues of S protein itself 603.And after this cysteine is removed,
RBD then only forms monomer (E379-E589).And then, respectively using the RBD albumen dimer and monomer of purifying to Balb/c mouse
It is immunized.The RBD of dimerization of the invention overcomes the not enough shortcoming of RBD monomeric immunogenics, substantially increases mouse pin
Neutralizing antibody to MERS-CoV is produced.SARS-CoV, the RBD of present invention expression SARS-CoV are generalized to according to same thinking
(R294-F515), dimer is formed by the cysteine residues of S protein itself 512.SARS-CoV RBD bis- after purification
Aggressiveness is significantly improved than monomer (cysteine residues of 512 are mutated into serine) immunogenicity.
Brief description of the drawings
Fig. 1：MERS RBD protein purifications molecular sieves and SDS-PAGE scheme；M represents LMWP Marker, Dimer
Dimer is represented, Monomer represents monomer；
Fig. 2：SARS RBD protein purifications molecular sieves and SDS-PAGE scheme；M represents LMWP Marker, Dimer
Dimer is represented, Monomer represents monomer；
Fig. 3：Mouse immune strategy.
Specific embodiment
Embodiment 1：It is prepared by the recombinant baculovirus for expressing MERS and SARS antigens
By MERS-CoV RBD nucleotide sequences (such as SEQ ID NO:Shown in 3) and SARS-CoV RBD (such as SEQ ID
NO:Shown in 4) end of nucleotide sequence 3 ' plus translation termination codon rear clone to the pFastBac carriers comprising gp67 signal peptides
(pFastBac-SP) between EcoR I and Xho I restriction enzyme sites so that protein-coding region is behind signal peptide gp67 sequences
Amalgamation and expression, for the secretion of destination protein.Respectively obtain carrier pFastBac-SP-MERS-RBD and pFastBac-SP-
SARS-RBD。
And it is used to express the carrier of MERS monomers RBD then by S protein sequence (the upper GenBank of NCBI:AFS88936.1 in)
The nucleic acid fragment of 379 to 589 albumen of amino acid inserts EcoRI the and XhoI restriction enzyme sites of pFastBac-SP, and at 3 ' ends
Take 6 histidines and constitute.For expressing the carrier of SARS monomers RBD then by SEQ ID NO:Amino acid sequence shown in 2
The cysteine mutation of the 219th into serine (for GenBank on NCBI:The of the S protein of AAP30030.1
512), and take 6 histidines at 3 ' ends and constitute.
Recombinant plasmid transformed DH10Bac competent cells, 37 DEG C of incubated overnights are screened by blue hickie and PCR are identified
Positive colony.The baculovirus DNA (Bacmid) of restructuring is extracted, by the correct recon of sequencing identification.Bacmid is transfected
Insect cells Sf9, transfection collects culture supernatant after 3 days, obtains 1st generation recombinant baculovirus.Continuously expand malicious 3 generation, obtained for the 4th generation
Baculoviral carries out virus titer measure.Suitable virus MOI infection Sf9 or insect cell line are chosen according to titration results
Hi5 is expressed, and cell conditioned medium is collected by centrifugation after 48 hours, carries out cell purification.
Embodiment 2：The purifying of MERS-RBD and SARS-RBD
The cell conditioned medium of MERS-RBD and SARS-RBD will be expressed by 0.22 μm of membrane filtration, cell fragment is removed.
And the capture of destination protein is carried out by HiTrap QHP anion-exchange columns, washed by the buffer solution containing 1M NaCl immediately
It is de- carry out it is thick pure.By collecting and combining the component containing destination protein after SDS-PAGE identifications.As A liquid loading drainage columns
HiTrap Phenyl HP are combined.Preparing less salt eluent B liquid (20mM Tris-HCl PH 8.0) carries out gradient elution.
The RBD that will be eluted carries out sds gel electrophoresis testing goal albumen.Obtain containing washing for destination protein by the way that hydrophobic chromatography is slightly pure
De- peak merges, and retaining concentration tube (Millipore) by 10K carries out being concentrated into 5mL, then by Superdex200 Hiload
16/60 pillar (GE) carries out sieve chromatography and carries out further destination protein purifying.Sieve chromatography buffer solution is 20mM
Tris, 150mM NaCl, PH8.0.
