CN116426543B - 一种新型冠状病毒疫苗及其制备方法 - Google Patents
一种新型冠状病毒疫苗及其制备方法 Download PDFInfo
- Publication number
- CN116426543B CN116426543B CN202111653353.8A CN202111653353A CN116426543B CN 116426543 B CN116426543 B CN 116426543B CN 202111653353 A CN202111653353 A CN 202111653353A CN 116426543 B CN116426543 B CN 116426543B
- Authority
- CN
- China
- Prior art keywords
- mrna
- novel coronavirus
- coronavirus vaccine
- vaccine
- lipid
- 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.)
- Active
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 52
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 121
- 108091026890 Coding region Proteins 0.000 claims abstract description 11
- 150000002632 lipids Chemical class 0.000 claims description 18
- -1 cationic lipid Chemical class 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 13
- 238000009472 formulation Methods 0.000 claims description 11
- 239000002105 nanoparticle Substances 0.000 claims description 11
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 108020005345 3' Untranslated Regions Proteins 0.000 claims description 7
- 150000007523 nucleic acids Chemical group 0.000 claims description 7
- 108020004414 DNA Proteins 0.000 claims description 6
- 239000004094 surface-active agent Substances 0.000 claims description 6
- 108020003589 5' Untranslated Regions Proteins 0.000 claims description 5
- 102000053602 DNA Human genes 0.000 claims description 5
- 235000012000 cholesterol Nutrition 0.000 claims description 5
- DSNRWDQKZIEDDB-SQYFZQSCSA-N 1,2-dioleoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-SQYFZQSCSA-N 0.000 claims description 4
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 claims description 4
- 125000002091 cationic group Chemical group 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 3
- WTBFLCSPLLEDEM-JIDRGYQWSA-N 1,2-dioleoyl-sn-glycero-3-phospho-L-serine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC WTBFLCSPLLEDEM-JIDRGYQWSA-N 0.000 claims description 2
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 claims description 2
- AOBORMOPSGHCAX-UHFFFAOYSA-N Tocophersolan Chemical compound OCCOC(=O)CCC(=O)OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C AOBORMOPSGHCAX-UHFFFAOYSA-N 0.000 claims description 2
- 150000001841 cholesterols Chemical class 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- GLGLUQVVDHRLQK-WRBBJXAJSA-N n,n-dimethyl-2,3-bis[(z)-octadec-9-enoxy]propan-1-amine Chemical compound CCCCCCCC\C=C/CCCCCCCCOCC(CN(C)C)OCCCCCCCC\C=C/CCCCCCCC GLGLUQVVDHRLQK-WRBBJXAJSA-N 0.000 claims description 2
- 241001678559 COVID-19 virus Species 0.000 abstract description 16
- 102000036639 antigens Human genes 0.000 abstract description 7
- 108091007433 antigens Proteins 0.000 abstract description 7
- 108700021021 mRNA Vaccine Proteins 0.000 abstract description 6
- 229940126582 mRNA vaccine Drugs 0.000 abstract description 6
- 239000000427 antigen Substances 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 3
- 238000011282 treatment Methods 0.000 abstract description 3
- 230000028993 immune response Effects 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 description 27
- 239000002773 nucleotide Substances 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 16
- 241000700605 Viruses Species 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 238000006386 neutralization reaction Methods 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 11
- 102100027382 Membrane-bound transcription factor site-2 protease Human genes 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 102100031673 Corneodesmosin Human genes 0.000 description 8
- 101710139375 Corneodesmosin Proteins 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 230000003053 immunization Effects 0.000 description 8
- 238000002649 immunization Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 230000003472 neutralizing effect Effects 0.000 description 7
- 241001112090 Pseudovirus Species 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102000018697 Membrane Proteins Human genes 0.000 description 3
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 3
- 229940096437 Protein S Drugs 0.000 description 3
- 101710198474 Spike protein Proteins 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 229940023146 nucleic acid vaccine Drugs 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- 108060002716 Exonuclease Proteins 0.000 description 2
- 102100034349 Integrase Human genes 0.000 description 2
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 241000831652 Salinivibrio sharmensis Species 0.000 description 2
- 108091036066 Three prime untranslated region Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 102000013165 exonuclease Human genes 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 210000003141 lower extremity Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 241000180579 Arca Species 0.000 description 1
- 206010003757 Atypical pneumonia Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 108091005902 Hemoglobin subunit alpha Proteins 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 101001009007 Homo sapiens Hemoglobin subunit alpha Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 235000010676 Ocimum basilicum Nutrition 0.000 description 1
- 240000007926 Ocimum gratissimum Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 231100000645 Reed–Muench method Toxicity 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- AGWRKMKSPDCRHI-UHFFFAOYSA-K [[5-(2-amino-7-methyl-6-oxo-1H-purin-9-ium-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl] [[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl]oxy-5-(6-aminopurin-9-yl)-4-methoxyoxolan-2-yl]methoxy-oxidophosphoryl] phosphate Chemical compound COC1C(OP([O-])(=O)OCC2OC(C(O)C2O)N2C=NC3=C2N=C(N)NC3=O)C(COP([O-])(=O)OP([O-])(=O)OP([O-])(=O)OCC2OC(C(O)C2O)N2C=[N+](C)C3=C2N=C(N)NC3=O)OC1N1C=NC2=C1N=CN=C2N AGWRKMKSPDCRHI-UHFFFAOYSA-K 0.000 description 1