Taking eluting peak of the MERS-RBD albumen near elution volume 78mL carries out SDS-PAGE analyses.Destination protein is non-
Size is about 60Kd in the case of (being not added with DTT) under reducing condition；And (add DTT) under the reducing conditions, size is about 30Kd,
Prove the peak mainly dimer.Taking the eluting peak near elution volume 90mL carries out SDS-PAGE analyses, and destination protein is non-
Size is about 30Kd in the case of (being not added with DTT) under reducing condition, it was demonstrated that the peak is mainly RBD monomers.Same SARS-RBD
Eluting peak of the albumen near elution volume 82mL is mainly dimer (size about 55kd under non reducing conditions, under reducing condition
Size about 28Kd), and the eluting peak near elution volume 92mL is mainly RBD monomers, and (size is about under non reducing conditions
28kd)。
Typical MERS-RBD and SARS-RBD protein moleculars sieve collection of illustrative plates and SDS-PAGE analysis charts are respectively such as Fig. 1,2 institutes
Show.The eluting peak of RBD dimers is collected and combined, traveling one is entered again through the pillars of Superdex200 Hiload 16/60 (GE)
Step purifying, obtains dimer RBD albumen of the purity more than 99%.After destination protein concentration, aliquot is distributed into, it is cold rapidly with liquid nitrogen
Freeze after -80 DEG C of preservations.
MERS-RBD and SARS-RBD with His labels are carried out thick pure by nickel post.By the cell supernatant after filtering with
Histrap_HP in 4 DEG C of combinations overnight.Afterwards, washed first by buffer solution (20mM Tris, 150mM NaCl, pH8.0)
Wash, the foreign protein that removal is flowed through.Thereafter, by containing imidazole buffer (20mM Tris, 150mM NaCl, pH 8.0,300mM
Imidazole destination protein wash-out) is carried out.Retaining concentration tube (Millipore) using 10K carries out being concentrated into volume 5mL, then leads to
Crossing the pillars of Superdex200Hiload 16/60 (GE) and carrying out sieve chromatography carries out further destination protein purifying.Molecule
Sieve chromatography buffer solution is the solution of 20mM Tris-HCl, 150mM NaCl, PH8.0.MERS-RBD is near elution volume 90mL
Eluting peak be RBD monomers, size is about 28Kd.There is no dimer peak.Wash-outs of the SARS-RBD near elution volume 92mL
Peak is RBD monomers, and size is about 28Kd.There is no dimer peak.Destination protein is collected and combined, it is dense by concentration tube (Milipore)
After contracting, aliquot is distributed into, with liquid nitrogen quick freeze after -80 DEG C of preservations.
Embodiment 3：Mouse immune is tested
MERS the and SARS antigens that embodiment 2 is obtained dilute according to the method for table 1 in physiological saline, and enter with adjuvant
Row packet emulsification.Then packet is carried out to the Balb/C mouse of 4-6 week old immune.Immunization strategy such as Fig. 3, i.e., by leg muscle
The mode of injection, every mouse receives 3 vaccine immunities, the inoculum of each 100ul on the 42nd day respectively at the 0th day, the 21st day
Product.54th day (i.e. 3 exempt from after the 12nd day), carry out afterbody and take blood to mouse.Mice serum treats that serum is analysed in standing a period of time
After going out, it is centrifuged 10 minutes by 3000rpm and is obtained, and in -20 DEG C of Refrigerator stores, in pseudovirus and detects.
Table 1：Animal immune is grouped situation
| Antigen or control | Antigenic content | Adjuvant | Size of animal |
| PBS | - | MF59 | 4 |
| PBS | - | Aluminium adjuvant | 4 |
| RBD dimers | 3μg,10μg | MF59 | 3 each 4 of μ g, 10 μ g |
| RBD monomers | 3μg,10μg | MF59 | 3 each 4 of μ g, 10 μ g |
| RBD dimers | 10 μ g*, 30 μ g | Aluminium adjuvant | 10 each 4 of μ g, 30 μ g |
| RBD monomers | 10 μ g*, 30 μ g | Aluminium adjuvant | 10 each 4 of μ g, 30 μ g |
* it is used for the immune of SARS-RBD and is free of 10 μ g antigen groups.
Embodiment 4：Pseudovirus neutralization test
I) preparation of pseudovirus：By plasmid pCAGGS-MERS S (refer to the food in one's mouth of expressing MERS S by transfection reagent PEI
Newborn animal carrier pCAGGS, wherein SARS pseudovirus are prepared and are used pCAGGS-SARS S) and HIV pNL4-3.luc.RE
(Invitrogen) cotransfection 293T cells.Transfection 5 hours after, PBS washed cells 2 times, be changed to plasma-free DMEM medium after
Continuous culture.Supernatant, centrifugation removal cell fragment are collected after 48 hours.Pseudovirus is centrifuged by supernatant ultracentrifuge 30000rpm
Precipitated within 3 hours, dissolved MERS the and SARS pseudovirus for obtaining concentration with small size serum-free DMEM afterwards.Packing be stored in-
80 DEG C of conditions.
Ii) pseudovirus TCID50Determine
The virus liquid that previous step is collected is added in the human liver cancer Huh7 cells in 96 orifice plates by 5 doubling dilutions.Infection
After 4 hours, virus liquid is discarded, the chi of PBS washed cells 2 is changed to the DMEM complete mediums containing 10% serum.48 as a child, abandons
Fall culture medium, PBS is washed 2 times, adds cell pyrolysis liquid.After -80 DEG C of freeze thawing once, 20 μ l are taken per hole using GloMax 96
Microplate Luminometer (Promega) detect uciferase activity value.TCID is calculated by Reed-Muech methods50。
Iii) neutralization test
2 times of doubling dilutions of antibody that will be purified, with 100TCID50Pseudovirus mixes, and 37 DEG C are incubated 30 minutes altogether.Will mixing
Liquid is added in 96 orifice plates for being paved with Huh7 cells.After 37 DEG C are incubated 4 hours, virus liquid is discarded, PBS washed cells 2 times are changed
It is the complete medium DMEM containing 10% serum.After 48 hours, nutrient solution is discarded, PBS washed cells 2 times add cell cracking
Liquid, detects uciferase activity value.
Iv) interpretation of result
Positive criteria：Uciferase activity value<The serum of (positive control-negative control)/2, as there is neutralization activity
Positive serum.Serum dilution highest the positive hole extension rate be calculated as NT50 (suppress 50% virus serum in and drip
Degree).The pseudovirus neutralizing antibody level of dimer and the serum of monomer RBD animals following immunizations is compared, checks right using t
Significance difference analysis are carried out between group.Judge significance finally by double tail probabilities P values.It is 0.05 in significance
In the case of, double tail probabilities levels represent that the average of two groups of samples has significant difference when being less than 0.05；When probability P value is more than 0.05
When can consider that two groups of variances of sample without significant difference, that is, have passed through Levene variances and checked together.
Present invention discover that for MERS vaccines, with MF59 or aluminium adjuvant is shared, RBD dimers are immune can
Mouse is excited to produce the neutralizing antibody of very high-titer, wherein highest potency is up to more than 3000, and group difference is small.And RBD monomers
The neutralizing antibody of potency 260 is produced when only 3 μ g dosage are shared with MF59, is excited much smaller than the dimer group of parallel control
NAT, and significant difference.And other several dosage components and adjuvant of RBD monomers are shared and do not produce detectable
Neutralizing antibody (be less than 20), with parallel dimer group comparing difference significantly (table 2).This prompting MERS RBD dimer conduct
The immunogenicity of vaccine is significantly more than RBD monomers.It is same for SARS vaccines, the immune group of all use dimers
Neutralizing antibody level very high is all generated, all between 1900 to 3900, group difference is small.Although RBD monomers can excite small
Mouse produces the neutralizing antibody of certain level, but still significantly smaller than parallel RBD dimers (table 2).This prompting SARS RBD
Dimer can significantly improve the immunogenicity of SARS-RBD vaccines as vaccine.