- 101150087698 alpha gene Proteins 0.000 description 1
- 230000002238 attenuated effect Effects 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
- 238000007664 blowing Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000007499 fusion processing Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229940035893 uracil Drugs 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Optics & Photonics (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Dispersion Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nanotechnology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
Abstract
本发明公开了一种新型冠状病毒疫苗及其制备方法，涉及生物疫苗技术领域，本发明对编码SARS‑CoV‑2病毒抗原的mRNA的编码区序列改进，得到了序列如SEQ ID No.2～6中任意一项所示的编码区序列，使得制备出来的mRNA疫苗递送到体内可以产生免疫反应，限制提高疫苗对变异株B.1.351.1(Beta株)毒株的保护效率，为变异株B.1.351.1(Beta株)的研究和治疗提供了新的途径。
Description
技术领域
本发明涉及生物疫苗技术领域，具体而言，涉及一种新型冠状病毒疫苗及其制备方法。
背景技术
新冠肺炎疫情造成了全世界范围内社会和经济的极大损失。该种非典型肺炎被世界卫生组织定名为COVID-19，是由一种新型冠状病毒(SARS-CoV-2,或称2019-nCoV)引起的。针对新型冠状病毒(SARS-CoV-2，或称2019-nCoV)，目前没有特异的治疗方法，只能根据患者的临床状况进行一些对症处理。
近年来mRNA疫苗成为新兴热点，将体外合成的、带有特定抗原蛋白编码序列的mRNA递送进细胞后，使得细胞成为制造抗原蛋白的工厂，引发免疫反应，从而起到疫苗的防护作用。相比传统的灭活、减毒病毒疫苗和DNA疫苗，mRNA疫苗有很多优越性。以mRNA技术来生产疫苗，不仅可以极大缩短研发时间，降低研发成本，实现疫苗的标准化生产，还可以利用mRNA简便的生产工艺随用随制，降低贮存难度。更重要的是，和灭活病毒疫苗比起来，mRNA产品的制剂极大地提高了疫苗的稳定性，因此在产品质量和安全性上，mRNA疫苗具有极大的优势，可以解决传统疫苗行业的痛点，将为疫苗市场带来一场革新。新冠病毒作为RNA病毒，存在极易变异的特点。
新冠病毒作为RNA病毒，存在极易变异的特点。自2020年7月起，变异株相继出现，如包括：Alpha、Beta、Gamma和Delta等。5月31日，世界卫生组织(WHO)宣布，将改用希腊字母命名变异新冠病毒，以避免对最早检测和发现变异病毒的国家造成污名化。依照新命名系统，最早在英国发现的B117变种病毒将改称Alpha变种病毒，最早在南非发现的B1351将改称Beta变种病毒，最早在巴西发现的P1将改称Gamma变种病毒，而最早在印度发现的B1617有两个分支——属于应关注变种的B16172将改称Delta变种病毒，属于待观察变种的B16171将改称Kappa。
研究发现，现有疫苗均是针对2019年的新型冠状病毒SARS-CoV-2进行的抗原设计，对变异株的保护效果出现不同程度的下降。因此，针对变异株具有更好保护效果的新冠疫苗亟待研发。
鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种新型冠状病毒疫苗及其制备方法。
本发明是这样实现的：
第一方面，本发明实施例提供了一种分离的mRNA，其编码区核酸序列如SEQ IDNo.2～6中任一项所示，或与如SEQ ID No.2～6中任一项所示的核苷酸序列具有至少99％～80％同一性。
第二方面，本发明实施例提供了一种新型冠状病毒疫苗，其制备原料包括：如前述实施例所述的mRNA和递送制剂。
第三方面，本发明实施例提供了如前述实施例所述的新型冠状病毒疫苗的制备方法，其包括：将所述mRNA和递送制剂混合，以形成新型冠状病毒疫苗。
本发明具有以下有益效果：
本发明实施例对编码SARS-CoV-2病毒抗原的mRNA的编码区序列改进，使得制备出来的mRNA疫苗递送到体内可以产生免疫反应，限制提高疫苗对变异株B.1.351.1(Beta株)毒株的保护效率，为变异株B.1.351.1的研究和治疗提供了新的途径。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为SF-Beta系列mRNA转染细胞后的蛋白表达免疫印迹结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
冠状病毒基因组依次编码棘突蛋白(spike protein)、包膜蛋白(Envelopeprotein)、膜蛋白(Membrane protein)和核衣壳蛋白(Nucleocapsid)。其中棘突蛋白(spike protein，S)被认为是冠状病毒侵入细胞使用的最重要的病毒表面膜蛋白，含有两个亚基(subunit)，S1和S2。其中S1主要包含有受体结合区(receptor binding domain，RBD)，负责识别细胞的受体。S2含有膜融合过程所需的基本元件。核衣壳蛋白(N)是冠状病毒中含量最丰富的蛋白。
本发明实施例提供了一种分离的mRNA，其编码区核酸序列如SEQ ID No.2～6中任一项所示，或与如SEQ ID No.2～6中任一项所示的核苷酸序列具有至少99％～80％同一性。序列同一性具体可以为99％、98％、97％、96％、95％、90％、85％或80％中的任意一项或任意两项之间的范围，后续实施例可参照此例举，不再赘述。
发明人经一系列创造性劳动，对mRNA的编码序列进行了优化，针对变异株的编码区突变位点进行了调整，以增加对变异株的亲和能力；还增加了序列上的第2400-3000号位点的核苷酸(如Seq No.13、Seq No.14、Seq No.15所示的序列)中4-10个核苷酸的调整，以优化S蛋白的结构，增加S蛋白pre-fusion稳定性，得到了上述核酸序列。优化后的核苷酸序列与现有序列相比，针对变异株B.1.351.1(Beta株)有更高的抗体滴度，从而更好的保护机体不被新冠病毒变异株B.1.351.1(Beta株)所感染。
优选地，mRNA的编码区核酸序列如SEQ ID No.2或SEQ ID No.4所示，或与SEQ IDNo.2或SEQ ID No.4的核苷酸序列具有至少99％～80％同一性。
优选地，mRNA的编码区核酸序列如SEQ ID No.4所示，或与SEQ ID No.4的核苷酸序列具有至少99％～80％同一性。
优选地，所述mRNA还包括：5’端帽子、5’UTR、3’UTR以及3’端polyA尾中的至少一项。
5’端帽子可以增加mRNA的稳定性，避免mRNA被核酸外切酶降解，优选包括ARCA、m7G(5')ppp(5')(2'OMeA)pG、m7G(5')ppp(5')(2'OMeG)pG、m7(3'OMeG)(5')ppp(5')(2'OMeG)pG、m7(3'OMeG)(5')ppp(5')(2'OMeA)pG、mCAP、dmCAP、tmCAP或dmCAP。
3’端polyA尾能够避免mRNA被核酸外切酶降解，同时终结转录，其长度优选为50-200个A，例如可以为但不限于为50、80、100、120、150、180或200个A，更优选为80-200个A。
5’UTR对mRNA的翻译具有调控作用，其长度优选为10-200个核苷酸，例如可以为但不限于为10、15、50、100、120、150或200个核苷酸，更优选为15-100个核苷酸。5’UTR优选包括DNAH25’UTR序列或KOZAK序列。
3’UTR优选包括来源于提供稳定的mRNA的基因的3’UTR或来源于提供稳定的mRNA的基因的3’UTR的同源物，片段或变体。提供稳定的mRNA的基因优选包括白蛋白基因、α-珠蛋白基因、β-珠蛋白基因、酪氨酸羟化酶基因、脂氧合酶基因或胶原蛋白α基因。更优选地，3’UTR包括血红蛋白HBA2的3’UTR序列或β-珠蛋白3’-UTR序列。
所述mRNA含有经过修饰的核苷酸和/或未经修饰的核苷酸。具体地，所述mRNA的全序列均使用未经修饰的核苷酸。具体地，所述mRNA的全序列中含有经过修饰的核苷酸。含有经过修饰的核苷酸指的是mRNA中的核苷酸经过修饰，例如经2’-O-甲基化修饰核苷酸和/或L-核苷修饰的核苷酸，或含有修饰的UTP和/或CTP，或者使用其他种类的核苷酸替换mRNA中常规的核苷酸，例如使用假尿嘧啶(pseudo-UTP)替换mRNA中的常规核苷酸。修饰的核苷酸的位置例如可以为但不限于为编码区、5’-UTR区、3’-UTR区和帽子区中至少一种。
本发明实施例还提供了一种重组载体，其包括如转录前述任意实施例所述的分离的mRNA的DNA分子。
所述重组载体还可以为含有上述DNA分子的表达盒或载体，例如含有编码所述mRNA的质粒，以用来贮存所述mRNA的序列信息，或用于克隆所述DNA分子或表达DNA分子编码的蛋白。
本发明实施例还提供了一种宿主细胞，其含有如前述实施例所述的重组载体，经重组载体转化后，宿主系包括可以表达重组载体中目的基因，实现DNA分子的克隆。
本发明实施例提供了一种新型冠状病毒疫苗，其制备原料包括：如前述任一项实施例所述的mRNA和递送制剂。
可选地，核酸疫苗的制备方法可参照现有公开的mRNA疫苗的制备方法，只要采用本申请公开的核酸序列应用于新型冠状病毒核酸疫苗的制备，则属于本申请的保护范围内。
优选地，所述递送制剂包括阳离子脂质纳米颗粒或阳离子脂质体。
优选地，所述mRNA和阳离子脂质纳米颗粒的质量比为5～20：1，具体可以为5：1、7：1、10：1、13：1、15：1、17：1和20：1中的任意一项或任意两项之间的范围。
优选地，所述阳离子脂质纳米颗粒的组分包括：所述可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂中的至少一种。
优选地，按重量份数计，所述阳离子脂质纳米颗粒的组分包括：40～60份可质子化阳离子脂质、20～40份结构脂质、10～30份辅助脂质和0.5～5份表面活性剂。
优选地，所述可质子化阳离子脂质选自：Dlin-MC3-DMA、DODMA、C12-200和DlinDMA中的至少一种。
优选地，所述结构脂质包括胆固醇和/或胆固醇衍生物。
优选地，所述辅助脂质包括DSPC、DOPE、DOPC、DOPG和DOPS中的至少一种。
优选地，所述表面活性剂包括PEG-DMG、PEG-DSPE和TPGS中的至少一种。
此外，本发明实施例还提供了如前述任意实施例所述的新型冠状病毒疫苗的制备方法，其包括：将所述mRNA和递送制剂混合，以形成新型冠状病毒疫苗。
本发明实施例涉及的核苷酸序列如下表所述。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
提供了如表1所示的mRNA，信息见表1所示。
表1 mRNA序列
将表1所示的mRNA转染细胞，检测S全长蛋白在细胞中的表达，结果如图1所示。详细方法如下：转染各mRNA24小时后的HEK293细胞进行裂解，按10μg的总蛋白在SDS-PAGE胶上样，用抗SARS-S1蛋白抗体进行免疫印迹，使用羊抗鼠-HRP二抗进行标记，然后显色。蛋白表达量使用内参b-actin进行标化定量，比较不同mRNA转染细胞后表达的蛋白量差异。未转染mRNA的细胞作为阴性对照。S蛋白全长、S1亚基均可以检测到表达。综合6个不同序列设计的mRNA转染后的蛋白表达效果可得出以下结论：
6个不同序列设计的mRNA转染细胞中的S蛋白表达均有表达，根据分子量大小均可见S蛋白全长和S1亚基这两个条带。
基于SF mRNA序列，S6P mRNA分别在第2400-2800号核苷酸位点上进行了5个核苷酸的优化、在第2910-2920号核苷酸位点上进行了4个核苷酸的优化，相对的翻译后氨基酸序列中6个位点的氨基酸残基被脯氨酸残基取代，具体来说，优化后的S6P mRNA翻译的氨基酸序列在出现了F817P、A882P、A889P、A942P、K986P、V987P共计6个位点的突变。
基于SF mRNA序列，S2P mRNA仅在第2910-2920号核苷酸位点上进行了4个核苷酸的优化，相对的翻译后氨基酸序列中2个位点的氨基酸残基被脯氨酸残基取代，具体来说，优化后的S2P mRNA翻译出的氨基酸序列出现了K986P、V987P共计2个位点的突变。
基于SF mRNA序列，S2P-Beta mRNA和S6P-Beta mRNA分别包含了S2P mRNA和S6PmRNA的优化序列，且增加了在第1200-190号核苷酸位点上进行了4个核苷酸的优化。