MERS-CoV the and SARS-CoV RBD vaccines of table 2 excite the detection of mouse neutralizing antibody level
* representing that dimer immune group is checked with the neutralizing antibody level of parallel monomer immune group by t- carries out statistics
Analysis, significant difference P<0.05
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes with modification, therefore protection model of the invention
Enclose being defined of being defined by claims.
Claims (10)
1. it is a kind of improve antigen immunogenicity method, methods described be express dimerization MERS-CoV albumen or SARS-
The RBD of CoV albumen is used as antigen；The amino acid sequence of the RBD such as SEQ ID NO:1 or SEQ ID NO:Shown in 2.
2. the Bovine Coronavirus Antigen that a kind of immunogenicity is improved, the antigen is the MERS-CoV albumen or SARS- of dimerization
The RBD of CoV albumen；The amino acid sequence of the RBD is such as shown in (1) or (2)：
(1) such as SEQ ID NO:1 or SEQ ID NO:Amino acid sequence shown in 2；
(2) amino acid sequence in (1) is substituted, lacks or adds an amino acid or several amino acid and with identical anti-
The polypeptide as derived from (1) of originality or its analog, and the albumen itself of its coding can form dimer.
3. Bovine Coronavirus Antigen according to claim 2, it is characterised in that the nucleotide sequence of the RBD such as SEQ ID
NO:3 or SEQ ID NO:Shown in 4.
4. the recombinant vector or expression cassette or transgenic cell line of the claim any Bovine Coronavirus Antigens of 2-3 are expressed
Or recombinant bacterium.
5. a kind of preparation method of Bovine Coronavirus Antigen, it is characterised in that methods described is in SEQ ID NO:3 or SEQ ID
NO:3 ' ends of nucleotide sequence shown in 4 add translation termination codon, carry out clonal expression, screen correct recon, then
Transfection insect cell is expressed, and cell conditioned medium is collected after expression, and purifying obtains Bovine Coronavirus Antigen.
6. method according to claim 5, it is characterised in that the insect cell is that Insect cells Sf9 or insect are thin
Born of the same parents system Hi5.
7. it is a kind of purify the claim any Bovine Coronavirus Antigens of 2-3 method, it is characterised in that methods described is by table
Cell supernatant up to the antigen is filtered to remove cell fragment, and carries out purpose egg by HiTrap QHP anion-exchange columns
White capture, is carried out thick pure by the buffer solution elution containing 1M NaCl immediately, and the component loading containing destination protein is dredged afterwards
Water column HiTrap Phenyl HP, carry out gradient elution, and the eluting peak containing target protein that will be obtained merges, then 10K retentions
Concentration, then carries out sieve chromatography and is purified using the pillars of Superdex200Hiload 16/60 (GE).
8. application of any Bovine Coronavirus Antigens of claim 2-3 in terms of anti-coronavirus medicine is prepared.
9. application according to claim 8, it is characterised in that the application be by the Bovine Coronavirus Antigen and MF59 or
Person's aluminium adjuvant is shared.