含有S6P mRNA和S6P-Beta mRNA转染细胞后，同等情况下的S蛋白含量远远低于其他mRNA转染后的样品，说明S6P mRNA和S6P-Beta mRNA包含的优化位点会导致S蛋白表达量下降，S6P mRNA和S6P-Beta mRNA不适合作为潜在的疫苗选择；SF mRNA、SF-Beta mRNA、S2PmRNA和S2P-Beta mRNA四个序列在HEK293细胞中表达量都较强，这四个mRNA适合做为潜在的疫苗选择。
本实施例还结合其他公开报道对SarS-COV-2的全长S蛋白的其他位点进行了突变，如G614S等，并以SF-Beta mRNA序列为基础设计了相应的mRNA序列，检测mRNA转染细胞后的蛋白表达免疫印迹结果，发现这些突变以及对应的mRNA序列的优化，相比于SF-BetamRNA和S2P-Beta mRNA序列会导致表达量的降低。
参考WOUS21032609、GB2002166、WOUS21016979等公开的SARS-COV-2的全长S蛋白mRNA，以及SF-Beta mRNA、S2P-Beta mRNA、S6P-Beta mRNA的优化方式进行设计，检测mRNA转染细胞后的蛋白表达免疫印迹结果，发现这些突变以及对应的mRNA序列的优化，相比于SF-Beta mRNA和S2P-Beta mRNA序列会导致表达量的降低。
实施例2
实验方案A假病毒病毒株的中和活性实验。
将SF mRNA、S2P-Beta mRNA两种mRNA分别制备成LNP制剂(Dlin-MC3-DMA 50％，cholesterol 29％，DOPC 20％，PEG-DMG 1％。Lipid/mRNA(w/w)＝10:1)，LNP制剂经检测包封率均90％以上，粒径约为70nm。
将所得2组疫苗制剂用于C57小鼠免疫实验，每只小鼠通过后肢外侧大腿肌肉注射5微克(以mRNA计)，在间隔7天后进行二次免疫。首次免疫后14天抽取小鼠血清送第三方实验室进行SARS-CoV-2假病毒中和活性测试。
在96孔板内将小鼠血清按照不同比例稀释后(初始稀释倍数为30)加入可感染SARS-CoV-2假病毒，并同时设置细胞对照和病毒对照，孵育1小时候，加入事先准备好的细胞，于细胞培养箱中培养20～28小时，吸弃一部分上清，加入荧光素酶检测试剂，室温避光反应后，反复吹吸孔内细胞，使细胞充分裂解后，置于化学发光检测仪中读取发光值。确保病毒对照和细胞对照成立的前提下，采用Reed-Muench法计算ID50值。血清对应分组编号及结果如表2所示。
表2 小鼠血清对不同假病毒病毒株的中和活性
由表2可知，SF mRNA和S2P-Beta mRNA两种mRNA制备而成疫苗制剂均可刺激小鼠产生对武汉株(WT)和B.1.351.1(Beta株)株两种假病毒具有中和能力的抗体，但包含S2P-Beta mRNA的疫苗制剂更优，产生的抗体对两种毒株假病毒的中和活性均比SF mRNA强。
实验方案B活病毒病毒株的中和活性实验。
为进一步验证抗原设计中S2P对免疫原性的影响，同时验证小鼠血清对活病毒的中和活性，本实施例增加了一组抗原为S2P的疫苗制剂，同样的免疫方式、免疫程序以及采血时间，对免疫后小鼠血清经ELISA法检测后，根据ELISA结果，随机选取3～4份阳性血清送第三方P3实验室按照新型冠状病毒“微量血清中和试验(CPE法)”技术规范进行操作，同时检测血清对SARS-CoV-2武汉株(WT株)和B.1.351.1(Beta株)株活病毒的中和活性。结果如表3所示。
表3 小鼠血清对不同活病毒病毒株的中和活性
由表3可知，1)SF mRNA、S2P mRNA、S2P-Beta mRNA三种mRNA制备而成疫苗制剂均可刺激小鼠产生对武汉株(WT)和B.1.351.1(Beta株)株两种活病毒具有中和能力的抗体；
2)对比SF mRNA和S2P mRNA的中和活性结果可知，包含SF mRNA的疫苗制剂更优，产生的抗体对两种毒株活病毒的中和活性均比S2P mRNA强。对比SF mRNA和S2P-Beta mRNA的中和活性结果可知，包含S2P-Beta mRNA的疫苗制剂更优，产生的抗体对两种毒株活病毒的中和活性均比S2P mRNA强。
实施方案A的假病毒数据和实验方案B的活病毒中和数据呈现直接对应性。
实施例3
将SF mRNA、SF-Beta mRNA、S2P-Beta mRNA分别和递送制剂制成疫苗，将不同组别mRNA混合物分别制备成LNP制剂(Dlin-MC3-DMA50％，cholesterol 29％，DOPC 20％，PEG-DMG 1％。Lipid/mRNA(w/w)＝10:1)，LNP制剂经检测包封率均90％以上，粒径约为70nm。
将所得三组疫苗制剂用于C57小鼠免疫实验，每只小鼠通过后肢外侧大腿肌肉注射5微克(以mRNA计)，在间隔7天后进行二次免疫。首次免疫后14天抽取小鼠血清送第三方P3实验室按照新型冠状病毒“微量血清中和试验(FRNT法)”技术规范进行操作，同时检测血清对SARS-CoV-2武汉株(WH株)、B.1.351.1(Beta株)株的中和活性。结果如表4所示。
表4 小鼠血清对不同活病毒病毒株的中和活性
实验结果显示：1)含有这3种mRNA的疫苗均能够使小鼠体内产生抗体，该抗体对SARS-CoV-2原始毒株(WT)和变异毒株(Beta毒株)均有中和效果；
2)SF mRNA的疫苗在小鼠体内产生对SARS-CoV-2原始毒株(WT)抗体的抗体滴度高于对SARS-CoV-2变异毒株(Beta毒株)抗体的抗体滴度；SF-Beta mRNA和S2P-Beta mRNA的疫苗在小鼠体内产生对SARS-CoV-2原始毒株(WT)和变异毒株(Beta毒株)抗体的抗体滴度相当；但是综合而言，S2P-Beta mRNA的疫苗针对2个病毒株产生抗体的抗体滴度最优高，可以同时对SARS-CoV-2原始毒株(WT)和变异毒株(Beta毒株)产生较好的中和效果。
实施例4
本实施例提供了一种mRNA核酸疫苗，利用脂质纳米颗粒包裹如SEQ ID NO.1所示的mRNA，其中脂质纳米颗粒按照摩尔百分比计包括Dlin-MC3-DMA 50％、DOPG 10％、胆固醇38.5％和PEG-DMG 1.5％。
制备方法如下：
(a)将如SEQ ID NO.1所示的mRNA溶解于pH4的柠檬酸盐缓冲液，将浓度调整为0.1mg/ml，得到水相。
(b)按照配方量将Dlin-MC3-DMA、DOPG、胆固醇和PEG-DMG按照配方量溶解于无水乙醇，将有机相中的脂质成分的浓度调整至6mg/mL，得到有机相。
(c)将步骤(a)的水相和步骤(b)的有机相按照1:3的体积比，使用微流控设备，按照12mL/min的流速混合，混合液立刻用pH7.4的PBS溶液稀释100倍，并使用切向流过滤(TFF)除去溶液中乙醇成分，然后浓缩至体系中的mRNA的浓度为100μg/ml，得到包裹如SEQID NO.1所示的mRNA的脂质纳米颗粒。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 珠海丽凡达生物技术有限公司
<120> 一种新型冠状病毒疫苗及其制备方法
<141> 2021-12-30
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3777
<212> RNA
<213> 人工序列
<400> 1
gugaaucuga caaccagaac ccagcugcca ccagccuaua caaacuccuu cacacggggc 60
guguauuacc cugacaaggu guuucgcucu agcgugcugc acagcaccca ggaucuguuu 120
cugccauucu uuucuaacgu gacaugguuc cacgccaucc acguguccgg caccaacggc 180
acaaagcggu uugacaaccc ugugcugccc uuuaaugacg gcguguauuu cgccuccaca 240
gagaagagca auaucaucag gggcuggauc uuuggcacaa cccuggacuc uaagacccag 300
agccugcuga ucgugaacaa ugccaccaau gugguaauca aggugugcga guuccaguuu 360
ugcaaugacc ccuuccuggg cguguacuau cacaagaaca acaagucuug gauggagucc 420
gaguucagag uguacuccag cgccaauaac uguaccuucg aguacguguc ucagccauuu 480
cugauggauc uggagggcaa gcagggcaau uucaagaacc ugagagaguu cguguuuaag 540
aacaucgaug gcuacuucaa gaucuauucc aagcacaccc caaucaaccu ggugcgggau 600
cugccucagg gcuucucugc ccuggagcca cugguggacc ugccuaucgg caucaacauc 660
acacgguuuc agacacugcu ggcccugcac agguccuauc ugaccccagg cgacuccucu 720
ucuggcugga cagccggcgc cgccgccuac uaugugggcu accugcagcc uagaacauuc 780
cugcugaagu acaaugagaa cggcaccauc accgacgccg uggauugugc ccuggauccc 840
cugucugaga caaaguguac ccugaagucc uuuacagugg agaagggcau cuaucagacc 900
uccaacuuca gggugcagcc caccgagucu aucgugcggu uuccuaacau cacaaaucug 960
ugccccuuug gcgagguguu uaacgccacc agguuugccu ccguguaugc cuggaacagg 1020
aagcgcaucu cuaacugcgu ggccgauuac agcgugcugu acaauagcgc cagcuuuucc 1080
accuuuaagu guuauggcgu gucuccaaca aagcugaacg aucugugcuu caccaacgug 1140
uaugccgaca gcuuugugau ccgcggcgac gaggugaggc agaucgcccc cggccagacc 1200
ggcaagaucg ccgauuauaa cuacaagcug ccugacgacu uuaccggcug cgugaucgcc 1260
uggaacagca acaaccugga uucuaaggug ggcggcaacu auaauuaccu guaucgccug 1320
uuccggaagu cuaaccugaa gccuuucgag cgcgacaucu ccaccgagau cuaucaggcc 1380
ggcuccaccc cauguaaugg cguggagggc uucaacuguu acuuuccucu gcaguccuac 1440
ggcuuucagc cuacaaaugg cgugggcuac cagccuuaua gggugguggu gcugucuuuu 1500
gagcugcugc acgccccugc caccgugugc ggccccaaga agagcacaaa ccuggugaag 1560
aacaagugcg ugaauuucaa cuuuaauggc cugacaggca caggcgugcu gaccgagucc 1620
aauaagaagu uucugccuuu ccagcaguuu ggccgggaca ucgccgauac caccgacgcc 1680
gugagagacc cacagacacu ggagauccug gacaucacac cuugcuccuu cggcggcgug 1740
uccgugauca caccuggcac aaauaccucc aaucaggugg ccgugcugua ucaggacgug 1800
aacugcacag aggugccugu ggccauccac gccgaccagc ugacaccaac cuggcgggug 1860
uauucuaccg gcuccaacgu guuccagaca cgcgccggcu gccugaucgg cgccgagcac 1920
gugaauaacu cuuacgagug ugacaucccu aucggcgccg gcaucugugc cagcuaccag 1980
acccagacca acagcccacg cagagccagg uccguggccu cccagucuau caucgccuac 2040
accaugagcc ugggcgccga gaauagcgug gccuacucca auaauuccau cgccauccca 2100
accaacuuca caaucucugu gacaaccgag auccugcccg uguccaugac caagacaagc 2160
guggacugca caauguauau cuguggcgac ucuacagagu gcucuaaucu gcugcugcag 2220
uauggcuccu uuuguaccca gcugaacaga gcccugaccg gcaucgccgu ggagcaggac 2280
aagaacaccc aggagguguu cgcccaggug aagcagaucu acaagacccc accuaucaag 2340
gacuucggcg gcuucaauuu uucccagauc cugcccgacc caucuaagcc cagcaagcgg 2400
uccuuuaucg aggaucugcu guucaauaag gugacacugg ccgaugccgg cuuuaucaag 2460
caguacggcg acugucuggg cgauaucgcc gccagagacc ugaucugcgc ccagaaguuc 2520
aacggccuga ccgugcugcc cccacugcug accgaugaga ugaucgccca guacaccagc 2580
gcccugcugg ccggcaccau cacaucuggc uggaccuuug gcgccggcgc cgcccugcag 2640
aucccauuug ccaugcagau ggccuaucgc uucaacggca ucggcgugac ccagaaugug 2700
cuguaugaga aucagaagcu gaucgccaau caguuuaaua gcgccaucgg caagauccag 2760
gauucucuga gcuccacagc cucugcccug ggcaagcugc aggacguggu gaaucagaac 2820
gcccaggccc ugaauacccu ggugaagcag cuguccucua auuuuggcgc caucuccagc 2880
gugcugaacg acauccuguc ccggcuggau aagguggagg ccgaggugca gaucgacagg 2940
cugaucacag gcagacugca gucccugcag accuacguga cacagcagcu gauccgcgcc 3000
gccgagaucc gggccagcgc caaucuggcc gccaccaaga uguccgagug cgugcugggc 3060
cagagcaaga ggguggacuu uugcggcaag ggcuaccacc ugauguccuu uccacagagc 3120
gccccccacg gcgugguguu ccugcacgug acauacgugc cugcccagga gaagaacuuu 3180
acaaccgccc cagccaucug ucacgacggc aaggcccacu uuccccgcga gggcguguuc 3240
guguccaaug gcacccacug guucgugacc cagaggaauu ucuaugagcc acagaucauc 3300
acaaccgaca auacauucgu gucuggcaau ugcgacgugg uaaucggcau cgugaauaau 3360
acaguguacg acccucugca gcccgagcug gauuccuuca aggaggagcu ggacaaguau 3420
uucaagaacc acaccucccc agacguggac cugggcgaca ucagcggcau caaugccagc 3480
guggugaaca uccagaagga gaucgaucgc cugaaugagg uggccaagaa ccugaaugag 3540
ucccugaucg accugcagga gcugggcaag uacgagcagu auaucaagug gcccugguac 3600
aucuggcugg gcuuuaucgc cggccugauc gccaucguga uggugacaau caugcugugc 3660
uguaugaccu ccuguugcag cugccugaag ggcuguuguu ccugcggcag cuguuguaag 3720
uuugacgagg augacucuga gccugugcug aagggcguga agcugcacua uaccuga 3777
<210> 2
<211> 3777
<212> RNA
<213> 人工序列
<400> 2
gugaaucuga caaccagaac ccagcugcca ccagccuaua caaacuccuu cacacggggc 60
guguauuacc cugacaaggu guuucgcucu agcgugcugc acagcaccca ggaucuguuu 120
cugccauucu uuucuaacgu gacaugguuc cacgccaucc acguguccgg caccaacggc 180
acaaagcggu uugacaaccc ugugcugccc uuuaaugacg gcguguauuu cgccuccaca 240
gagaagagca auaucaucag gggcuggauc uuuggcacaa cccuggacuc uaagacccag 300
agccugcuga ucgugaacaa ugccaccaau gugguaauca aggugugcga guuccaguuu 360
ugcaaugacc ccuuccuggg cguguacuau cacaagaaca acaagucuug gauggagucc 420
gaguucagag uguacuccag cgccaauaac uguaccuucg aguacguguc ucagccauuu 480
cugauggauc uggagggcaa gcagggcaau uucaagaacc ugagagaguu cguguuuaag 540
aacaucgaug gcuacuucaa gaucuauucc aagcacaccc caaucaaccu ggugcgggau 600
cugccucagg gcuucucugc ccuggagcca cugguggacc ugccuaucgg caucaacauc 660
acacgguuuc agacacugcu ggcccugcac agguccuauc ugaccccagg cgacuccucu 720
ucuggcugga cagccggcgc cgccgccuac uaugugggcu accugcagcc uagaacauuc 780
cugcugaagu acaaugagaa cggcaccauc accgacgccg uggauugugc ccuggauccc 840
cugucugaga caaaguguac ccugaagucc uuuacagugg agaagggcau cuaucagacc 900
uccaacuuca gggugcagcc caccgagucu aucgugcggu uuccuaacau cacaaaucug 960
ugccccuuug gcgagguguu uaacgccacc agguuugccu ccguguaugc cuggaacagg 1020
aagcgcaucu cuaacugcgu ggccgauuac agcgugcugu acaauagcgc cagcuuuucc 1080
accuuuaagu guuauggcgu gucuccaaca aagcugaacg aucugugcuu caccaacgug 1140
uaugccgaca gcuuugugau ccgcggcgac gaggugaggc agaucgcccc cggccagacc 1200
ggcaacaucg ccgauuauaa cuacaagcug ccugacgacu uuaccggcug cgugaucgcc 1260
uggaacagca acaaccugga uucuaaggug ggcggcaacu auaauuaccu guaucgccug 1320
uuccggaagu cuaaccugaa gccuuucgag cgcgacaucu ccaccgagau cuaucaggcc 1380
ggcuccaccc cauguaaugg cgugaagggc uucaacuguu acuuuccucu gcaguccuac 1440
ggcuuucagc cuacauaugg cgugggcuac cagccuuaua gggugguggu gcugucuuuu 1500
gagcugcugc acgccccugc caccgugugc ggccccaaga agagcacaaa ccuggugaag 1560
aacaagugcg ugaauuucaa cuuuaauggc cugacaggca caggcgugcu gaccgagucc 1620
aauaagaagu uucugccuuu ccagcaguuu ggccgggaca ucgccgauac caccgacgcc 1680
gugagagacc cacagacacu ggagauccug gacaucacac cuugcuccuu cggcggcgug 1740
uccgugauca caccuggcac aaauaccucc aaucaggugg ccgugcugua ucagggcgug 1800
aacugcacag aggugccugu ggccauccac gccgaccagc ugacaccaac cuggcgggug 1860
uauucuaccg gcuccaacgu guuccagaca cgcgccggcu gccugaucgg cgccgagcac 1920
gugaauaacu cuuacgagug ugacaucccu aucggcgccg gcaucugugc cagcuaccag 1980
acccagacca acagcccacg cagagccagg uccguggccu cccagucuau caucgccuac 2040
accaugagcc ugggcgccga gaauagcgug gccuacucca auaauuccau cgccauccca 2100
accaacuuca caaucucugu gacaaccgag auccugcccg uguccaugac caagacaagc 2160
guggacugca caauguauau cuguggcgac ucuacagagu gcucuaaucu gcugcugcag 2220
uauggcuccu uuuguaccca gcugaacaga gcccugaccg gcaucgccgu ggagcaggac 2280
aagaacaccc aggagguguu cgcccaggug aagcagaucu acaagacccc accuaucaag 2340
gacuucggcg gcuucaauuu uucccagauc cugcccgacc caucuaagcc cagcaagcgg 2400
uccuuuaucg aggaucugcu guucaauaag gugacacugg ccgaugccgg cuuuaucaag 2460
caguacggcg acugucuggg cgauaucgcc gccagagacc ugaucugcgc ccagaaguuc 2520
aacggccuga ccgugcugcc cccacugcug accgaugaga ugaucgccca guacaccagc 2580
gcccugcugg ccggcaccau cacaucuggc uggaccuuug gcgccggcgc cgcccugcag 2640
aucccauuug ccaugcagau ggccuaucgc uucaacggca ucggcgugac ccagaaugug 2700
cuguaugaga aucagaagcu gaucgccaau caguuuaaua gcgccaucgg caagauccag 2760
gauucucuga gcuccacagc cucugcccug ggcaagcugc aggacguggu gaaucagaac 2820
gcccaggccc ugaauacccu ggugaagcag cuguccucua auuuuggcgc caucuccagc 2880
gugcugaacg acauccuguc ccggcuggau aagguggagg ccgaggugca gaucgacagg 2940
cugaucacag gcagacugca gucccugcag accuacguga cacagcagcu gauccgcgcc 3000
gccgagaucc gggccagcgc caaucuggcc gccaccaaga uguccgagug cgugcugggc 3060
cagagcaaga ggguggacuu uugcggcaag ggcuaccacc ugauguccuu uccacagagc 3120
gccccccacg gcgugguguu ccugcacgug acauacgugc cugcccagga gaagaacuuu 3180
acaaccgccc cagccaucug ucacgacggc aaggcccacu uuccccgcga gggcguguuc 3240
guguccaaug gcacccacug guucgugacc cagaggaauu ucuaugagcc acagaucauc 3300
acaaccgaca auacauucgu gucuggcaau ugcgacgugg uaaucggcau cgugaauaau 3360
acaguguacg acccucugca gcccgagcug gauuccuuca aggaggagcu ggacaaguau 3420