10. application according to claim 8 or claim 9, it is characterised in that the application is for reagent preparation box；The examination
Contain the antigen in agent box, or encode the DNA molecular of the antigen, or the recombinant vector/expression for expressing the antigen
Box/transgenic cell line/recombinant bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511021535.8A CN106928326B (en) | 2015-12-31 | 2015-12-31 | Coronavirus vaccine based on dimerized receptor binding domain subunit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511021535.8A CN106928326B (en) | 2015-12-31 | 2015-12-31 | Coronavirus vaccine based on dimerized receptor binding domain subunit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106928326A true CN106928326A (en) | 2017-07-07 |
| CN106928326B CN106928326B (en) | 2019-12-24 |
Family
ID=59442401
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511021535.8A Active CN106928326B (en) | 2015-12-31 | 2015-12-31 | Coronavirus vaccine based on dimerized receptor binding domain subunit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106928326B (en) |
Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111217918A (en) * | 2020-03-04 | 2020-06-02 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase |
| CN111471088A (en) * | 2020-04-21 | 2020-07-31 | 北京中科微盾生物科技有限责任公司 | Polypeptide for inhibiting SARS-COV-2 infection, composition and use thereof |
| CN111560074A (en) * | 2020-03-20 | 2020-08-21 | 中山大学 | Novel coronavirus S protein single-region subunit nano vaccine based on helicobacter pylori ferritin |
| CN111592602A (en) * | 2020-02-10 | 2020-08-28 | 中国科学院微生物研究所 | Beta coronavirus antigen, preparation method and application thereof |
| CN111607002A (en) * | 2020-02-24 | 2020-09-01 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on helicobacter pylori ferritin |
| CN112048005A (en) * | 2020-09-04 | 2020-12-08 | 江苏省中国科学院植物研究所 | Novel coronavirus S protein fragment polyploid, preparation method thereof, detection kit, vaccine and medicament |
| CN112500498A (en) * | 2020-02-26 | 2021-03-16 | 四川大学 | Novel coronavirus vaccine and preparation method and application thereof |
| CN113230395A (en) * | 2020-05-29 | 2021-08-10 | 中国科学院微生物研究所 | Beta coronavirus antigen, beta coronavirus bivalent vaccine, and preparation method and application thereof |
| WO2021169434A1 (en) * | 2020-02-28 | 2021-09-02 | 深圳市亚辉龙生物科技股份有限公司 | Sars-cov-2 recombinant rbd protein, and preparation method therefor |
| CN113355288A (en) * | 2020-03-06 | 2021-09-07 | 河北森朗生物科技有限公司 | Preparation method and application of universal chimeric antigen receptor T cell for treating COVID-19 |
| CN113527446A (en) * | 2021-07-14 | 2021-10-22 | 河南省农业科学院动物免疫学重点实验室 | MERS-CoV S-RBD linear B cell epitope and specific recognition monoclonal antibody and application thereof |
| WO2021237516A1 (en) * | 2020-05-27 | 2021-12-02 | 上海济煜医药科技有限公司 | Sars-cov-2 antibody and application thereof |
| CN113769080A (en) * | 2021-09-17 | 2021-12-10 | 清华大学 | Polypeptide immunoconjugates and uses thereof |
| WO2021249116A1 (en) * | 2020-06-10 | 2021-12-16 | Sichuan Clover Biopharmaceuticals, Inc. | Coronavirus vaccine compositions, methods, and uses thereof |
| WO2021253172A1 (en) * | 2020-06-15 | 2021-12-23 | 上海市公共卫生临床中心 | Method for inducing anti-novel coronavirus neutralizing antibody using receptor recognition domain |
| WO2021243149A3 (en) * | 2020-05-28 | 2021-12-30 | President And Fellows Of Harvard College | Protein covariance networks reveal interactions important to the emergence of sars coronaviruses as human pathogens |
| WO2022089471A1 (en) * | 2020-10-27 | 2022-05-05 | 中国科学院微生物研究所 | β-CORONAVIRUS MULTIMERIC ANTIGEN, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF |
| CN116113431A (en) * | 2020-04-01 | 2023-05-12 | 迪奥辛瓦克斯有限公司 | Coronavirus vaccine |