uucaagaacc acaccucccc agacguggac cugggcgaca ucagcggcau caaugccagc 3480
guggugaaca uccagaagga gaucgaucgc cugaaugagg uggccaagaa ccugaaugag 3540
ucccugaucg accugcagga gcugggcaag uacgagcagu auaucaagug gcccugguac 3600
aucuggcugg gcuuuaucgc cggccugauc gccaucguga uggugacaau caugcugugc 3660
uguaugaccu ccuguugcag cugccugaag ggcuguuguu ccugcggcag cuguuguaag 3720
uuugacgagg augacucuga gccugugcug aagggcguga agcugcacua uaccuga 3777
<210> 3
<211> 3777
<212> RNA
<213> 人工序列
<400> 3
gugaaucuga caaccagaac ccagcugcca ccagccuaua caaacuccuu cacacggggc 60
guguauuacc cugacaaggu guuucgcucu agcgugcugc acagcaccca ggaucuguuu 120
cugccauucu uuucuaacgu gacaugguuc cacgccaucc acguguccgg caccaacggc 180
acaaagcggu uugacaaccc ugugcugccc uuuaaugacg gcguguauuu cgccuccaca 240
gagaagagca auaucaucag gggcuggauc uuuggcacaa cccuggacuc uaagacccag 300
agccugcuga ucgugaacaa ugccaccaau gugguaauca aggugugcga guuccaguuu 360
ugcaaugacc ccuuccuggg cguguacuau cacaagaaca acaagucuug gauggagucc 420
gaguucagag uguacuccag cgccaauaac uguaccuucg aguacguguc ucagccauuu 480
cugauggauc uggagggcaa gcagggcaau uucaagaacc ugagagaguu cguguuuaag 540
aacaucgaug gcuacuucaa gaucuauucc aagcacaccc caaucaaccu ggugcgggau 600
cugccucagg gcuucucugc ccuggagcca cugguggacc ugccuaucgg caucaacauc 660
acacgguuuc agacacugcu ggcccugcac agguccuauc ugaccccagg cgacuccucu 720
ucuggcugga cagccggcgc cgccgccuac uaugugggcu accugcagcc uagaacauuc 780
cugcugaagu acaaugagaa cggcaccauc accgacgccg uggauugugc ccuggauccc 840
cugucugaga caaaguguac ccugaagucc uuuacagugg agaagggcau cuaucagacc 900
uccaacuuca gggugcagcc caccgagucu aucgugcggu uuccuaacau cacaaaucug 960
ugccccuuug gcgagguguu uaacgccacc agguuugccu ccguguaugc cuggaacagg 1020
aagcgcaucu cuaacugcgu ggccgauuac agcgugcugu acaauagcgc cagcuuuucc 1080
accuuuaagu guuauggcgu gucuccaaca aagcugaacg aucugugcuu caccaacgug 1140
uaugccgaca gcuuugugau ccgcggcgac gaggugaggc agaucgcccc cggccagacc 1200
ggcaagaucg ccgauuauaa cuacaagcug ccugacgacu uuaccggcug cgugaucgcc 1260
uggaacagca acaaccugga uucuaaggug ggcggcaacu auaauuaccu guaucgccug 1320
uuccggaagu cuaaccugaa gccuuucgag cgcgacaucu ccaccgagau cuaucaggcc 1380
ggcuccaccc cauguaaugg cguggagggc uucaacuguu acuuuccucu gcaguccuac 1440
ggcuuucagc cuacaaaugg cgugggcuac cagccuuaua gggugguggu gcugucuuuu 1500
gagcugcugc acgccccugc caccgugugc ggccccaaga agagcacaaa ccuggugaag 1560
aacaagugcg ugaauuucaa cuuuaauggc cugacaggca caggcgugcu gaccgagucc 1620
aauaagaagu uucugccuuu ccagcaguuu ggccgggaca ucgccgauac caccgacgcc 1680
gugagagacc cacagacacu ggagauccug gacaucacac cuugcuccuu cggcggcgug 1740
uccgugauca caccuggcac aaauaccucc aaucaggugg ccgugcugua ucaggacgug 1800
aacugcacag aggugccugu ggccauccac gccgaccagc ugacaccaac cuggcgggug 1860
uauucuaccg gcuccaacgu guuccagaca cgcgccggcu gccugaucgg cgccgagcac 1920
gugaauaacu cuuacgagug ugacaucccu aucggcgccg gcaucugugc cagcuaccag 1980
acccagacca acagcccacg cagagccagg uccguggccu cccagucuau caucgccuac 2040
accaugagcc ugggcgccga gaauagcgug gccuacucca auaauuccau cgccauccca 2100
accaacuuca caaucucugu gacaaccgag auccugcccg uguccaugac caagacaagc 2160
guggacugca caauguauau cuguggcgac ucuacagagu gcucuaaucu gcugcugcag 2220
uauggcuccu uuuguaccca gcugaacaga gcccugaccg gcaucgccgu ggagcaggac 2280
aagaacaccc aggagguguu cgcccaggug aagcagaucu acaagacccc accuaucaag 2340
gacuucggcg gcuucaauuu uucccagauc cugcccgacc caucuaagcc cagcaagcgg 2400
uccuuuaucg aggaucugcu guucaauaag gugacacugg ccgaugccgg cuuuaucaag 2460
caguacggcg acugucuggg cgauaucgcc gccagagacc ugaucugcgc ccagaaguuc 2520
aacggccuga ccgugcugcc cccacugcug accgaugaga ugaucgccca guacaccagc 2580
gcccugcugg ccggcaccau cacaucuggc uggaccuuug gcgccggcgc cgcccugcag 2640
aucccauuug ccaugcagau ggccuaucgc uucaacggca ucggcgugac ccagaaugug 2700
cuguaugaga aucagaagcu gaucgccaau caguuuaaua gcgccaucgg caagauccag 2760
gauucucuga gcuccacagc cucugcccug ggcaagcugc aggacguggu gaaucagaac 2820
gcccaggccc ugaauacccu ggugaagcag cuguccucua auuuuggcgc caucuccagc 2880
gugcugaacg acauccuguc ccggcuggau ccgccggagg ccgaggugca gaucgacagg 2940
cugaucacag gcagacugca gucccugcag accuacguga cacagcagcu gauccgcgcc 3000
gccgagaucc gggccagcgc caaucuggcc gccaccaaga uguccgagug cgugcugggc 3060
cagagcaaga ggguggacuu uugcggcaag ggcuaccacc ugauguccuu uccacagagc 3120
gccccccacg gcgugguguu ccugcacgug acauacgugc cugcccagga gaagaacuuu 3180
acaaccgccc cagccaucug ucacgacggc aaggcccacu uuccccgcga gggcguguuc 3240
guguccaaug gcacccacug guucgugacc cagaggaauu ucuaugagcc acagaucauc 3300
acaaccgaca auacauucgu gucuggcaau ugcgacgugg uaaucggcau cgugaauaau 3360
acaguguacg acccucugca gcccgagcug gauuccuuca aggaggagcu ggacaaguau 3420
uucaagaacc acaccucccc agacguggac cugggcgaca ucagcggcau caaugccagc 3480
guggugaaca uccagaagga gaucgaucgc cugaaugagg uggccaagaa ccugaaugag 3540
ucccugaucg accugcagga gcugggcaag uacgagcagu auaucaagug gcccugguac 3600
aucuggcugg gcuuuaucgc cggccugauc gccaucguga uggugacaau caugcugugc 3660
uguaugaccu ccuguugcag cugccugaag ggcuguuguu ccugcggcag cuguuguaag 3720
uuugacgagg augacucuga gccugugcug aagggcguga agcugcacua uaccuga 3777
<210> 4
<211> 3777
<212> RNA
<213> 人工序列
<400> 4
gugaaucuga caaccagaac ccagcugcca ccagccuaua caaacuccuu cacacggggc 60
guguauuacc cugacaaggu guuucgcucu agcgugcugc acagcaccca ggaucuguuu 120
cugccauucu uuucuaacgu gacaugguuc cacgccaucc acguguccgg caccaacggc 180
acaaagcggu uugacaaccc ugugcugccc uuuaaugacg gcguguauuu cgccuccaca 240
gagaagagca auaucaucag gggcuggauc uuuggcacaa cccuggacuc uaagacccag 300
agccugcuga ucgugaacaa ugccaccaau gugguaauca aggugugcga guuccaguuu 360
ugcaaugacc ccuuccuggg cguguacuau cacaagaaca acaagucuug gauggagucc 420
gaguucagag uguacuccag cgccaauaac uguaccuucg aguacguguc ucagccauuu 480
cugauggauc uggagggcaa gcagggcaau uucaagaacc ugagagaguu cguguuuaag 540
aacaucgaug gcuacuucaa gaucuauucc aagcacaccc caaucaaccu ggugcgggau 600
cugccucagg gcuucucugc ccuggagcca cugguggacc ugccuaucgg caucaacauc 660
acacgguuuc agacacugcu ggcccugcac agguccuauc ugaccccagg cgacuccucu 720
ucuggcugga cagccggcgc cgccgccuac uaugugggcu accugcagcc uagaacauuc 780
cugcugaagu acaaugagaa cggcaccauc accgacgccg uggauugugc ccuggauccc 840
cugucugaga caaaguguac ccugaagucc uuuacagugg agaagggcau cuaucagacc 900
uccaacuuca gggugcagcc caccgagucu aucgugcggu uuccuaacau cacaaaucug 960
ugccccuuug gcgagguguu uaacgccacc agguuugccu ccguguaugc cuggaacagg 1020
aagcgcaucu cuaacugcgu ggccgauuac agcgugcugu acaauagcgc cagcuuuucc 1080
accuuuaagu guuauggcgu gucuccaaca aagcugaacg aucugugcuu caccaacgug 1140
uaugccgaca gcuuugugau ccgcggcgac gaggugaggc agaucgcccc cggccagacc 1200
ggcaacaucg ccgauuauaa cuacaagcug ccugacgacu uuaccggcug cgugaucgcc 1260
uggaacagca acaaccugga uucuaaggug ggcggcaacu auaauuaccu guaucgccug 1320
uuccggaagu cuaaccugaa gccuuucgag cgcgacaucu ccaccgagau cuaucaggcc 1380
ggcuccaccc cauguaaugg cgugaagggc uucaacuguu acuuuccucu gcaguccuac 1440
ggcuuucagc cuacauaugg cgugggcuac cagccuuaua gggugguggu gcugucuuuu 1500
gagcugcugc acgccccugc caccgugugc ggccccaaga agagcacaaa ccuggugaag 1560
aacaagugcg ugaauuucaa cuuuaauggc cugacaggca caggcgugcu gaccgagucc 1620
aauaagaagu uucugccuuu ccagcaguuu ggccgggaca ucgccgauac caccgacgcc 1680
gugagagacc cacagacacu ggagauccug gacaucacac cuugcuccuu cggcggcgug 1740
uccgugauca caccuggcac aaauaccucc aaucaggugg ccgugcugua ucagggcgug 1800
aacugcacag aggugccugu ggccauccac gccgaccagc ugacaccaac cuggcgggug 1860
uauucuaccg gcuccaacgu guuccagaca cgcgccggcu gccugaucgg cgccgagcac 1920
gugaauaacu cuuacgagug ugacaucccu aucggcgccg gcaucugugc cagcuaccag 1980
acccagacca acagcccacg cagagccagg uccguggccu cccagucuau caucgccuac 2040
accaugagcc ugggcgccga gaauagcgug gccuacucca auaauuccau cgccauccca 2100
accaacuuca caaucucugu gacaaccgag auccugcccg uguccaugac caagacaagc 2160
guggacugca caauguauau cuguggcgac ucuacagagu gcucuaaucu gcugcugcag 2220
uauggcuccu uuuguaccca gcugaacaga gcccugaccg gcaucgccgu ggagcaggac 2280
aagaacaccc aggagguguu cgcccaggug aagcagaucu acaagacccc accuaucaag 2340
gacuucggcg gcuucaauuu uucccagauc cugcccgacc caucuaagcc cagcaagcgg 2400
uccuuuaucg aggaucugcu guucaauaag gugacacugg ccgaugccgg cuuuaucaag 2460
caguacggcg acugucuggg cgauaucgcc gccagagacc ugaucugcgc ccagaaguuc 2520
aacggccuga ccgugcugcc cccacugcug accgaugaga ugaucgccca guacaccagc 2580
gcccugcugg ccggcaccau cacaucuggc uggaccuuug gcgccggcgc cgcccugcag 2640
aucccauuug ccaugcagau ggccuaucgc uucaacggca ucggcgugac ccagaaugug 2700
cuguaugaga aucagaagcu gaucgccaau caguuuaaua gcgccaucgg caagauccag 2760
gauucucuga gcuccacagc cucugcccug ggcaagcugc aggacguggu gaaucagaac 2820
gcccaggccc ugaauacccu ggugaagcag cuguccucua auuuuggcgc caucuccagc 2880
gugcugaacg acauccuguc ccggcuggau ccgccggagg ccgaggugca gaucgacagg 2940
cugaucacag gcagacugca gucccugcag accuacguga cacagcagcu gauccgcgcc 3000
gccgagaucc gggccagcgc caaucuggcc gccaccaaga uguccgagug cgugcugggc 3060
cagagcaaga ggguggacuu uugcggcaag ggcuaccacc ugauguccuu uccacagagc 3120
gccccccacg gcgugguguu ccugcacgug acauacgugc cugcccagga gaagaacuuu 3180
acaaccgccc cagccaucug ucacgacggc aaggcccacu uuccccgcga gggcguguuc 3240
guguccaaug gcacccacug guucgugacc cagaggaauu ucuaugagcc acagaucauc 3300
acaaccgaca auacauucgu gucuggcaau ugcgacgugg uaaucggcau cgugaauaau 3360
acaguguacg acccucugca gcccgagcug gauuccuuca aggaggagcu ggacaaguau 3420
uucaagaacc acaccucccc agacguggac cugggcgaca ucagcggcau caaugccagc 3480
guggugaaca uccagaagga gaucgaucgc cugaaugagg uggccaagaa ccugaaugag 3540
ucccugaucg accugcagga gcugggcaag uacgagcagu auaucaagug gcccugguac 3600
aucuggcugg gcuuuaucgc cggccugauc gccaucguga uggugacaau caugcugugc 3660
uguaugaccu ccuguugcag cugccugaag ggcuguuguu ccugcggcag cuguuguaag 3720
uuugacgagg augacucuga gccugugcug aagggcguga agcugcacua uaccuga 3777
<210> 5
<211> 3777
<212> RNA
<213> 人工序列
<400> 5
gugaaucuga caaccagaac ccagcugcca ccagccuaua caaacuccuu cacacggggc 60
guguauuacc cugacaaggu guuucgcucu agcgugcugc acagcaccca ggaucuguuu 120
cugccauucu uuucuaacgu gacaugguuc cacgccaucc acguguccgg caccaacggc 180
acaaagcggu uugacaaccc ugugcugccc uuuaaugacg gcguguauuu cgccuccaca 240
gagaagagca auaucaucag gggcuggauc uuuggcacaa cccuggacuc uaagacccag 300
agccugcuga ucgugaacaa ugccaccaau gugguaauca aggugugcga guuccaguuu 360
ugcaaugacc ccuuccuggg cguguacuau cacaagaaca acaagucuug gauggagucc 420
gaguucagag uguacuccag cgccaauaac uguaccuucg aguacguguc ucagccauuu 480
cugauggauc uggagggcaa gcagggcaau uucaagaacc ugagagaguu cguguuuaag 540
aacaucgaug gcuacuucaa gaucuauucc aagcacaccc caaucaaccu ggugcgggau 600
cugccucagg gcuucucugc ccuggagcca cugguggacc ugccuaucgg caucaacauc 660
acacgguuuc agacacugcu ggcccugcac agguccuauc ugaccccagg cgacuccucu 720
ucuggcugga cagccggcgc cgccgccuac uaugugggcu accugcagcc uagaacauuc 780
cugcugaagu acaaugagaa cggcaccauc accgacgccg uggauugugc ccuggauccc 840
cugucugaga caaaguguac ccugaagucc uuuacagugg agaagggcau cuaucagacc 900
uccaacuuca gggugcagcc caccgagucu aucgugcggu uuccuaacau cacaaaucug 960
ugccccuuug gcgagguguu uaacgccacc agguuugccu ccguguaugc cuggaacagg 1020
aagcgcaucu cuaacugcgu ggccgauuac agcgugcugu acaauagcgc cagcuuuucc 1080
accuuuaagu guuauggcgu gucuccaaca aagcugaacg aucugugcuu caccaacgug 1140
uaugccgaca gcuuugugau ccgcggcgac gaggugaggc agaucgcccc cggccagacc 1200
ggcaagaucg ccgauuauaa cuacaagcug ccugacgacu uuaccggcug cgugaucgcc 1260
uggaacagca acaaccugga uucuaaggug ggcggcaacu auaauuaccu guaucgccug 1320
uuccggaagu cuaaccugaa gccuuucgag cgcgacaucu ccaccgagau cuaucaggcc 1380
ggcuccaccc cauguaaugg cguggagggc uucaacuguu acuuuccucu gcaguccuac 1440
ggcuuucagc cuacaaaugg cgugggcuac cagccuuaua gggugguggu gcugucuuuu 1500
gagcugcugc acgccccugc caccgugugc ggccccaaga agagcacaaa ccuggugaag 1560
aacaagugcg ugaauuucaa cuuuaauggc cugacaggca caggcgugcu gaccgagucc 1620
aauaagaagu uucugccuuu ccagcaguuu ggccgggaca ucgccgauac caccgacgcc 1680
gugagagacc cacagacacu ggagauccug gacaucacac cuugcuccuu cggcggcgug 1740
uccgugauca caccuggcac aaauaccucc aaucaggugg ccgugcugua ucaggacgug 1800
aacugcacag aggugccugu ggccauccac gccgaccagc ugacaccaac cuggcgggug 1860