| WO2023250110A1 (en) | 2022-06-22 | 2023-12-28 | Flagship Pioneering Innovations Vi, Llc | Combination therapies for the treatment of viral infections |
| WO2023250111A1 (en) | 2022-06-22 | 2023-12-28 | Flagship Pioneering Innovations Vi, Llc | Combination therapies for the treatment of viral infections |
| CN113797326B (en) * | 2020-06-17 | 2024-01-19 | 上海君实生物医药科技股份有限公司 | Vaccine for preventing diseases caused by coronaviruses |
| EP4240414A4 (en) * | 2020-11-03 | 2024-09-04 | Wistar Inst | Dna encoded nanoparticles and method of use thereof as a coronavirus disease 2019 (covid-19) vaccine |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2723008C9 (en) * | 2020-05-19 | 2021-02-09 | федеральное государственное бюджетное учреждение «Национальный исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф. Гамалеи» Министерства здравоохранения Российской Федерации | Method for producing chinese hamster ovary cell strain, producer of sars-cov-2 virus recombinant rbd protein, chinese hamster ovary cell strain, producer of recombinant rbd protein of sars-cov-2 virus, method of producing recombinant rbd protein of sars-cov-2 virus, a test system for enzyme-linked immunosorbent assay of human blood serum or plasma and its use |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852921A (en) * | 2003-07-21 | 2006-10-25 | 美国政府卫生及公众服务部国家卫生协会 | Soluble fragments of the SARS-cov spike glycoprotein |
| CN101370516A (en) * | 2006-01-19 | 2009-02-18 | 美国政府（由卫生和人类服务部国立卫生研究院的部长所代表） | Soluble fragments of the SARS-CoV spike glycoprotein |
| CN103864924A (en) * | 2014-02-14 | 2014-06-18 | 中国科学院微生物研究所 | Middle east and respiratory syndrome coronavirus antibody and preparation method thereof |
| CN104447986A (en) * | 2014-12-23 | 2015-03-25 | 中国科学院微生物研究所 | Middle East respiratory syndrome coronavirus (MERS-CoV) neutralizing antibody and preparation method thereof |
| US20150337029A1 (en) * | 2014-05-23 | 2015-11-26 | Regeneron Pharmaceuticals, Inc. | Human Antibodies to Middle East Respiratory Syndrome - Coronavirus Spike Protein |
-
2015
- 2015-12-31 CN CN201511021535.8A patent/CN106928326B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852921A (en) * | 2003-07-21 | 2006-10-25 | 美国政府卫生及公众服务部国家卫生协会 | Soluble fragments of the SARS-cov spike glycoprotein |
| CN101370516A (en) * | 2006-01-19 | 2009-02-18 | 美国政府（由卫生和人类服务部国立卫生研究院的部长所代表） | Soluble fragments of the SARS-CoV spike glycoprotein |
| CN103864924A (en) * | 2014-02-14 | 2014-06-18 | 中国科学院微生物研究所 | Middle east and respiratory syndrome coronavirus antibody and preparation method thereof |
| US20150337029A1 (en) * | 2014-05-23 | 2015-11-26 | Regeneron Pharmaceuticals, Inc. | Human Antibodies to Middle East Respiratory Syndrome - Coronavirus Spike Protein |
| CN104447986A (en) * | 2014-12-23 | 2015-03-25 | 中国科学院微生物研究所 | Middle East respiratory syndrome coronavirus (MERS-CoV) neutralizing antibody and preparation method thereof |
Cited By (32)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111592602B (en) * | 2020-02-10 | 2021-03-02 | 中国科学院微生物研究所 | Beta coronavirus antigen, preparation method and application thereof |
| CN113416259A (en) * | 2020-02-10 | 2021-09-21 | 中国科学院微生物研究所 | Beta coronavirus antigen, preparation method and application thereof |
| CN111592602A (en) * | 2020-02-10 | 2020-08-28 | 中国科学院微生物研究所 | Beta coronavirus antigen, preparation method and application thereof |
| CN111607002B (en) * | 2020-02-24 | 2021-07-09 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on helicobacter pylori ferritin |
| CN111607002A (en) * | 2020-02-24 | 2020-09-01 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on helicobacter pylori ferritin |
| CN112500498A (en) * | 2020-02-26 | 2021-03-16 | 四川大学 | Novel coronavirus vaccine and preparation method and application thereof |
| WO2021169434A1 (en) * | 2020-02-28 | 2021-09-02 | 深圳市亚辉龙生物科技股份有限公司 | Sars-cov-2 recombinant rbd protein, and preparation method therefor |
| CN111217918B (en) * | 2020-03-04 | 2020-11-10 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase |
| CN111217918A (en) * | 2020-03-04 | 2020-06-02 | 中山大学 | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase |
| CN113355288B (en) * | 2020-03-06 | 2023-01-06 | 河北森朗生物科技有限公司 | Preparation method and application of universal chimeric antigen receptor T cell for treating COVID-19 |
| CN113355288A (en) * | 2020-03-06 | 2021-09-07 | 河北森朗生物科技有限公司 | Preparation method and application of universal chimeric antigen receptor T cell for treating COVID-19 |
| CN111560074B (en) * | 2020-03-20 | 2021-07-09 | 中山大学 | Novel coronavirus S protein single-region subunit nano vaccine based on helicobacter pylori ferritin |
| CN111560074A (en) * | 2020-03-20 | 2020-08-21 | 中山大学 | Novel coronavirus S protein single-region subunit nano vaccine based on helicobacter pylori ferritin |
| CN116113431A (en) * | 2020-04-01 | 2023-05-12 | 迪奥辛瓦克斯有限公司 | Coronavirus vaccine |
| CN111471088A (en) * | 2020-04-21 | 2020-07-31 | 北京中科微盾生物科技有限责任公司 | Polypeptide for inhibiting SARS-COV-2 infection, composition and use thereof |
| WO2021237516A1 (en) * | 2020-05-27 | 2021-12-02 | 上海济煜医药科技有限公司 | Sars-cov-2 antibody and application thereof |
| WO2021243149A3 (en) * | 2020-05-28 | 2021-12-30 | President And Fellows Of Harvard College | Protein covariance networks reveal interactions important to the emergence of sars coronaviruses as human pathogens |
| WO2021239147A1 (en) * | 2020-05-29 | 2021-12-02 | 中国科学院微生物研究所 | β-CORONAVIRUS ANTIGEN, β-CORONAVIRUS BIVALENT VACCINE, PREPARATION METHODS THEREFOR, AND APPLICATIONS THEREOF |
| CN113230395B (en) * | 2020-05-29 | 2022-07-19 | 中国科学院微生物研究所 | Beta coronavirus antigen, beta coronavirus bivalent vaccine, preparation methods and applications thereof |
| CN113230395A (en) * | 2020-05-29 | 2021-08-10 | 中国科学院微生物研究所 | Beta coronavirus antigen, beta coronavirus bivalent vaccine, and preparation method and application thereof |
| WO2021249116A1 (en) * | 2020-06-10 | 2021-12-16 | Sichuan Clover Biopharmaceuticals, Inc. | Coronavirus vaccine compositions, methods, and uses thereof |
| US11389528B2 (en) | 2020-06-10 | 2022-07-19 | Sichuan Clover Biopharmaceuticals, Inc | Coronavirus vaccine compositions, methods, and uses thereof |
| WO2021253172A1 (en) * | 2020-06-15 | 2021-12-23 | 上海市公共卫生临床中心 | Method for inducing anti-novel coronavirus neutralizing antibody using receptor recognition domain |
| CN113797326B (en) * | 2020-06-17 | 2024-01-19 | 上海君实生物医药科技股份有限公司 | Vaccine for preventing diseases caused by coronaviruses |
| CN112048005A (en) * | 2020-09-04 | 2020-12-08 | 江苏省中国科学院植物研究所 | Novel coronavirus S protein fragment polyploid, preparation method thereof, detection kit, vaccine and medicament |
| WO2022089471A1 (en) * | 2020-10-27 | 2022-05-05 | 中国科学院微生物研究所 | β-CORONAVIRUS MULTIMERIC ANTIGEN, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF |
| EP4240414A4 (en) * | 2020-11-03 | 2024-09-04 | Wistar Inst | Dna encoded nanoparticles and method of use thereof as a coronavirus disease 2019 (covid-19) vaccine |
| CN113527446A (en) * | 2021-07-14 | 2021-10-22 | 河南省农业科学院动物免疫学重点实验室 | MERS-CoV S-RBD linear B cell epitope and specific recognition monoclonal antibody and application thereof |
| CN113769080B (en) * | 2021-09-17 | 2023-04-07 | 清华大学 | Polypeptide immunoconjugates and uses thereof |
| CN113769080A (en) * | 2021-09-17 | 2021-12-10 | 清华大学 | Polypeptide immunoconjugates and uses thereof |
| WO2023250110A1 (en) | 2022-06-22 | 2023-12-28 | Flagship Pioneering Innovations Vi, Llc | Combination therapies for the treatment of viral infections |
| WO2023250111A1 (en) | 2022-06-22 | 2023-12-28 | Flagship Pioneering Innovations Vi, Llc | Combination therapies for the treatment of viral infections |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106928326B (en) | 2019-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106928326A (en) | A kind of coronavirus vaccine of the receptor binding domain subunit based on dimerization | |
| CN113416259B (en) | Beta coronavirus antigen, preparation method and application thereof | |
| CN111560074B (en) | Novel coronavirus S protein single-region subunit nano vaccine based on helicobacter pylori ferritin | |
| CN111607002B (en) | Novel coronavirus S protein double-region subunit nano vaccine based on helicobacter pylori ferritin | |
| CN111217918B (en) | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase | |
| CN113230395B (en) | Beta coronavirus antigen, beta coronavirus bivalent vaccine, preparation methods and applications thereof | |
| CA2712605C (en) | Monoclonal antibodies for ebola and marburg viruses | |
| CN1914226B (en) | Antibodies against SARS-COV and methods of use thereof | |
| CN111217919A (en) | Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin | |
| CN102574897B (en) | Chimaeric protein | |
| Brian et al. | The coronavirus hemagglutinin esterase glycoprotein | |
| CN115246874B (en) | Recombinant novel coronavirus S-RBD trimer protein, preparation method and application thereof | |
| CN113943375B (en) | Recombinant fusion protein derived from HR region of novel coronavirus S2 protein and application thereof | |
| CN112794898B (en) | Fully human monoclonal antibody for resisting novel coronavirus and application thereof | |
| CA2960102C (en) | Flavivirus virus like particle | |
| CN107619819A (en) | A kind of recombinant cell lines, vaccine and the application of stable expression Porcine epidemic diarrhea virus S1 albumen | |
| CN101235085B (en) | Monoclonal antibody of membrane protein E for resisting West Nile virus and application thereof | |
| CA2548942A1 (en) | Anti-sars monoclonal antibodies | |
| CN102406929A (en) | Co-expression molecular adjuvant enhanced bivalent foot-and-mouth disease protein engineering vaccine | |
| CN116769048A (en) | Polymeric molecules, single and multiple structures including the same | |
| CN115975042B (en) | Beta coronavirus heteromultimeric antigen, preparation method and application thereof | |
| Da Silva | Studies on the Fusion and Attachment Envelope Glycoproteins of Mojiang henipavirus: Function, Structure and Serologic Applications in Rodents | |
| CN101016547A (en) | Method of preparing ocean double RNA virus MABV recombination albumen and application thereof | |
| CN118440190A (en) | Humanized rabies virus nanobody, recombinant nanobody protein and application thereof in preparation of rabies prevention and treatment products | |
| CN113308440A (en) | N7-methyltransferase defective coronavirus attenuated vaccine strain and preparation method and application thereof |
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 | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180725 Address after: No. 3, No. 1, Beichen West Road, Beichen, Beijing Applicant after: INSTITUTE OF MICROBIOLOGY, CHINESE ACADEMY OF SCIENCES Address before: No. 5, No. 1, Beichen West Road, Beichen, Beijing Applicant before: INSTITUTE OF ZOOLOGY, CHINESE ACADEMY OF SCIENCES Applicant before: Institute of Microbiology, Chinese Academy of Sciences |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210209 Address after: 230000 No.100 Fushan Road, high tech Zone, Hefei City, Anhui Province Patentee after: ANHUI ZHIFEI LONGCOM BIOPHARMACEUTICAL Co.,Ltd. Address before: 100101 No. 1, West Beichen Road, Beijing, Chaoyang District, 3 Patentee before: INSTITUTE OF MICROBIOLOGY, CHINESE ACADEMY OF SCIENCES |
|
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 230000, No. 5008 Mingzhu Avenue, Hefei City, Anhui Province Patentee after: ANHUI ZHIFEI LONGCOM BIOPHARMACEUTICAL Co.,Ltd. Address before: 230000 No.100 Fushan Road, high tech Zone, Hefei City, Anhui Province Patentee before: ANHUI ZHIFEI LONGCOM BIOPHARMACEUTICAL Co.,Ltd. |