uauucuaccg gcuccaacgu guuccagaca cgcgccggcu gccugaucgg cgccgagcac 1920
gugaauaacu cuuacgagug ugacaucccu aucggcgccg gcaucugugc cagcuaccag 1980
acccagacca acagcccacg cagagccagg uccguggccu cccagucuau caucgccuac 2040
accaugagcc ugggcgccga gaauagcgug gccuacucca auaauuccau cgccauccca 2100
accaacuuca caaucucugu gacaaccgag auccugcccg uguccaugac caagacaagc 2160
guggacugca caauguauau cuguggcgac ucuacagagu gcucuaaucu gcugcugcag 2220
uauggcuccu uuuguaccca gcugaacaga gcccugaccg gcaucgccgu ggagcaggac 2280
aagaacaccc aggagguguu cgcccaggug aagcagaucu acaagacccc accuaucaag 2340
gacuucggcg gcuucaauuu uucccagauc cugcccgacc caucuaagcc cagcaagcgg 2400
uccccuaucg aggaucugcu guucaauaag gugacacugg ccgaugccgg cuuuaucaag 2460
caguacggcg acugucuggg cgauaucgcc gccagagacc ugaucugcgc ccagaaguuc 2520
aacggccuga ccgugcugcc cccacugcug accgaugaga ugaucgccca guacaccagc 2580
gcccugcugg ccggcaccau cacaucuggc uggaccuuug gcgccggccc cgcccugcag 2640
aucccauuuc ccaugcagau ggccuaucgc uucaacggca ucggcgugac ccagaaugug 2700
cuguaugaga aucagaagcu gaucgccaau caguuuaaua gcgccaucgg caagauccag 2760
gauucucuga gcuccacacc cucugcccug ggcaagcugc aggacguggu gaaucagaac 2820
gcccaggccc ugaauacccu ggugaagcag cuguccucua auuuuggcgc caucuccagc 2880
gugcugaacg acauccuguc ccggcuggau ccgccggagg ccgaggugca gaucgacagg 2940
cugaucacag gcagacugca gucccugcag accuacguga cacagcagcu gauccgcgcc 3000
gccgagaucc gggccagcgc caaucuggcc gccaccaaga uguccgagug cgugcugggc 3060
cagagcaaga ggguggacuu uugcggcaag ggcuaccacc ugauguccuu uccacagagc 3120
gccccccacg gcgugguguu ccugcacgug acauacgugc cugcccagga gaagaacuuu 3180
acaaccgccc cagccaucug ucacgacggc aaggcccacu uuccccgcga gggcguguuc 3240
guguccaaug gcacccacug guucgugacc cagaggaauu ucuaugagcc acagaucauc 3300
acaaccgaca auacauucgu gucuggcaau ugcgacgugg uaaucggcau cgugaauaau 3360
acaguguacg acccucugca gcccgagcug gauuccuuca aggaggagcu ggacaaguau 3420
uucaagaacc acaccucccc agacguggac cugggcgaca ucagcggcau caaugccagc 3480
guggugaaca uccagaagga gaucgaucgc cugaaugagg uggccaagaa ccugaaugag 3540
ucccugaucg accugcagga gcugggcaag uacgagcagu auaucaagug gcccugguac 3600
aucuggcugg gcuuuaucgc cggccugauc gccaucguga uggugacaau caugcugugc 3660
uguaugaccu ccuguugcag cugccugaag ggcuguuguu ccugcggcag cuguuguaag 3720
uuugacgagg augacucuga gccugugcug aagggcguga agcugcacua uaccuga 3777
<210> 6
<211> 3777
<212> RNA
<213> 人工序列
<400> 6
gugaaucuga caaccagaac ccagcugcca ccagccuaua caaacuccuu cacacggggc 60
guguauuacc cugacaaggu guuucgcucu agcgugcugc acagcaccca ggaucuguuu 120
cugccauucu uuucuaacgu gacaugguuc cacgccaucc acguguccgg caccaacggc 180
acaaagcggu uugacaaccc ugugcugccc uuuaaugacg gcguguauuu cgccuccaca 240
gagaagagca auaucaucag gggcuggauc uuuggcacaa cccuggacuc uaagacccag 300
agccugcuga ucgugaacaa ugccaccaau gugguaauca aggugugcga guuccaguuu 360
ugcaaugacc ccuuccuggg cguguacuau cacaagaaca acaagucuug gauggagucc 420
gaguucagag uguacuccag cgccaauaac uguaccuucg aguacguguc ucagccauuu 480
cugauggauc uggagggcaa gcagggcaau uucaagaacc ugagagaguu cguguuuaag 540
aacaucgaug gcuacuucaa gaucuauucc aagcacaccc caaucaaccu ggugcgggau 600
cugccucagg gcuucucugc ccuggagcca cugguggacc ugccuaucgg caucaacauc 660
acacgguuuc agacacugcu ggcccugcac agguccuauc ugaccccagg cgacuccucu 720
ucuggcugga cagccggcgc cgccgccuac uaugugggcu accugcagcc uagaacauuc 780
cugcugaagu acaaugagaa cggcaccauc accgacgccg uggauugugc ccuggauccc 840
cugucugaga caaaguguac ccugaagucc uuuacagugg agaagggcau cuaucagacc 900
uccaacuuca gggugcagcc caccgagucu aucgugcggu uuccuaacau cacaaaucug 960
ugccccuuug gcgagguguu uaacgccacc agguuugccu ccguguaugc cuggaacagg 1020
aagcgcaucu cuaacugcgu ggccgauuac agcgugcugu acaauagcgc cagcuuuucc 1080
accuuuaagu guuauggcgu gucuccaaca aagcugaacg aucugugcuu caccaacgug 1140
uaugccgaca gcuuugugau ccgcggcgac gaggugaggc agaucgcccc cggccagacc 1200
ggcaacaucg ccgauuauaa cuacaagcug ccugacgacu uuaccggcug cgugaucgcc 1260
uggaacagca acaaccugga uucuaaggug ggcggcaacu auaauuaccu guaucgccug 1320
uuccggaagu cuaaccugaa gccuuucgag cgcgacaucu ccaccgagau cuaucaggcc 1380
ggcuccaccc cauguaaugg cgugaagggc uucaacuguu acuuuccucu gcaguccuac 1440
ggcuuucagc cuacauaugg cgugggcuac cagccuuaua gggugguggu gcugucuuuu 1500
gagcugcugc acgccccugc caccgugugc ggccccaaga agagcacaaa ccuggugaag 1560
aacaagugcg ugaauuucaa cuuuaauggc cugacaggca caggcgugcu gaccgagucc 1620
aauaagaagu uucugccuuu ccagcaguuu ggccgggaca ucgccgauac caccgacgcc 1680
gugagagacc cacagacacu ggagauccug gacaucacac cuugcuccuu cggcggcgug 1740
uccgugauca caccuggcac aaauaccucc aaucaggugg ccgugcugua ucagggcgug 1800
aacugcacag aggugccugu ggccauccac gccgaccagc ugacaccaac cuggcgggug 1860
uauucuaccg gcuccaacgu guuccagaca cgcgccggcu gccugaucgg cgccgagcac 1920
gugaauaacu cuuacgagug ugacaucccu aucggcgccg gcaucugugc cagcuaccag 1980
acccagacca acagcccacg cagagccagg uccguggccu cccagucuau caucgccuac 2040
accaugagcc ugggcgccga gaauagcgug gccuacucca auaauuccau cgccauccca 2100
accaacuuca caaucucugu gacaaccgag auccugcccg uguccaugac caagacaagc 2160
guggacugca caauguauau cuguggcgac ucuacagagu gcucuaaucu gcugcugcag 2220
uauggcuccu uuuguaccca gcugaacaga gcccugaccg gcaucgccgu ggagcaggac 2280
aagaacaccc aggagguguu cgcccaggug aagcagaucu acaagacccc accuaucaag 2340
gacuucggcg gcuucaauuu uucccagauc cugcccgacc caucuaagcc cagcaagcgg 2400
uccccuaucg aggaucugcu guucaauaag gugacacugg ccgaugccgg cuuuaucaag 2460
caguacggcg acugucuggg cgauaucgcc gccagagacc ugaucugcgc ccagaaguuc 2520
aacggccuga ccgugcugcc cccacugcug accgaugaga ugaucgccca guacaccagc 2580
gcccugcugg ccggcaccau cacaucuggc uggaccuuug gcgccggccc cgcccugcag 2640
aucccauuuc ccaugcagau ggccuaucgc uucaacggca ucggcgugac ccagaaugug 2700
cuguaugaga aucagaagcu gaucgccaau caguuuaaua gcgccaucgg caagauccag 2760
gauucucuga gcuccacacc cucugcccug ggcaagcugc aggacguggu gaaucagaac 2820
gcccaggccc ugaauacccu ggugaagcag cuguccucua auuuuggcgc caucuccagc 2880
gugcugaacg acauccuguc ccggcuggau ccgccggagg ccgaggugca gaucgacagg 2940
cugaucacag gcagacugca gucccugcag accuacguga cacagcagcu gauccgcgcc 3000
gccgagaucc gggccagcgc caaucuggcc gccaccaaga uguccgagug cgugcugggc 3060
cagagcaaga ggguggacuu uugcggcaag ggcuaccacc ugauguccuu uccacagagc 3120
gccccccacg gcgugguguu ccugcacgug acauacgugc cugcccagga gaagaacuuu 3180
acaaccgccc cagccaucug ucacgacggc aaggcccacu uuccccgcga gggcguguuc 3240
guguccaaug gcacccacug guucgugacc cagaggaauu ucuaugagcc acagaucauc 3300
acaaccgaca auacauucgu gucuggcaau ugcgacgugg uaaucggcau cgugaauaau 3360
acaguguacg acccucugca gcccgagcug gauuccuuca aggaggagcu ggacaaguau 3420
uucaagaacc acaccucccc agacguggac cugggcgaca ucagcggcau caaugccagc 3480
guggugaaca uccagaagga gaucgaucgc cugaaugagg uggccaagaa ccugaaugag 3540
ucccugaucg accugcagga gcugggcaag uacgagcagu auaucaagug gcccugguac 3600
aucuggcugg gcuuuaucgc cggccugauc gccaucguga uggugacaau caugcugugc 3660
uguaugaccu ccuguugcag cugccugaag ggcuguuguu ccugcggcag cuguuguaag 3720
uuugacgagg augacucuga gccugugcug aagggcguga agcugcacua uaccuga 3777
<210> 7
<211> 34
<212> RNA
<213> 人工序列
<400> 7
gagacccaag cuggcuagcg ggagaaagcu uacc 34
<210> 8
<211> 109
<212> RNA
<213> 人工序列
<400> 8
gcuggagccu cgguagccgu uccuccugcc cgcugggccu cccaacgggc ccuccucccc 60
uccuugcacc ggcccuuccu ggucuuugaa uaaagucuga gugggcagc 109
Claims (14)
1.一种分离的mRNA，其特征在于，其编码区核酸序列如SEQ ID No.2或4所示。
2.根据权利要求1所述的mRNA，其特征在于，所述mRNA还包括：5’端帽子、5’UTR、3’UTR以及3’端polyA尾中的至少一项。
3.一种重组载体，其特征在于，其包括转录如权利要求1或2所述的分离的mRNA的DNA分子。
4.一种宿主细胞，其特征在于，其含有如权利要求3所述的重组载体。
5.一种新型冠状病毒疫苗，其特征在于，其制备原料包括：如权利要求1或2所述的mRNA和递送制剂。
6.根据权利要求5所述的新型冠状病毒疫苗，其特征在于，所述递送制剂包括阳离子脂质纳米颗粒或阳离子脂质体。
7.根据权利要求6所述的新型冠状病毒疫苗，其特征在于，所述mRNA和阳离子脂质纳米颗粒的质量比为5~20：1。
8.根据权利要求6所述的新型冠状病毒疫苗，其特征在于，所述阳离子脂质纳米颗粒的组分包括：可质子化阳离子脂质、结构脂质、辅助脂质和表面活性剂。
9.根据权利要求8所述的新型冠状病毒疫苗，其特征在于，按重量份数计，所述阳离子脂质纳米颗粒的组分包括：40~60份可质子化阳离子脂质、20~40份结构脂质、10~30份辅助脂质和0.5~5份表面活性剂。
10.根据权利要求9所述的新型冠状病毒疫苗，其特征在于，所述可质子化阳离子脂质选自：Dlin-MC3-DMA、DODMA、C12-200和DlinDMA中的至少一种。
11.根据权利要求9所述的新型冠状病毒疫苗，其特征在于，所述结构脂质包括胆固醇和/或胆固醇衍生物。
12.根据权利要求9所述的新型冠状病毒疫苗，其特征在于，所述辅助脂质包括DSPC、DOPE、DOPC、DOPG和DOPS中的至少一种。
13.根据权利要求9所述的新型冠状病毒疫苗，其特征在于，所述表面活性剂包括PEG-DMG、PEG-DSPE和TPGS中的至少一种。
14.如权利要求5~9任一项所述的新型冠状病毒疫苗的制备方法，其特征在于，其包括：将所述mRNA和递送制剂混合，以形成新型冠状病毒疫苗。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111653353.8A CN116426543B (zh) | 2021-12-30 | 2021-12-30 | 一种新型冠状病毒疫苗及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111653353.8A CN116426543B (zh) | 2021-12-30 | 2021-12-30 | 一种新型冠状病毒疫苗及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116426543A CN116426543A (zh) | 2023-07-14 |
| CN116426543B true CN116426543B (zh) | 2024-03-22 |
Family
ID=87084195
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111653353.8A Active CN116426543B (zh) | 2021-12-30 | 2021-12-30 | 一种新型冠状病毒疫苗及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116426543B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114934056B (zh) * | 2022-06-24 | 2023-10-20 | 仁景(苏州)生物科技有限公司 | 一种基于新型冠状病毒奥密克戎突变株的mRNA疫苗 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218458A (zh) * | 2020-02-27 | 2020-06-02 | 珠海丽凡达生物技术有限公司 | 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法 |
| CN113322348A (zh) * | 2021-03-27 | 2021-08-31 | 中国人民解放军军事科学院军事医学研究院 | 一种高灵敏新型冠状病毒2019-nCoV核酸检测试剂盒及使用方法 |
| WO2021261993A1 (en) * | 2020-06-24 | 2021-12-30 | Unthreadt B.V. | Pharmaceutical composition for treatment of covid-19 and related pathologies |
-
2021
- 2021-12-30 CN CN202111653353.8A patent/CN116426543B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218458A (zh) * | 2020-02-27 | 2020-06-02 | 珠海丽凡达生物技术有限公司 | 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法 |
| WO2021261993A1 (en) * | 2020-06-24 | 2021-12-30 | Unthreadt B.V. | Pharmaceutical composition for treatment of covid-19 and related pathologies |
| CN113322348A (zh) * | 2021-03-27 | 2021-08-31 | 中国人民解放军军事科学院军事医学研究院 | 一种高灵敏新型冠状病毒2019-nCoV核酸检测试剂盒及使用方法 |
Non-Patent Citations (3)
| Title |
|---|
| Phase 1 randomized trial of a plant-derived virus-like particle vaccine for COVID-19;Brian J. Ward等;《Nature Medicine》;第27卷;第1079页左栏第1段 * |
| The SARS-CoV-2 spike protein: balancing stability and infectivity;Imre Berger等;《Cell Research》;第30卷;第1059–1060页 * |
| 基于新冠病毒RBD的广谱性mRNA疫苗设计与评价;杨璐;《中国优秀硕士学位论文全文数据库医药卫生科技辑》(第02期);第E059-399页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116426543A (zh) | 2023-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111218458B (zh) | 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法 | |
| US20240000917A1 (en) | Influenza vaccine | |
| US20230108894A1 (en) | Coronavirus rna vaccines | |
| US20230355743A1 (en) | Multi-proline-substituted coronavirus spike protein vaccines | |
| US20240293534A1 (en) | Coronavirus glycosylation variant vaccines | |
| US20240358819A1 (en) | Pan-human coronavirus domain vaccines | |
| CN115551545A (zh) | SARS-COV-2 mRNA结构域疫苗 | |
| US20240207392A1 (en) | Epstein-barr virus mrna vaccines | |
| US20240285754A1 (en) | Mrna vaccines encoding flexible coronavirus spike proteins | |
| EP3713601A1 (en) | Epstein-barr virus vaccines | |
| CN114507675B (zh) | 一种新型冠状病毒mRNA疫苗及其制备方法 | |
| WO2023283642A2 (en) | Pan-human coronavirus concatemeric vaccines | |
| EP4366768A1 (en) | Pan-human coronavirus vaccines | |
| CN115297892A (zh) | 抗新型冠状病毒感染的mRNA疫苗的组合物和方法 | |
| WO2023092069A1 (en) | Sars-cov-2 mrna domain vaccines and methods of use | |
| WO2023051701A1 (zh) | 抗SARS-CoV-2感染的mRNA、蛋白以及抗SARS-CoV-2感染的疫苗 | |
| CN116426543B (zh) | 一种新型冠状病毒疫苗及其制备方法 | |
| WO2023098679A1 (zh) | 预防突变株的新型冠状病毒mRNA疫苗 | |
| CN116808191A (zh) | 新型冠状病毒疫苗及其制备方法和应用 | |
| CN117205309B (zh) | 一种流感免疫原组合物和制备方法及其用途 | |
| CN116904489B (zh) | 一种鸭坦布苏病毒核酸疫苗及应用 | |
| CN116925195B (zh) | 一种基于新型冠状病毒的mRNA疫苗 | |
| CN116510001B (zh) | 一种水产养殖用mRNA疫苗及其制备方法 | |
| US20240366750A1 (en) | Viral antigen display compositions and methods | |
| WO2023172547A1 (en) | Nucleic acid-lipid nanoparticle and method using the same |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |