WO2023051701A1 - Mrna, protein and vaccine against sars-cov-2 infection - Google Patents

Mrna, protein and vaccine against sars-cov-2 infection Download PDF

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WO2023051701A1
WO2023051701A1 PCT/CN2022/122626 CN2022122626W WO2023051701A1 WO 2023051701 A1 WO2023051701 A1 WO 2023051701A1 CN 2022122626 W CN2022122626 W CN 2022122626W WO 2023051701 A1 WO2023051701 A1 WO 2023051701A1
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seq
mrna
protein
cov
sars
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魏霞蔚
宋相容
魏于全
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成都威斯津生物医药科技有限公司
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    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine, in particular, the invention relates to anti-SARS-CoV-2 infection mRNA, protein and anti-SARS-CoV-2 infection vaccine.
  • the new coronavirus (SARS-CoV-2, referred to as the new coronavirus) is an enveloped, non-segmented, single-stranded positive-sense RNA virus, belonging to the new coronavirus of the genus ⁇ .
  • the length of each genome of the virus is about 30,000 nucleotides, and its gene sequence is similar to that of the coronavirus (MERS-CoV or SARS-CoV) found in Chinese horseshoe bats, but there are some obvious differences; current research shows The homology with bat SARS-like coronavirus (bat-SL-CoVZC45) is more than 85%.
  • ACE2 angiotensin-converting enzyme 2
  • S protein spike protein
  • an object of the present invention is to provide anti-SARS-CoV-2 infected mRNA.
  • Another object of the present invention is to provide a vaccine prepared by using the mRNA for preventing and/or treating SARS-CoV-2 infection.
  • the new crown belongs to RNA virus, RNA virus is unstable, and natural mutations will occur during virus replication. In some cases, viruses will acquire trait changes, such as changes in transmissibility, pathogenicity, and environmental tolerance.
  • SARS-Cov-2 several key mutant strains have been produced and reported recently, one of which is the Beta mutant strain B.1.351 (20H/501Y.V2), and the other is the Gamma mutant strain P.1 (20J /501Y.V3), and the other ones are Delta mutants B.1.617.1 series and B.1.617.2 series. The emergence of mutant strains led to increased infectivity of the virus and immune escape.
  • the antibody titer produced by the recovered person's serum to the Gamma mutant strain was reduced by an average of 8 times. Therefore, it is urgent to make some changes to the sequence of the vaccine antigen to adapt to the mutation of the spike protein on the mutant strain.
  • Traditional vaccines include live attenuated vaccines, inactivated vaccines and subunit vaccines (recombinant protein vaccines or polypeptide vaccines).
  • the use of vaccines has successfully prevented many diseases worldwide, such as smallpox virus has been completely eradicated, and the incidence of hepatitis B, polio, measles and other childhood diseases has been greatly reduced around the world.
  • these vaccines have different development and application shortcomings.
  • the development cycle of live attenuated vaccines or inactivated vaccines is relatively long. Because they contain pathogens, there is a risk of causing host infection.
  • the routine test items in the test are complicated and time-consuming. Therefore, it is difficult for traditional vaccines to play their role in a timely manner in the fight against SARS-CoV-2, Ebola virus, Zika virus and other sudden viruses that have caused great harm to human health.
  • new vaccines include nucleic acid vaccines and virus vector vaccines, among which nucleic acid vaccines (mRNA or DNA) have a very obvious timeliness advantage as emergency vaccines.
  • mRNA is a type of single-stranded ribonucleic acid that carries genetic information and guides protein synthesis. At present, it is mainly synthesized by in vitro transcription using DNA as a template.
  • the working principle of the mRNA vaccine is: the mRNA encoding a specific antigen protein is directly introduced into the somatic cells, and the antigen protein is synthesized through the expression system of the host cell, and the host immune system is induced to produce a specific immune response to the antigen, effectively exerting the effect of the disease. Therapeutic and preventive effects.
  • mRNA does not need to enter the nucleus, it can be translated in the cytoplasm, and the effect is faster; there is no risk of integrating the host genome, and it will be automatically degraded in the body; it can simulate the natural infection process of the virus to activate the immune system, which can stimulate a potentially stronger immunity reaction.
  • the invention provides an mRNA.
  • the template DNA of the mRNA includes an antigen coding region; the antigen coding region encodes the SARS-CoV-2 virus Delta mutant strain S protein without a signal peptide, and the SARS-CoV-2 virus without a signal peptide
  • the S protein of the CoV-2 viral Delta mutant strain has at least one of the K986P and V987P mutations.
  • the antigenic protein synthesized by the mRNA of the present invention has strong stability and strong immunogenicity, especially can effectively prevent and treat wild-type SARS-CoV-2 virus (also known as wild-type new coronavirus or wild-type virus) and its Mutant strains (SARS-CoV-2 virus mutant strain Alpha (referred to as Alpha virus), SARS-CoV-2 virus mutant strain Beta (referred to as Beta virus), SARS-CoV-2 virus mutant strain Gamma (referred to as Gamma virus), SARS- The infection of CoV-2 virus variant Delta (abbreviated as Delta virus) and SARS-CoV-2 virus variant strain Omicron (abbreviated as Omicron virus) has broad-spectrum antiviral infection ability.
  • SARS-CoV-2 virus mutant strain Alpha referred to as Alpha virus
  • SARS-CoV-2 virus mutant strain Beta referred to as Beta virus
  • Gamma virus SARS-CoV-2 virus mutant strain Gamma
  • the mRNA may further comprise at least one of the following technical features:
  • the antigen coding region encodes a protein having an amino acid sequence as shown in SEQ ID NO:7.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 7 is B.1.617.1 after removing the 1-19 signal peptide sequence (SEQ ID NO: 2) of the B.1.617.1 mutant S protein
  • SEQ ID NO: 2 The full length of the S protein of the mutant strain, the S protein of the B.1.617.1 mutant strain undergoes K986P and V987P mutations (ie, SEQ ID NO: 4).
  • the S protein of the SARS-CoV-2 virus Delta mutant strain further has at least one of the following mutations: F817P, A892P, A899P and A942P. Therefore, the antigenic protein synthesized by the mRNA has strong stability and strong immunogenicity, thereby improving the prevention and treatment effect on wild-type SARS-CoV-2 virus and its mutant strain infection.
  • the antigen coding region encodes a protein having an amino acid sequence as shown in SEQ ID NO: 8.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 8 is the B.1.617.1 mutation that removes the 1-19 signal peptide sequence (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain.
  • SEQ ID NO: 2 The full length of the S protein of the strain, the S protein of the B.1.617.1 mutant strain undergoes F817P, A892P, A899P, A942P, K986P and V987P mutations (ie, SEQ ID NO: 5).
  • the antigen coding region further encodes the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with a mutation, the mutation being C538S.
  • amino acid sequence of the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with mutation is shown in SEQ ID NO: 6.
  • amino acid sequence of the protein shown in SEQ ID NO: 6 is the RBD (319-545) sequence of the wild-type SARS-CoV-2 virus S protein with a C538S mutation.
  • the C-terminus of the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with a mutation and the S protein of the Delta mutant strain of the SARS-CoV-2 virus without a signal peptide The N-terminus is connected. That is, the RBD domain of the wild-type S protein with the C538S mutation is fused to the nitrogen terminal of the Delta mutant S protein.
  • the template DNA of the mRNA further includes a signal peptide coding region.
  • the signal peptide coding region encodes the signal peptide of the Delta mutant strain of SARS-CoV-2 virus.
  • amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
  • the signal peptide whose amino acid sequence is shown in SEQ ID NO: 2 is the 1-19 signal peptide of the S protein of the Delta mutant strain B.1.617.1.
  • MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 2).
  • the amino acid sequence encoded by the antigen coding region of the template DNA of the mRNA is such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16. At least one of SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 30 .
  • the protein whose amino acid sequence is shown in SEQ ID NO: 4 is the full length of the S protein of the B.1.617.1 mutant strain with K986P and V987P mutations.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 5 is the full length of the S protein of the B.1.617.1 mutant strain with F817P, A892P, A899P, A942P, K986P and V987P mutations.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 16 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the wild The fusion protein obtained by direct fusion of RBD (319-545) (SEQ ID NO: 6) of the type S protein and the S protein (SEQ ID NO: 7) of the B.1.617.1 mutant strain with the 1-19 signal peptide removed .
  • the mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and B.1.617.1 mutant SARS-CoV-2.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 17 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the wild The fusion protein obtained by direct fusion of RBD (319-545) (SEQ ID NO: 6) of the type S protein and the S protein (SEQ ID NO: 8) of the B.1.617.1 mutant strain with the 1-19 signal peptide removed .
  • the mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and B.1.617.1 mutant SARS-CoV-2.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 22 is the full-length S protein of the Delta mutant strain B.1.617.2 with K986P and V987P mutations. Therefore, the antigenic protein produced by the mRNA has strong stability, and the mRNA vaccine prepared by the mRNA has a broad spectrum.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 23 is the full length of the S protein of the Delta mutant strain B.1.617.2 with F817P, A892P, A899P, A942P, K986P and V987P mutations.
  • the antigenic protein produced by the mRNA has strong stability and can enhance the expression level of the antigenic protein.
  • the mRNA vaccine prepared from the mRNA has a broad spectrum.
  • SEQ ID NO: 25 the protein whose amino acid sequence is shown in SEQ ID NO: 25 is the S protein (SEQ ID NO: twenty two).
  • SEQ ID NO: 26 the protein whose amino acid sequence is shown in SEQ ID NO: 26 is the S protein (SEQ ID NO: twenty three).
  • the protein whose amino acid sequence is shown in SEQ ID NO: 29 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, which has a C538S mutation
  • the fusion obtained by direct fusion of the RBD (319-545, SEQ ID NO: 6) of the wild-type S protein and the S protein (SEQ ID NO: 25) of the Delta mutant strain B.1.617.2 that removes the 1-19 signal peptide protein.
  • the mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and Delta mutant B.1.617.2 SARS-CoV-2.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 29 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, which has a C538S mutation
  • the fusion obtained by direct fusion of the RBD (319-545, SEQ ID NO: 6) of the wild-type S protein and the S protein (SEQ ID NO: 26) of the Delta mutant strain B.1.617.2 that removes the 1-19 signal peptide protein.
  • the mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and Delta mutant B.1.617.2 SARS-CoV-2.
  • the promoter of the template DNA is a T7 or SP6 promoter.
  • the template DNA further includes a 5' untranslated region.
  • nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
  • the template DNA further includes a 3' untranslated region.
  • nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
  • the 3' end of the template DNA is further connected to polyA.
  • nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
  • the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
  • the template DNA is the promoter, the 5'-terminal untranslated region, the signal peptide coding region, the antigen coding region, the 3'-terminal untranslated region and polyA.
  • the signal peptide coding region and the antigen coding region can be provided in one sequence, or can be provided in two separate sequences; in the present invention, when the signal peptide coding region and the antigen coding region are provided in one sequence, it can be referred to as for the antigen coding region.
  • the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
  • the present invention provides an mRNA.
  • the template DNA of the mRNA comprises an antigen coding region; the antigen coding region encodes the NTD_RBD domain of the SARS-CoV-2 virus Delta mutant strain, and the SARS-CoV-2 virus Delta mutant strain
  • the NTD_RBD domain has a C538S mutation.
  • the mRNA of the present invention has stronger immunogenicity, can produce higher neutralizing antibody after injecting this mRNA, can effectively prevent and treat wild-type SARS-CoV-2 virus and mutant strain thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
  • the mRNA may further comprise at least one of the following technical features:
  • amino acid sequence of the SARS-CoV-2 virus Delta mutant is shown in SEQ ID NO: 9.
  • NTD_RBD 20-545 of the S protein of the B.1.617.1 mutant strain with a C538S mutation.
  • the antigen coding region further encodes the RBD domain of wild-type SARS-CoV-2 with a mutation, the mutation being C538S.
  • amino acid sequence of the RBD domain of the mutated wild-type SARS-CoV-2 is shown in SEQ ID NO: 6.
  • the antigen coding region further encodes a Foldon fragment.
  • the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
  • GYIPEAPRDGQAYVRKDGEWVFLSTFL (SEQ ID NO: 11).
  • the antigen coding region further encodes a linker.
  • the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 or (GSG) 10 .
  • GGGGS SEQ ID NO: 43;
  • the amino acid sequence of (GGGGS) 3 is: GGGGSGGGGSGGGGS (SEQ ID NO: 44);
  • the amino acid sequence of (GGGGS) 6 is: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 10);
  • the amino acid sequence of (GGS) 10 is: GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 45);
  • the amino acid sequence of (GSG) 10 is: GSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSG (SEQ ID NO: 46).
  • the template DNA further includes a signal peptide coding region.
  • the signal peptide coding region encodes the signal peptide of the Delta mutant strain of SARS-CoV-2 virus.
  • amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
  • the amino acid sequence encoded by the template DNA of the mRNA is such as SEQ ID NO: 9, SEQ ID NO: 18, or SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 31 or SEQ ID NO: At least one of ID NO: 32 is shown.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 18 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the B .
  • the protein whose amino acid sequence is shown in SEQ ID NO: 19 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the B .1.617.1 NTD_RBD (20-545) sequence (SEQ ID NO: 9), wild-type RBD (SEQ ID NO: 6) with C538S mutation, sequence is (GGGGS) 6 Linker and Foldon fragment (SEQ ID NO: 11) fusion protein obtained by fusion.
  • NTD_RBD 20-545 of the S protein of the Delta mutant strain B.1.617.2 with the C538S mutation.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 31 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the C538S mutation A fusion protein obtained by fusing the NTD_RBD sequence (20-545, SEQ ID NO: 27) of the Delta mutant strain B.1.617.2, and the Linker and Foldon fragments (SEQ ID NO: 11) of (GGGGS) 6 .
  • the protein whose amino acid sequence is shown in SEQ ID NO: 32 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the C538S mutation
  • the promoter of the template DNA is a T7 or SP6 promoter.
  • the template DNA further includes a 5' untranslated region.
  • nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
  • the template DNA further includes a 3' untranslated region.
  • nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
  • the 3' end of the template DNA is further connected to polyA.
  • nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
  • the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
  • the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
  • the invention provides an mRNA.
  • the template DNA of the mRNA comprises an antigen coding region; the antigen coding region encodes the RBD domain of the SARS-CoV-2 virus Delta mutant strain and the NTD_RBD structure of the SARS-CoV-2 virus Gamma mutant strain At least one of the domains; wherein, the RBD domain of the SARS-CoV-2 virus Delta mutant has a C538S mutation; the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant has a D80A mutation, a R246I mutation and C538S mutation, as well as increased ⁇ 242-244 of the Beta mutant.
  • the mRNA of the present invention has stronger immunogenicity, can produce higher neutralizing antibody after injecting this mRNA, can effectively prevent and treat wild-type SARS-CoV-2 virus and mutant strain thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
  • the mRNA may further comprise at least one of the following technical features:
  • amino acid sequence of the RBD domain of the SARS-CoV-2 virus Delta mutant is shown in SEQ ID NO: 14 or SEQ ID NO: 28.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 14 is the RBD (319-545) of the S protein of the B.1.617.1 mutant strain with the C538S mutation.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 28 is NTD_RBD of the S protein of the B.1.617.2 mutant strain with a C538S mutation.
  • the antigen coding region further encodes the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant strain, and the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant strain has D80A mutation, R246I mutation and C538S mutation, as well as increased ⁇ 242-244 of the Beta mutant.
  • the mRNA synthesis can correspond to the antigenic protein of the Beta mutant strain and the Gamma mutant strain at the same time, and can be subsequently used to construct a bivalent mRNA vaccine capable of simultaneously targeting the Beta mutant and Gamma mutant SARS-CoV-2.
  • ⁇ 242-244 refers to the deletion mutation of the three amino acids “LAL” at positions 242-244 of the Gamma mutant strain.
  • amino acid sequence of the NTD_RBD domain of the ARS-CoV-2 virus Gamma mutant is shown in SEQ ID NO: 13.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 13 is the NTD_RBD domain of the Gamma mutant strain with D80A mutation, R246I mutation and C538S mutation and increased ⁇ 242-244 of the Beta mutant strain, referred to as the Beta&Gamma double mutation strain.
  • the antigen coding region further encodes the RBD domain of the wild-type SARS-CoV-2 virus with a mutation, the mutation being C538S.
  • the amino acid sequence of the RBD domain of the mutated wild-type SARS-CoV-2 virus is shown in SEQ ID NO: 6.
  • the antigen coding region encodes the RBD domain of the SARS-CoV-2 virus Delta mutant strain, the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant strain, and the wild-type SARS-CoV- 2 The RBD domain of the virus.
  • the antigen coding region further encodes a Foldon fragment.
  • the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
  • the antigen coding region further encodes a linker.
  • the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
  • the template DNA of the mRNA further contains a signal peptide coding region.
  • the signal peptide coding region encodes the signal peptide of the Beta mutant strain of SARS-CoV-2 virus.
  • the amino acid sequence of the signal peptide of the SARS-CoV-2 virus Beta mutant strain is shown in SEQ ID NO: 3.
  • amino acid sequence of the signal peptide shown in SEQ ID NO: 3 is the 1-19 signal peptide of the S protein of the Beta mutant strain.
  • the amino acid sequence encoded by the antigen coding region is such as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO : 34, shown in at least one of SEQ ID NO: 33.
  • amino acid sequence shown in SEQ ID NO: 20 is the sequence of the 1-19 signal peptide (SEQ ID NO: 3) of the S protein of the Beta mutant strain, the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant strain : 13), the RBD (SEQ ID NO: 14) of the B.1.617.1 mutant strain with the C538S mutation and the wild-type RBD (SEQ ID NO: 6) with the C538S mutation are fused to obtain the protein.
  • amino acid sequence shown in SEQ ID NO: 20 is the signal peptide at positions 1-19 of the Beta mutant S protein (SEQ ID NO: 3), the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant : 13), the RBD (SEQ ID NO: 14) of the B.1.617.1 mutant strain with the C538S mutation, the wild-type RBD (SEQ ID NO: 6) with the C538S mutation, the Linker and Foldon fragments of (GGGGS) 6 ( The fusion protein obtained by fusion of SEQ ID NO: 11).
  • amino acid sequence as shown in SEQ ID NO: 33 is the 1-19 signal peptide (SEQ ID NO: 3) of the S protein of the Beta mutant strain, the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant strain : 13), the RBD (SEQ ID NO: 28) of the Delta mutant strain B.1.617.2 with the C538S mutation and the wild-type RBD (SEQ ID NO: 6) with the C538S mutation are fused to obtain a fusion protein.
  • amino acid sequence shown in SEQ ID NO: 34 is the 1-19 signal peptide (SEQ ID NO: 3) of the S protein of the Beta mutant strain, the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant strain : 13), the RBD (SEQ ID NO: 28) of the Delta mutant strain B.1.617.2 with the C538S mutation, the wild-type RBD (SEQ ID NO: 6) with the C538S mutation, the Linker whose sequence is (GGGGS) 6 and A fusion protein obtained by fusing the Foldon fragment (SEQ ID NO: 11).
  • the promoter of the template DNA is a T7 or SP6 promoter.
  • the template DNA further includes a 5' untranslated region.
  • nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
  • the template DNA further includes a 3' untranslated region.
  • nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
  • the 3' end of the template DNA is further connected to polyA.
  • nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
  • the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
  • the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
  • the invention provides an mRNA.
  • the template DNA of the mRNA comprises an antigen coding region; the antigen coding region encodes the RBD domain of the S protein of the SARS-CoV-2 virus Delta mutant strain, the SARS-CoV-2 virus Beta mutant strain.
  • the mRNA of the present invention has stronger immunogenicity, can produce higher neutralizing antibody after injecting this mRNA, can effectively prevent and treat wild-type SARS-CoV-2 virus and mutant strain thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
  • the RBD domain of the S protein of the SARS-CoV-2 virus Delta mutant strain the RBD domain of the S protein of the SARS-CoV-2 virus Beta mutant strain, and the SARS-CoV-2 virus Gamma mutation
  • the connection sequence of the RBD domain of the strain S protein and the RBD domain of the S protein of the wild-type SARS-CoV-2 virus is not specifically limited, as long as the above four RBD domains can be encoded simultaneously.
  • the N-terminal to the C-terminal of the template DNA is the RBD domain of the S protein of the SARS-CoV-2 virus Delta mutant strain, the RBD domain of the S protein of the SARS-CoV-2 virus Beta mutant strain, and the SARS-CoV -2 virus Gamma mutant strain S protein RBD domain and wild-type SARS-CoV-2 virus S protein RBD domain.
  • the mRNA may further comprise at least one of the following technical features:
  • the antigen coding region further encodes a Foldon fragment.
  • the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
  • the antigen coding region further encodes a linker.
  • the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
  • the template DNA further includes a signal peptide coding region.
  • the signal peptide coding region encodes the signal peptide of the Beta mutant strain of SARS-CoV-2 virus.
  • the amino acid sequence of the signal peptide of the SARS-CoV-2 virus Beta mutant strain is shown in SEQ ID NO: 3.
  • the amino acid sequence encoded by the template DNA is at least one of SEQ ID NO: 39 and SEQ ID NO: 40.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 39 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the Delta mutant strain B RBD (SEQ ID NO: 35) of the S protein of 1.617.2, the RBD (SEQ ID NO: 36) of the Beta mutant strain S protein, the RBD (SEQ ID NO: 37) of the Gamma mutant strain S protein and the wild-type S protein.
  • the protein whose amino acid sequence is shown in SEQ ID NO: 40 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the Delta mutant strain B
  • the promoter of the template DNA is a T7 or SP6 promoter.
  • the template DNA further includes a 5' untranslated region.
  • nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
  • the template DNA further includes a 3' untranslated region.
  • nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
  • the 3' end of the template DNA is further connected to polyA.
  • nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
  • the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
  • the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
  • the present invention provides an mRNA vaccine.
  • the mRNA vaccine comprises: the aforementioned mRNA, and optionally pharmaceutically acceptable adjuvants or auxiliary components.
  • the mRNA vaccine of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and its mutants (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
  • the mRNA vaccine may further comprise at least one of the following technical features:
  • the auxiliary component is a nanocarrier carrying the mRNA; and/or, the auxiliary material includes at least one selected from injection buffer medium, lyophilization or cryoprotectant.
  • the nanocarrier includes at least one selected from liposomes, nanoparticles, microspheres and lipid nanocarriers.
  • the nanocarrier is prepared by using at least one of the following lipid materials: DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and its derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, Phosphatidyl Ethanolamine (PE), Phosphatidylcholine (PC) and Cholesterol (Chol).
  • DOTAP DOTAP
  • DOTIM DOTIM
  • DDA DC-Chol
  • CCS diC14-amidine
  • DOTPA DOSPA
  • DTAB DOSPA
  • DTAB TTAB
  • CTAB CTAB
  • DORIE DORIE
  • DOGS DOSC
  • DOSC DO
  • the lipid material:mRNA mass ratio is (0.5-50):1, preferably (2-10):1.
  • the mRNA vaccine is formed by self-assembly of the mRNA and lipid material using a microfluidic device; or, the mRNA vaccine is formed by incubating the nanocarrier with the mRNA.
  • the present invention proposes a preparation method of the aforementioned mRNA vaccine.
  • the method includes: mixing the nanocarrier and mRNA in a solution, so as to obtain the mRNA vaccine.
  • the preparation process of the method of the invention is simple, and the prepared mRNA vaccine has strong immunogenicity.
  • the lipid material of the nanocarrier is a cationic lipid material.
  • the invention provides a protein.
  • the protein is encoded by the template DNA of the aforementioned mRNA.
  • the protein of the present invention can be used to stimulate the body to produce antibodies that specifically target the wild-type SARS-CoV-2 virus and its mutants, and can be used to prepare protein vaccines.
  • the protein may further include at least one of the following technical features:
  • the protein comprises the S protein of the Indian mutant strain of SARS-CoV-2 virus without a signal peptide, and at least one of the K986P or V987P mutations is carried out.
  • amino acid sequence of the S protein of the Indian mutant strain of SARS-CoV-2 virus without signal peptide described in the above protein is shown in SEQ ID NO:7.
  • the S protein of the Indian mutant strain of SARS-CoV-2 described in the above protein has also undergone at least one mutation among the four mutations of F817P, A892P, A899P or A942P.
  • amino acid sequence of the SARS-CoV-2 Indian mutant strain S protein without signal peptide described in the above protein is shown in SEQ ID NO:8.
  • the RBD domain of the wild-type S protein is also fused with the above protein, and the RBD domain of the wild-type S protein is mutated at position 538 (C538S).
  • amino acid sequence of the RBD domain of the wild-type S protein described in the above protein is shown in SEQ ID NO 6.
  • the RBD domain of the wild-type S protein at the mutation site 538 (C538S) in the above protein is fused to the nitrogen terminal of the Indian mutant S protein.
  • the above protein is also fused with a signal peptide.
  • the signal peptide described in the above protein is the signal peptide of the Indian mutant strain of SARS-CoV-2 virus.
  • amino acid sequence of the signal peptide of the Indian mutant strain of SARS-CoV-2 virus described in the above protein is shown in SEQ ID NO:2.
  • the above protein contains such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: At least one of the peptides shown in ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 30.
  • the protein comprises the NTD_RBD domain of the Indian mutant strain of SARS-CoV-2 virus, and the NTD_RBD domain of the Indian mutant strain has a mutation at position 538 (C538S).
  • amino acid sequence of the NTD_RBD domain of the Indian mutant described in the above protein is shown in SEQ ID NO: 9.
  • the RBD domain of the wild-type SARS-CoV-2 is also fused to the above protein, and the 538 site (C538S) of this domain is mutated.
  • amino acid sequence of the RBD domain of the wild-type SARS-CoV-2 described in the above protein is shown in SEQ ID NO:6.
  • the above protein is further fused with a Foldon fragment.
  • the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
  • the above protein is also fused with a linker.
  • amino acid sequence of the linker in the above protein is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 or (GSG) 10 .
  • the above protein is also fused with a signal peptide.
  • its signal peptide is the signal peptide of the Indian mutant strain of SARS-CoV-2 virus.
  • the amino acid sequence of the signal peptide of the Indian mutant strain of SARS-CoV-2 virus is shown in SEQ ID NO:2.
  • the above-mentioned protein contains an amino acid sequence such as SEQ ID NO: 9, SEQ ID NO: 18, or at least one of SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 31 or SEQ ID NO: 32.
  • the indicated peptides are amino acid sequences such as SEQ ID NO: 9, SEQ ID NO: 18, or at least one of SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 31 or SEQ ID NO: 32.
  • the protein comprises the RBD domain of the Indian mutant strain of SARS-CoV-2 virus; the RBD domain of the Indian mutant strain has a mutation at position 538 (C538S).
  • amino acid sequence of the RBD domain of the Indian mutant described in the above protein is shown in SEQ ID NO: 14.
  • the NTD_RBD sequence of the Brazilian mutant strain is also fused to the above protein; the NTD_RBD sequence of the Brazilian mutant strain is mutated at position 538 (C538S), and three mutations of D80A, ⁇ 242-244 and R246I on the South African mutant strain are also added.
  • amino acid sequence of the NTD_RBD sequence of the Brazilian mutant strain with three mutations added to the South African mutant strain described in the above protein is shown in SEQ ID NO: 13.
  • the RBD domain of the wild-type SARS-CoV-2 virus is also fused to the above protein, and the 538 site (C538S) of this domain is mutated.
  • amino acid sequence of the RBD domain of the wild-type S protein described in the above protein is shown in SEQ ID NO 6.
  • the above-mentioned protein also contains a Foldon fragment.
  • the amino acid sequence of the Foldon is shown in SEQ ID NO: 11.
  • the above protein also contains a linker.
  • the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
  • the above-mentioned protein also contains a signal peptide coding region.
  • the signal peptide described in the above protein is the signal peptide of the South African mutant strain of SARS-CoV-2 virus.
  • amino acid sequence of the signal peptide of the SARS-CoV-2 virus South African mutant strain described in the above protein is shown in SEQ ID NO: 3.
  • the above protein contains amino acid sequences such as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO: 34 , at least one of the peptides shown in SEQ ID NO: 33.
  • the protein contains the RBD domain of the S protein of the Indian mutant strain of SARS-CoV-2 virus, the RBD domain of the S protein of the South African mutant strain, and the RBD domain of the S protein of the Brazilian mutant strain and the RBD domain of the wild-type S protein.
  • amino acid sequence of the RBD of the S protein of the Indian mutant strain that is, the RBD of the S protein of the Delta mutant strain B.1.617.2 is as shown in SEQ ID NO: 35:
  • amino acid sequence of the RBD of the wild-type S protein is as shown in SEQ ID NO: 38:
  • the above protein also contains a Foldon fragment.
  • the amino acid sequence of the Foldon is shown in SEQ ID NO: 11.
  • the above protein also contains a linker.
  • the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
  • the above-mentioned protein also contains a signal peptide.
  • the signal peptide of the above protein is the signal peptide of the South African mutant strain of SARS-CoV-2 virus. Further, the amino acid sequence of the signal peptide of the South African mutant strain of SARS-CoV-2 virus is shown in SEQ ID NO: 3.
  • amino acid sequence of the above protein is at least one of SEQ ID NO: 39 and SEQ ID NO: 40.
  • the invention provides a protein.
  • the protein has such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22.
  • SEQ ID NO: 23 SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 39 and SEQ ID NO: 40 at least one of the amino acid sequences shown; or, the protein has the same as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19.
  • the same or similar functions refer to preventing and/or treating SARS-CoV-2 infection.
  • the invention provides an antibody.
  • the antibody has the aforementioned protein binding activity.
  • the antibody of the present invention can effectively bind to the aforementioned protein, especially specifically target the aforementioned protein, and can be used to detect the aforementioned protein in vivo or in vitro.
  • the antibody is a polyclonal antibody or a monoclonal antibody.
  • the antibody has the activity of specifically binding to the protein.
  • the present invention provides a conjugate.
  • the conjugate comprises: the aforementioned antibody; and a coupling moiety, the coupling moiety being linked to the antibody.
  • the conjugate of the present invention can effectively bind to the aforementioned proteins, especially specifically target the aforementioned proteins, and can be used to detect the aforementioned proteins in vivo or in vitro.
  • the coupling moiety includes, but is not limited to, at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modification, an imaging molecule, and a chemotherapeutic agent.
  • the carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can accommodate or carry a liquid phase (for example, plates, membranes, etc.) , test tubes and other supports, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, monolithic columns, etc.); it can also be a label for labeling antibodies or antigen-binding fragments, recombinant proteins or multispecific antibodies
  • Carriers such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, ⁇ -galactosidase), affinity substances (e.g., streptavidin and One, one of the nucleic acids of the sense strand and the antisense strand complementary to each other), fluorescent substances (for example, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), lumin
  • the drug is a small molecule drug that can bind to an antibody, and the specific type is not limited.
  • the toxin includes at least one selected from abrin, ricin A, Pseudomonas exotoxin and diphtheria toxin.
  • the cytokines include at least one selected from IL-10, VEGF, EpCAM, GM2 and RANKL.
  • the protein tags include but are not limited to His tags, Flag tags, GST tags, MBP tags, SUMO tags and C-Myc tags.
  • the modification should be understood in a broad sense, and may refer to substances used to modify proteins. Exemplarily, it may be polyethylene glycol or its derivatives.
  • chemotherapeutic agents refer to nab-paclitaxel, cyclophosphamide, ifosfamide, melphalan, methotrexate, fluorouracil, actinomycin D, vincristine.
  • binding method of the coupling moiety and the antibody can be used for the binding method of the coupling moiety and the antibody.
  • a physical adsorption method for example, a covalent binding method, a method using an affinity substance (for example, biotin, streptavidin), and an ion binding method are mentioned.
  • an affinity substance for example, biotin, streptavidin
  • the present invention provides a protein or polypeptide vaccine.
  • the protein or polypeptide vaccine comprises: the aforementioned protein as an antigenic component.
  • the protein or polypeptide vaccine of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and mutants thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
  • pharmaceutically acceptable excipients or auxiliary components are further included.
  • an immune adjuvant is further included.
  • the immune adjuvant includes Freund's incomplete adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant and microorganism at least one of the adjuvants.
  • the invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the aforementioned protein, aforementioned antibody or aforementioned conjugate.
  • the nucleic acid molecules according to the embodiments of the present invention encode the aforementioned proteins, antibodies or conjugates.
  • the nucleic acid molecule is DNA.
  • nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
  • the invention proposes a carrier.
  • the vector carries the aforementioned nucleic acid molecule.
  • the vector shown is an expression vector for expressing the aforementioned protein, antibody or conjugate.
  • the nucleic acid molecule can be directly or indirectly linked to the control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
  • the nucleic acid molecule is operably linked to a control element.
  • "Operably linked” herein refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function.
  • Commonly used vectors can be, for example, plasmids, phages and the like. After the vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, under the mediation of the regulatory system, the expression of the aforementioned proteins, antibodies or conjugates can be effectively realized, and then the expression of the proteins, antibodies or conjugates can be realized. obtained in large quantities in vitro.
  • the vector is a eukaryotic vector or a prokaryotic vector.
  • the vector includes at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.
  • the present invention proposes a vector vaccine.
  • the carrier vaccine includes an active ingredient; the active ingredient is obtained by loading the antigen coding region of the template DNA of the aforementioned mRNA, or the aforementioned nucleic acid molecule, into the aforementioned carrier.
  • the vector vaccine of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and its mutants (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
  • the adenoviral vector is a replication-defective adenoviral vector.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes: the aforementioned mRNA, the aforementioned mRNA vaccine, the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine or the aforementioned carrier vaccine.
  • the pharmaceutical composition of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and its mutants (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
  • pharmaceutically acceptable auxiliary materials are further included.
  • the present invention provides the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine, the aforementioned carrier Use of the vaccine or the aforementioned pharmaceutical composition in the preparation of medicines for preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection.
  • the present invention provides the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine, the aforementioned carrier Use of the vaccine or the aforementioned pharmaceutical composition in preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection.
  • the present invention provides the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine, the aforementioned carrier
  • the vaccine or the aforementioned pharmaceutical composition is used to prevent SARS-CoV-2 infection and/or prevent and/or treat related diseases caused by SARS-CoV-2 infection.
  • the present invention proposes a method for preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection.
  • the method includes: administering to the subject a pharmaceutically acceptable amount of the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned The protein or polypeptide vaccine, the aforementioned carrier vaccine or the aforementioned pharmaceutical composition.
  • the method of the present invention can effectively prevent SARS-CoV-2 infection, and can also effectively prevent and/or treat related diseases caused by SARS-CoV-2 infection.
  • the effective amount of the mRNA, mRNA vaccine, protein or polypeptide vaccine, carrier vaccine, conjugate or pharmaceutical composition of the present invention may vary with the mode of administration and the severity of the disease to be treated.
  • the selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials).
  • the factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, drug administration way etc. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.
  • the mRNA, mRNA vaccine, protein or polypeptide vaccine, vector vaccine or pharmaceutical composition of the present invention may be incorporated into a drug suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular
  • These drugs can be prepared in various forms.
  • liquid, semi-solid and solid dosage forms, etc. including but not limited to liquid solutions (eg, injection solutions and infusion solutions) or lyophilized powders.
  • the administration route of the method includes injection, nasal drop or inhalation.
  • the administration method of injection includes at least one selected from intramuscular injection, subcutaneous injection and intravenous injection; or the administration method of inhalation is selected from at least one of powder inhalation and nebulized inhalation .
  • the number of administrations is 1-20 times, preferably 1-10 times, more preferably 1, 2, 3, 4, 5 or 6 times.
  • the method further includes: after administering the mRNA, mRNA vaccine, protein or polypeptide vaccine, vector vaccine or pharmaceutical composition to a person infected with SARS-CoV-2 or a person at risk of exposure to SARS-CoV-2, Detect the antibody titer of the SARS-CoV-2 infected person or SARS-CoV-2 exposure risk person.
  • the beneficial effect of the present invention is that starting from the design of the antigen coding sequence, a variety of coding sequences have been designed. According to the sequences and mutation sites designed in the present invention, in addition to designing mRNA vaccines, further protein, polypeptide, DNA , Circular RNA and the application of viral vector vaccine design to achieve the preventive effect against SARS-CoV-2 infection.
  • the mRNA provided by the present invention has strong immunogenicity, and the prepared mRNA vaccine can be used to resist the infection of new coronavirus and its mutant strains; especially the Delta mutant strain B.1.617.2 series and its mutation evolution Female parent B.1.617.1 etc.
  • the mRNA vaccine provided by the present invention improves the structure of traditional mRNA, so that the mRNA obtained by this method not only enhances the immunogenicity of the vaccine, but also allows the method to be easily extended to the design of other high-risk mRNAs, which has universal sex.
  • the protein vaccine provided by the present invention has the advantages of safety, high efficiency and large-scale production. There have been successful precedents for the protein vaccine route, and the more successful genetically engineered subunit vaccine is the hepatitis B surface antigen vaccine.
  • viral vector vaccines The advantage of viral vector vaccines is that the gene efficiency is high, and in vitro experiments are usually close to 100% transduction efficiency; different types of human tissue cells can be transduced, regardless of whether the target cells are dividing cells; high titers can be easily obtained Viral vectors do not integrate into the host cell genome when they enter cells, and are only expressed transiently, with high safety. In terms of production capacity, it can also be easily industrialized through cell culture.
  • This vaccine has a successful precedent: Previously, the "recombinant Ebola virus disease vaccine" jointly developed by Academician Chen Wei's team and Tianjin Kangxinuo Biotechnology Co., Ltd. also used adenovirus as a carrier.
  • DNA vaccines such as plasmid DNA
  • DNA vaccines have a simple structure, and the process of purifying plasmid DNA is simple, so the production cost is low, and it is suitable for mass production; DNA molecular cloning is relatively easy, so that DNA vaccines can be updated at any time as needed
  • the DNA molecule is very stable, so it is easy to transport and store; the DNA vaccine can activate cytotoxic T lymphocytes to induce cellular immunity; the plasmid DNA itself can also be used as an adjuvant, so the use of the DNA vaccine does not need to add an adjuvant, which not only reduces the cost but also is convenient to use.
  • Fig. 1 is the electrophoresis detection result of plasmid and linearized DNA template in Example 2 of the present invention, wherein, swimming lane 1: Delta S-2P linearized DNA template; swimming lane 2: Delta S-2P plasmid; swimming lane 3: Delta S-6P plasmid ;lane 4: Delta S-6P linearized DNA template; lane 5: RBD-Delta S-2P plasmid; lane 6: RBD-Delta S-2P linearized DNA template; lane 7: RBD-Delta S-6P plasmid; 8: RBD-Delta S-6P linearized DNA template; lane 9: B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD linearized DNA template; lane 10: B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD plasmid; lane 11 : B.1.617.2_RBD-Beta_RBD-
  • Fig. 2 is different mRNA vaccine immunization BALB/c mouse Day 14 serum binding antibody titers in the embodiment of the present invention 4;
  • Fig. 3 is different mRNA vaccine immunization BALB/c mouse Day 14 serum binding antibody titers in the embodiment of the present invention 4;
  • Fig. 4 is different mRNA vaccine immunization BALB/c mice Day 28 serum binding antibody titer in the embodiment of the present invention 4;
  • Fig. 5 is different mRNA vaccine immunization BALB/c mouse Day 35 serum pseudovirus neutralizing antibody titer in the embodiment of the present invention 4;
  • Fig. 6 is different mRNA vaccine immunization BALB/c mouse Day 42 serum binding antibody titer in the embodiment of the present invention 5;
  • Fig. 7 is different mRNA vaccine immunization BALB/c mice Day 14 serum pseudovirus neutralizing antibody titers in the embodiment of the present invention 5;
  • Fig. 8 is different mRNA vaccine immunization BALB/c mice Day 14 serum pseudovirus neutralizing antibody titers in the embodiment of the present invention 5;
  • Fig. 9 is different mRNA vaccine immunization BALB/c mouse Day 14 serum pseudovirus neutralizing antibody titer in the embodiment of the present invention 5;
  • Figure 10 is the 12-month serum binding antibody titer of different mRNA vaccines immunized BALB/c mice in Example 6 of the present invention.
  • Fig. 11 is the expression result of in vitro dodecamer SARS-CoV-2 RBD mRNA immunogen in Example 7 of the present invention, wherein, (A) is the flow chart of experimental design, through codon optimization, synthesized in the DNA of plasmid hCD2 containing Various RBDs (R391-D541) of the SARS-CoV-2 Spike protein tagged with N-terminal tPA signal peptide (SP), linker peptide (LP) and C-terminal Foldon (FD) trimer.
  • SP N-terminal tPA signal peptide
  • LP linker peptide
  • FD C-terminal Foldon
  • Fig. 12 is the 4N4T self-assembled SARS-CoV-2RBD dodecamer immune BALB/c mouse Day 14 serum pseudovirus neutralizing antibody titer in the embodiment 7 of the present invention;
  • Fig. 13 is a flow chart of experimental design in Example 7 of the present invention.
  • Figure 14 is the SARS-CoV-2 specific T cell immune response of self-assembled SARS-CoV-2 RBD dodecamer immunized mice in Example 7 of the present invention, wherein (A) is flow cytometry detection covering SARS-CoV-2 SARS-CoV-2 RBD-specific CD4+ and CD8+ T cells in splenocytes after stimulation with a peptide pool of the CoV-2 RBD; (B) CD4+ and CD8+ T cell generation after stimulation with a peptide pool covering the SARS-CoV-2 RBD for ICS SARS-CoV-2 RBD-specific cytokines IL-4, IL-2 and IFN- ⁇ ; (C) ELISpot method for the detection of IFN- ⁇ in splenocytes, HRBD: 10 ⁇ g heterologous RBD, HRBD-F: 10 ⁇ g heterologous RBD Source RBD Foldon, 5HRBD: 5 ⁇ g heterologous RBD, 5HRBD-F: 5 ⁇ g heterologous RBD Fold
  • the terms “optionally”, “optionally” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes the circumstances in which it occurs, and The circumstances in which the event or condition did not occur.
  • the present invention indicates that the sequence number of the mutation site is numbered according to the SARS-CoV-2 virus prototype strain (NC_045512.2), and the amino acid number is numbered according to the EU numbering system.
  • the 986th position refers to the 366th position in the EU numbering system
  • the "K986P” means that the lysine at the 986th position in the EU numbering system is replaced by proline
  • the 987th valine is replaced by proline
  • the "C538S” means that the 538th cysteine is replaced by serine according to the EU numbering system.
  • (XXXX)n means that n XXXX are connected, and X means an amino acid.
  • (GGGGS) 3 means GGGGSGGGGSGGGGS.
  • fragment refers to a protein or polypeptide of interest, as well as a protein or polypeptide of interest with N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and/or internal deletions.
  • a Foldon fragment can be a Foldon sequence or a partial sequence.
  • Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996 )); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and Intelligenetics, Mountain View, California CLUSTAL in the PC/Gene program.
  • GCG Genetics Computing Group
  • the term "at least 80% identity” means at least 80%, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the respective reference sequence , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.
  • antibody generally refers to an antibody that can recognize one or more epitopes, including but not limited to monoclonal antibody, polyclonal antibody, multimeric antibody, nanobody and CDR-grafted antibody.
  • the term "vector” generally refers to a nucleic acid molecule capable of being inserted into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having various functions as described above.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.
  • the term "pharmaceutical composition” generally refers to unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both.
  • the term "pharmaceutically acceptable excipients” may include any solvents, solid excipients, diluents or other liquid excipients, etc., which are suitable for the specific target dosage form. Except to the extent that any conventional excipients are incompatible with the compounds of the present invention, such as any adverse biological effects produced or interacted in a deleterious manner with any other components of the pharmaceutically acceptable composition, their Use is also within the scope of the present invention.
  • administering means introducing a predetermined amount of a substance into a patient by some suitable means.
  • the antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention can be administered through any common route as long as it can reach the intended tissue.
  • Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these exemplified modes of administration.
  • the compositions of the present invention are administered intravenously or subcutaneously.
  • treatment is used to refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects caused by the disease.
  • Treatment encompasses disease in mammals, especially humans, including: (a) preventing the disease or condition in a predisposed but undiagnosed individual; (b) inhibiting the disease, e.g., arresting its progression; Or (c) ameliorating the disease, eg, alleviating symptoms associated with the disease.
  • Treatment encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit a disease in that individual, including but not limited to administering a drug comprising a compound described herein to an individual in need thereof.
  • SARS-CoV-2 virus Beta mutant strain and “Beta mutant strain” are synonymous;
  • SARS-CoV-2 virus Gamma mutant strain and “Gamma mutant strain” are synonymous;
  • SARS-CoV-2 Virus Delta mutant and "Delta mutant” are synonymous.
  • the new coronavirus is an RNA virus, and the RNA virus is unstable, and natural mutations will occur during virus replication.
  • SARS-CoV-2 continues to mutate, two key mutant strains have attracted people's attention recently, one is the Beta mutant strain B.1.351 (20H/501Y.V2), and the other is the Gamma mutant strain P.1 (20J/ 501Y.V3), and the latest Delta mutants B.1.617.2 series and B.1.617.1 series.
  • the emergence of mutations leads to increased infectivity of the virus and immune escape. Therefore, a new design is required for the sequence of the vaccine antigen to produce a better effect against the mutation of the spike protein on the mutant strain.
  • the present invention is a newer generation of new crown vaccine designed for the above-mentioned mutations.
  • the active ingredient design of the new crown vaccine of the present invention has four directions:
  • the K986P and V987P mutations are performed simultaneously.
  • a preferred method is to carry out at least one mutation among the four mutations F817P, A892P, A899P or A942P.
  • the RBD domain of the wild-type S protein with the mutated 538 position (C538S) can be linked at its nitrogen or carbon terminus.
  • the nitrogen terminal of the above fusion protein can also be connected with a signal peptide.
  • a signal peptide for example, the signal peptide of the Delta mutant strain of SARS-CoV-2 virus.
  • the RBD domain of the wild-type S protein with the mutated 538 position (C538S) can be linked at its nitrogen or carbon terminus.
  • Foldon fragments are connected to the carbon-terminal of the above proteins to facilitate the formation of transmembrane trimers after antigen translation, so that the antigen proteins can be located on the cell surface and more easily recognized by immune cells.
  • the Foldon segment flexible Linker is connected to the main body.
  • the Linker of the present invention is preferably (GGGGS) 6 .
  • NTD_RBD domain of the above-mentioned Gamma mutant strain also increased three mutations of D80A, ⁇ 242-244 and R246I of the Beta mutant strain.
  • the RBD domain of the wild-type SARS-CoV-2 virus can also be fused to the nitrogen or carbon terminal of the above-mentioned domain, and the 538-site (C538S) of this domain is mutated.
  • the present invention further provides the structure of the NTD_RBD domain of the Gamma mutant + the RBD domain of the Delta mutant + the wild-type RBD domain.
  • the signal peptide of the S protein of the Beta mutant strain can be used as the signal peptide for the above structural domain.
  • Foldon fragments are connected to the carbon-terminal of the above proteins to facilitate the formation of transmembrane trimers after antigen translation, so that the antigen proteins can be located on the cell surface and more easily recognized by immune cells.
  • the Foldon segment flexible Linker is connected to the main body.
  • the Linker of the present invention is preferably (GGGGS) 6 .
  • the Delta mutant described in the above mRNA is the B.1.617.2 or B.1.617.1 mutant.
  • the RBD domain of the S protein of the Delta mutant strain B.1.617.2 or B.1.617.1 the RBD domain of the S protein of the Beta mutant strain, the RBD domain of the S protein of the Gamma mutant strain, and the wild-type S protein
  • the RBD domain of the protein is fused to obtain a new protein structure.
  • the signal peptide of the S protein of the Delta mutant strain B.1.617.2 or B.1.617.1 can be used as the signal peptide for the above domain.
  • the Foldon fragment can be connected to the carbon-terminus of the above-mentioned protein, so that after the antigen is translated, a transmembrane trimer can be formed, so that the antigen protein can be located on the cell surface and more easily recognized by immune cells.
  • the Foldon segment flexible Linker is connected to the main body.
  • the Linker of the present invention is preferably (GGGGS) 6 .
  • the RBD domain of the S protein of the Delta mutant strain B.1.617.2 or B.1.617.1 in the above structure the RBD domain of the S protein of the Beta mutant strain, the RBD domain of the S protein of the Gamma mutant strain, and the wild-type S protein
  • the sequence of the RBD domains can be adjusted as needed.
  • amino acid sequences such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO : 18, SEQ ID NO: 19, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20 or SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 , SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ The antigen shown in any one of ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 40; and an antigen comprising these amino acid sequences as part of itself.
  • a protein having the same or similar function as the above-mentioned protein containing peptides obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence of each of the above peptides.
  • the same or similar functions refer to preventing and/or treating SARS-CoV-2 infection.
  • the SARS-CoV-2 is at least one of wild type, Beta mutant, Gamma mutant or Delta mutant.
  • the expression "a protein with the same or similar function as the above-mentioned protein of the peptide obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence of each peptide” includes but is not limited to Several (usually 1-20, preferably 1-10, more preferably 1-5, most preferably 1-3) amino acid deletions, insertions and/or substitutions, and at the C-terminal and/or Or add one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the N-terminus.
  • substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of said proteins.
  • peptides obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence of each peptide also includes, but is not limited to, at most 10 (that is, one or several), preferably at most 8, more preferably at most 5 (5, 4, 3, 2 or 1) amino acids are replaced by amino acids with similar or similar properties to form a polypeptide, that is, a conservative variant polypeptide. Further, these conservative variant polypeptides can be produced by substitution according to the following table.
  • the above-mentioned proteins can be used as active ingredients to prepare drugs for preventing and/or treating SARS-CoV-2 infection.
  • those skilled in the art can use the above-mentioned proteins as antigenic active ingredients to prepare protein or polypeptide vaccines for preventing and/or treating SARS-CoV-2 infection.
  • the vaccine uses the above-mentioned proteins as antigenic components, and pharmaceutically acceptable adjuvants or auxiliary components.
  • immune adjuvants are often added to enhance the body's immune response to the vaccine.
  • the immune adjuvant is incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant, microbial adjuvant and the like.
  • the above-mentioned antibodies are polyclonal antibodies or monoclonal antibodies; preferably monoclonal antibodies.
  • the above-mentioned antibodies can also form conjugates with a coupling moiety.
  • the coupling moiety is one or more selected from radionuclides, drugs, toxins, cytokines, enzymes, fluoresceins, carrier proteins or biotin.
  • Antibodies that can specifically bind to the aforementioned proteins can be used to prepare drugs for the prevention and/or treatment of SARS-CoV-2 infection on the one hand to resist SARS-CoV-2 virus infection, and on the other hand can be used for SARS-CoV-2 virus-related immune detection.
  • the present invention also includes the genes encoding the above proteins.
  • the gene encoding the above protein can be used to express and prepare the above protein or antibody; on the other hand, it can also be operably loaded in a vector, and then can be prepared into a vector vaccine or a vector drug.
  • Expression can be selected among commonly used vectors such as plasmid vectors, adenoviral vectors, lentiviral vectors or adeno-associated viral vectors. When an adenoviral vector is used, generally a replication-deficient adenoviral vector is used.
  • the present invention designs a series of mRNAs encoding the above antigens.
  • These mRNAs contain coding regions encoding the above-mentioned antigens, and may or may not contain signal peptide sequence coding regions.
  • the above mRNA may also contain a 5' untranslated region, a signal peptide sequence coding region, an antigen coding region, and a 3' untranslated region connected in sequence.
  • the above-mentioned mRNA can be transcribed through a DNA template sequentially connected with a promoter, a 5' untranslated region, a signal peptide sequence, an antigen coding region, and a 3' untranslated region.
  • the transcription process can be performed by existing in vitro transcription methods and related kits.
  • the transcribed mRNA can be prepared into an mRNA vaccine together with pharmaceutically acceptable adjuvants or auxiliary components.
  • the auxiliary component can be a nano-carrier carrying the mRNA.
  • the nanocarriers are usually lipid nanocarriers.
  • lipid nanocarriers prepared from at least one of the following raw materials: DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE, and Derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, Phosphatidylethanolamine (PE), Phosphatidyl Choline (PC), Cholesterol (Chol).
  • PE Phosphatidylethanol
  • the lipid material used to prepare the nanocarrier is an amphiphilic lipid material or a cationic lipid material, because this type of lipid material has a positive charge on the surface under acidic conditions and can be electrostatically adsorbed with the phosphate radical of the nucleic acid.
  • the role is to wrap mRNA molecules inside to form mRNA-lipid complexes.
  • the mRNA-lipid complex can be adsorbed by the negatively charged cell membrane on the surface, and then through the fusion of the membrane or the endocytosis of the cell, the mRNA is delivered into the cell for further expression, and exerts the immune effect of the vaccine.
  • corresponding DNA vaccines, proteins, protein or polypeptide vaccines, conjugates and viral vectors can be prepared.
  • the preparation of proteins, viral vectors and DNA vaccines in the present invention requires the construction of plasmid DNA first to realize the expression of antigenic proteins at different levels.
  • the signal peptide sequence, antigen coding region, and Foldon sequence of the present invention are inserted into the corresponding positions of the plasmid DNA vector, and the plasmid DNA for constructing recombinant protein vaccines, virus vector vaccines, and DNA vaccines can be obtained after sequencing and verifying that the sequences are correct.
  • the conventional technology in the field of vaccine preparation in the present invention the specific method is not limited, and the corresponding protein vaccine, virus vector vaccine and DNA vaccine can be prepared by pharmacy.
  • the preparation of the mRNA stock solution usually includes the cultivation and amplification of engineering Escherichia coli, the extraction and purification of plasmid DNA, the preparation and purification of linearized DNA template, and the preparation and purification of mRNA.
  • the technicians of the present invention prepare intermediates such as plasmid DNA and linearized DNA template through a multi-step process, and use the linearized DNA template as the starting material to prepare the mRNA solution of the present invention. Further, the key quality indicators of intermediates such as plasmid DNA and linearized DNA templates and mRNA solutions are tested to ensure that they can be used for subsequent experimental research.
  • an mRNA-LNP preparation was further prepared for pharmacodynamic evaluation experiments.
  • Embodiment 1 Construction of mRNA transcription DNA template
  • the inventors designed two mRNA sequences, Delta S-2P and Delta S-6P, based on the S protein of the Delta variant. At the same time, considering that SARS-CoV-2 is constantly mutating, the inventor added the RBD sequence of the S protein of the wild-type strain to the N-terminus of the S protein of the Delta mutant strain, thereby constructing a bivalent new crown mRNA vaccine.
  • the template DNA for mRNA transcription is sequentially connected by the promoter, 5' untranslated region, signal peptide sequence, antigen coding region, 3' untranslated region, and Poly(A), and connected into the corresponding position of the plasmid.
  • the sequence is verified to be correct by sequencing.
  • mRNA transcription template DNA can be provided as two sequences respectively, or can be provided in the same sequence.
  • the amino acid sequence of the signal peptide of the present invention is SEQ ID NO: 24, core
  • the nucleotide sequence is SEQ ID NO: 15, which is the 1-19 signal peptide of the S protein of the Delta mutant strain B.1.617.2; when there is a signal peptide at the N-terminal of the antigen coding region in Table 1, it does not need to be provided separately signal peptide.
  • the promoter is T7 or SP6 promoter.
  • the nucleotide sequence of the 5' untranslated region of the mRNA sequence is SEQ ID NO: 1.
  • the nucleotide sequence of the 3' untranslated region of the mRNA sequence is SEQ ID NO: 41.
  • the nucleotide sequence of polyA of the mRNA sequence is SEQ ID NO: 42.
  • nucleotide sequence of each antigen coding region in Table 1 is as follows:
  • SEQ ID NO: 47 is used to encode SEQ ID NO: 22:
  • SEQ ID NO: 48 is used to encode SEQ ID NO: 23:
  • SEQ ID NO: 49 is used to encode SEQ ID NO: 29:
  • SEQ ID NO:50 is used to encode SEQ ID NO:30:
  • SEQ ID NO:51 is used to encode SEQ ID NO:12:
  • SEQ ID NO:52 is used to encode SEQ ID NO:13:
  • SEQ ID NO:53 is used to encode SEQ ID NO:31:
  • SEQ ID NO:54 is used to encode SEQ ID NO:32:
  • SEQ ID NO:55 is used to encode SEQ ID NO:34:
  • SEQ ID NO:56 is used to encode SEQ ID NO:39:
  • SEQ ID NO:57 is used to encode SEQ ID NO:40:
  • SEQ ID NO: 15 is used to encode SEQ ID NO: 24:
  • Insert the mRNA transcription template DNA of Example 1 into the pUC57 vector to prepare plasmid DNA refer to "Molecular Cloning Experiment Guide (Fourth Edition)", commercially available restriction endonucleases and DNA purification kit product instructions, for plasmid DNA Carry out enzyme digestion, process into a linearized plasmid DNA template, and then purify the linearized plasmid DNA template. The concentration and purity of plasmid DNA and linearized DNA template were detected by spectrophotometry and gel electrophoresis, and the linearization was confirmed by electrophoresis verification.
  • RNA polymerase NTP and cap analogs to synthesize and prepare mRNA.
  • In vitro transcription of pre-mRNA was performed according to the operating instructions of a commercially available RNA in vitro transcription kit (Novazyme TR101-02). details as follows:
  • the purity of mRNA was detected by agarose gel electrophoresis or nucleic acid fragment analyzer, and the 11 kinds of mRNA in Table 1 were respectively obtained, namely Delta S-2P, Delta S-6P, RBD-Delta S-2P, RBD-Delta S- 6P, Gamma NTD_RBD, BetaGamma NTD_RBD, B.1.617.2_NTD_RBD-Foldon, B.1.617.2_NTD_RBD-RBD-Foldon, BetaGamma NTD_RBD-B.1.617.2_RBD-RBD-Foldon, B.1.617.2_RBD-Beta_RBD-Gamma_RBD , B.1.617.2_RBD-Beta_RBD-Gamma_RBD , B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon.
  • the agarose gel electrophoresis is shown
  • Embodiment 3 the preparation of mRNA lipid nanoparticle (LNP)
  • the mRNA lipid nanoparticles can be selected according to the mRNA, the target organ, etc., with a suitable prescription, and after screening, recipe 1 is finally selected to prepare the mRNA lipid nanoparticles.
  • the lipid material in the prescription is dissolved in ethanol solution and then mixed with mRNA solution to make it self-assemble to form mRNA-lipid nanoparticles, and then remove ethanol through a tangential flow system.
  • the specific method is as follows:
  • Solution preparation MC3, DSPC, cholesterol (Chol), and DMG-PEG2000 were dissolved in absolute ethanol so that the concentration of ionizable lipid MC3 was 10 mg/mL to obtain a lipid solution.
  • the molar ratio of MC3, DSPC, cholesterol (Chol), and DMG-PEG2000 is 50:10:38.5:1.5.
  • the mRNA was diluted with PBS buffer (made with RNase-free water) to an appropriate concentration for use.
  • LNP preparation The ionizable lipid solution obtained in step (1) was mixed with the mRNA solution, and the mass ratio of cationic ionizable lipid to mRNA was shown in Table 4 to obtain the initial preparation of LNP.
  • the mixing was carried out in a microfluidic device (Maina (Shanghai) Instrument Technology Co., Ltd.), the process parameters of the microfluidic device: the volume ratio of the ethanol phase to the water phase was 1:3, and the flow rate was 9mL/min.
  • Ionizable or Cationic Liposome Carrier Solutions Mass ratio of ionizable or cationic lipid material to mRNA 1 MC3, DSPC, DMG-PEG, cholesterol 15:1 2 DTAB, DSPC, DMG-PEG, cholesterol 10:1 3 DC-Chol, DOPE, DMG-PEG, Cholesterol 25:1 4 CTAB, DOPE, DMG-PEG, cholesterol 30:1 5 DOTMA, DSPC, DMG-PEG, cholesterol 50:1 6 DDA, DOPE, DMG-PEG, cholesterol 5:1 7 DOTAP, DOPE, DMG-PEG, cholesterol 15:1
  • the specific detection method is as follows:
  • the test results show that the prepared mRNA lipid nanoparticles have a particle size of 60-150nm and a potential of 0 ⁇ 35mV, which is a typical structure of lipid nanoparticles.
  • Encapsulation efficiency detection Quant-iT TM RiboGreen TM kit was used to detect the encapsulation efficiency. The test results show that the prepared mRNALNP vaccine has good nano-preparation properties, the encapsulation rate is above 80%, and has good protection to mRNA.
  • the source of the wild-type, Alpha, Beta, Gamma, B.1.617.1, Delta (B.1.617.2), Omicron_BA.1 and Omicron_BA.2 pseudoviruses in the following examples of the present invention is Jiman Biology. Wherein, unless otherwise specified, the Delta strains in the following examples all refer to the B.1.617.2 strain.
  • Example 4 In vivo immune activity evaluation of mRNA lipid nanoparticles
  • This example evaluates the immune activity of the mRNA lipid nanoparticles prepared in Example 3 in mice.
  • the coding amino acid sequence of the present invention is as the mRNA of the antigen coding region shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 30 (abbreviated as SEQ ID NO: 22, 23 , 29 or 30 mRNA) the binding antibody titer can reach more than 10 4 .
  • the Ctrl group is the solvent control group without adding any mRNA lipid nanoparticles and drugs.
  • the dosage concentration 10 ⁇ g, 30 ⁇ g, 50 ⁇ g.
  • mice 6-8 weeks old Balb/c male mice were randomly divided into 80 groups, 6 mice in each group, and different concentrations of administration doses were used to immunize mice in different groups by intramuscular injection.
  • the mouse serum was collected 14 days and 28 days after the first immunization, and the binding antibody titer to the wild type (referred to as "WT"), Beta, Gamma, B.1.617.1, and Delta strains in the serum was detected by ELISA method;
  • WT binding antibody titer to the wild type
  • Beta Beta
  • Gamma B.1.617.1
  • Delta strains in the serum was detected by ELISA method
  • the mouse serum was collected 35 days after the first immunization, and the level of neutralizing antibodies to wild type (denoted as "WT") and Delta strains in the serum was detected by the pseudovirus neutralizing antibody detection method.
  • the results are shown in Fig. 3, Fig. 4 and Fig. 5.
  • the mRNA lipid nanoparticles containing Delta S-2P (SEQ ID NO: 22) antigen prepared in Example 3 were selected to further investigate the broad-spectrum antiviral infection performance of the mRNA vaccine of the present invention.
  • mice 36 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group, 0.1 ⁇ g dose group, 1 ⁇ g dose group, 5 ⁇ g dose group, 10 ⁇ g dose group and 30 ⁇ g dose group according to random block method, with 6 rats in each group.
  • the Ctrl group was given intramuscular injection of 0.1 mL/mouse of solvent (PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, the first administration 42 days after immunization, blood was collected and serum was separated, and the titers of binding antibodies against the S protein of each mutant strain in the 42-day serum of each dose group were detected by ELISA, and the results are shown in Figure 6.
  • solvent PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity
  • the mRNA vaccine of the present invention can produce the high titer of specific binding to Alpha, Beta, Gamma, Delta, Omicron_BA.1 and Omicron_BA.2 strains after the continuous intramuscular injection of BALB/c mouse twice The antibody has good immunogenicity in mice, further suggesting that the mRNA vaccine of the present invention has a broad-spectrum antiviral infection ability covering Omicron.
  • the inventors compared the mRNA vaccines of the Delta S-2P (SEQ ID NO: 22) prepared in Example 4 and the RBD-Delta S-2P (SEQ ID NO: 29) antigen against Delta and Omicron strains. Neutralizing antibody levels.
  • the inventors used the multivalent mRNA vaccine (SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 39) prepared in Example 4 against Prototype, Beta, Gamma, Delta and Omicron. Virus neutralizing antibody titers.
  • mice 30 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group and 1 ⁇ g dose group according to random block method, with 6 rats in each group.
  • the Ctrl group was given intramuscular injection of 0.1 mL/rat of solvent (PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration
  • Blood was collected 14 days after immunization and serum was separated, and the neutralizing antibody titers against Prototype, Beta, Gamma, Delta and Omicron pseudoviruses were detected in the serum by pseudovirus neutralization test, the results are shown in Figure 8. It was found that compared with the mRNAs of SEQ ID NO: 31 and SEQ ID NO: 32, the mRNAs of SEQ ID NO: 34 and SEQ ID NO: 39 had higher
  • BetaGamma NTD_RBD (SEQ ID NO: 13) after adding D80A, ⁇ 242-244 and R246I three Beta mutation sites on the basis of Gamma NTD_RBD (SEQ ID NO: 12), and further investigated Example 4
  • the prepared Gamma NTD_RBD and BetaGamma NTD_RBD antigen mRNA vaccines are aimed at the neutralizing antibody levels of Gamma and Beta strains.
  • mice 18 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group and 1 ⁇ g dose group according to random block method, with 6 rats in each group.
  • the Ctrl group was given intramuscular injection of 0.1 mL/mouse of solvent (PBS solution to adjust the preparation mRNA concentration to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration
  • Blood was collected 14 days after immunization and the serum was separated, and the neutralizing antibody titers against Gamma, Beta and Delta pseudoviruses in the serum were detected by the pseudovirus neutralization test, and the results were shown in Figure 9.
  • the mRNA of SEQ ID NO: 13 had a higher titer of pseudovirus neutralizing antibodies, and its titer of pseudovirus neutralizing antibodies was about 10 3 .
  • the inventors further investigated the persistent performance of the mRNA lipid nanoparticles containing the Delta S-2P (SEQ ID NO: 22) antigen prepared in Example 3 against novel coronavirus infection.
  • the inventors were surprised to find that the specific binding antibody titer against the Delta strain can be maintained between 10 5 and 10 6 within 12 months after two consecutive intramuscular injections of the mRNA vaccine of the present invention to immunize BALB/c mice twice ( FIG. 10 ), suggesting that the mRNA vaccine of the present invention has relatively durable antiviral infection ability.
  • Example 7 Antigen expression and immune performance investigation of heterologous RBD fusion protein antigen multivalent mRNA vaccine
  • the RBD immunogen was integrated into the open reading frame of a plasmid (hCD2.4) containing a T7 promoter and poly A tail, SP, LP and C-terminal Foldon (FD) trimer tags (the experimental scheme is shown in Figure 11A shown).
  • the expression of the heterologous fusion RBD protein in HEK293T cells was confirmed by using immunoblotting under reducing conditions (the experimental results are shown in FIG. 11B ).
  • Example 4 the inventor encapsulates fusion RBD mRNA into ionizable lipid nanoparticles MC3-LNPs to form a self-assembled SARS-CoV-2 RBD dodecamer universal mRNA vaccine (HRBD dodecamer) , and investigated the neutralizing antibody titers against wild-type (WT), Beta, Delta and Omicron pseudoviruses.
  • mice 30 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. According to random block method, they were divided into Ctrl group and 5 ⁇ g and 10 ⁇ g dose groups, with 6 rats in each group.
  • the Ctrl group was given intramuscular injection of 0.1 mL/rat of solvent (PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration
  • Blood was collected and serum was collected at 0.5, 1, 2, 4, 6, 9, and 12 months after immunization, blood was collected and serum was separated 35 days after the last dose of immunization, and serum was tested for wild-type ( The neutralizing antibody titers of WT), Beta, Delta and Omicron pseudoviruses, the experimental scheme is shown in Figure 13, and the results are shown in Figure 12.
  • the self-assembled SARS-CoV-2RBD dodecamer of the present invention induces neutralizing antibodies against wild type (WT), Beta, Delta and Omicron, can be used as a general mRNA vaccine, and can induce strong specificity RBD neutralizing antibody against SARS-CoV-2 immune escape caused by evolutionary mutation.
  • WT wild type
  • Beta Beta
  • Delta Delta
  • Omicron wild type
  • the self-assembled SARS-CoV-2 RBD with the Foldon structure at the C-terminus showed higher levels of neutralizing antibodies against Delta and Omicron.
  • SARS-CoV-2 RBD-specific CD4 and CD8 T cells in splenocytes were then detected by flow cytometry (Fig. 14A).
  • the results showed that specific CD4 and CD8 effector T cells in splenocytes inoculated with self-assembled SARS-CoV-2 RBD dodecamers were smaller than those inoculated with heterologous RBD after stimulation with a peptide pool covering the SARS-CoV-2 RBD Rats increased significantly.

Abstract

An mRNA against SARS-CoV-2 infection. The template DNA of the mRNA comprises an antigen encoding region. The antigen encoding region encodes a signal peptide-free S protein of a SARS-CoV-2 Delta mutant, wherein the S protein of the Delta mutant has at least one mutation of K986P and V987P mutations; the antigen encoding region encodes an NTD_RBD domain of a SARS-CoV-2 Delta mutant, wherein the NTD_RBD domain of the Delta mutant has a C538S mutation; the antigen encoding region encodes at least one of a RBD domain of a SARS-CoV-2 Delta mutant and an NTD_RBD domain of a SARS-CoV-2 Gamma mutant, wherein the RBD domain of the SARS-CoV-2 Delta mutant has a C538S mutation, and the NTD_RBD domain of the SARS-CoV-2 Gamma mutant has a D80A mutation, an R246I mutation and a C538S mutation, and has Δ242-244 of a Beta mutant added thereto; or the antigen encoding region encodes an RBD domain of the S protein of a SARS-CoV-2 Delta mutant, an RBD domain of the S protein of a SARS-CoV-2 Beta mutant, an RBD domain of the S protein of a SARS-CoV-2 Gamma mutant and an RBD domain of the S protein of wild-type SARS-CoV-2.

Description

抗SARS-CoV-2感染的mRNA、蛋白以及抗SARS-CoV-2感染的疫苗Anti-SARS-CoV-2 infection mRNA, protein and anti-SARS-CoV-2 infection vaccine
本申请要求于2021年9月29日提交的、申请号为202111156124.5的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims priority to a Chinese patent application with application number 202111156124.5 filed on September 29, 2021, the entire contents of which are incorporated herein by reference.
技术领域technical field
本发明属于生物医药领域，具体地，本发明涉及抗SARS-CoV-2感染的mRNA、蛋白以及抗SARS-CoV-2感染的疫苗。The invention belongs to the field of biomedicine, in particular, the invention relates to anti-SARS-CoV-2 infection mRNA, protein and anti-SARS-CoV-2 infection vaccine.
背景技术Background technique
新型冠状病毒(SARS-CoV-2，简称新冠病毒)，是一种具有包膜的、不分节段的单股正链RNA病毒，属于β属的新型冠状病毒。病毒每组基因组长度约30000个核苷酸左右，其基因序列显示与在中华菊头蝠中发现的冠状病毒(MERS-CoV或SARS-CoV)相似，但又有一些明显的区别；目前研究显示与蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)同源性达85％以上。它可以通过病毒表面的刺突蛋白(S蛋白)与人类呼吸道上皮细胞表面的血管紧张素转换酶2(ACE2)结合，进入人体呼吸道上皮细胞进行复制。因此，亟需开发一种新的疫苗以预防SARS-CoV-2感染。The new coronavirus (SARS-CoV-2, referred to as the new coronavirus) is an enveloped, non-segmented, single-stranded positive-sense RNA virus, belonging to the new coronavirus of the genus β. The length of each genome of the virus is about 30,000 nucleotides, and its gene sequence is similar to that of the coronavirus (MERS-CoV or SARS-CoV) found in Chinese horseshoe bats, but there are some obvious differences; current research shows The homology with bat SARS-like coronavirus (bat-SL-CoVZC45) is more than 85%. It can bind to angiotensin-converting enzyme 2 (ACE2) on the surface of human respiratory epithelial cells through the spike protein (S protein) on the surface of the virus, and enter human respiratory epithelial cells for replication. Therefore, there is an urgent need to develop a new vaccine to prevent SARS-CoV-2 infection.
发明内容Contents of the invention
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提供抗SARS-CoV-2感染的mRNA。本发明的另一目的在于提供利用所述mRNA制备的用于预防和/或治疗SARS-CoV-2感染的疫苗。The present invention aims to solve at least one of the technical problems existing in the prior art. For this reason, an object of the present invention is to provide anti-SARS-CoV-2 infected mRNA. Another object of the present invention is to provide a vaccine prepared by using the mRNA for preventing and/or treating SARS-CoV-2 infection.
本发明是基于发明人的下列发现而完成的：The present invention has been accomplished based on the following findings of the inventors:
新冠属于RNA病毒，RNA病毒不稳定，病毒复制中会发生自然突变。在一些情况下病毒会获得性状改变，如传播能力的变化、致病性的变化、对环境耐受力的变化等。随着SARS-Cov-2不断变异，近期陆续产生和报道了几个关键的突变株，其中一个是Beta突变株B.1.351(20H/501Y.V2)，一个是Gamma突变株P.1(20J/501Y.V3)，还有一个是Delta突变株B.1.617.1系列和B.1.617.2系列。突变株的出现导致病毒的传染力增强，并且出现了免疫逃逸现象。相比于对野生型病毒株的中和抗体滴度，康复者血清对Gamma变异株产生的抗体滴度平均降低了8倍。因此，亟需对疫苗抗原的序列做出一些改变，以适应变异株上的刺突蛋白的突变。The new crown belongs to RNA virus, RNA virus is unstable, and natural mutations will occur during virus replication. In some cases, viruses will acquire trait changes, such as changes in transmissibility, pathogenicity, and environmental tolerance. With the continuous mutation of SARS-Cov-2, several key mutant strains have been produced and reported recently, one of which is the Beta mutant strain B.1.351 (20H/501Y.V2), and the other is the Gamma mutant strain P.1 (20J /501Y.V3), and the other ones are Delta mutants B.1.617.1 series and B.1.617.2 series. The emergence of mutant strains led to increased infectivity of the virus and immune escape. Compared with the neutralizing antibody titer to the wild-type virus strain, the antibody titer produced by the recovered person's serum to the Gamma mutant strain was reduced by an average of 8 times. Therefore, it is urgent to make some changes to the sequence of the vaccine antigen to adapt to the mutation of the spike protein on the mutant strain.
传统疫苗包括减毒活疫苗、灭活疫苗和亚单位疫苗(重组蛋白疫苗或多肽疫苗)。疫苗的使用在全球范围内成功预防了多种疾病，如天花病毒已被彻底根除，乙肝、小儿麻痹症、麻疹和其他儿童疾病的发病率在世界各地已大幅度降低。然而，这些疫苗存在着不同的开发和应用短板，如减毒活疫苗或灭活疫苗的开发周期就相对较长，因含有病原体，有导致宿主感染的风险，为了保证安全性，在研发过程中的常规测试项目繁复，耗时较长。因此，在对抗SARS-CoV-2、埃博拉病毒和寨卡病毒等这些对人类健康造成巨大危害的突发病毒时，传统疫苗难以及时地发挥其作用。Traditional vaccines include live attenuated vaccines, inactivated vaccines and subunit vaccines (recombinant protein vaccines or polypeptide vaccines). The use of vaccines has successfully prevented many diseases worldwide, such as smallpox virus has been completely eradicated, and the incidence of hepatitis B, polio, measles and other childhood diseases has been greatly reduced around the world. However, these vaccines have different development and application shortcomings. For example, the development cycle of live attenuated vaccines or inactivated vaccines is relatively long. Because they contain pathogens, there is a risk of causing host infection. In order to ensure safety, during the research and development process The routine test items in the test are complicated and time-consuming. Therefore, it is difficult for traditional vaccines to play their role in a timely manner in the fight against SARS-CoV-2, Ebola virus, Zika virus and other sudden viruses that have caused great harm to human health.
目前，新型疫苗包括核酸疫苗以及病毒载体等疫苗，其中，核酸疫苗(mRNA或DNA)作为应急疫苗则具有非常明显的时效性优势。mRNA是携带有遗传信息并指导蛋白质合成的一类单链核糖核酸，目前主要是以DNA作为模板，通过体外转录方法合成制得。mRNA疫苗的工作原理是：将编码特定抗原蛋白的mRNA直接导入体细胞内，并通过宿主细胞的表达***合成抗原蛋白，诱导宿主免疫***产生对该抗原的特异性免疫应答，有效的发挥疾病的治疗与预防作用。mRNA无需进入细胞核，在细胞质内即可翻译，起效更快；没有整合宿主基因组的风险，且会在体内自动降解；能够模拟病毒的天然感染过程来激活免疫***，可激发潜在更强力的免疫反应。At present, new vaccines include nucleic acid vaccines and virus vector vaccines, among which nucleic acid vaccines (mRNA or DNA) have a very obvious timeliness advantage as emergency vaccines. mRNA is a type of single-stranded ribonucleic acid that carries genetic information and guides protein synthesis. At present, it is mainly synthesized by in vitro transcription using DNA as a template. The working principle of the mRNA vaccine is: the mRNA encoding a specific antigen protein is directly introduced into the somatic cells, and the antigen protein is synthesized through the expression system of the host cell, and the host immune system is induced to produce a specific immune response to the antigen, effectively exerting the effect of the disease. Therapeutic and preventive effects. mRNA does not need to enter the nucleus, it can be translated in the cytoplasm, and the effect is faster; there is no risk of integrating the host genome, and it will be automatically degraded in the body; it can simulate the natural infection process of the virus to activate the immune system, which can stimulate a potentially stronger immunity reaction.
因此，在本发明的一个方面，本发明提出了一种mRNA。根据本发明的实施例，所述mRNA的模板DNA包含抗原编码区；所述抗原编码区编码不含信号肽的SARS-CoV-2病毒Delta突变株S蛋白，所述不含信号肽的SARS-CoV-2病毒Delta突变株S蛋白具有K986P和V987P突变中的至少一个突变。本发明的mRNA合成的抗原蛋白稳定性强，且具有较强的免疫原性，尤其是可有效预防和治疗野生型SARS-CoV-2病毒(又称野生型新冠病毒或野生型病毒)及其突变株(SARS-CoV-2病毒变异株Alpha(简称Alpha病毒)、SARS-CoV-2病毒变异株Beta(简称Beta病毒)、SARS-CoV-2病毒变异株Gamma(简称Gamma病毒)、SARS-CoV-2病毒变异株Delta(简称Delta病毒)和SARS-CoV-2病毒变异株Omicron(简称Omicron病毒))的感染，具有广谱的抗病毒感染能力。Thus, in one aspect of the invention, the invention provides an mRNA. According to an embodiment of the present invention, the template DNA of the mRNA includes an antigen coding region; the antigen coding region encodes the SARS-CoV-2 virus Delta mutant strain S protein without a signal peptide, and the SARS-CoV-2 virus without a signal peptide The S protein of the CoV-2 viral Delta mutant strain has at least one of the K986P and V987P mutations. The antigenic protein synthesized by the mRNA of the present invention has strong stability and strong immunogenicity, especially can effectively prevent and treat wild-type SARS-CoV-2 virus (also known as wild-type new coronavirus or wild-type virus) and its Mutant strains (SARS-CoV-2 virus mutant strain Alpha (referred to as Alpha virus), SARS-CoV-2 virus mutant strain Beta (referred to as Beta virus), SARS-CoV-2 virus mutant strain Gamma (referred to as Gamma virus), SARS- The infection of CoV-2 virus variant Delta (abbreviated as Delta virus) and SARS-CoV-2 virus variant strain Omicron (abbreviated as Omicron virus) has broad-spectrum antiviral infection ability.
根据本发明的实施例，所述mRNA还可以进一步包含如下技术特征的至少之一：According to an embodiment of the present invention, the mRNA may further comprise at least one of the following technical features:
根据本发明的实施例，所述抗原编码区编码氨基酸序列如SEQ ID NO：7所示的蛋白。According to an embodiment of the present invention, the antigen coding region encodes a protein having an amino acid sequence as shown in SEQ ID NO:7.
需要说明的是，氨基酸序列如SEQ ID NO：7所示的蛋白为去掉B.1.617.1突变株S蛋白的1-19位信号肽序列(SEQ ID NO：2)后的B.1.617.1突变株的S蛋白的全长，该B.1.617.1突变株的S蛋白进行K986P和V987P突变(即为SEQ ID NO：4)。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 7 is B.1.617.1 after removing the 1-19 signal peptide sequence (SEQ ID NO: 2) of the B.1.617.1 mutant S protein The full length of the S protein of the mutant strain, the S protein of the B.1.617.1 mutant strain undergoes K986P and V987P mutations (ie, SEQ ID NO: 4).
Figure PCTCN2022122626-appb-000001
Figure PCTCN2022122626-appb-000001
根据本发明的实施例，所述SARS-CoV-2病毒Delta突变株S蛋白进一步具有如下突变的至少之一：F817P、A892P、A899P和A942P。由此，该mRNA合成的抗原蛋白稳定性强，且具有较强的免疫原性，从而提高对野生型SARS-CoV-2病毒及其突变株感染的预防和治疗效果。According to an embodiment of the present invention, the S protein of the SARS-CoV-2 virus Delta mutant strain further has at least one of the following mutations: F817P, A892P, A899P and A942P. Therefore, the antigenic protein synthesized by the mRNA has strong stability and strong immunogenicity, thereby improving the prevention and treatment effect on wild-type SARS-CoV-2 virus and its mutant strain infection.
根据本发明的实施例，所述抗原编码区编码氨基酸序列如SEQ ID NO：8所示的蛋白。According to an embodiment of the present invention, the antigen coding region encodes a protein having an amino acid sequence as shown in SEQ ID NO: 8.
需要说明的是，氨基酸序列如SEQ ID NO：8所示的蛋白为去掉B.1.617.1突变株S蛋白的1-19位信号肽序列(SEQ ID NO：2)的B.1.617.1突变株的S蛋白的全长，该B.1.617.1突变株的S蛋白进行F817P、A892P、A899P、A942P、K986P和V987P突变(即为SEQ ID NO：5)。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 8 is the B.1.617.1 mutation that removes the 1-19 signal peptide sequence (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain The full length of the S protein of the strain, the S protein of the B.1.617.1 mutant strain undergoes F817P, A892P, A899P, A942P, K986P and V987P mutations (ie, SEQ ID NO: 5).
Figure PCTCN2022122626-appb-000002
Figure PCTCN2022122626-appb-000002
根据本发明的实施例，所述抗原编码区进一步编码具有突变的野生型SARS-CoV-2病毒的S蛋白的RBD结构域，所述突变为C538S。According to an embodiment of the present invention, the antigen coding region further encodes the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with a mutation, the mutation being C538S.
根据本发明的实施例，所述具有突变的野生型SARS-CoV-2病毒的S蛋白的RBD结构域的氨基酸序列如SEQ ID NO：6所示。According to an embodiment of the present invention, the amino acid sequence of the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with mutation is shown in SEQ ID NO: 6.
需要说明的是，氨基酸序列如SEQ ID NO：6所示的蛋白为具有C538S突变的野生型SARS-CoV-2病毒S蛋白的RBD(319-545)序列。It should be noted that the amino acid sequence of the protein shown in SEQ ID NO: 6 is the RBD (319-545) sequence of the wild-type SARS-CoV-2 virus S protein with a C538S mutation.
Figure PCTCN2022122626-appb-000003
Figure PCTCN2022122626-appb-000003
根据本发明的实施例，所述具有突变的野生型SARS-CoV-2病毒的S蛋白的RBD结构域的C端与所述不含信号肽的SARS-CoV-2病毒Delta突变株S蛋白的N端相连。即为，所述具有C538S突变的野生型S蛋白的RBD结构域融合在所述Delta突变株S蛋白的氮端。According to an embodiment of the present invention, the C-terminus of the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with a mutation and the S protein of the Delta mutant strain of the SARS-CoV-2 virus without a signal peptide The N-terminus is connected. That is, the RBD domain of the wild-type S protein with the C538S mutation is fused to the nitrogen terminal of the Delta mutant S protein.
根据本发明的实施例，所述mRNA的模板DNA进一步包含信号肽编码区。According to an embodiment of the present invention, the template DNA of the mRNA further includes a signal peptide coding region.
根据本发明的实施例，所述信号肽编码区编码SARS-CoV-2病毒Delta突变株的信号肽。According to an embodiment of the present invention, the signal peptide coding region encodes the signal peptide of the Delta mutant strain of SARS-CoV-2 virus.
根据本发明的实施例，所述信号肽的氨基酸序列如SEQ ID NO：2所示。According to an embodiment of the present invention, the amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
需要说明的是，氨基酸序列如SEQ ID NO：2所示的信号肽为Delta突变株B.1.617.1的S蛋白的1-19位信号肽。It should be noted that the signal peptide whose amino acid sequence is shown in SEQ ID NO: 2 is the 1-19 signal peptide of the S protein of the Delta mutant strain B.1.617.1.
MFVFLVLLPLVSSQCVNLT(SEQ ID NO：2)。MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 2).
根据本发明的实施例，所述mRNA的模板DNA的抗原编码区编码的氨基酸序列如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：29和SEQ ID NO：30中的至少一种所示。According to an embodiment of the present invention, the amino acid sequence encoded by the antigen coding region of the template DNA of the mRNA is such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16. At least one of SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 30 .
需要说明的是，氨基酸序列如SEQ ID NO：4所示的蛋白为进行了K986P和V987P突变的B.1.617.1突变株的S蛋白的全长。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 4 is the full length of the S protein of the B.1.617.1 mutant strain with K986P and V987P mutations.
Figure PCTCN2022122626-appb-000004
Figure PCTCN2022122626-appb-000004
需要说明的是，氨基酸序列如SEQ ID NO：5所示的蛋白为进行了F817P、A892P、A899P、A942P、K986P和V987P突变的B.1.617.1突变株的S蛋白的全长。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 5 is the full length of the S protein of the B.1.617.1 mutant strain with F817P, A892P, A899P, A942P, K986P and V987P mutations.
Figure PCTCN2022122626-appb-000005
Figure PCTCN2022122626-appb-000005
需要说明的是，氨基酸序列如SEQ ID NO：16所示的蛋白为，将B.1.617.1突变株的S蛋白的1-19位信号肽(SEQ ID  NO：2)、具有C538S突变的野生型S蛋白的RBD(319-545)(SEQ ID NO：6)以及去掉1-19位信号肽的B.1.617.1突变株的S蛋白(SEQ ID NO：7)进行直接融合得到的融合蛋白。该mRNA可用于构建能够同时针对野生型和B.1.617.1突变型的SARS-CoV-2的二价mRNA疫苗。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 16 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the wild The fusion protein obtained by direct fusion of RBD (319-545) (SEQ ID NO: 6) of the type S protein and the S protein (SEQ ID NO: 7) of the B.1.617.1 mutant strain with the 1-19 signal peptide removed . The mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and B.1.617.1 mutant SARS-CoV-2.
Figure PCTCN2022122626-appb-000006
Figure PCTCN2022122626-appb-000006
需要说明的是，氨基酸序列如SEQ ID NO：17所示的蛋白为，将B.1.617.1突变株的S蛋白的1-19位信号肽(SEQ ID NO：2)、具有C538S突变的野生型S蛋白的RBD(319-545)(SEQ ID NO：6)以及去掉1-19位信号肽的B.1.617.1突变株的S蛋白(SEQ ID NO：8)进行直接融合得到的融合蛋白。该mRNA可用于构建能够同时针对野生型和B.1.617.1突变型的SARS-CoV-2的二价mRNA疫苗。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 17 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the wild The fusion protein obtained by direct fusion of RBD (319-545) (SEQ ID NO: 6) of the type S protein and the S protein (SEQ ID NO: 8) of the B.1.617.1 mutant strain with the 1-19 signal peptide removed . The mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and B.1.617.1 mutant SARS-CoV-2.
Figure PCTCN2022122626-appb-000007
Figure PCTCN2022122626-appb-000007
需要说明的是，氨基酸序列如SEQ ID NO：22所示的蛋白为具有进行了K986P和V987P突变的Delta突变株B.1.617.2的S蛋白全长。由此，该mRNA产生的抗原蛋白稳定性强，且该mRNA制备的mRNA疫苗具有广谱性。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 22 is the full-length S protein of the Delta mutant strain B.1.617.2 with K986P and V987P mutations. Therefore, the antigenic protein produced by the mRNA has strong stability, and the mRNA vaccine prepared by the mRNA has a broad spectrum.
Figure PCTCN2022122626-appb-000008
Figure PCTCN2022122626-appb-000008
Figure PCTCN2022122626-appb-000009
Figure PCTCN2022122626-appb-000009
需要说明的是，氨基酸序列如SEQ ID NO：23所示的蛋白为具有进行了F817P、A892P、A899P、A942P、K986P和V987P突变的Delta突变株B.1.617.2的S蛋白的全长。由此，该mRNA产生的抗原蛋白稳定性强，且能够增强抗原蛋白的表达量。并且，该mRNA制备的mRNA疫苗具有广谱性。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 23 is the full length of the S protein of the Delta mutant strain B.1.617.2 with F817P, A892P, A899P, A942P, K986P and V987P mutations. Thus, the antigenic protein produced by the mRNA has strong stability and can enhance the expression level of the antigenic protein. Moreover, the mRNA vaccine prepared from the mRNA has a broad spectrum.
Figure PCTCN2022122626-appb-000010
Figure PCTCN2022122626-appb-000010
需要说明的是，氨基酸序列如SEQ ID NO：25所示的蛋白为，去掉1-19位信号肽(SEQ ID NO：2)的Delta突变株B.1.617.2的S蛋白(SEQ ID NO：22)。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 25 is the S protein (SEQ ID NO: twenty two).
Figure PCTCN2022122626-appb-000011
Figure PCTCN2022122626-appb-000011
Figure PCTCN2022122626-appb-000012
Figure PCTCN2022122626-appb-000012
需要说明的是，氨基酸序列如SEQ ID NO：26所示的蛋白为，去掉1-19位信号肽(SEQ ID NO：2)的Delta突变株B.1.617.2的S蛋白(SEQ ID NO：23)。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 26 is the S protein (SEQ ID NO: twenty three).
Figure PCTCN2022122626-appb-000013
Figure PCTCN2022122626-appb-000013
需要说明的是，氨基酸序列如SEQ ID NO：29所示的蛋白为，将Delta突变株B.1.617.2的S蛋白的1-19位信号肽(SEQ ID NO：24)、具有C538S突变的野生型S蛋白的RBD(319-545，SEQ ID NO：6)和去掉1-19位信号肽的Delta突变株B.1.617.2的S蛋白(SEQ ID NO：25)进行直接融合得到的融合蛋白。该mRNA可用于构建能够同时针对野生型和Delta突变型B.1.617.2的SARS-CoV-2的二价mRNA疫苗。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 29 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, which has a C538S mutation The fusion obtained by direct fusion of the RBD (319-545, SEQ ID NO: 6) of the wild-type S protein and the S protein (SEQ ID NO: 25) of the Delta mutant strain B.1.617.2 that removes the 1-19 signal peptide protein. The mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and Delta mutant B.1.617.2 SARS-CoV-2.
MFVFLVLLPLVSSQCVNLR(SEQ ID NO：24)；MFVFLVLLPLVSSQCVNLR (SEQ ID NO: 24);
Figure PCTCN2022122626-appb-000014
Figure PCTCN2022122626-appb-000014
需要说明的是，氨基酸序列如SEQ ID NO：29所示的蛋白为，将Delta突变株B.1.617.2的S蛋白的1-19位信号肽(SEQ ID NO：24)、具有C538S突变的野生型S蛋白的RBD(319-545，SEQ ID NO：6)和去掉1-19位信号肽的Delta突变 株B.1.617.2的S蛋白(SEQ ID NO：26)进行直接融合得到的融合蛋白。该mRNA可用于构建能够同时针对野生型和Delta突变型B.1.617.2的SARS-CoV-2的二价mRNA疫苗。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 29 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, which has a C538S mutation The fusion obtained by direct fusion of the RBD (319-545, SEQ ID NO: 6) of the wild-type S protein and the S protein (SEQ ID NO: 26) of the Delta mutant strain B.1.617.2 that removes the 1-19 signal peptide protein. The mRNA can be used to construct a bivalent mRNA vaccine capable of simultaneously targeting wild-type and Delta mutant B.1.617.2 SARS-CoV-2.
Figure PCTCN2022122626-appb-000015
Figure PCTCN2022122626-appb-000015
根据本发明的实施例，所述模板DNA的启动子为T7或SP6启动子。According to an embodiment of the present invention, the promoter of the template DNA is a T7 or SP6 promoter.
根据本发明的实施例，所述模板DNA进一步包含有5’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 5' untranslated region.
根据本发明的实施例，所述5’端非翻译区的核苷酸序列如SEQ ID NO：1所示。According to an embodiment of the present invention, the nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
Figure PCTCN2022122626-appb-000016
Figure PCTCN2022122626-appb-000016
根据本发明的实施例，所述模板DNA进一步包含3’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 3' untranslated region.
根据本发明的实施例，所述的3’端非翻译区的核苷酸序列如SEQ ID NO：41所示。According to an embodiment of the present invention, the nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
Figure PCTCN2022122626-appb-000017
Figure PCTCN2022122626-appb-000017
根据本发明的实施例，所述模板DNA的3’端进一步连接polyA。According to an embodiment of the present invention, the 3' end of the template DNA is further connected to polyA.
根据本发明的实施例，所述polyA的核苷酸序列如SEQ ID NO：42所示。According to an embodiment of the present invention, the nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
Figure PCTCN2022122626-appb-000018
Figure PCTCN2022122626-appb-000018
根据本发明的实施例，所述模板DNA由所述启动子、5’端非翻译区、信号肽编码区、抗原编码区、3’端非翻译区和polyA连接组成。According to an embodiment of the present invention, the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
需要说明的是，模板DNA从N端到C端依次为启动子、5’端非翻译区、信号肽编码区、抗原编码区、3’端非翻译区和polyA。在本发明中，信号肽编码区和抗原编码区可以以一条序列提供，也可分开以两条序列提供；在本发明中，当信号肽编码区和抗原编码区以一条序列提供时，可简称为抗原编码区。It should be noted that, from the N-terminus to the C-terminus, the template DNA is the promoter, the 5'-terminal untranslated region, the signal peptide coding region, the antigen coding region, the 3'-terminal untranslated region and polyA. In the present invention, the signal peptide coding region and the antigen coding region can be provided in one sequence, or can be provided in two separate sequences; in the present invention, when the signal peptide coding region and the antigen coding region are provided in one sequence, it can be referred to as for the antigen coding region.
根据本发明的实施例，所述SARS-CoV-2病毒Delta突变株为B.1.617.1突变株或B.1.617.2突变株。According to an embodiment of the present invention, the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
在本发明的又一方面，本发明提出了一种mRNA。根据本发明的实施例，所述mRNA的模板DNA包含抗原编码区；所述抗原编码区编码SARS-CoV-2病毒Delta突变株的NTD_RBD结构域，所述SARS-CoV-2病毒Delta突变株的NTD_RBD结构域具有C538S突变。本发明的mRNA具有较强的免疫原性，注射该mRNA后可产生较高的中和抗体，可有效预防和治疗野生型SARS-CoV-2病毒及其突变株(Alpha病毒、Beta病毒、Gamma病毒、Delta病毒和Omicron病毒)的感染。In yet another aspect of the present invention, the present invention provides an mRNA. According to an embodiment of the present invention, the template DNA of the mRNA comprises an antigen coding region; the antigen coding region encodes the NTD_RBD domain of the SARS-CoV-2 virus Delta mutant strain, and the SARS-CoV-2 virus Delta mutant strain The NTD_RBD domain has a C538S mutation. The mRNA of the present invention has stronger immunogenicity, can produce higher neutralizing antibody after injecting this mRNA, can effectively prevent and treat wild-type SARS-CoV-2 virus and mutant strain thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
根据本发明的实施例，所述mRNA还可以进一步包含如下技术特征的至少之一：According to an embodiment of the present invention, the mRNA may further comprise at least one of the following technical features:
根据本发明的实施例，所述SARS-CoV-2病毒Delta突变株的氨基酸序列如SEQ ID NO：9所示。According to an embodiment of the present invention, the amino acid sequence of the SARS-CoV-2 virus Delta mutant is shown in SEQ ID NO: 9.
需要说明的是，氨基酸序列如SEQ ID NO：9所示的蛋白为具有C538S突变的B.1.617.1突变株的S蛋白的NTD_RBD(20-545)。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 9 is NTD_RBD (20-545) of the S protein of the B.1.617.1 mutant strain with a C538S mutation.
Figure PCTCN2022122626-appb-000019
Figure PCTCN2022122626-appb-000019
根据本发明的实施例，所述抗原编码区进一步编码具有突变的野生型SARS-CoV-2的RBD结构域，所述突变为C538S。According to an embodiment of the present invention, the antigen coding region further encodes the RBD domain of wild-type SARS-CoV-2 with a mutation, the mutation being C538S.
根据本发明的实施例，所述具有突变的野生型SARS-CoV-2的RBD结构域的氨基酸序列如SEQ ID NO：6所示。According to an embodiment of the present invention, the amino acid sequence of the RBD domain of the mutated wild-type SARS-CoV-2 is shown in SEQ ID NO: 6.
根据本发明的实施例，所述抗原编码区进一步编码Foldon片段。由此，有利于编码抗原翻译后，形成跨膜三聚体。According to an embodiment of the present invention, the antigen coding region further encodes a Foldon fragment. Thus, it is beneficial to form a transmembrane trimer after the encoded antigen is translated.
根据本发明的实施例，所述Foldon片段的氨基酸序列如SEQ ID NO：11所示。According to an embodiment of the present invention, the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
GYIPEAPRDGQAYVRKDGEWVFLSTFL(SEQ ID NO：11)。GYIPEAPRDGQAYVRKDGEWVFLSTFL (SEQ ID NO: 11).
根据本发明的实施例，所述抗原编码区进一步编码linker。According to an embodiment of the present invention, the antigen coding region further encodes a linker.
根据本发明的实施例，所述linker的氨基酸序列为GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10或(GSG) 10中的至少一种。 According to an embodiment of the present invention, the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 or (GSG) 10 .
GGGGS的氨基酸序列为：GGGGS(SEQ ID NO：43)；The amino acid sequence of GGGGS is: GGGGS (SEQ ID NO: 43);
(GGGGS) 3的氨基酸序列为：GGGGSGGGGSGGGGS(SEQ ID NO：44)； The amino acid sequence of (GGGGS) 3 is: GGGGSGGGGSGGGGS (SEQ ID NO: 44);
(GGGGS) 6的氨基酸序列为：GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：10)； The amino acid sequence of (GGGGS) 6 is: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 10);
(GGS) 10的氨基酸序列为：GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS(SEQ ID NO：45)； The amino acid sequence of (GGS) 10 is: GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 45);
(GSG) 10的氨基酸序列为：GSGGSGGSGGSGGSGGSGGSGGSGGSGGSG(SEQ ID NO：46)。 The amino acid sequence of (GSG) 10 is: GSGGSGGSGGSGGSGGSGGSGGSGGSGGSG (SEQ ID NO: 46).
根据本发明的实施例，所述模板DNA进一步包含信号肽编码区。According to an embodiment of the present invention, the template DNA further includes a signal peptide coding region.
根据本发明的实施例，所述信号肽编码区编码SARS-CoV-2病毒Delta突变株的信号肽。According to an embodiment of the present invention, the signal peptide coding region encodes the signal peptide of the Delta mutant strain of SARS-CoV-2 virus.
根据本发明的实施例，所述信号肽的氨基酸序列如SEQ ID NO：2所示。According to an embodiment of the present invention, the amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
根据本发明的实施例，所述mRNA的模板DNA编码的氨基酸序列如SEQ ID NO：9、SEQ ID NO：18、或SEQ ID NO：19、SEQ ID NO：27、SEQ ID NO：31或SEQ ID NO：32中的至少一种所示。According to an embodiment of the present invention, the amino acid sequence encoded by the template DNA of the mRNA is such as SEQ ID NO: 9, SEQ ID NO: 18, or SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 31 or SEQ ID NO: At least one of ID NO: 32 is shown.
需要说明的是，氨基酸序列如SEQ ID NO：18所示的蛋白为，将B.1.617.1突变株的S蛋白的1-19位信号肽(SEQ ID NO：2)、具有C538S突变的B.1.617.1突变株的NTD_RBD(20-545)序列(SEQ ID NO：9)、序列为(GGGGS) 6的Linker和Foldon片段(SEQ ID NO：11)进行融合得到的融合蛋白。 It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 18 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the B . A fusion protein obtained by fusing the NTD_RBD (20-545) sequence (SEQ ID NO: 9) of the 1.617.1 mutant strain, the Linker and Foldon fragments (SEQ ID NO: 11) whose sequence is (GGGGS) 6 .
Figure PCTCN2022122626-appb-000020
Figure PCTCN2022122626-appb-000020
需要说明的是，氨基酸序列如SEQ ID NO：19所示的蛋白为，将B.1.617.1突变株的S蛋白的1-19位信号肽(SEQ ID NO：2)、具有C538S突变的B.1.617.1突变株的NTD_RBD(20-545)序列(SEQ ID NO：9)、具有C538S突变的野生型RBD(SEQ ID NO：6)、序列为(GGGGS) 6的Linker和Foldon片段(SEQ ID NO：11)进行融合得到的融合蛋白。 It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 19 is the 1-19 signal peptide (SEQ ID NO: 2) of the S protein of the B.1.617.1 mutant strain, the B .1.617.1 NTD_RBD (20-545) sequence (SEQ ID NO: 9), wild-type RBD (SEQ ID NO: 6) with C538S mutation, sequence is (GGGGS) 6 Linker and Foldon fragment (SEQ ID NO: 11) fusion protein obtained by fusion.
Figure PCTCN2022122626-appb-000021
Figure PCTCN2022122626-appb-000021
Figure PCTCN2022122626-appb-000022
Figure PCTCN2022122626-appb-000022
需要说明的是，氨基酸序列如SEQ ID NO：27所示的蛋白为具有C538S突变的Delta突变株B.1.617.2的S蛋白的NTD_RBD(20-545)。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 27 is NTD_RBD (20-545) of the S protein of the Delta mutant strain B.1.617.2 with the C538S mutation.
Figure PCTCN2022122626-appb-000023
Figure PCTCN2022122626-appb-000023
需要说明的是，氨基酸序列如SEQ ID NO：31所示的蛋白为，将Delta突变株B.1.617.2的S蛋白的1-19位信号肽(SEQ ID NO：24)、具有C538S突变的Delta突变株B.1.617.2的NTD_RBD序列(20-545，SEQ ID NO：27)、(GGGGS) 6的Linker和Foldon片段(SEQ ID NO：11)进行融合得到的融合蛋白。 It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 31 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the C538S mutation A fusion protein obtained by fusing the NTD_RBD sequence (20-545, SEQ ID NO: 27) of the Delta mutant strain B.1.617.2, and the Linker and Foldon fragments (SEQ ID NO: 11) of (GGGGS) 6 .
Figure PCTCN2022122626-appb-000024
Figure PCTCN2022122626-appb-000024
需要说明的是，氨基酸序列如SEQ ID NO：32所示的蛋白为，将Delta突变株B.1.617.2的S蛋白的1-19位信号肽(SEQ ID NO：24)、具有C538S突变的Delta突变株B.1.617.2的NTD_RBD序列(20-545，SEQ ID NO：27)、具有C538S突变的野生型RBD(SEQ ID NO：6)、(GGGGS) 6的Linker和Foldon片段(SEQ ID NO：11)进行融合得到的融合蛋白。 It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 32 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the C538S mutation The NTD_RBD sequence (20-545, SEQ ID NO: 27) of the Delta mutant strain B.1.617.2, the wild-type RBD (SEQ ID NO: 6) with the C538S mutation, the Linker and Foldon fragments of (GGGGS) 6 (SEQ ID NO: 11) Fusion protein obtained by fusion.
Figure PCTCN2022122626-appb-000025
Figure PCTCN2022122626-appb-000025
根据本发明的实施例，所述模板DNA的启动子为T7或SP6启动子。According to an embodiment of the present invention, the promoter of the template DNA is a T7 or SP6 promoter.
根据本发明的实施例，所述模板DNA进一步包含有5’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 5' untranslated region.
根据本发明的实施例，所述5’端非翻译区的核苷酸序列如SEQ ID NO：1所示。According to an embodiment of the present invention, the nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
根据本发明的实施例，所述模板DNA进一步包含3’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 3' untranslated region.
根据本发明的实施例，所述的3’端非翻译区的核苷酸序列如SEQ ID NO：41所示。According to an embodiment of the present invention, the nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
根据本发明的实施例，所述模板DNA的3’端进一步连接polyA。According to an embodiment of the present invention, the 3' end of the template DNA is further connected to polyA.
根据本发明的实施例，所述polyA的核苷酸序列如SEQ ID NO：42所示。According to an embodiment of the present invention, the nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
根据本发明的实施例，所述模板DNA由所述启动子、5’端非翻译区、信号肽编码区、抗原编码区、3’端非翻译区和polyA连接组成。According to an embodiment of the present invention, the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
根据本发明的实施例，所述SARS-CoV-2病毒Delta突变株为B.1.617.1突变株或B.1.617.2突变株。According to an embodiment of the present invention, the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
在本发明的另一方面，本发明提出了一种mRNA。根据本发明的实施例，所述mRNA的模板DNA包含抗原编码区；所述抗原编码区编码SARS-CoV-2病毒Delta突变株的RBD结构域和SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域中的至少之一；其中，所述SARS-CoV-2病毒Delta突变株的RBD结构域具有C538S突变；所述SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域具有D80A突变、R246I突变和C538S突变，以及增加Beta突变株的Δ242-244。本发明的mRNA具有较强的免疫原性，注射该mRNA后可产生较高的中和抗体，可有效预防和治疗野生型SARS-CoV-2病毒及其突变株(Alpha病毒、Beta病毒、Gamma病毒、Delta病毒和Omicron病毒)的感染。In another aspect of the invention, the invention provides an mRNA. According to an embodiment of the present invention, the template DNA of the mRNA comprises an antigen coding region; the antigen coding region encodes the RBD domain of the SARS-CoV-2 virus Delta mutant strain and the NTD_RBD structure of the SARS-CoV-2 virus Gamma mutant strain At least one of the domains; wherein, the RBD domain of the SARS-CoV-2 virus Delta mutant has a C538S mutation; the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant has a D80A mutation, a R246I mutation and C538S mutation, as well as increased Δ242-244 of the Beta mutant. The mRNA of the present invention has stronger immunogenicity, can produce higher neutralizing antibody after injecting this mRNA, can effectively prevent and treat wild-type SARS-CoV-2 virus and mutant strain thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
根据本发明的实施例，所述mRNA还可以进一步包含如下技术特征的至少之一：According to an embodiment of the present invention, the mRNA may further comprise at least one of the following technical features:
根据本发明的实施例，所述SARS-CoV-2病毒Delta突变株的RBD结构域的氨基酸序列如SEQ ID NO：14或SEQ ID NO：28所示。According to an embodiment of the present invention, the amino acid sequence of the RBD domain of the SARS-CoV-2 virus Delta mutant is shown in SEQ ID NO: 14 or SEQ ID NO: 28.
需要说明的是，氨基酸序列如SEQ ID NO：14所示的蛋白为具有C538S突变的B.1.617.1突变株的S蛋白的RBD(319-545)。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 14 is the RBD (319-545) of the S protein of the B.1.617.1 mutant strain with the C538S mutation.
Figure PCTCN2022122626-appb-000026
Figure PCTCN2022122626-appb-000026
需要说明的是，氨基酸序列如SEQ ID NO：28所示的蛋白为具有C538S突变的B.1.617.2突变株的S蛋白的NTD_RBD。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 28 is NTD_RBD of the S protein of the B.1.617.2 mutant strain with a C538S mutation.
Figure PCTCN2022122626-appb-000027
Figure PCTCN2022122626-appb-000027
根据本发明的实施例，所述抗原编码区进一步编码SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域，所述SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域具有D80A突变、R246I突变和C538S突变，以及增加Beta突变株的Δ242-244。由此，该mRNA合成能够同时对应Beta突变株和Gamma突变株的抗原蛋白，后续可用于构建能够同时针对Beta突变型和Gamma突变型的SARS-CoV-2的二价mRNA疫苗。According to an embodiment of the present invention, the antigen coding region further encodes the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant strain, and the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant strain has D80A mutation, R246I mutation and C538S mutation, as well as increased Δ242-244 of the Beta mutant. Thus, the mRNA synthesis can correspond to the antigenic protein of the Beta mutant strain and the Gamma mutant strain at the same time, and can be subsequently used to construct a bivalent mRNA vaccine capable of simultaneously targeting the Beta mutant and Gamma mutant SARS-CoV-2.
需要说明的是，在本文中，“Δ242-244”是指在将Gamma突变株的第242位-第244位的三个氨基酸“LAL”进行缺失突变。It should be noted that, in this article, "Δ242-244" refers to the deletion mutation of the three amino acids "LAL" at positions 242-244 of the Gamma mutant strain.
根据本发明的实施例，所述ARS-CoV-2病毒Gamma突变株的NTD_RBD结构域的氨基酸序列如SEQ ID NO：13所示。According to an embodiment of the present invention, the amino acid sequence of the NTD_RBD domain of the ARS-CoV-2 virus Gamma mutant is shown in SEQ ID NO: 13.
需要说明的是，氨基酸序列如SEQ ID NO：13所示的蛋白为，具有D80A突变、R246I突变和C538S突变以及增加Beta突变株的Δ242-244的Gamma突变株的NTD_RBD结构域，简称Beta&Gamma双突变株。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 13 is the NTD_RBD domain of the Gamma mutant strain with D80A mutation, R246I mutation and C538S mutation and increased Δ242-244 of the Beta mutant strain, referred to as the Beta&Gamma double mutation strain.
Figure PCTCN2022122626-appb-000028
Figure PCTCN2022122626-appb-000028
根据本发明的实施例，所述抗原编码区进一步编码具有突变的野生型SARS-CoV-2病毒的RBD结构域，所述突变为C538S。According to an embodiment of the present invention, the antigen coding region further encodes the RBD domain of the wild-type SARS-CoV-2 virus with a mutation, the mutation being C538S.
根据本发明的实施例，所述具有突变的野生型SARS-CoV-2病毒的RBD结构域的氨基酸序列如SEQ ID NO：6所示。According to an embodiment of the present invention, the amino acid sequence of the RBD domain of the mutated wild-type SARS-CoV-2 virus is shown in SEQ ID NO: 6.
根据本发明的实施例，所述抗原编码区编码SARS-CoV-2病毒Delta突变株的RBD结构域、SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域和具有突变的野生型SARS-CoV-2病毒的RBD结构域。According to an embodiment of the present invention, the antigen coding region encodes the RBD domain of the SARS-CoV-2 virus Delta mutant strain, the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant strain, and the wild-type SARS-CoV- 2 The RBD domain of the virus.
根据本发明的实施例，所述抗原编码区进一步编码Foldon片段。According to an embodiment of the present invention, the antigen coding region further encodes a Foldon fragment.
根据本发明的实施例，所述Foldon片段的氨基酸序列如SEQ ID NO：11所示。According to an embodiment of the present invention, the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
根据本发明的实施例，所述抗原编码区进一步编码linker。According to an embodiment of the present invention, the antigen coding region further encodes a linker.
根据本发明的实施例，所述linker的氨基酸序列如GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10和(GSG) 10中的至少一 种所示。 According to an embodiment of the present invention, the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
根据本发明的实施例，所述mRNA的模板DNA进一步含有信号肽编码区。According to an embodiment of the present invention, the template DNA of the mRNA further contains a signal peptide coding region.
根据本发明的实施例，所述信号肽编码区编码SARS-CoV-2病毒Beta突变株的信号肽。According to an embodiment of the present invention, the signal peptide coding region encodes the signal peptide of the Beta mutant strain of SARS-CoV-2 virus.
根据本发明的实施例，所述SARS-CoV-2病毒Beta突变株的信号肽的氨基酸序列如SEQ ID NO：3所示。According to an embodiment of the present invention, the amino acid sequence of the signal peptide of the SARS-CoV-2 virus Beta mutant strain is shown in SEQ ID NO: 3.
需要说明的是，的氨基酸序列如SEQ ID NO：3所示的信号肽为Beta突变株S蛋白的1-19位信号肽。It should be noted that the amino acid sequence of the signal peptide shown in SEQ ID NO: 3 is the 1-19 signal peptide of the S protein of the Beta mutant strain.
MFVFLVLLPLVSSQCVNFT(SEQ ID NO：3)。MFVFLVLLPLVSSQCVNFT (SEQ ID NO: 3).
根据本发明的实施例，所述抗原编码区编码的氨基酸序列如SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：28、SEQ ID NO：34、SEQ ID NO：33中的至少一种所示。According to an embodiment of the present invention, the amino acid sequence encoded by the antigen coding region is such as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO : 34, shown in at least one of SEQ ID NO: 33.
需要说明的是，氨基酸序列如SEQ ID NO：20所示的序列为，将Beta突变株S蛋白的1-19位信号肽(SEQ ID NO：3)、Beta&Gamma双突变株的NTD_RBD(SEQ ID NO：13)、具有C538S突变的B.1.617.1突变株的RBD(SEQ ID NO：14)和具有C538S突变的野生型RBD(SEQ ID NO：6)进行融合得到的蛋白。It should be noted that the amino acid sequence shown in SEQ ID NO: 20 is the sequence of the 1-19 signal peptide (SEQ ID NO: 3) of the S protein of the Beta mutant strain, the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant strain : 13), the RBD (SEQ ID NO: 14) of the B.1.617.1 mutant strain with the C538S mutation and the wild-type RBD (SEQ ID NO: 6) with the C538S mutation are fused to obtain the protein.
Figure PCTCN2022122626-appb-000029
Figure PCTCN2022122626-appb-000029
需要说明的是，氨基酸序列如SEQ ID NO：20所示的序列为，将Beta突变株S蛋白的1-19位信号肽(SEQ ID NO：3)、Beta&Gamma双突变株的NTD_RBD(SEQ ID NO：13)、具有C538S突变的B.1.617.1突变株的RBD(SEQ ID NO：14)、具有C538S突变的野生型RBD(SEQ ID NO：6)、(GGGGS) 6的Linker和Foldon片段(SEQ ID NO：11)进行融合得到的融合蛋白。 It should be noted that the amino acid sequence shown in SEQ ID NO: 20 is the signal peptide at positions 1-19 of the Beta mutant S protein (SEQ ID NO: 3), the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant : 13), the RBD (SEQ ID NO: 14) of the B.1.617.1 mutant strain with the C538S mutation, the wild-type RBD (SEQ ID NO: 6) with the C538S mutation, the Linker and Foldon fragments of (GGGGS) 6 ( The fusion protein obtained by fusion of SEQ ID NO: 11).
Figure PCTCN2022122626-appb-000030
Figure PCTCN2022122626-appb-000030
需要说明的是，氨基酸序列如SEQ ID NO：33所示的序列为，将Beta突变株S蛋白的1-19位信号肽(SEQ ID NO：3)、Beta&Gamma双突变株的NTD_RBD(SEQ ID NO：13)、具有C538S突变的Delta突变株B.1.617.2的RBD(SEQ  ID NO：28)和具有C538S突变的野生型RBD(SEQ ID NO：6)进行融合得到的融合蛋白。It should be noted that the amino acid sequence as shown in SEQ ID NO: 33 is the 1-19 signal peptide (SEQ ID NO: 3) of the S protein of the Beta mutant strain, the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant strain : 13), the RBD (SEQ ID NO: 28) of the Delta mutant strain B.1.617.2 with the C538S mutation and the wild-type RBD (SEQ ID NO: 6) with the C538S mutation are fused to obtain a fusion protein.
Figure PCTCN2022122626-appb-000031
Figure PCTCN2022122626-appb-000031
需要说明的是，氨基酸序列如SEQ ID NO：34所示的序列为，将Beta突变株S蛋白的1-19位信号肽(SEQ ID NO：3)、Beta&Gamma双突变株的NTD_RBD(SEQ ID NO：13)、具有C538S突变的Delta突变株B.1.617.2的RBD(SEQ ID NO：28)、具有C538S突变的野生型RBD(SEQ ID NO：6)、序列为(GGGGS) 6的Linker和Foldon片段(SEQ ID NO：11)进行融合得到的融合蛋白。 It should be noted that the amino acid sequence shown in SEQ ID NO: 34 is the 1-19 signal peptide (SEQ ID NO: 3) of the S protein of the Beta mutant strain, the NTD_RBD (SEQ ID NO: 3) of the Beta & Gamma double mutant strain : 13), the RBD (SEQ ID NO: 28) of the Delta mutant strain B.1.617.2 with the C538S mutation, the wild-type RBD (SEQ ID NO: 6) with the C538S mutation, the Linker whose sequence is (GGGGS) 6 and A fusion protein obtained by fusing the Foldon fragment (SEQ ID NO: 11).
Figure PCTCN2022122626-appb-000032
Figure PCTCN2022122626-appb-000032
根据本发明的实施例，所述模板DNA的启动子为T7或SP6启动子。According to an embodiment of the present invention, the promoter of the template DNA is a T7 or SP6 promoter.
根据本发明的实施例，所述模板DNA进一步包含有5’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 5' untranslated region.
根据本发明的实施例，所述5’端非翻译区的核苷酸序列如SEQ ID NO：1所示。According to an embodiment of the present invention, the nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
根据本发明的实施例，所述模板DNA进一步包含3’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 3' untranslated region.
根据本发明的实施例，所述的3’端非翻译区的核苷酸序列如SEQ ID NO：41所示。According to an embodiment of the present invention, the nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
根据本发明的实施例，所述模板DNA的3’端进一步连接polyA。According to an embodiment of the present invention, the 3' end of the template DNA is further connected to polyA.
根据本发明的实施例，所述polyA的核苷酸序列如SEQ ID NO：42所示。According to an embodiment of the present invention, the nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
根据本发明的实施例，所述模板DNA由所述启动子、5’端非翻译区、信号肽编码区、抗原编码区、3’端非翻译区和polyA连接组成。According to an embodiment of the present invention, the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
根据本发明的实施例，所述SARS-CoV-2病毒Delta突变株为B.1.617.1突变株或B.1.617.2突变株。According to an embodiment of the present invention, the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
在本发明的另一方面，本发明提出了一种mRNA。根据本发明的实施例，所述mRNA的模板DNA包含抗原编码区；所述抗原编码区编码SARS-CoV-2病毒Delta突变株的S蛋白的RBD结构域、SARS-CoV-2病毒Beta突变株S蛋白的RBD 结构域、SARS-CoV-2病毒Gamma突变株S蛋白的RBD结构域和野生型SARS-CoV-2病毒的S蛋白的RBD结构域。本发明的mRNA具有较强的免疫原性，注射该mRNA后可产生较高的中和抗体，可有效预防和治疗野生型SARS-CoV-2病毒及其突变株(Alpha病毒、Beta病毒、Gamma病毒、Delta病毒和Omicron病毒)的感染。In another aspect of the invention, the invention provides an mRNA. According to an embodiment of the present invention, the template DNA of the mRNA comprises an antigen coding region; the antigen coding region encodes the RBD domain of the S protein of the SARS-CoV-2 virus Delta mutant strain, the SARS-CoV-2 virus Beta mutant strain The RBD domain of the S protein, the RBD domain of the S protein of the SARS-CoV-2 virus Gamma mutant strain, and the RBD domain of the S protein of the wild-type SARS-CoV-2 virus. The mRNA of the present invention has stronger immunogenicity, can produce higher neutralizing antibody after injecting this mRNA, can effectively prevent and treat wild-type SARS-CoV-2 virus and mutant strain thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
需要说明的是，本发明中，SARS-CoV-2病毒Delta突变株的S蛋白的RBD结构域、SARS-CoV-2病毒Beta突变株S蛋白的RBD结构域、SARS-CoV-2病毒Gamma突变株S蛋白的RBD结构域和野生型SARS-CoV-2病毒的S蛋白的RBD结构域的连接顺序不受具体限制，只要可同时编码上述4种RBD结构域即可。It should be noted that in the present invention, the RBD domain of the S protein of the SARS-CoV-2 virus Delta mutant strain, the RBD domain of the S protein of the SARS-CoV-2 virus Beta mutant strain, and the SARS-CoV-2 virus Gamma mutation The connection sequence of the RBD domain of the strain S protein and the RBD domain of the S protein of the wild-type SARS-CoV-2 virus is not specifically limited, as long as the above four RBD domains can be encoded simultaneously.
示例性地，模板DNA的N端至C端依次为SARS-CoV-2病毒Delta突变株的S蛋白的RBD结构域、SARS-CoV-2病毒Beta突变株S蛋白的RBD结构域、SARS-CoV-2病毒Gamma突变株S蛋白的RBD结构域和野生型SARS-CoV-2病毒的S蛋白的RBD结构域。Exemplarily, the N-terminal to the C-terminal of the template DNA is the RBD domain of the S protein of the SARS-CoV-2 virus Delta mutant strain, the RBD domain of the S protein of the SARS-CoV-2 virus Beta mutant strain, and the SARS-CoV -2 virus Gamma mutant strain S protein RBD domain and wild-type SARS-CoV-2 virus S protein RBD domain.
根据本发明的实施例，所述mRNA还可以进一步包含如下技术特征的至少之一：According to an embodiment of the present invention, the mRNA may further comprise at least one of the following technical features:
根据本发明的实施例，所述抗原编码区进一步编码Foldon片段。According to an embodiment of the present invention, the antigen coding region further encodes a Foldon fragment.
根据本发明的实施例，所述Foldon片段的氨基酸序列如SEQ ID NO：11所示。According to an embodiment of the present invention, the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
根据本发明的实施例，所述抗原编码区进一步编码linker。According to an embodiment of the present invention, the antigen coding region further encodes a linker.
根据本发明的实施例，所述linker的氨基酸序列如GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10和(GSG) 10中的至少一种所示。 According to an embodiment of the present invention, the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
根据本发明的实施例，所述模板DNA进一步包含信号肽编码区。According to an embodiment of the present invention, the template DNA further includes a signal peptide coding region.
根据本发明的实施例，所述信号肽编码区编码SARS-CoV-2病毒Beta突变株的信号肽。According to an embodiment of the present invention, the signal peptide coding region encodes the signal peptide of the Beta mutant strain of SARS-CoV-2 virus.
根据本发明的实施例，所述SARS-CoV-2病毒Beta突变株的信号肽的氨基酸序列如SEQ ID NO：3所示。According to an embodiment of the present invention, the amino acid sequence of the signal peptide of the SARS-CoV-2 virus Beta mutant strain is shown in SEQ ID NO: 3.
根据本发明的实施例，所述模板DNA编码的氨基酸序列如SEQ ID NO：39和SEQ ID NO：40中的至少一种所示。According to an embodiment of the present invention, the amino acid sequence encoded by the template DNA is at least one of SEQ ID NO: 39 and SEQ ID NO: 40.
需要说明的是，氨基酸序列如SEQ ID NO：39所示的蛋白为，将Delta突变株B.1.617.2的S蛋白的1-19位信号肽(SEQ ID NO：24)、Delta突变株B.1.617.2的S蛋白的RBD(SEQ ID NO：35)、Beta突变株S蛋白的RBD(SEQ ID NO：36)、Gamma突变株S蛋白的RBD(SEQ ID NO：37)和野生型S蛋白的RBD(SEQ ID NO：38)进行融合得到的蛋白。It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 39 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the Delta mutant strain B RBD (SEQ ID NO: 35) of the S protein of 1.617.2, the RBD (SEQ ID NO: 36) of the Beta mutant strain S protein, the RBD (SEQ ID NO: 37) of the Gamma mutant strain S protein and the wild-type S protein The protein obtained by fusing the RBD (SEQ ID NO: 38) of the protein.
Figure PCTCN2022122626-appb-000033
Figure PCTCN2022122626-appb-000033
需要说明的是，氨基酸序列如SEQ ID NO：40所示的蛋白为，将Delta突变株B.1.617.2的S蛋白的1-19位信号肽(SEQ ID NO：24)、Delta突变株B.1.617.2的S蛋白的RBD(SEQ ID NO：35)、Beta突变株S蛋白的RBD(SEQ ID NO：36)、Gamma突变株S蛋白的RBD(SEQ ID NO：37)、野生型S蛋白的RBD(SEQ ID NO：38)、序列为(GGGGS) 6的Linker和Foldon片段(SEQ ID NO：11)进行融合得到的蛋白。 It should be noted that the protein whose amino acid sequence is shown in SEQ ID NO: 40 is the 1-19 signal peptide (SEQ ID NO: 24) of the S protein of the Delta mutant strain B.1.617.2, the Delta mutant strain B The RBD (SEQ ID NO: 35) of the S protein of 1.617.2, the RBD (SEQ ID NO: 36) of the S protein of the Beta mutant strain, the RBD (SEQ ID NO: 37) of the S protein of the Gamma mutant strain, the wild type S protein A protein obtained by fusing the RBD (SEQ ID NO: 38) of the protein, the Linker and Foldon fragments (SEQ ID NO: 11) whose sequence is (GGGGS) 6 .
Figure PCTCN2022122626-appb-000034
Figure PCTCN2022122626-appb-000034
Figure PCTCN2022122626-appb-000035
Figure PCTCN2022122626-appb-000035
根据本发明的实施例，所述模板DNA的启动子为T7或SP6启动子。According to an embodiment of the present invention, the promoter of the template DNA is a T7 or SP6 promoter.
根据本发明的实施例，所述模板DNA进一步包含有5’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 5' untranslated region.
根据本发明的实施例，所述5’端非翻译区的核苷酸序列如SEQ ID NO：1所示。According to an embodiment of the present invention, the nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1.
根据本发明的实施例，所述模板DNA进一步包含3’端非翻译区。According to an embodiment of the present invention, the template DNA further includes a 3' untranslated region.
根据本发明的实施例，所述的3’端非翻译区的核苷酸序列如SEQ ID NO：41所示。According to an embodiment of the present invention, the nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 41.
根据本发明的实施例，所述模板DNA的3’端进一步连接polyA。According to an embodiment of the present invention, the 3' end of the template DNA is further connected to polyA.
根据本发明的实施例，所述polyA的核苷酸序列如SEQ ID NO：42所示。According to an embodiment of the present invention, the nucleotide sequence of the polyA is shown in SEQ ID NO: 42.
根据本发明的实施例，所述模板DNA由所述启动子、5’端非翻译区、信号肽编码区、抗原编码区、3’端非翻译区和polyA连接组成。According to an embodiment of the present invention, the template DNA is composed of the promoter, 5' untranslated region, signal peptide coding region, antigen coding region, 3' untranslated region and polyA junction.
根据本发明的实施例，所述SARS-CoV-2病毒Delta突变株为B.1.617.1突变株或B.1.617.2突变株。According to an embodiment of the present invention, the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
在本发明的另一方面，本发明提出了一种mRNA疫苗。根据本发明的实施例，所述mRNA疫苗包含：前述的mRNA，以及任选地药学上可接受的辅料或者辅助性成分。本发明的mRNA疫苗可有效预防和治疗野生型SARS-CoV-2病毒及其突变株(Alpha病毒、Beta病毒、Gamma病毒、Delta病毒和Omicron病毒)的感染。In another aspect of the present invention, the present invention provides an mRNA vaccine. According to an embodiment of the present invention, the mRNA vaccine comprises: the aforementioned mRNA, and optionally pharmaceutically acceptable adjuvants or auxiliary components. The mRNA vaccine of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and its mutants (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
根据本发明的实施例，所述mRNA疫苗还可以进一步包含如下技术特征的至少之一：According to an embodiment of the present invention, the mRNA vaccine may further comprise at least one of the following technical features:
根据本发明的实施例，所述辅助性成分为运载所述mRNA的纳米载体；和/或，所述辅料包括选自注射剂缓冲介质、冻干或冷冻保护剂中的至少之一。According to an embodiment of the present invention, the auxiliary component is a nanocarrier carrying the mRNA; and/or, the auxiliary material includes at least one selected from injection buffer medium, lyophilization or cryoprotectant.
根据本发明的实施例，所述纳米载体包括选自脂质体、纳米粒、微球和脂质纳米载体中的至少之一。According to an embodiment of the present invention, the nanocarrier includes at least one selected from liposomes, nanoparticles, microspheres and lipid nanocarriers.
根据本发明的实施例，所述纳米载体是采用以下至少一种脂质材料制备而成：DOTAP、DOTMA、DOTIM、DDA、DC-Chol、CCS、diC14-脒、DOTPA、DOSPA、DTAB、TTAB、CTAB、DORI、DORIE及其衍生物、DPRIE、DSRIE、DMRIE、DOGS、DOSC、LPLL、DODMA、DDAB、Dlin-MC3-DMA、CKK-E12、C12-200、DSPC、DMG-PEG、DOPE、磷脂酰乙醇胺(PE)、磷脂酰胆碱(PC)与胆固醇(Chol)。According to an embodiment of the present invention, the nanocarrier is prepared by using at least one of the following lipid materials: DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and its derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, Phosphatidyl Ethanolamine (PE), Phosphatidylcholine (PC) and Cholesterol (Chol).
根据本发明的实施例，所述脂质材料：mRNA的质量比为(0.5～50):1，优选为(2～10):1。According to an embodiment of the present invention, the lipid material:mRNA mass ratio is (0.5-50):1, preferably (2-10):1.
根据本发明的实施例，所述mRNA疫苗是采用微流控设备将所述mRNA和脂质材料自组装形成；或者，所述mRNA疫苗是通过所述纳米载体与mRNA进行孵育形成的。According to an embodiment of the present invention, the mRNA vaccine is formed by self-assembly of the mRNA and lipid material using a microfluidic device; or, the mRNA vaccine is formed by incubating the nanocarrier with the mRNA.
本领域技术人员能够理解的是，前面针对mRNA所描述的特征和优点，同样适用于该mRNA疫苗，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA are also applicable to the mRNA vaccine, and will not be repeated here.
在本发明的另一方面，本发明提出了一种前述mRNA疫苗的制备方法。根据本发明的实施例，所述包括：将所述纳米载体和mRNA在溶液进行混合处理，以便获得所述mRNA疫苗。本发明的方法制备工艺简单，制得的mRNA疫苗具有较强的免疫原性。In another aspect of the present invention, the present invention proposes a preparation method of the aforementioned mRNA vaccine. According to an embodiment of the present invention, the method includes: mixing the nanocarrier and mRNA in a solution, so as to obtain the mRNA vaccine. The preparation process of the method of the invention is simple, and the prepared mRNA vaccine has strong immunogenicity.
根据本发明的实施例，所述纳米载体的脂质材料为阳离子脂质材料。According to an embodiment of the present invention, the lipid material of the nanocarrier is a cationic lipid material.
本领域技术人员能够理解的是，前面针对mRNA疫苗所描述的特征和优点，同样适用于该方法，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for the mRNA vaccine are also applicable to this method, and will not be repeated here.
在本发明的另一方面，本发明提出了一种蛋白。根据本发明的实施例，所述蛋白由权前述mRNA的模板DNA编码形成的。本发明的蛋白可用于抗原刺激机体产生特异性靶向野生型SARS-CoV-2病毒及其突变株的抗体，可用于制备蛋白疫苗。In another aspect of the invention, the invention provides a protein. According to an embodiment of the present invention, the protein is encoded by the template DNA of the aforementioned mRNA. The protein of the present invention can be used to stimulate the body to produce antibodies that specifically target the wild-type SARS-CoV-2 virus and its mutants, and can be used to prepare protein vaccines.
根据本发明的实施例，所述蛋白还可以进一步包含如下技术特征中的至少之一：According to an embodiment of the present invention, the protein may further include at least one of the following technical features:
在本发明的一个实施例中，所述蛋白包含不含信号肽的SARS-CoV-2病毒印度突变株S蛋白，并进行了K986P或V987P突变中的至少一个突变。In one embodiment of the present invention, the protein comprises the S protein of the Indian mutant strain of SARS-CoV-2 virus without a signal peptide, and at least one of the K986P or V987P mutations is carried out.
其中，上述蛋白中所述的不含信号肽的SARS-CoV-2病毒印度突变株S蛋白的氨基酸序列如SEQ ID NO：7所示。Wherein, the amino acid sequence of the S protein of the Indian mutant strain of SARS-CoV-2 virus without signal peptide described in the above protein is shown in SEQ ID NO:7.
其中，上述蛋白中所述的SARS-CoV-2印度突变株S蛋白还进行了F817P、A892P、A899P或A942P突变四个突变中的至少一个突变。Wherein, the S protein of the Indian mutant strain of SARS-CoV-2 described in the above protein has also undergone at least one mutation among the four mutations of F817P, A892P, A899P or A942P.
其中，上述蛋白中所述的不含信号肽的SARS-CoV-2印度突变株S蛋白的氨基酸序列如SEQ ID NO：8所示。Wherein, the amino acid sequence of the SARS-CoV-2 Indian mutant strain S protein without signal peptide described in the above protein is shown in SEQ ID NO:8.
其中，上述蛋白中所述的还融合有野生型S蛋白的RBD结构域，且该野生型S蛋白的RBD结构域突变了538位点 (C538S)。Wherein, the RBD domain of the wild-type S protein is also fused with the above protein, and the RBD domain of the wild-type S protein is mutated at position 538 (C538S).
其中，上述蛋白中所述的野生型S蛋白的RBD结构域的氨基酸序列如SEQ ID NO 6所示。Wherein, the amino acid sequence of the RBD domain of the wild-type S protein described in the above protein is shown in SEQ ID NO 6.
其中，上述蛋白中所述的突变538位点(C538S)的野生型S蛋白的RBD结构域融合在所述印度突变株S蛋白的氮端。Wherein, the RBD domain of the wild-type S protein at the mutation site 538 (C538S) in the above protein is fused to the nitrogen terminal of the Indian mutant S protein.
其中，上述蛋白中还融合有信号肽。Wherein, the above protein is also fused with a signal peptide.
其中，上述蛋白中所述的信号肽为SARS-CoV-2病毒印度突变株的信号肽。Wherein, the signal peptide described in the above protein is the signal peptide of the Indian mutant strain of SARS-CoV-2 virus.
其中，上述蛋白中所述的SARS-CoV-2病毒印度突变株的信号肽的氨基酸序列如SEQ ID NO：2所示。Wherein, the amino acid sequence of the signal peptide of the Indian mutant strain of SARS-CoV-2 virus described in the above protein is shown in SEQ ID NO:2.
进一步的，上述蛋白含有如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：29或SEQ ID NO：30中的至少一种所示的肽段。Further, the above protein contains such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: At least one of the peptides shown in ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 30.
在本发明的另一个实施例中，所述蛋白包含SARS-CoV-2病毒印度突变株的NTD_RBD结构域，所述印度突变株的NTD_RBD结构域，突变了538位点(C538S)。In another embodiment of the present invention, the protein comprises the NTD_RBD domain of the Indian mutant strain of SARS-CoV-2 virus, and the NTD_RBD domain of the Indian mutant strain has a mutation at position 538 (C538S).
其中，上述蛋白中所述的印度突变株的NTD_RBD结构域的氨基酸序列为SEQ ID NO：9所示。Wherein, the amino acid sequence of the NTD_RBD domain of the Indian mutant described in the above protein is shown in SEQ ID NO: 9.
其中，上述蛋白中还融合了野生型SARS-CoV-2的RBD结构域，该结构域突变了538位点(C538S)。Among them, the RBD domain of the wild-type SARS-CoV-2 is also fused to the above protein, and the 538 site (C538S) of this domain is mutated.
其中，上述蛋白中所述的野生型SARS-CoV-2的RBD结构域的氨基酸序列为SEQ ID NO：6所示。Wherein, the amino acid sequence of the RBD domain of the wild-type SARS-CoV-2 described in the above protein is shown in SEQ ID NO:6.
其中，上述蛋白中还融合有Foldon片段。优选的，所述的Foldon片段的氨基酸序列如SEQ ID NO：11所示。Wherein, the above protein is further fused with a Foldon fragment. Preferably, the amino acid sequence of the Foldon fragment is shown in SEQ ID NO: 11.
其中，上述蛋白中还融合有linker。Wherein, the above protein is also fused with a linker.
其中，上述蛋白中所述的linker的氨基酸序列为GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10或(GSG) 10中的至少一种所示。 Wherein, the amino acid sequence of the linker in the above protein is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 or (GSG) 10 .
其中，上述蛋白中还融合有信号肽。进一步的，其信号肽为SARS-CoV-2病毒印度突变株的信号肽。更进一步的，所述SARS-CoV-2病毒印度突变株的信号肽的氨基酸序列如SEQ ID NO：2所示。Wherein, the above protein is also fused with a signal peptide. Further, its signal peptide is the signal peptide of the Indian mutant strain of SARS-CoV-2 virus. Furthermore, the amino acid sequence of the signal peptide of the Indian mutant strain of SARS-CoV-2 virus is shown in SEQ ID NO:2.
其中，上述蛋白含有氨基酸序列如SEQ ID NO：9、SEQ ID NO：18、或SEQ ID NO：19、SEQ ID NO：27、SEQ ID NO：31或SEQ ID NO：32中的至少一种所示的肽段。Wherein, the above-mentioned protein contains an amino acid sequence such as SEQ ID NO: 9, SEQ ID NO: 18, or at least one of SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 31 or SEQ ID NO: 32. The indicated peptides.
在本发明的另一个实施例中，所述蛋白包含SARS-CoV-2病毒印度突变株的RBD结构域；所述印度突变株的RBD结构域，突变了538位点(C538S)。In another embodiment of the present invention, the protein comprises the RBD domain of the Indian mutant strain of SARS-CoV-2 virus; the RBD domain of the Indian mutant strain has a mutation at position 538 (C538S).
其中，上述蛋白中所述的印度突变株的RBD结构域的氨基酸序列为SEQ ID NO：14所示。Wherein, the amino acid sequence of the RBD domain of the Indian mutant described in the above protein is shown in SEQ ID NO: 14.
其中，上述蛋白中还融合了巴西突变株的NTD_RBD序列；所述巴西突变株的NTD_RBD序列突变了538位点(C538S)，还增加南非突变株上的D80A、Δ242-244和R246I三个突变。Among them, the NTD_RBD sequence of the Brazilian mutant strain is also fused to the above protein; the NTD_RBD sequence of the Brazilian mutant strain is mutated at position 538 (C538S), and three mutations of D80A, Δ242-244 and R246I on the South African mutant strain are also added.
其中，上述蛋白中所述的增加了南非突变株三个突变的巴西突变株的NTD_RBD序列的氨基酸序列为SEQ ID NO：13所示。Wherein, the amino acid sequence of the NTD_RBD sequence of the Brazilian mutant strain with three mutations added to the South African mutant strain described in the above protein is shown in SEQ ID NO: 13.
其中，上述蛋白中还融合了野生型SARS-CoV-2病毒的RBD结构域，该结构域突变了538位点(C538S)。Among them, the RBD domain of the wild-type SARS-CoV-2 virus is also fused to the above protein, and the 538 site (C538S) of this domain is mutated.
其中，上述蛋白中所述的野生型S蛋白的RBD结构域的氨基酸序列如SEQ ID NO 6所示。Wherein, the amino acid sequence of the RBD domain of the wild-type S protein described in the above protein is shown in SEQ ID NO 6.
其中，上述蛋白中还含有Foldon片段。优选的，所述的Foldon的氨基酸序列如SEQ ID NO：11所示。Wherein, the above-mentioned protein also contains a Foldon fragment. Preferably, the amino acid sequence of the Foldon is shown in SEQ ID NO: 11.
其中，上述蛋白中还含有linker。进一步的，所述的linker的氨基酸序列为GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10和(GSG) 10中的至少一种。 Wherein, the above protein also contains a linker. Further, the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
其中，上述蛋白还含有信号肽编码区。Wherein, the above-mentioned protein also contains a signal peptide coding region.
其中，上述蛋白中所述的信号肽为SARS-CoV-2病毒南非突变株的信号肽。Wherein, the signal peptide described in the above protein is the signal peptide of the South African mutant strain of SARS-CoV-2 virus.
其中，上述蛋白中所述SARS-CoV-2病毒南非突变株的信号肽的氨基酸序列如SEQ ID NO：3所示。Wherein, the amino acid sequence of the signal peptide of the SARS-CoV-2 virus South African mutant strain described in the above protein is shown in SEQ ID NO: 3.
进一步的，上述蛋白含有氨基酸序列如SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：6、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：28、SEQ ID NO：34、SEQ ID NO：33的至少一种所示的肽段。Further, the above protein contains amino acid sequences such as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO: 34 , at least one of the peptides shown in SEQ ID NO: 33.
在本发明的另一个实施例中，所述蛋白含有SARS-CoV-2病毒印度突变株的S蛋白的RBD结构域、南非突变株S蛋白的RBD结构域、巴西突变株S蛋白的RBD结构域和野生型S蛋白的RBD结构域。In another embodiment of the present invention, the protein contains the RBD domain of the S protein of the Indian mutant strain of SARS-CoV-2 virus, the RBD domain of the S protein of the South African mutant strain, and the RBD domain of the S protein of the Brazilian mutant strain and the RBD domain of the wild-type S protein.
其中，上述蛋白中，所述印度突变株的S蛋白的RBD(即为Delta突变株B.1.617.2的S蛋白的RBD)氨基酸序列如SEQ ID NO：35所示：Wherein, among the above-mentioned proteins, the amino acid sequence of the RBD of the S protein of the Indian mutant strain (that is, the RBD of the S protein of the Delta mutant strain B.1.617.2) is as shown in SEQ ID NO: 35:
Figure PCTCN2022122626-appb-000036
Figure PCTCN2022122626-appb-000036
其中，上述蛋白中，所述南非突变株S蛋白的RBD(Beta突变株S蛋白的RBD)氨基酸序列如SEQ ID NO：36所示：Wherein, among the above proteins, the amino acid sequence of the RBD of the South African mutant S protein (the RBD of the Beta mutant S protein) is shown in SEQ ID NO: 36:
Figure PCTCN2022122626-appb-000037
Figure PCTCN2022122626-appb-000037
其中，上述蛋白中，所述巴西突变株S蛋白的RBD(Gamma突变株S蛋白的RBD)氨基酸序列如SEQ ID NO：37所示：Wherein, among the above proteins, the amino acid sequence of the RBD of the Brazilian mutant S protein (RBD of the Gamma mutant S protein) is shown in SEQ ID NO: 37:
Figure PCTCN2022122626-appb-000038
Figure PCTCN2022122626-appb-000038
其中，上述蛋白中，所述野生型S蛋白的RBD(野生型S蛋白的RBD)氨基酸序列如SEQ ID NO：38所示：Wherein, among the above proteins, the amino acid sequence of the RBD of the wild-type S protein (RBD of the wild-type S protein) is as shown in SEQ ID NO: 38:
Figure PCTCN2022122626-appb-000039
Figure PCTCN2022122626-appb-000039
进一步的，上述蛋白中还含有Foldon片段。优选的，所述的Foldon的氨基酸序列如SEQ ID NO：11所示。Further, the above protein also contains a Foldon fragment. Preferably, the amino acid sequence of the Foldon is shown in SEQ ID NO: 11.
进一步的，上述蛋白中还含有linker。进一步的，上述的linker的氨基酸序列为GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10和(GSG) 10中的至少一种。 Further, the above protein also contains a linker. Further, the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
其中，上述蛋白中还含有信号肽。Wherein, the above-mentioned protein also contains a signal peptide.
其中，上述蛋白中的其信号肽为SARS-CoV-2病毒南非突变株的信号肽。进一步的，所述SARS-CoV-2病毒南非突变株的信号肽的氨基酸序列如SEQ ID NO：3所示。Wherein, the signal peptide of the above protein is the signal peptide of the South African mutant strain of SARS-CoV-2 virus. Further, the amino acid sequence of the signal peptide of the South African mutant strain of SARS-CoV-2 virus is shown in SEQ ID NO: 3.
进一步的，上述蛋白的氨基酸序列如SEQ ID NO：39、SEQ ID NO：40中的至少一种。Further, the amino acid sequence of the above protein is at least one of SEQ ID NO: 39 and SEQ ID NO: 40.
本领域技术人员能够理解的是，前面针对mRNA所描述的特征和优点，同样适用于该蛋白，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA are also applicable to this protein, and will not be repeated here.
在本发明的另一方面，本发明提出了一种蛋白。根据本发明的实施例，所述蛋白具有如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：9、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：6、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：39和SEQ ID NO：40中的至少一种所示的氨基酸序列；或者，所述蛋白具有如与SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：9、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：6、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：39和SEQ ID NO：40中的至少一种具有至少80％同一性的氨基酸序列；或者，含有上述各肽段的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的肽段的与上述蛋白的功能相同或相似的蛋白。优选的，所述的功能相同或相似，是指能预防和/或治疗SARS-CoV-2感染。In another aspect of the invention, the invention provides a protein. According to an embodiment of the present invention, the protein has such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22. SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 39 and SEQ ID NO: 40 at least one of the amino acid sequences shown; or, the protein has the same as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19. SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID At least one of NO: 34, SEQ ID NO: 39 and SEQ ID NO: 40 has an amino acid sequence with at least 80% identity; or, the amino acid sequence containing each of the above peptides has been substituted and/or deleted and/or A protein having the same or similar function as the above-mentioned protein, which is a peptide segment obtained by adding at least one amino acid. Preferably, the same or similar functions refer to preventing and/or treating SARS-CoV-2 infection.
本领域技术人员能够理解的是，前面针对mRNA所描述的特征和优点，同样适用于该蛋白，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA are also applicable to this protein, and will not be repeated here.
在本发明的另一方面，本发明提出了一种抗体。根据本发明的实施例，所述抗体具有前述的蛋白结合活性。本发明的抗体可有效结合前述的蛋白，尤其是特异性靶向结合前述的蛋白，可用于与体内或体外检测前述蛋白。In another aspect of the invention, the invention provides an antibody. According to an embodiment of the present invention, the antibody has the aforementioned protein binding activity. The antibody of the present invention can effectively bind to the aforementioned protein, especially specifically target the aforementioned protein, and can be used to detect the aforementioned protein in vivo or in vitro.
根据本发明的实施例，所述抗体为多克隆抗体或单克隆抗体。According to an embodiment of the present invention, the antibody is a polyclonal antibody or a monoclonal antibody.
根据本发明的实施例，所述抗体具有特异性结合所述蛋白的活性。According to an embodiment of the present invention, the antibody has the activity of specifically binding to the protein.
本领域技术人员能够理解的是，前面针对蛋白所描述的特征和优点，同样适用于该抗体，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for the protein are also applicable to the antibody and will not be repeated here.
在本发明的另一方面，本发明提出了一种偶联物。根据本发明的实施例，所述偶联物包含：前述的抗体；以及偶联部分，所述偶联部分与所述抗体相连。本发明的偶联物可有效结合前述的蛋白，尤其是特异性靶向结合前述的蛋白，可用于与体内或体外检测前述蛋白。In another aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the aforementioned antibody; and a coupling moiety, the coupling moiety being linked to the antibody. The conjugate of the present invention can effectively bind to the aforementioned proteins, especially specifically target the aforementioned proteins, and can be used to detect the aforementioned proteins in vivo or in vitro.
根据本发明的实施例，所述偶联部分包括但不限于载体、药物、毒素、细胞因子、蛋白标签、修饰物、成像分子和化疗剂中的至少之一。According to an embodiment of the present invention, the coupling moiety includes, but is not limited to, at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modification, an imaging molecule, and a chemotherapeutic agent.
在本文中，所述载体可以为能够在液相中悬浊或分散的物质(例如，粒子、磁珠等固相载体)，或者为能够收容或搭载液相的固相(例如，板、膜、试管等支持体，以及孔板、微流路、玻璃毛细管、纳米柱、整体柱等的容器)；也可以为用于对抗体或抗原结合片段、重组蛋白或多特异性抗体进行标记的标记载体，例如酶(例如，过氧化物酶、碱性磷酸酶、虫萤光素酶(luciferin)、β半乳糖苷酶)、亲和性物质(例如，链霉亲和素和生物素中的一者，相互互补的正义链和反义链的核酸中的一者)、荧光物质(例如，荧光素、异硫氰酸荧光素、罗丹明、绿色荧光蛋白质、红色荧光蛋白质)、发光物质(例如，虫萤光素、水母发光蛋白(Aequorin)、吖啶酯、三(2,2'联吡啶)钌、鲁米诺)、放射性同位素(例如， 3H、 14C、 32P、 35S、 125I)以及金胶体等。 Herein, the carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can accommodate or carry a liquid phase (for example, plates, membranes, etc.) , test tubes and other supports, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, monolithic columns, etc.); it can also be a label for labeling antibodies or antigen-binding fragments, recombinant proteins or multispecific antibodies Carriers, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), affinity substances (e.g., streptavidin and One, one of the nucleic acids of the sense strand and the antisense strand complementary to each other), fluorescent substances (for example, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances ( For example, luciferin, aequorin, acridinium esters, tris(2,2' bipyridine) ruthenium, luminol), radioactive isotopes (for example, 3 H, 14 C, 32 P, 35 S , 125 I) and gold colloid, etc.
根据本发明的实施例，所述药物为可与抗体结合的小分子药物，具体类型不受限制。According to an embodiment of the present invention, the drug is a small molecule drug that can bind to an antibody, and the specific type is not limited.
根据本发明的实施例，所述毒素包括选自相思豆毒蛋白、蓖麻毒蛋白A、假单胞菌外毒素和白喉毒素中的至少之一。According to an embodiment of the present invention, the toxin includes at least one selected from abrin, ricin A, Pseudomonas exotoxin and diphtheria toxin.
根据本发明的实施例，所述细胞因子包括选自IL-10、VEGF、EpCAM、GM2和RANKL中的至少之一。According to an embodiment of the present invention, the cytokines include at least one selected from IL-10, VEGF, EpCAM, GM2 and RANKL.
根据本发明的实施例，所述蛋白标签包括但不限His标签、Flag标签、GST标签、MBP标签、SUMO标签和C-Myc标签。According to an embodiment of the present invention, the protein tags include but are not limited to His tags, Flag tags, GST tags, MBP tags, SUMO tags and C-Myc tags.
根据本发明的实施例，所述修饰物应作广义理解，可以指用于修饰蛋白的物质。示例性地，可以为聚乙二醇或其衍生物。According to the embodiments of the present invention, the modification should be understood in a broad sense, and may refer to substances used to modify proteins. Exemplarily, it may be polyethylene glycol or its derivatives.
在本文中，化疗剂是指白蛋白紫杉醇、环磷酰胺、异环磷酰胺、苯丙氨酸氮芥、甲氨蝶呤、氟尿嘧啶、放线菌素D、长春新碱。Herein, chemotherapeutic agents refer to nab-paclitaxel, cyclophosphamide, ifosfamide, melphalan, methotrexate, fluorouracil, actinomycin D, vincristine.
需要说明的是，偶联部分和抗体的结合方法，可以使用本领域公知的方法。例如，可以举出物理吸附法、共价结合法、利用亲和性物质(例如，生物素、链霉亲和素)的方法及离子结合法。It should be noted that methods known in the art can be used for the binding method of the coupling moiety and the antibody. For example, a physical adsorption method, a covalent binding method, a method using an affinity substance (for example, biotin, streptavidin), and an ion binding method are mentioned.
本领域技术人员能够理解的是，前面针对蛋白和抗体所描述的特征和优点，同样适用于该偶联物，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for proteins and antibodies are also applicable to the conjugate and will not be repeated here.
在本发明的另一方面，本发明提出了一种蛋白或多肽疫苗。根据本发明的实施例，所述蛋白或多肽疫苗包含：前述的蛋白作为抗原成分。本发明的蛋白或多肽疫苗可有效预防和治疗野生型SARS-CoV-2病毒及其突变株(Alpha病毒、Beta病毒、Gamma病毒、Delta病毒和Omicron病毒)的感染。In another aspect of the present invention, the present invention provides a protein or polypeptide vaccine. According to an embodiment of the present invention, the protein or polypeptide vaccine comprises: the aforementioned protein as an antigenic component. The protein or polypeptide vaccine of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and mutants thereof (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
根据本发明的实施例，进一步包括药学上可接受的辅料或者辅助性成分。According to the embodiments of the present invention, pharmaceutically acceptable excipients or auxiliary components are further included.
根据本发明的实施例，进一步包含免疫佐剂。According to an embodiment of the present invention, an immune adjuvant is further included.
根据本发明的实施例，所述免疫佐剂包括选自弗氏不完全佐剂、完全弗氏佐剂、氢氧化铝佐剂、磷酸铝佐剂、乳佐剂、脂质体佐剂和微生物佐剂中的至少之一。According to an embodiment of the present invention, the immune adjuvant includes Freund's incomplete adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant and microorganism at least one of the adjuvants.
本领域技术人员能够理解的是，前面针对蛋白所描述的特征和优点，同样适用于该蛋白或多肽疫苗，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for the protein are also applicable to the protein or polypeptide vaccine, and will not be repeated here.
在本发明的另一方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码前述的蛋白、前述的抗体或前述的偶联物。根据本发明实施例的核酸分子所编码前述的蛋白、抗体或偶联物。In another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned protein, aforementioned antibody or aforementioned conjugate. The nucleic acid molecules according to the embodiments of the present invention encode the aforementioned proteins, antibodies or conjugates.
根据本发明的实施例，所述核酸分子为DNA。According to an embodiment of the present invention, the nucleic acid molecule is DNA.
需要说明的是，对于本文中所提及的核酸分子，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本文中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。It should be noted that, for the nucleic acid molecule mentioned herein, those skilled in the art should understand that it actually includes any one or both of the complementary double strands. For convenience, in this paper, although only one chain is given in most cases, the other chain that is complementary to it is actually also disclosed. In addition, the nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
在本发明的另一方面，本发明提出了一种载体。根据本发明的实施例，所述载体携带前述的核酸分子。In another aspect of the invention, the invention proposes a carrier. According to an embodiment of the present invention, the vector carries the aforementioned nucleic acid molecule.
需要说明的是，只要携带前述的核酸分子的载体均在本申请的保护范围内，具体不受限制。It should be noted that as long as the vectors carrying the aforementioned nucleic acid molecules are within the protection scope of the present application, there is no specific limitation.
示例性地，所示载体为表达载体，用于表达前述的蛋白、抗体或偶联物。Exemplarily, the vector shown is an expression vector for expressing the aforementioned protein, antibody or conjugate.
在将上述核酸分子连接到载体上时，可以将所述核酸分子与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制所述核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。当然，所述核酸分子与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。常用的载体例如可以为质粒、噬菌体等等。根据本发明的一些具体实施例的载体导入合适的受体细胞后，可在调控***的介导下，有效实现前述的蛋白、抗体或偶联物的表达，进而实现蛋白、抗体或偶联物的体外大量获得。When linking the above-mentioned nucleic acid molecule to the carrier, the nucleic acid molecule can be directly or indirectly linked to the control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, it is sufficient that the nucleic acid molecule is operably linked to a control element. "Operably linked" herein refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function. Commonly used vectors can be, for example, plasmids, phages and the like. After the vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, under the mediation of the regulatory system, the expression of the aforementioned proteins, antibodies or conjugates can be effectively realized, and then the expression of the proteins, antibodies or conjugates can be realized. obtained in large quantities in vitro.
根据本发明的实施例，所述载体为真核载体或原核载体。According to an embodiment of the present invention, the vector is a eukaryotic vector or a prokaryotic vector.
根据本发明的实施例，所述载体包括选自质粒载体、腺病毒载体、慢病毒载体和腺相关病毒载体中的至少之一。According to an embodiment of the present invention, the vector includes at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.
在本发明的另一方面，本发明提出了一种载体疫苗。根据本发明的实施例，所述载体疫苗包括活性成分；所述活性成分是通过将前述mRNA的模板DNA的抗原编码区，或者前述的核酸分子装载到前述的载体得到的。本发明的载体疫苗可有效预防和治疗野生型SARS-CoV-2病毒及其突变株(Alpha病毒、Beta病毒、Gamma病毒、Delta病毒和Omicron病毒)的感染。In another aspect of the present invention, the present invention proposes a vector vaccine. According to an embodiment of the present invention, the carrier vaccine includes an active ingredient; the active ingredient is obtained by loading the antigen coding region of the template DNA of the aforementioned mRNA, or the aforementioned nucleic acid molecule, into the aforementioned carrier. The vector vaccine of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and its mutants (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
根据本发明的实施例，所述腺病毒载体为复制缺陷型腺病毒载体。According to an embodiment of the present invention, the adenoviral vector is a replication-defective adenoviral vector.
本领域技术人员能够理解的是，前面针对mRNA所描述的特征和优点，同样适用于该载体疫苗，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA are also applicable to the vector vaccine, and will not be repeated here.
在本发明的另一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前述mRNA、前述mRNA疫苗、依据前述的方法制备的mRNA疫苗、前述的蛋白、前述的偶联物、前述的蛋白或多肽疫苗或前述的载体疫苗。本发明的药物组合物可有效预防和治疗野生型SARS-CoV-2病毒及其突变株(Alpha病毒、Beta病毒、Gamma病毒、Delta病毒和Omicron病毒)的感染。In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the aforementioned mRNA, the aforementioned mRNA vaccine, the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine or the aforementioned carrier vaccine. The pharmaceutical composition of the present invention can effectively prevent and treat the infection of wild-type SARS-CoV-2 virus and its mutants (Alpha virus, Beta virus, Gamma virus, Delta virus and Omicron virus).
根据本发明的实施例，进一步包括药学上可接受的辅料。According to an embodiment of the present invention, pharmaceutically acceptable auxiliary materials are further included.
本领域技术人员能够理解的是，前面针对mRNA、mRNA疫苗、蛋白、蛋白或多肽疫苗、偶联物所描述的特征和优点，同样适用于该药物组合物，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA, mRNA vaccines, proteins, protein or polypeptide vaccines, and conjugates are also applicable to the pharmaceutical composition and will not be repeated here.
在本发明的另一方面，本发明提出了一种前述mRNA、前述mRNA疫苗或依据前述的方法制备的mRNA疫苗、前述的蛋白、前述的偶联物、前述的蛋白或多肽疫苗、前述的载体疫苗或前述的药物组合物在制备预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病的药物中的用途。In another aspect of the present invention, the present invention provides the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine, the aforementioned carrier Use of the vaccine or the aforementioned pharmaceutical composition in the preparation of medicines for preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection.
本领域技术人员能够理解的是，前面针对mRNA、mRNA疫苗、蛋白、蛋白或多肽疫苗、偶联物和药物组合物所描述的特征和优点，同样适用于该用途，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA, mRNA vaccines, proteins, protein or polypeptide vaccines, conjugates and pharmaceutical compositions are also applicable to this application and will not be repeated here.
在本发明的另一方面，本发明提出了一种前述mRNA、前述mRNA疫苗或依据前述的方法制备的mRNA疫苗、前述的蛋白、前述的偶联物、前述的蛋白或多肽疫苗、前述的载体疫苗或前述的药物组合物在预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病中的用途。In another aspect of the present invention, the present invention provides the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine, the aforementioned carrier Use of the vaccine or the aforementioned pharmaceutical composition in preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection.
本领域技术人员能够理解的是，前面针对mRNA、mRNA疫苗、蛋白、蛋白或多肽疫苗、偶联物和药物组合物所描述的特征和优点，同样适用于该用途，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA, mRNA vaccines, proteins, protein or polypeptide vaccines, conjugates and pharmaceutical compositions are also applicable to this application and will not be repeated here.
在本发明的另一方面，本发明提出了一种前述mRNA、前述mRNA疫苗或依据前述的方法制备的mRNA疫苗、前述的蛋白、前述的偶联物、前述的蛋白或多肽疫苗、前述的载体疫苗或前述的药物组合物，用于预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病。In another aspect of the present invention, the present invention provides the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned protein or polypeptide vaccine, the aforementioned carrier The vaccine or the aforementioned pharmaceutical composition is used to prevent SARS-CoV-2 infection and/or prevent and/or treat related diseases caused by SARS-CoV-2 infection.
本领域技术人员能够理解的是，前面针对mRNA、mRNA疫苗、蛋白、蛋白或多肽疫苗、偶联物和药物组合物所描述的特征和优点，同样适用于该mRNA、mRNA疫苗、蛋白、蛋白或多肽疫苗、偶联物和药物组合物，在此不再赘述。Those skilled in the art will understand that the features and advantages described above for mRNA, mRNA vaccines, proteins, protein or polypeptide vaccines, conjugates and pharmaceutical compositions are also applicable to the mRNA, mRNA vaccines, proteins, protein or polypeptide vaccines. Polypeptide vaccines, conjugates and pharmaceutical compositions will not be described in detail here.
在本发明的另一方面，本发明提出了一种预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病的方法。根据本申请的实施例，所述方法包括：向受试者使用药学上可接受量的前述mRNA、前述mRNA疫苗或依据前述的方法制备的mRNA疫苗、前述的蛋白、前述的偶联物、前述的蛋白或多肽疫苗、前述的载体疫苗或前述的药物组合物。本发明的方法可有效预防SARS-CoV-2感染，还可以有效预防和/或治疗SARS-CoV-2感染引起的相关疾病。In another aspect of the present invention, the present invention proposes a method for preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection. According to an embodiment of the present application, the method includes: administering to the subject a pharmaceutically acceptable amount of the aforementioned mRNA, the aforementioned mRNA vaccine or the mRNA vaccine prepared according to the aforementioned method, the aforementioned protein, the aforementioned conjugate, the aforementioned The protein or polypeptide vaccine, the aforementioned carrier vaccine or the aforementioned pharmaceutical composition. The method of the present invention can effectively prevent SARS-CoV-2 infection, and can also effectively prevent and/or treat related diseases caused by SARS-CoV-2 infection.
本发明所述的mRNA、mRNA疫苗、蛋白或多肽疫苗、载体疫苗、偶联物或药物组合物的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the mRNA, mRNA vaccine, protein or polypeptide vaccine, carrier vaccine, conjugate or pharmaceutical composition of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, drug administration way etc. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.
本发明的mRNA、mRNA疫苗、蛋白或多肽疫苗、载体疫苗或药物组合物可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物中。这些药物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)或冻干粉。The mRNA, mRNA vaccine, protein or polypeptide vaccine, vector vaccine or pharmaceutical composition of the present invention may be incorporated into a drug suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semi-solid and solid dosage forms, etc., including but not limited to liquid solutions (eg, injection solutions and infusion solutions) or lyophilized powders.
根据本申请的实施例，所述方法的给药途径包括选自注射、滴鼻或吸入。According to an embodiment of the present application, the administration route of the method includes injection, nasal drop or inhalation.
根据本申请的实施例，所述注射的施用方式包括选自肌肉注射、皮下注射和静脉注射中的至少一种；或者所述吸入的施用方式选自粉末吸入和雾化吸入中的至少一种。According to an embodiment of the present application, the administration method of injection includes at least one selected from intramuscular injection, subcutaneous injection and intravenous injection; or the administration method of inhalation is selected from at least one of powder inhalation and nebulized inhalation .
根据本申请的实施例，所述施用的次数为1～20次，优选为1～10次，更优选为1、2、3、4、5或6次。According to an embodiment of the present application, the number of administrations is 1-20 times, preferably 1-10 times, more preferably 1, 2, 3, 4, 5 or 6 times.
根据本申请的实施例，所述方法进一步包含：SARS-CoV-2感染者或者SARS-CoV-2暴露风险者施用所述mRNA、mRNA疫苗、蛋白或多肽疫苗、载体疫苗或药物组合物后，检测所述SARS-CoV-2感染者或者SARS-CoV-2暴露风险者抗体滴度。According to an embodiment of the present application, the method further includes: after administering the mRNA, mRNA vaccine, protein or polypeptide vaccine, vector vaccine or pharmaceutical composition to a person infected with SARS-CoV-2 or a person at risk of exposure to SARS-CoV-2, Detect the antibody titer of the SARS-CoV-2 infected person or SARS-CoV-2 exposure risk person.
本领域技术人员能够理解的是，前面针对mRNA、mRNA疫苗、蛋白、蛋白或多肽疫苗、偶联物和药物组合物所描述的特征和优点，同样适用于该方法，在此不再赘述。Those skilled in the art can understand that the features and advantages described above for mRNA, mRNA vaccines, proteins, protein or polypeptide vaccines, conjugates and pharmaceutical compositions are also applicable to this method and will not be repeated here.
本发明的有益效果在于从抗原编码序列设计出发，进行了多种编码序列的设计，根据本发明所设计的序列及突变位点，除了可以设计mRNA疫苗，还可以进一步地进行蛋白、多肽、DNA、环状RNA以及病毒载体疫苗设计的应用，实现抗SARS-CoV-2感染的预防作用。The beneficial effect of the present invention is that starting from the design of the antigen coding sequence, a variety of coding sequences have been designed. According to the sequences and mutation sites designed in the present invention, in addition to designing mRNA vaccines, further protein, polypeptide, DNA , Circular RNA and the application of viral vector vaccine design to achieve the preventive effect against SARS-CoV-2 infection.
1)本发明提供的mRNA具有较强的免疫原性，所制备的mRNA疫苗可用于抗新型冠状病毒及其突变株的感染；尤其是Delta突变株B.1.617.2系列及其突变进化上的母本B.1.617.1等。1) The mRNA provided by the present invention has strong immunogenicity, and the prepared mRNA vaccine can be used to resist the infection of new coronavirus and its mutant strains; especially the Delta mutant strain B.1.617.2 series and its mutation evolution Female parent B.1.617.1 etc.
2)本发明提供的mRNA疫苗改进了传统mRNA的结构，使得该方法得到的mRNA不仅增强的疫苗的免疫原性，还使该方法可简便的拓展到其他高风险型mRNA的设计中，具有通用性。2) The mRNA vaccine provided by the present invention improves the structure of traditional mRNA, so that the mRNA obtained by this method not only enhances the immunogenicity of the vaccine, but also allows the method to be easily extended to the design of other high-risk mRNAs, which has universal sex.
3)本发明提供的蛋白疫苗具有安全、高效、可规模化生产的优点。蛋白疫苗路线已经有成功先例，比较成功的基因工程亚单位疫苗是乙型肝炎表面抗原疫苗。3) The protein vaccine provided by the present invention has the advantages of safety, high efficiency and large-scale production. There have been successful precedents for the protein vaccine route, and the more successful genetically engineered subunit vaccine is the hepatitis B surface antigen vaccine.
4)病毒载体疫苗的优点是基因效率高，体外实验通常接近100％的转导效率；可转导不同类型的人组织细胞，不受靶细胞是否为***细胞所限；容易制得高滴度病毒载体，进入细胞内并不整合到宿主细胞基因组，仅瞬间表达，安全性高。在产能上，也可以通过细胞培养易于实现产业化。这种疫苗有成功先例：此前，由陈薇院士团队和天津康希诺生物技术有限公司联合自主研制的“重组埃博拉病毒病疫苗”也是用腺病毒作载体。4) The advantage of viral vector vaccines is that the gene efficiency is high, and in vitro experiments are usually close to 100% transduction efficiency; different types of human tissue cells can be transduced, regardless of whether the target cells are dividing cells; high titers can be easily obtained Viral vectors do not integrate into the host cell genome when they enter cells, and are only expressed transiently, with high safety. In terms of production capacity, it can also be easily industrialized through cell culture. This vaccine has a successful precedent: Previously, the "recombinant Ebola virus disease vaccine" jointly developed by Academician Chen Wei's team and Tianjin Kangxinuo Biotechnology Co., Ltd. also used adenovirus as a carrier.
5)DNA疫苗的核心组分如质粒DNA的结构简单，提纯质粒DNA的工艺简便，因而生产成本较低，且适于大批量生产；DNA分子克隆比较容易，使得DNA疫苗能根据需要随时进行更新；DNA分子很稳定，因而便于运输和保存；DNA疫苗能激活细胞毒性T淋巴细胞而诱导细胞免疫；质粒DNA本身还可作为佐剂，因此使用DNA疫苗不用加佐剂，既降低成本又方便使用。5) The core components of DNA vaccines, such as plasmid DNA, have a simple structure, and the process of purifying plasmid DNA is simple, so the production cost is low, and it is suitable for mass production; DNA molecular cloning is relatively easy, so that DNA vaccines can be updated at any time as needed The DNA molecule is very stable, so it is easy to transport and store; the DNA vaccine can activate cytotoxic T lymphocytes to induce cellular immunity; the plasmid DNA itself can also be used as an adjuvant, so the use of the DNA vaccine does not need to add an adjuvant, which not only reduces the cost but also is convenient to use.
附图说明Description of drawings
图1为本发明实施例2中质粒和线性化DNA模板电泳检测结果，其中，泳道1：Delta S-2P线性化DNA模板；泳道2：Delta S-2P质粒；泳道3：Delta S-6P质粒；泳道4：Delta S-6P线性化DNA模板；泳道5：RBD-Delta S-2P质粒；泳道6：RBD-Delta S-2P线性化DNA模板；泳道7：RBD-Delta S-6P质粒；泳道8：RBD-Delta S-6P线性化DNA模板；泳道9：B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD线性化DNA模板；泳道10：B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD质粒；泳道11：B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon质粒；泳道12：B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon线性化DNA模板；Fig. 1 is the electrophoresis detection result of plasmid and linearized DNA template in Example 2 of the present invention, wherein, swimming lane 1: Delta S-2P linearized DNA template; Swimming lane 2: Delta S-2P plasmid; Swimming lane 3: Delta S-6P plasmid ;lane 4: Delta S-6P linearized DNA template; lane 5: RBD-Delta S-2P plasmid; lane 6: RBD-Delta S-2P linearized DNA template; lane 7: RBD-Delta S-6P plasmid; 8: RBD-Delta S-6P linearized DNA template; lane 9: B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD linearized DNA template; lane 10: B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD plasmid; lane 11 : B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon plasmid; lane 12: B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon linearized DNA template;
图2为本发明实施例4中不同mRNA疫苗免疫BALB/c小鼠Day 14血清结合抗体滴度；Fig. 2 is different mRNA vaccine immunization BALB/c mouse Day 14 serum binding antibody titers in the embodiment of the present invention 4;
图3为本发明实施例4中不同mRNA疫苗免疫BALB/c小鼠Day 14血清结合抗体滴度；Fig. 3 is different mRNA vaccine immunization BALB/c mouse Day 14 serum binding antibody titers in the embodiment of the present invention 4;
图4为本发明实施例4中不同mRNA疫苗免疫BALB/c小鼠Day 28血清结合抗体滴度；Fig. 4 is different mRNA vaccine immunization BALB/c mice Day 28 serum binding antibody titer in the embodiment of the present invention 4;
图5为本发明实施例4中不同mRNA疫苗免疫BALB/c小鼠Day 35血清假病毒中和抗体滴度；Fig. 5 is different mRNA vaccine immunization BALB/c mouse Day 35 serum pseudovirus neutralizing antibody titer in the embodiment of the present invention 4;
图6为本发明实施例5中不同mRNA疫苗免疫BALB/c小鼠Day 42血清结合抗体滴度；Fig. 6 is different mRNA vaccine immunization BALB/c mouse Day 42 serum binding antibody titer in the embodiment of the present invention 5;
图7为本发明实施例5中不同mRNA疫苗免疫BALB/c小鼠Day 14血清假病毒中和抗体滴度；Fig. 7 is different mRNA vaccine immunization BALB/c mice Day 14 serum pseudovirus neutralizing antibody titers in the embodiment of the present invention 5;
图8为本发明实施例5中不同mRNA疫苗免疫BALB/c小鼠Day 14血清假病毒中和抗体滴度；Fig. 8 is different mRNA vaccine immunization BALB/c mice Day 14 serum pseudovirus neutralizing antibody titers in the embodiment of the present invention 5;
图9为本发明实施例5中不同mRNA疫苗免疫BALB/c小鼠Day 14血清假病毒中和抗体滴度；Fig. 9 is different mRNA vaccine immunization BALB/c mouse Day 14 serum pseudovirus neutralizing antibody titer in the embodiment of the present invention 5;
图10为本发明实施例6中不同mRNA疫苗免疫BALB/c小鼠12个月血清结合抗体滴度；Figure 10 is the 12-month serum binding antibody titer of different mRNA vaccines immunized BALB/c mice in Example 6 of the present invention;
图11为本发明实施例7中体外十二聚体SARS-CoV-2RBD mRNA免疫原表达结果，其中，(A)为实验设计流程图，通过密码子优化，在质粒hCD2的DNA中合成了含有N端tPA信号肽(SP)、连接肽(LP)和C端Foldon(FD)三聚体标签的SARS-CoV-2刺突蛋白的各种RBD(R391-D541)。通过体外转录(IVT)合成修饰的mRNA；(B)在还原条件下，通过western blotting和ELISA定量全细胞裂解液(WCL)和上清液(C)中十二聚体SARS-CoV-2RBD蛋白的表达；(D)在非还原条件下，通过western印迹分析十二聚体SARS-CoV-2RBD蛋白的表达，HRBD：异源RBD mRNA 抗原，HRBD-F：异源RBD Foldon mRNA抗原；数据以平均值±标准误差平均值(SEM)的形式呈现，由生物三重样本确定，与对照组相比，通过单因素方差分析进行分析(*p<0.05，**p<0.01，***p<0.001，***p<0.0001)；Fig. 11 is the expression result of in vitro dodecamer SARS-CoV-2 RBD mRNA immunogen in Example 7 of the present invention, wherein, (A) is the flow chart of experimental design, through codon optimization, synthesized in the DNA of plasmid hCD2 containing Various RBDs (R391-D541) of the SARS-CoV-2 Spike protein tagged with N-terminal tPA signal peptide (SP), linker peptide (LP) and C-terminal Foldon (FD) trimer. Synthesis of modified mRNA by in vitro transcription (IVT); (B) Quantification of dodecamer SARS-CoV-2 RBD protein in whole cell lysate (WCL) and supernatant (C) by western blotting and ELISA under reducing conditions (D) Under non-reducing conditions, the expression of the dodecamer SARS-CoV-2 RBD protein was analyzed by western blot, HRBD: heterologous RBD mRNA antigen, HRBD-F: heterologous RBD Foldon mRNA antigen; data in Presented as mean ± standard error of the mean (SEM), determined from biological triplicate samples compared to controls, analyzed by one-way ANOVA (*p<0.05, **p<0.01, ***p< 0.001, ***p<0.0001);
图12为本发明实施例7中4N4T自组装SARS-CoV-2RBD十二聚体免疫BALB/c小鼠Day 14血清假病毒中和抗体滴度；Fig. 12 is the 4N4T self-assembled SARS-CoV-2RBD dodecamer immune BALB/c mouse Day 14 serum pseudovirus neutralizing antibody titer in the embodiment 7 of the present invention;
图13为本发明实施例7中实验设计流程图；Fig. 13 is a flow chart of experimental design in Example 7 of the present invention;
图14为本发明实施例7中自组装SARS-CoV-2RBD十二聚体免疫小鼠的SARS-CoV-2特异性T细胞免疫应答，其中，(A)为流式细胞仪检测覆盖SARS-CoV-2RBD的肽池刺激后脾细胞中SARS-CoV-2RBD特异性CD4+和CD8+T细胞；(B)为ICS检测覆盖SARS-CoV-2RBD的肽池刺激后，CD4+和CD8+T细胞产生的SARS-CoV-2RBD特异性细胞因子IL-4、IL-2和IFN-γ；(C)为ELISpot法检测脾细胞中的IFN-γ，HRBD：10μg异源RBD，HRBD-F：10μg异源RBD Foldon，5HRBD:5μg异源RBD，5HRBD-F:5μg异源RBD Foldon。数据以平均值±标准误差平均值(SEM)的形式呈现，由生物三重样本确定，与对照组相比，通过单因素方差分析进行分析(*p<0.05，**p<0.01，***p<0.001，***p<0.0001)。Figure 14 is the SARS-CoV-2 specific T cell immune response of self-assembled SARS-CoV-2 RBD dodecamer immunized mice in Example 7 of the present invention, wherein (A) is flow cytometry detection covering SARS-CoV-2 SARS-CoV-2 RBD-specific CD4+ and CD8+ T cells in splenocytes after stimulation with a peptide pool of the CoV-2 RBD; (B) CD4+ and CD8+ T cell generation after stimulation with a peptide pool covering the SARS-CoV-2 RBD for ICS SARS-CoV-2 RBD-specific cytokines IL-4, IL-2 and IFN-γ; (C) ELISpot method for the detection of IFN-γ in splenocytes, HRBD: 10 μg heterologous RBD, HRBD-F: 10 μg heterologous RBD Source RBD Foldon, 5HRBD: 5 μg heterologous RBD, 5HRBD-F: 5 μg heterologous RBD Foldon. Data are presented as mean ± standard error of the mean (SEM), determined from biological triplicate samples compared to controls, analyzed by one-way ANOVA (*p<0.05, **p<0.01, ***p<0.01, *** p<0.001, ***p<0.0001).
具体实施方式Detailed ways
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention.
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。In this article, the term "comprising" or "comprising" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally", "optionally" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes the circumstances in which it occurs, and The circumstances in which the event or condition did not occur.
在本文中，本发明表示突变位点的序号是根据SARS-CoV-2病毒原型株(NC_045512.2)进行编号的，氨基酸编号为根据EU编号***编号。例如，第986位是指按EU编号***编号第366位；所述“K986P”是指按EU编号***编号第986位的赖氨酸被脯氨酸替代；“V987P”是指按EU编号***编号第987位的缬氨酸被脯氨酸替代；所述“C538S”是指按EU编号***编号第538位的半胱氨酸被丝氨酸替代。Herein, the present invention indicates that the sequence number of the mutation site is numbered according to the SARS-CoV-2 virus prototype strain (NC_045512.2), and the amino acid number is numbered according to the EU numbering system. For example, the 986th position refers to the 366th position in the EU numbering system; the "K986P" means that the lysine at the 986th position in the EU numbering system is replaced by proline; The 987th valine is replaced by proline; the "C538S" means that the 538th cysteine is replaced by serine according to the EU numbering system.
在本文中，术语“(XXXX)n”表示n个XXXX相连接，X表示氨基酸。例如“(GGGGS) 3”表示GGGGSGGGGSGGGGS。 As used herein, the term "(XXXX)n" means that n XXXX are connected, and X means an amino acid. For example "(GGGGS) 3 " means GGGGSGGGGSGGGGS.
在本文中，术语“片段”是指目标蛋白或多肽，以及具有N-末端(N端)或C-末端(C端)截短、和/或内部删除的目标蛋白或多肽。例如，Foldon片段可为Foldon序列或部分序列。Herein, the term "fragment" refers to a protein or polypeptide of interest, as well as a protein or polypeptide of interest with N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and/or internal deletions. For example, a Foldon fragment can be a Foldon sequence or a partial sequence.
在本文中，术语“同一性”、“同源性”或“相似相”均用于描述相对于参考序列的氨基酸序列或核酸序列时，采用通过常规的方法进行确定两个氨基酸序列或核酸序列之间的相同氨基酸或核苷酸的百分比，例如参见，Ausubel等，编著(1995)，Current Protocols in Molecular Biology，第19章(Greene Publishing and Wiley-Interscience，New York)；和ALIGN程序(Dayhoff(1978)，Atlas of Protein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation，Washington，D.C.)。关于比对序列和测定序列同一性有很多算法，包括，Needleman等(1970)J.Mol.Biol.48：443的同源性比对算法；Smith等(1981)Adv.Appl.Math.2：482的局部同源性算法；Pearson等(1988)Proc.Natl.Acad.Sci.85：2444的相似性搜索方法；Smith-Waterman算法(Meth.Mol.Biol.70：173-187(1997)；和BLASTP，BLASTN，和BLASTX算法(参见Altschul等(1990)J.Mol.Biol.215：403-410)。利用这些算法的计算机程序也是可获得的，并且包括但不限于：ALIGN或Megalign(DNASTAR)软件，或者WU-BLAST-2(Altschul等，Meth.Enzym.，266：460-480(1996))；或者GAP，BESTFIT，BLAST Altschul等，上文，FASTA，和TFASTA，在Genetics Computing Group(GCG)包，8版，Madison，Wisconsin，USA中可获得；和Intelligenetics，Mountain View，California提供的PC/Gene程序中的CLUSTAL。In this document, when the terms "identity", "homology" or "similarity" are used to describe the amino acid sequence or nucleic acid sequence relative to a reference sequence, two amino acid sequences or nucleic acid sequences are determined by conventional methods. See, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff ( 1978), Atlas of Protein Sequence and Structure 5: Suppl.3 (National Biomedical Research Foundation, Washington, D.C.). There are many algorithms for comparing sequences and determining sequence identity, including, Needleman et al. (1970) J.Mol.Biol .48:443 homology comparison algorithm; Smith et al. (1981) Adv.Appl.Math.2:482 local homology algorithm; Pearson et al. (1988) Proc.Natl.Acad.Sci.85:2444 Similarity search methods; Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403- 410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996 )); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and Intelligenetics, Mountain View, California CLUSTAL in the PC/Gene program.
在本文中，术语“至少80％同一性”指与各参考序列至少为80％，可为80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％的同一性。As used herein, the term "at least 80% identity" means at least 80%, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the respective reference sequence , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.
在本文中，术语“抗体”通常是指可识别一种或多种抗原表位的抗体，包括但不限于单克隆抗体、多克隆抗体、多聚体抗体、纳米抗体和CDR移植抗体。Herein, the term "antibody" generally refers to an antibody that can recognize one or more epitopes, including but not limited to monoclonal antibody, polyclonal antibody, multimeric antibody, nanobody and CDR-grafted antibody.
在本文中，术语“载体”通常是指能够***在合适的宿主中自我复制的核酸分子，其将***的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA***细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。Herein, the term "vector" generally refers to a nucleic acid molecule capable of being inserted into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having various functions as described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.
在本文中，术语“药物组合物”通常是指单位剂量形式，并且可以通过制药领域中熟知的方法的任何一种进行制备。所有的方法包括使活性成分与构成一种或多种附属成分的载体相结合的步骤。通常，通过均匀并充分地使活性化合物与液体载体、细碎固体载体或这两者相结合，制备组合物。As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both.
在本文中，术语“药学上可接受的辅料”均可包括任何溶剂、固体赋形剂、稀释剂或其他液体赋形剂等等，适合于特 有的目标剂型。除了任何常规的辅料与本发明的化合物不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所考虑的范围。Herein, the term "pharmaceutically acceptable excipients" may include any solvents, solid excipients, diluents or other liquid excipients, etc., which are suitable for the specific target dosage form. Except to the extent that any conventional excipients are incompatible with the compounds of the present invention, such as any adverse biological effects produced or interacted in a deleterious manner with any other components of the pharmaceutically acceptable composition, their Use is also within the scope of the present invention.
在本文中，术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的抗体或抗原结合片段、重组蛋白、多特异性抗体、偶联物或药物组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜、静脉注射、肌肉注射、皮下注射等等，但是本发明不限于这些已举例的给药方式。优选地，本发明的组合物采用静脉注射或皮下注射方式被给药。As used herein, the term "administering" means introducing a predetermined amount of a substance into a patient by some suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention can be administered through any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered intravenously or subcutaneously.
在本文中，术语“治疗”是指用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或化合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述化合物的药物给予有需要的个体。As used herein, the term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects caused by the disease. "Treatment" as used herein encompasses disease in mammals, especially humans, including: (a) preventing the disease or condition in a predisposed but undiagnosed individual; (b) inhibiting the disease, e.g., arresting its progression; Or (c) ameliorating the disease, eg, alleviating symptoms associated with the disease. "Treatment" as used herein encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit a disease in that individual, including but not limited to administering a drug comprising a compound described herein to an individual in need thereof.
在本文中，“SARS-CoV-2病毒Beta突变株”和“Beta突变株”同义；“SARS-CoV-2病毒Gamma突变株”和“Gamma突变株”同义；“SARS-CoV-2病毒Delta突变株”和“Delta突变株”同义。In this paper, "SARS-CoV-2 virus Beta mutant strain" and "Beta mutant strain" are synonymous; "SARS-CoV-2 virus Gamma mutant strain" and "Gamma mutant strain" are synonymous; "SARS-CoV-2 Virus Delta mutant" and "Delta mutant" are synonymous.
在本文中，“碳端”和“C端”同义；“氮端”和“N端”同义。Herein, "carbon terminal" and "C terminal" are synonymous; "nitrogen terminal" and "N terminal" are synonymous.
蛋白及其新冠疫苗protein and its new crown vaccine
新冠病毒属于RNA病毒，RNA病毒不稳定，病毒复制中会发生自然突变。随着SARS-CoV-2不断变异，近期两个关键的突变株引起了人们的注意，一个是Beta突变株B.1.351(20H/501Y.V2)，一个是Gamma突变株P.1(20J/501Y.V3)，还有最新的Delta突变株B.1.617.2系列及B.1.617.1系列等。突变的出现导致病毒的传染力增强，并且出现了免疫逃逸现象。因此疫苗抗原的序列需要作出新的设计，以针对变异株上的刺突蛋白的突变产生更好的效果。本发明正是针对上述的变异设计的更新一代的新冠疫苗。The new coronavirus is an RNA virus, and the RNA virus is unstable, and natural mutations will occur during virus replication. As SARS-CoV-2 continues to mutate, two key mutant strains have attracted people's attention recently, one is the Beta mutant strain B.1.351 (20H/501Y.V2), and the other is the Gamma mutant strain P.1 (20J/ 501Y.V3), and the latest Delta mutants B.1.617.2 series and B.1.617.1 series. The emergence of mutations leads to increased infectivity of the virus and immune escape. Therefore, a new design is required for the sequence of the vaccine antigen to produce a better effect against the mutation of the spike protein on the mutant strain. The present invention is a newer generation of new crown vaccine designed for the above-mentioned mutations.
本发明新冠疫苗的活性成分设计有四个方向：The active ingredient design of the new crown vaccine of the present invention has four directions:
第一：首先设计了SARS-CoV-2病毒Delta突变株S蛋白并进行K986P或V987P突变中的至少一个突变的结构域。优选为同时进行K986P和V987P突变。First: first design the S protein of the SARS-CoV-2 virus Delta mutant strain and carry out at least one mutated domain in the K986P or V987P mutation. Preferably, the K986P and V987P mutations are performed simultaneously.
而在此基础上的一种优选是进行F817P、A892P、A899P或A942P突变四个突变中的至少一个突变。On this basis, a preferred method is to carry out at least one mutation among the four mutations F817P, A892P, A899P or A942P.
然后，可以在其氮端或碳端连接突变了538位点(C538S)的野生型S蛋白的RBD结构域。Then, the RBD domain of the wild-type S protein with the mutated 538 position (C538S) can be linked at its nitrogen or carbon terminus.
上述融合蛋白的氮端还可以连接信号肽。比如SARS-CoV-2病毒Delta突变株的信号肽。The nitrogen terminal of the above fusion protein can also be connected with a signal peptide. For example, the signal peptide of the Delta mutant strain of SARS-CoV-2 virus.
第二：首先设计了Delta突变株S蛋白的1-19位信号肽+Delta突变株的NTD_RBD结构域(20-545，突变538位点)的结构。Second: first design the structure of the 1-19 signal peptide of the S protein of the Delta mutant + the NTD_RBD domain (20-545, mutation 538) of the Delta mutant.
然后，可以在其氮端或碳端连接突变了538位点(C538S)的野生型S蛋白的RBD结构域。Then, the RBD domain of the wild-type S protein with the mutated 538 position (C538S) can be linked at its nitrogen or carbon terminus.
进一步的，在上述蛋白的碳端连接Foldon片段，以便于抗原翻译后，形成跨膜三聚体，使抗原蛋白能定位到细胞表面，更容易被免疫细胞识别。优选的，所述Foldon片段柔性Linker与主体相连。本发明的Linker优选为(GGGGS) 6。 Furthermore, Foldon fragments are connected to the carbon-terminal of the above proteins to facilitate the formation of transmembrane trimers after antigen translation, so that the antigen proteins can be located on the cell surface and more easily recognized by immune cells. Preferably, the Foldon segment flexible Linker is connected to the main body. The Linker of the present invention is preferably (GGGGS) 6 .
第三：首先确定Delta突变株的RBD结构域(20-545，突变538位点)的结构。Third: first determine the structure of the RBD domain (20-545, mutation 538) of the Delta mutant.
还融合Gamma突变株的NTD_RBD结构域，其突变了538位点(C538S)。Also fused to the NTD_RBD domain of the Gamma mutant, which mutated position 538 (C538S).
进一步的，上述Gamma突变株的NTD_RBD结构域还增加Beta突变株的D80A、Δ242-244和R246I三个突变。Further, the NTD_RBD domain of the above-mentioned Gamma mutant strain also increased three mutations of D80A, Δ242-244 and R246I of the Beta mutant strain.
再进一步的，还可以在上述结构域的氮端或碳端融合野生型SARS-CoV-2病毒的RBD结构域，该结构域突变了538位点(C538S)。Furthermore, the RBD domain of the wild-type SARS-CoV-2 virus can also be fused to the nitrogen or carbon terminal of the above-mentioned domain, and the 538-site (C538S) of this domain is mutated.
比如，本发明进一步提供了Gamma突变株的NTD_RBD结构域+Delta突变株的RBD结构域+野生型RBD结构域的结构。从而能够构建同时针对Beta突变型、Gamma突变型和Delta突变株的SARS-CoV-2的四价疫苗。For example, the present invention further provides the structure of the NTD_RBD domain of the Gamma mutant + the RBD domain of the Delta mutant + the wild-type RBD domain. Thereby can construct the quadrivalent vaccine of the SARS-CoV-2 of Beta mutant type, Gamma mutant type and Delta mutant strain simultaneously.
上述结构域可用Beta突变株S蛋白的信号肽作为信号肽。The signal peptide of the S protein of the Beta mutant strain can be used as the signal peptide for the above structural domain.
进一步的，在上述蛋白的碳端连接Foldon片段，以便于抗原翻译后，形成跨膜三聚体，使抗原蛋白能定位到细胞表面，更容易被免疫细胞识别。优选的，所述Foldon片段柔性Linker与主体相连。本发明的Linker优选为(GGGGS) 6。 Furthermore, Foldon fragments are connected to the carbon-terminal of the above proteins to facilitate the formation of transmembrane trimers after antigen translation, so that the antigen proteins can be located on the cell surface and more easily recognized by immune cells. Preferably, the Foldon segment flexible Linker is connected to the main body. The Linker of the present invention is preferably (GGGGS) 6 .
上述mRNA中所述的Delta突变株为B.1.617.2或B.1.617.1突变株。The Delta mutant described in the above mRNA is the B.1.617.2 or B.1.617.1 mutant.
第四：首先将Delta突变株B.1.617.2或B.1.617.1的S蛋白的RBD结构域、Beta突变株S蛋白的RBD结构域、Gamma突变株S蛋白的RBD结构域、野生型S蛋白的RBD结构域进行融合得到新的蛋白结构。Fourth: First, the RBD domain of the S protein of the Delta mutant strain B.1.617.2 or B.1.617.1, the RBD domain of the S protein of the Beta mutant strain, the RBD domain of the S protein of the Gamma mutant strain, and the wild-type S protein The RBD domain of the protein is fused to obtain a new protein structure.
上述结构域可用Delta突变株B.1.617.2或B.1.617.1的S蛋白的信号肽作为信号肽。The signal peptide of the S protein of the Delta mutant strain B.1.617.2 or B.1.617.1 can be used as the signal peptide for the above domain.
然后，可在上述蛋白的碳端连接Foldon片段，以便于抗原翻译后，形成跨膜三聚体，使抗原蛋白能定位到细胞表面，更容易被免疫细胞识别。优选的，所述Foldon片段柔性Linker与主体相连。本发明的Linker优选为(GGGGS) 6。 Then, the Foldon fragment can be connected to the carbon-terminus of the above-mentioned protein, so that after the antigen is translated, a transmembrane trimer can be formed, so that the antigen protein can be located on the cell surface and more easily recognized by immune cells. Preferably, the Foldon segment flexible Linker is connected to the main body. The Linker of the present invention is preferably (GGGGS) 6 .
上述结构中的Delta突变株B.1.617.2或B.1.617.1的S蛋白的RBD结构域、Beta突变株S蛋白的RBD结构域、Gamma突变株S蛋白的RBD结构域、野生型S蛋白的RBD结构域的前后顺序可以根据需要进行调整。The RBD domain of the S protein of the Delta mutant strain B.1.617.2 or B.1.617.1 in the above structure, the RBD domain of the S protein of the Beta mutant strain, the RBD domain of the S protein of the Gamma mutant strain, and the wild-type S protein The sequence of the RBD domains can be adjusted as needed.
通过上述方案，能得到一系列的针对SARS-Cov-2，尤其是主要针对Delta突变株的抗原。比如，氨基酸序列如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：9、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：6、SEQ ID NO：20或SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：39、SEQ ID NO：40中任一项所示的抗原；以及含有这些氨基酸序列并作为自身的一部分的抗原。Through the above scheme, a series of antigens against SARS-Cov-2, especially mainly against the Delta mutant strain, can be obtained. For example, amino acid sequences such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO : 18, SEQ ID NO: 19, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20 or SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 , SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ The antigen shown in any one of ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 40; and an antigen comprising these amino acid sequences as part of itself.
或者，含有上述各肽段的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的肽段的与上述蛋白的的功能相同或相似的蛋白。进一步的，所述的功能相同或相似，是指能预防和/或治疗SARS-CoV-2感染。更进一步的，所述的SARS-CoV-2为野生型、Beta突变株、Gamma突变株或Delta突变株中的至少一种。Alternatively, a protein having the same or similar function as the above-mentioned protein containing peptides obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence of each of the above peptides. Further, the same or similar functions refer to preventing and/or treating SARS-CoV-2 infection. Furthermore, the SARS-CoV-2 is at least one of wild type, Beta mutant, Gamma mutant or Delta mutant.
在本发明中，“各肽段的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的肽段的与上述蛋白的的功能相同或相似的蛋白”的表述包括但并不限于若干个(通常为1-20个，较佳地1-10个，更佳地1-5个，最佳地1-3个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括所述蛋白的活性片段和活性衍生物。In the present invention, the expression "a protein with the same or similar function as the above-mentioned protein of the peptide obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence of each peptide" includes but is not limited to Several (usually 1-20, preferably 1-10, more preferably 1-5, most preferably 1-3) amino acid deletions, insertions and/or substitutions, and at the C-terminal and/or Or add one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the N-terminus. For example, in the protein, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of said proteins.
“各肽段的氨基酸序列中经过取代和/或缺失和/或添加至少一个氨基酸所得的肽段”的表述还包括但并不限于有至多10个(即一个或几个)，较佳地至多8个，更佳地至多5个(5个、4个、3个、2个或1个)氨基酸被性质相似或相近的氨基酸所替换而形成多肽，即保守性变异多肽。进一步的，这些保守性变异多肽可根据下表进行替换而产生。The expression "peptides obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence of each peptide" also includes, but is not limited to, at most 10 (that is, one or several), preferably at most 8, more preferably at most 5 (5, 4, 3, 2 or 1) amino acids are replaced by amino acids with similar or similar properties to form a polypeptide, that is, a conservative variant polypeptide. Further, these conservative variant polypeptides can be produced by substitution according to the following table.
最初的残基initial residue 代表性的取代representative replacement 优选的取代preferred substitution
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Lys；ArgGln; His; Lys; Arg GlnGln
Asp(D)Asp(D) GluGlu GluGlu
Cys(C)Cys(C) SerSer SerSer
Gln(Q)Gln(Q) AsnAsn AsnAsn
Glu(E)Glu(E) AspAsp AspAsp
Gly(G)Gly(G) Pro；AlaPro; AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile (I) Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe LeuLeu
Leu(L)Leu(L) Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr LeuLeu
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) SerSer SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; LeuLeu
上述的蛋白可以作为活性成分，制备预防和/或治疗SARS-CoV-2感染的药物。一般来说，本领域技术人员可将上述的蛋白可以作为抗原活性成分制备预防和/或治疗SARS-CoV-2感染的蛋白或多肽疫苗。该疫苗以上述的蛋白作为抗原成分，以及药学上可接受的辅料或者辅助性成分。The above-mentioned proteins can be used as active ingredients to prepare drugs for preventing and/or treating SARS-CoV-2 infection. Generally speaking, those skilled in the art can use the above-mentioned proteins as antigenic active ingredients to prepare protein or polypeptide vaccines for preventing and/or treating SARS-CoV-2 infection. The vaccine uses the above-mentioned proteins as antigenic components, and pharmaceutically acceptable adjuvants or auxiliary components.
制备疫苗时，经常会添加免疫佐剂以增强机体对疫苗的免疫响应。其中，所述的免疫佐剂为弗氏不完全佐剂、完全弗氏佐剂、氢氧化铝佐剂、磷酸铝佐剂、乳佐剂、脂质体佐剂、微生物佐剂等。When preparing vaccines, immune adjuvants are often added to enhance the body's immune response to the vaccine. Wherein, the immune adjuvant is incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant, liposome adjuvant, microbial adjuvant and the like.
自然的，本领域在本发明记载的蛋白的基础上，容易得到抗上述蛋白的抗体。上述的抗体为多克隆抗体或单克隆抗体；优选为单克隆抗体。上述抗体还可与偶联部分形成偶联物。进一步的，所述偶联部分为选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白或生物素中的一种或多种。可特异性结合前述的蛋白的抗体一方面可用于制备预防和/或治疗SARS-CoV-2感染的药物以抗SARS-CoV-2病毒的感染，另一方面可用于SARS-CoV-2病毒相关的免疫检测。Naturally, on the basis of the protein described in the present invention, it is easy to obtain antibodies against the above-mentioned protein in the art. The above-mentioned antibodies are polyclonal antibodies or monoclonal antibodies; preferably monoclonal antibodies. The above-mentioned antibodies can also form conjugates with a coupling moiety. Further, the coupling moiety is one or more selected from radionuclides, drugs, toxins, cytokines, enzymes, fluoresceins, carrier proteins or biotin. Antibodies that can specifically bind to the aforementioned proteins can be used to prepare drugs for the prevention and/or treatment of SARS-CoV-2 infection on the one hand to resist SARS-CoV-2 virus infection, and on the other hand can be used for SARS-CoV-2 virus-related immune detection.
此外，本发明也包含了上述蛋白的编码基因。上述蛋白的编码基因一方面可以用于表达制备上述的蛋白或抗体外；另一方面还可以可操作地装载在载体中，进而可制备成载体疫苗或载体药物的。表达可在质粒载体、腺病毒载体、慢病毒载体或腺相关病毒载体等常用载体中选择。当使用腺病毒载体时，一般采用复制缺陷型腺病毒载体。In addition, the present invention also includes the genes encoding the above proteins. On the one hand, the gene encoding the above protein can be used to express and prepare the above protein or antibody; on the other hand, it can also be operably loaded in a vector, and then can be prepared into a vector vaccine or a vector drug. Expression can be selected among commonly used vectors such as plasmid vectors, adenoviral vectors, lentiviral vectors or adeno-associated viral vectors. When an adenoviral vector is used, generally a replication-deficient adenoviral vector is used.
mRNAmRNA
同时，本发明设计一系列的编码上述抗原的mRNA。这些mRNA含有编码上述抗原的编码区，还可以包含或不包含信号肽序列编码区。At the same time, the present invention designs a series of mRNAs encoding the above antigens. These mRNAs contain coding regions encoding the above-mentioned antigens, and may or may not contain signal peptide sequence coding regions.
进一步的，上述mRNA还可以含有5’端非翻译区、信号肽序列编码区、抗原编码区、3’端非翻译区依次连接。通过启动子、5’端非翻译区、信号肽序列、抗原编码区、3’端非翻译区依次连接的DNA模板，可以转录获得上述mRNA。转录过程可以通过现有的体外转录方法和相关试剂盒进行。Further, the above mRNA may also contain a 5' untranslated region, a signal peptide sequence coding region, an antigen coding region, and a 3' untranslated region connected in sequence. The above-mentioned mRNA can be transcribed through a DNA template sequentially connected with a promoter, a 5' untranslated region, a signal peptide sequence, an antigen coding region, and a 3' untranslated region. The transcription process can be performed by existing in vitro transcription methods and related kits.
转录获得的mRNA可以与药学上可接受的辅料或者辅助性成分一起制备成mRNA疫苗。所述的辅助性成分可为运载所述mRNA的纳米载体。所述的纳米载体常用脂质纳米载体。比如采用采用以下至少一种原料制备而成的脂质纳米载体：DOTAP、DOTMA、DOTIM、DDA、DC-Chol、CCS、diC14-脒、DOTPA、DOSPA、DTAB、TTAB、CTAB、DORI、DORIE及其衍生物、DPRIE、DSRIE、DMRIE、DOGS、DOSC、LPLL、DODMA、DDAB、Dlin-MC3-DMA、CKK-E12、C12-200、DSPC、DMG-PEG、DOPE、磷脂酰乙醇胺(PE)、磷脂酰胆碱(PC)、胆固醇(Chol)。The transcribed mRNA can be prepared into an mRNA vaccine together with pharmaceutically acceptable adjuvants or auxiliary components. The auxiliary component can be a nano-carrier carrying the mRNA. The nanocarriers are usually lipid nanocarriers. For example, using lipid nanocarriers prepared from at least one of the following raw materials: DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE, and Derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, Phosphatidylethanolamine (PE), Phosphatidyl Choline (PC), Cholesterol (Chol).
优选的，制备纳米载体所用的脂质材料为两亲性脂质材料或阳离子脂质材料，这是因为这类脂质材料在酸性条件下表面带正电荷，能与核酸的磷酸根通过静电吸附作用将mRNA分子包裹入内，形成mRNA-脂复合体。mRNA-脂复合体能被表面带负电荷的细胞膜吸附，再通过膜的融合或细胞的内吞作用，将mRNA传递进入细胞再进一步表达，发挥疫苗的免疫作用。Preferably, the lipid material used to prepare the nanocarrier is an amphiphilic lipid material or a cationic lipid material, because this type of lipid material has a positive charge on the surface under acidic conditions and can be electrostatically adsorbed with the phosphate radical of the nucleic acid. The role is to wrap mRNA molecules inside to form mRNA-lipid complexes. The mRNA-lipid complex can be adsorbed by the negatively charged cell membrane on the surface, and then through the fusion of the membrane or the endocytosis of the cell, the mRNA is delivered into the cell for further expression, and exerts the immune effect of the vaccine.
在本发明的一个实施例中，基于上述的mRNA，可制备相应的DNA疫苗、蛋白、蛋白或多肽疫苗、偶联物及病毒载体。In one embodiment of the present invention, based on the aforementioned mRNA, corresponding DNA vaccines, proteins, protein or polypeptide vaccines, conjugates and viral vectors can be prepared.
需要说明的是，本发明中的蛋白、病毒载体及DNA疫苗的制备要首先构建质粒DNA来实现在不同层次的抗原蛋白表达。具体地，将本发明中的信号肽序列、抗原编码区、Foldon序列***质粒DNA载体的相应位置，测序验证序列正确后即得构建重组蛋白疫苗、病毒载体疫苗及DNA疫苗的质粒DNA。其中，本发明中的疫苗制备领域常规技术，具体方法不受限制，制药可制备得到相应的蛋白疫苗、病毒载体疫苗及DNA疫苗即可。It should be noted that the preparation of proteins, viral vectors and DNA vaccines in the present invention requires the construction of plasmid DNA first to realize the expression of antigenic proteins at different levels. Specifically, the signal peptide sequence, antigen coding region, and Foldon sequence of the present invention are inserted into the corresponding positions of the plasmid DNA vector, and the plasmid DNA for constructing recombinant protein vaccines, virus vector vaccines, and DNA vaccines can be obtained after sequencing and verifying that the sequences are correct. Among them, the conventional technology in the field of vaccine preparation in the present invention, the specific method is not limited, and the corresponding protein vaccine, virus vector vaccine and DNA vaccine can be prepared by pharmacy.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
在本发明的实施例中，mRNA原液的制备通常包括工程大肠杆菌的培养扩增、质粒DNA的提取与纯化、线性化DNA模板的制备与纯化和mRNA的制备与纯化等过程。本发明技术人员从基因合成得到的原始质粒出发，通过多步工艺过程制备得到质粒DNA和线性化DNA模板等中间体，并以线性化DNA模板为起始物制备本发明的mRNA溶液。进一步地，对质粒DNA、线性化DNA模板等中间体以及mRNA溶液的关键质量指标进行检测，确保其可以用于后续的实验研究。进一步地，利用制备的本发明的mRNA溶液，进一步制备了mRNA-LNP制剂，以进行药效学评价实验。In the embodiment of the present invention, the preparation of the mRNA stock solution usually includes the cultivation and amplification of engineering Escherichia coli, the extraction and purification of plasmid DNA, the preparation and purification of linearized DNA template, and the preparation and purification of mRNA. Starting from the original plasmid obtained by gene synthesis, the technicians of the present invention prepare intermediates such as plasmid DNA and linearized DNA template through a multi-step process, and use the linearized DNA template as the starting material to prepare the mRNA solution of the present invention. Further, the key quality indicators of intermediates such as plasmid DNA and linearized DNA templates and mRNA solutions are tested to ensure that they can be used for subsequent experimental research. Furthermore, using the prepared mRNA solution of the present invention, an mRNA-LNP preparation was further prepared for pharmacodynamic evaluation experiments.
实施例1：mRNA转录DNA模板的构建Embodiment 1: Construction of mRNA transcription DNA template
发明人基于Delta变异株的S蛋白设计了Delta S-2P和Delta S-6P两种mRNA序列。同时，考虑到SARS-CoV-2处于不断的变异中，发明人在Delta变异株的S蛋白的N端添加了野生型毒株的S蛋白的RBD序列，从而构建了二价的新冠mRNA疫苗。The inventors designed two mRNA sequences, Delta S-2P and Delta S-6P, based on the S protein of the Delta variant. At the same time, considering that SARS-CoV-2 is constantly mutating, the inventor added the RBD sequence of the S protein of the wild-type strain to the N-terminus of the S protein of the Delta mutant strain, thereby constructing a bivalent new crown mRNA vaccine.
mRNA转录模板DNA由启动子、5’端非翻译区、信号肽序列、抗原编码区、3’端非翻译区、Poly(A)依次连接，并连入质粒相应位置，测序验证序列正确，得到mRNA转录模板DNA。其中，信号肽序列和抗原编码区可以分别以两条序列提供、也可以位于同一序列提供，具体参见表1中的各mRNA疫苗中抗原编码区对应氨基酸序列，各抗原编码区的核苷酸序列分别如SEQ ID NO：47～57所示。需要说明的是，当表1中抗原编码区的N端不包含信号肽时(如SEQ ID NO：13)，则需要提供信号肽，本发明信号肽的氨基酸序列为SEQ ID NO：24、核苷酸序列为SEQ ID NO：15，是Delta突变株B.1.617.2的S蛋白的1-19位信号肽；当表1中抗原编码区的N端存在信号肽时，则不需要单独提供信号肽。 启动子为T7或SP6启动子。The template DNA for mRNA transcription is sequentially connected by the promoter, 5' untranslated region, signal peptide sequence, antigen coding region, 3' untranslated region, and Poly(A), and connected into the corresponding position of the plasmid. The sequence is verified to be correct by sequencing. mRNA transcription template DNA. Among them, the signal peptide sequence and the antigen coding region can be provided as two sequences respectively, or can be provided in the same sequence. For details, refer to the amino acid sequence corresponding to the antigen coding region in each mRNA vaccine in Table 1, and the nucleotide sequence of each antigen coding region Respectively as shown in SEQ ID NO: 47～57. It should be noted that when the N-terminal of the antigen coding region in Table 1 does not contain a signal peptide (such as SEQ ID NO: 13), a signal peptide needs to be provided. The amino acid sequence of the signal peptide of the present invention is SEQ ID NO: 24, core The nucleotide sequence is SEQ ID NO: 15, which is the 1-19 signal peptide of the S protein of the Delta mutant strain B.1.617.2; when there is a signal peptide at the N-terminal of the antigen coding region in Table 1, it does not need to be provided separately signal peptide. The promoter is T7 or SP6 promoter.
mRNA序列的5'非翻译区的核苷酸序列为SEQ ID NO：1。The nucleotide sequence of the 5' untranslated region of the mRNA sequence is SEQ ID NO: 1.
mRNA序列的3'非翻译区的核苷酸序列为SEQ ID NO：41。The nucleotide sequence of the 3' untranslated region of the mRNA sequence is SEQ ID NO: 41.
mRNA序列的polyA的核苷酸序列为SEQ ID NO：42。The nucleotide sequence of polyA of the mRNA sequence is SEQ ID NO: 42.
表1：mRNA抗原对应氨基酸序列及突变位点Table 1: Amino acid sequence and mutation site corresponding to mRNA antigen
Figure PCTCN2022122626-appb-000040
Figure PCTCN2022122626-appb-000040
表1中各抗原编码区的核苷酸序列如下所示：The nucleotide sequence of each antigen coding region in Table 1 is as follows:
SEQ ID NO：47用于编码SEQ ID NO:22：SEQ ID NO: 47 is used to encode SEQ ID NO: 22:
Figure PCTCN2022122626-appb-000041
Figure PCTCN2022122626-appb-000041
Figure PCTCN2022122626-appb-000042
Figure PCTCN2022122626-appb-000042
SEQ ID NO：48用于编码SEQ ID NO:23：SEQ ID NO: 48 is used to encode SEQ ID NO: 23:
Figure PCTCN2022122626-appb-000043
Figure PCTCN2022122626-appb-000043
Figure PCTCN2022122626-appb-000044
Figure PCTCN2022122626-appb-000044
SEQ ID NO：49用于编码SEQ ID NO:29：SEQ ID NO: 49 is used to encode SEQ ID NO: 29:
Figure PCTCN2022122626-appb-000045
Figure PCTCN2022122626-appb-000045
Figure PCTCN2022122626-appb-000046
Figure PCTCN2022122626-appb-000046
Figure PCTCN2022122626-appb-000047
Figure PCTCN2022122626-appb-000047
SEQ ID NO：50用于编码SEQ ID NO:30：SEQ ID NO:50 is used to encode SEQ ID NO:30:
Figure PCTCN2022122626-appb-000048
Figure PCTCN2022122626-appb-000048
Figure PCTCN2022122626-appb-000049
Figure PCTCN2022122626-appb-000049
SEQ ID NO：51用于编码SEQ ID NO:12：SEQ ID NO:51 is used to encode SEQ ID NO:12:
Figure PCTCN2022122626-appb-000050
Figure PCTCN2022122626-appb-000050
SEQ ID NO：52用于编码SEQ ID NO:13：SEQ ID NO:52 is used to encode SEQ ID NO:13:
Figure PCTCN2022122626-appb-000051
Figure PCTCN2022122626-appb-000051
Figure PCTCN2022122626-appb-000052
Figure PCTCN2022122626-appb-000052
SEQ ID NO：53用于编码SEQ ID NO:31：SEQ ID NO:53 is used to encode SEQ ID NO:31:
Figure PCTCN2022122626-appb-000053
Figure PCTCN2022122626-appb-000053
SEQ ID NO：54用于编码SEQ ID NO:32：SEQ ID NO:54 is used to encode SEQ ID NO:32:
Figure PCTCN2022122626-appb-000054
Figure PCTCN2022122626-appb-000054
Figure PCTCN2022122626-appb-000055
Figure PCTCN2022122626-appb-000055
SEQ ID NO：55用于编码SEQ ID NO:34：SEQ ID NO:55 is used to encode SEQ ID NO:34:
Figure PCTCN2022122626-appb-000056
Figure PCTCN2022122626-appb-000056
Figure PCTCN2022122626-appb-000057
Figure PCTCN2022122626-appb-000057
SEQ ID NO：56用于编码SEQ ID NO:39：SEQ ID NO:56 is used to encode SEQ ID NO:39:
Figure PCTCN2022122626-appb-000058
Figure PCTCN2022122626-appb-000058
Figure PCTCN2022122626-appb-000059
Figure PCTCN2022122626-appb-000059
SEQ ID NO：57用于编码SEQ ID NO:40：SEQ ID NO:57 is used to encode SEQ ID NO:40:
Figure PCTCN2022122626-appb-000060
Figure PCTCN2022122626-appb-000060
Figure PCTCN2022122626-appb-000061
Figure PCTCN2022122626-appb-000061
SEQ ID NO：15用于编码SEQ ID NO:24：SEQ ID NO: 15 is used to encode SEQ ID NO: 24:
Figure PCTCN2022122626-appb-000062
Figure PCTCN2022122626-appb-000062
实施例2：mRNA的体外转录(mRNA原液的制备)Example 2: In vitro transcription of mRNA (preparation of mRNA stock solution)
将实施例1的mRNA转录模板DNA***到pUC57载体中制备质粒DNA，参考《分子克隆实验指南(第四版)》、市售限制性内切酶和DNA纯化试剂盒产品使用说明书，对质粒DNA进行酶切，处理成线性化质粒DNA模板，然后纯化线性化质粒DNA模板。通过分光光度法和凝胶电泳检测质粒DNA及线性化DNA模板的浓度、纯度，通过电泳验证确认是否线性化完全。Insert the mRNA transcription template DNA of Example 1 into the pUC57 vector to prepare plasmid DNA, refer to "Molecular Cloning Experiment Guide (Fourth Edition)", commercially available restriction endonucleases and DNA purification kit product instructions, for plasmid DNA Carry out enzyme digestion, process into a linearized plasmid DNA template, and then purify the linearized plasmid DNA template. The concentration and purity of plasmid DNA and linearized DNA template were detected by spectrophotometry and gel electrophoresis, and the linearization was confirmed by electrophoresis verification.
选用RNA聚合酶、NTP以及帽子类似物等合成制备mRNA。根据市售RNA体外转录试剂盒(诺唯赞TR101-02)操作指南进行前体mRNA的体外转录。具体如下：Use RNA polymerase, NTP and cap analogs to synthesize and prepare mRNA. In vitro transcription of pre-mRNA was performed according to the operating instructions of a commercially available RNA in vitro transcription kit (Novazyme TR101-02). details as follows:
按剂量和顺序分别加入相关反应物(具体见表2)至1.5mL离心管中，移液枪吸放3次或手指轻敲管底使混匀，简单离心使反应液收于管底。然后将反应液置37℃水浴中孵育4h，然后加入1μL DNase，37℃孵化15min；再加入无核酸酶的水179μL，然后加入200μL的苯酚/氯仿/异戊醇(25:24:1)溶液进行mRNA的萃取。混匀之后以15000rpm的转速离心10min，然后取水相。然后加入200μL的5M醋酸铵溶液，4℃放置过夜进行mRNA的沉淀。然后取出在4℃、15000rpm离心10min，小心移去上清液。加入1mL 70％乙醇洗涤沉淀，4℃、15000rpm离心10min，小心移去上清液，风干沉淀。然后加20μL无核酸酶的水复溶即得mRNA溶液。Add the relevant reactants (see Table 2 for details) into 1.5mL centrifuge tubes according to the dose and order, pipette three times or tap the bottom of the tube with your fingers to mix well, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Then incubate the reaction solution in a water bath at 37°C for 4 hours, then add 1 μL of DNase, and incubate at 37°C for 15 minutes; then add 179 μL of nuclease-free water, and then add 200 μL of phenol/chloroform/isoamyl alcohol (25:24:1) solution Carry out mRNA extraction. After mixing, centrifuge at 15,000 rpm for 10 min, and then take the aqueous phase. Then, 200 μL of 5M ammonium acetate solution was added, and left overnight at 4° C. to precipitate mRNA. Then take it out and centrifuge at 15000rpm for 10min at 4°C, and carefully remove the supernatant. Add 1 mL of 70% ethanol to wash the precipitate, centrifuge at 15,000 rpm for 10 min at 4°C, carefully remove the supernatant, and air-dry the precipitate. Then add 20 μL of nuclease-free water to redissolve to obtain the mRNA solution.
采用琼脂糖凝胶电泳或核酸片段分析仪检测mRNA的纯度，分别得到表1中的11种mRNA，即为Delta S-2P、Delta S-6P、RBD-Delta S-2P、RBD-Delta S-6P、Gamma NTD_RBD、BetaGamma NTD_RBD、B.1.617.2_NTD_RBD-Foldon、B.1.617.2_NTD_RBD-RBD-Foldon、BetaGamma NTD_RBD-B.1.617.2_RBD-RBD-Foldon、B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD、B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon。琼脂糖凝胶电泳如图1所示，线性化DNA模板的浓度检测结果如表3所示。The purity of mRNA was detected by agarose gel electrophoresis or nucleic acid fragment analyzer, and the 11 kinds of mRNA in Table 1 were respectively obtained, namely Delta S-2P, Delta S-6P, RBD-Delta S-2P, RBD-Delta S- 6P, Gamma NTD_RBD, BetaGamma NTD_RBD, B.1.617.2_NTD_RBD-Foldon, B.1.617.2_NTD_RBD-RBD-Foldon, BetaGamma NTD_RBD-B.1.617.2_RBD-RBD-Foldon, B.1.617.2_RBD-Beta_RBD-Gamma_RBD , B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon. The agarose gel electrophoresis is shown in Figure 1, and the concentration detection results of the linearized DNA template are shown in Table 3.
表2：mRNA转录反应液处方Table 2: Recipe of mRNA transcription reaction solution
Figure PCTCN2022122626-appb-000063
Figure PCTCN2022122626-appb-000063
Figure PCTCN2022122626-appb-000064
Figure PCTCN2022122626-appb-000064
表3：线性化DNA模板的浓度检测结果Table 3: Concentration detection results of linearized DNA templates
Figure PCTCN2022122626-appb-000065
Figure PCTCN2022122626-appb-000065
实施例3：mRNA脂质纳米粒(LNP)的制备Embodiment 3: the preparation of mRNA lipid nanoparticle (LNP)
如表4所示，mRNA脂质纳米粒可以根据mRNA、靶向器官等选用合适的处方，通过筛选，最终选择处方1制备mRNA脂质纳米粒。As shown in Table 4, the mRNA lipid nanoparticles can be selected according to the mRNA, the target organ, etc., with a suitable prescription, and after screening, recipe 1 is finally selected to prepare the mRNA lipid nanoparticles.
本实施例采用MC3(DLin-MC3-DMA，MW＝642.09)作为可电离脂质，选用处方1制备mRNA脂质纳米粒。将处方中的脂质材料溶于乙醇溶液后再与mRNA溶液混合，使其自组装形成mRNA-脂质纳米粒，然后通过切向流***除去乙醇，即得。具体方法如下：In this example, MC3 (DLin-MC3-DMA, MW=642.09) was used as the ionizable lipid, and recipe 1 was selected to prepare mRNA lipid nanoparticles. The lipid material in the prescription is dissolved in ethanol solution and then mixed with mRNA solution to make it self-assemble to form mRNA-lipid nanoparticles, and then remove ethanol through a tangential flow system. The specific method is as follows:
(1)溶液配制：将MC3、DSPC、胆固醇(Chol)、DMG-PEG2000溶于无水乙醇中，使可电离脂质MC3的浓度为10mg/mL，得到脂质溶液。MC3、DSPC、胆固醇(Chol)、DMG-PEG2000摩尔比为50:10:38.5:1.5。mRNA以PBS缓冲液(RNase-free水配制)稀释至适宜浓度待用。(1) Solution preparation: MC3, DSPC, cholesterol (Chol), and DMG-PEG2000 were dissolved in absolute ethanol so that the concentration of ionizable lipid MC3 was 10 mg/mL to obtain a lipid solution. The molar ratio of MC3, DSPC, cholesterol (Chol), and DMG-PEG2000 is 50:10:38.5:1.5. The mRNA was diluted with PBS buffer (made with RNase-free water) to an appropriate concentration for use.
(2)LNP制备：将步骤(1)获得的可电离脂质溶液与mRNA溶液进行混合，阳离子可电离脂质的质量与mRNA质量比参见表4，以获得LNP初制剂。混合在微流控装置(迈安纳(上海)仪器科技有限公司)中进行，微流控装置的工艺参数：乙醇相和水相的体积比为1:3，流速为9mL/min。(2) LNP preparation: The ionizable lipid solution obtained in step (1) was mixed with the mRNA solution, and the mass ratio of cationic ionizable lipid to mRNA was shown in Table 4 to obtain the initial preparation of LNP. The mixing was carried out in a microfluidic device (Maina (Shanghai) Instrument Technology Co., Ltd.), the process parameters of the microfluidic device: the volume ratio of the ethanol phase to the water phase was 1:3, and the flow rate was 9mL/min.
(3)超滤：以PBS缓冲液将LNP初制剂稀释25倍，经超滤杯超滤至初始体积即为LNP终制剂，超滤过程中初制剂中的乙醇被除去，制得mRNALNP。超滤工艺参数：滤膜100kDa，气压0.2MPa，转速100-200rpm。(3) Ultrafiltration: the initial LNP preparation was diluted 25 times with PBS buffer solution, and the final LNP preparation was obtained by ultrafiltration through an ultrafiltration cup to the initial volume. During the ultrafiltration process, the ethanol in the initial preparation was removed to obtain mRNA LNP. Ultrafiltration process parameters: filter membrane 100kDa, air pressure 0.2MPa, speed 100-200rpm.
表4：mRNA脂质纳米粒的处方Table 4: Recipe for mRNA lipid nanoparticles
处方prescription 可电离或阳离子脂质体载体溶液Ionizable or Cationic Liposome Carrier Solutions 可电离或阳离子脂质材料与mRNA的质量比Mass ratio of ionizable or cationic lipid material to mRNA
11 MC3、DSPC、DMG-PEG、胆固醇MC3, DSPC, DMG-PEG, cholesterol 15:115:1
22 DTAB、DSPC、DMG-PEG、胆固醇DTAB, DSPC, DMG-PEG, cholesterol 10:110:1
33 DC-Chol、DOPE、DMG-PEG、胆固醇DC-Chol, DOPE, DMG-PEG, Cholesterol 25:125:1
44 CTAB、DOPE、DMG-PEG、胆固醇CTAB, DOPE, DMG-PEG, cholesterol 30:130:1
55 DOTMA、DSPC、DMG-PEG、胆固醇DOTMA, DSPC, DMG-PEG, cholesterol 50:150:1
66 DDA、DOPE、DMG-PEG、胆固醇DDA, DOPE, DMG-PEG, cholesterol 5:15:1
77 DOTAP、DOPE、DMG-PEG、胆固醇DOTAP, DOPE, DMG-PEG, cholesterol 15:115:1
然后对制备得到表4中编号1的mRNA脂质纳米粒的粒径和电位以及mRNA包封率进行检测。具体检测方法如下：Then the particle size, potential and mRNA encapsulation efficiency of the prepared mRNA lipid nanoparticles No. 1 in Table 4 were detected. The specific detection method is as follows:
量取一定体积的mRNA疫苗胶体溶液，用纯化水稀释至mRNA浓度为0.01mg/ml，于激光粒度分析仪中测定mRNA疫苗的粒径和电位(n＝3，即每个处方测定3次)。检测结果显示，制备得到的mRNA脂质纳米粒粒径为60-150nm，电位为0±35mV，为脂质纳米粒典型结构。Measure a certain volume of mRNA vaccine colloid solution, dilute it with purified water to an mRNA concentration of 0.01 mg/ml, and measure the particle size and potential of the mRNA vaccine in a laser particle size analyzer (n=3, that is, each prescription is measured 3 times) . The test results show that the prepared mRNA lipid nanoparticles have a particle size of 60-150nm and a potential of 0±35mV, which is a typical structure of lipid nanoparticles.
包封率检测：Quant-iT TM RiboGreen TM试剂盒检测包封率。检测结果表明：制备得到的mRNALNP疫苗具有良好的纳米制剂性质，包封率在80％以上，对mRNA有良好的保护性。 Encapsulation efficiency detection: Quant-iT TM RiboGreen TM kit was used to detect the encapsulation efficiency. The test results show that the prepared mRNALNP vaccine has good nano-preparation properties, the encapsulation rate is above 80%, and has good protection to mRNA.
本发明下述实施例中的野生型、Alpha、Beta、Gamma、B.1.617.1、Delta(B.1.617.2)、Omicron_BA.1和Omicron_BA.2假病毒的来源为吉满生物。其中，无特殊说明，下述实施例中的Delta毒株均是指B.1.617.2毒株。The source of the wild-type, Alpha, Beta, Gamma, B.1.617.1, Delta (B.1.617.2), Omicron_BA.1 and Omicron_BA.2 pseudoviruses in the following examples of the present invention is Jiman Biology. Wherein, unless otherwise specified, the Delta strains in the following examples all refer to the B.1.617.2 strain.
实施例4：mRNA脂质纳米粒的体内免疫活性评价Example 4: In vivo immune activity evaluation of mRNA lipid nanoparticles
本实施例评价了实施例3制备的mRNA脂质纳米粒在小鼠体内的免疫活性。This example evaluates the immune activity of the mRNA lipid nanoparticles prepared in Example 3 in mice.
对编码氨基酸序列如SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：29、SEQ ID NO：30所示的抗原编码区的mRNA脂质纳米粒进行体内免疫活性检测。In vivo immune activity detection was performed on mRNA lipid nanoparticles encoding amino acid sequences such as the antigen coding regions shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 29, and SEQ ID NO: 30.
1、方法：取6-8周Balb/c雄性小鼠，随机分为20组，每组6只，采用5μg的给药剂量，通过肌肉注射的方式对小鼠进行免疫给药，14天后采集小鼠血清，通过ELISA法检测血清中对野生型(记为“WT”)、Beta(记为“ZA”)、Gamma(记为“BR”)、B.1.617.1(记为“IN”)和Delta(B.1.617.2)(记为“IN2”)毒株的S蛋白的RBD的抗体滴度，结果如图2所示。结果表明，本发明的编码氨基酸序列如SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：29、SEQ ID NO：30所示的抗原编码区的mRNA(简称SEQ ID NO：22、23、29或30的mRNA)的结合抗体滴度可达到10 4以上。Ctrl组为不添加任何mRNA脂质纳米粒和药物的溶剂对照组。 1. Method: Take 6-8 week old Balb/c male mice and randomly divide them into 20 groups, 6 mice in each group. The mice are immunized by intramuscular injection at a dosage of 5 μg, and collected after 14 days Mouse serum, detected by ELISA method for wild type (marked as "WT"), Beta (marked as "ZA"), Gamma (marked as "BR"), B.1.617.1 (marked as "IN" ) and Delta (B.1.617.2) (referred to as "IN2") strains of the S protein RBD antibody titers, the results are shown in Figure 2. The results show that the coding amino acid sequence of the present invention is as the mRNA of the antigen coding region shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 30 (abbreviated as SEQ ID NO: 22, 23 , 29 or 30 mRNA) the binding antibody titer can reach more than 10 4 . The Ctrl group is the solvent control group without adding any mRNA lipid nanoparticles and drugs.
2、评价了不同给药剂量对小鼠体内的免疫活性的影响，给药剂量浓度：10μg、30μg、50μg。2. Evaluated the effects of different dosages on the immune activity in mice, the dosage concentration: 10μg, 30μg, 50μg.
方法：取6-8周Balb/c雄性小鼠，随机分为80组，每组6只，采用不同浓度的给药剂量，通过肌肉注射的方式对不同组小鼠进行免疫给药，分别在首次免疫后14天、28天采集小鼠血清，通过ELISA法检测血清中对野生型(记为“WT”)、Beta、Gamma、B.1.617.1、Delta毒株的结合抗体滴度；在首次免疫后35天采集小鼠血清，通过假病毒中和抗体检测法检测血清中对野生型(记为“WT”)、Delta毒株的中和抗体水平，结果如图3、图4、图5所示。Methods: 6-8 weeks old Balb/c male mice were randomly divided into 80 groups, 6 mice in each group, and different concentrations of administration doses were used to immunize mice in different groups by intramuscular injection. The mouse serum was collected 14 days and 28 days after the first immunization, and the binding antibody titer to the wild type (referred to as "WT"), Beta, Gamma, B.1.617.1, and Delta strains in the serum was detected by ELISA method; The mouse serum was collected 35 days after the first immunization, and the level of neutralizing antibodies to wild type (denoted as "WT") and Delta strains in the serum was detected by the pseudovirus neutralizing antibody detection method. The results are shown in Fig. 3, Fig. 4 and Fig. 5.
实验结果表明，在首次免疫后14天(即单次免疫)后，序列SEQ ID NO：22、23、29、30的mRNA的结合抗体滴度可达到10 4～10 5以上(图3)。首次免疫后28天(即第二次免疫后14天)后，四种序列的mRNA疫苗的结合抗体滴度可达到10 5～10 6以上(图4)。首次免疫后35天后，四种序列的mRNA疫苗的假病毒中和抗体滴度为10 2～10 3(图5)。 Experimental results show that, 14 days after the first immunization (ie, single immunization), the titers of binding antibodies to mRNAs with sequences SEQ ID NO: 22, 23, 29, and 30 can reach above 10 4 -10 5 ( FIG. 3 ). Twenty-eight days after the first immunization (that is, 14 days after the second immunization), the binding antibody titers of the mRNA vaccines of the four sequences can reach above 10 5 -10 6 ( FIG. 4 ). Thirty-five days after the first immunization, the pseudovirus neutralizing antibody titers of the four sequences of mRNA vaccines were 10 2 -10 3 (Fig. 5).
以上实验结果显示，序列SEQ ID NO：22、23、29、30的mRNA疫苗在结合抗体滴度和中和抗体滴度方面都没有明显的差异。但是，发明人意外发现，上述四条序列的mRNA疫苗对野生型、Gamma、Beta、Delta、B.1.617.1等不同毒株，均表现了基本相当的血清结合抗体滴度，提示上述四种序列的mRNA疫苗可能具有广谱的抗病毒感染能力。The above experimental results show that the mRNA vaccines with sequences SEQ ID NO: 22, 23, 29, and 30 have no significant difference in binding antibody titers and neutralizing antibody titers. However, the inventor unexpectedly found that the mRNA vaccines of the above four sequences showed substantially equivalent serum-binding antibody titers to different strains such as wild type, Gamma, Beta, Delta, and B.1.617.1, suggesting that the above four sequences mRNA vaccines may have broad-spectrum antiviral infection capabilities.
实施例5：mRNA疫苗广谱的抗病毒感染性能考察Example 5: Investigation of broad-spectrum antiviral infection performance of mRNA vaccine
1、本实施例中选用实施例3制备的含有Delta S-2P(SEQ ID NO：22)抗原的mRNA脂质纳米粒进一步考察本发明mRNA疫苗广谱的抗病毒感染性能。1. In this embodiment, the mRNA lipid nanoparticles containing Delta S-2P (SEQ ID NO: 22) antigen prepared in Example 3 were selected to further investigate the broad-spectrum antiviral infection performance of the mRNA vaccine of the present invention.
方法：取检疫及适应性观察合格的BALB/c小鼠36只，周龄为6-8周，雄性。按随机区组法分为Ctrl组、0.1μg剂量组、1μg剂量组、5μg剂量组、10μg剂量组和30μg剂量组，每组6只。Ctrl组给予肌肉注射0.1mL/只的溶剂(PBS溶液调整制剂mRNA浓度至为0.1mg/mL，同时调整制剂的渗透压至等渗)，间隔3周给药，连续免疫两次，首次给药免疫后的42天采血并分离血清，通过ELISA检测各剂量组42天的血清中分别针对各变异株的S蛋白的结合抗体滴度，结果如图6所示。Methods: 36 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group, 0.1 μg dose group, 1 μg dose group, 5 μg dose group, 10 μg dose group and 30 μg dose group according to random block method, with 6 rats in each group. The Ctrl group was given intramuscular injection of 0.1 mL/mouse of solvent (PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, the first administration 42 days after immunization, blood was collected and serum was separated, and the titers of binding antibodies against the S protein of each mutant strain in the 42-day serum of each dose group were detected by ELISA, and the results are shown in Figure 6.
实验结果表明：本发明的mRNA疫苗连续肌肉注射免疫两次BALB/c小鼠后即可产生针对Alpha、Beta、Gamma、Delta、Omicron_BA.1和Omicron_BA.2毒株的特异性结合的高滴度抗体，在小鼠体内具有很好的免疫原性，进一步提示本发明的mRNA疫苗具有覆盖Omicron的广谱抗病毒感染能力。Experimental results show: the mRNA vaccine of the present invention can produce the high titer of specific binding to Alpha, Beta, Gamma, Delta, Omicron_BA.1 and Omicron_BA.2 strains after the continuous intramuscular injection of BALB/c mouse twice The antibody has good immunogenicity in mice, further suggesting that the mRNA vaccine of the present invention has a broad-spectrum antiviral infection ability covering Omicron.
2、进一步地，发明人对比了实施例4制备的Delta S-2P(SEQ ID NO：22)和RBD-Delta S-2P(SEQ ID NO：29)抗原的mRNA疫苗针对Delta和Omicron毒株的中和抗体水平。2. Further, the inventors compared the mRNA vaccines of the Delta S-2P (SEQ ID NO: 22) prepared in Example 4 and the RBD-Delta S-2P (SEQ ID NO: 29) antigen against Delta and Omicron strains. Neutralizing antibody levels.
方法：取检疫及适应性观察合格的BALB/c小鼠18只，周龄为6-8周，雄性。按随机区组法分为Ctrl组和1μg剂量组，每组6只。Ctrl组给予肌肉注射0.1mL/只的溶剂(PBS溶液调整制剂mRNA浓度至为0.1mg/mL，同时调整制剂的渗透压至等渗)，间隔3周给药，连续免疫两次，末次给药免疫后的14天采血并分离血清，通过假病毒中和试验检测血清中分别针对Delta和Omicron假病毒的中和抗体滴度，结果如图7所示。结果发现，针对Omicron毒株，RBD-Delta S-2P 相较于Delta S-2P具有更高的中和抗体水平。Methods: 18 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group and 1 μg dose group according to random block method, with 6 rats in each group. The Ctrl group was given intramuscular injection of 0.1 mL/rat of solvent (PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration Blood was collected 14 days after immunization and the serum was separated, and the neutralizing antibody titers against the Delta and Omicron pseudoviruses were detected in the serum by the pseudovirus neutralization test, and the results are shown in Figure 7. It was found that, against the Omicron strain, RBD-Delta S-2P had a higher level of neutralizing antibodies than Delta S-2P.
3、发明人将实施例4制备的多价mRNA疫苗(SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：34、SEQ ID NO：39)针对Prototype、Beta、Gamma、Delta和Omicron假病毒的中和抗体滴度。3. The inventors used the multivalent mRNA vaccine (SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 39) prepared in Example 4 against Prototype, Beta, Gamma, Delta and Omicron. Virus neutralizing antibody titers.
方法：取检疫及适应性观察合格的BALB/c小鼠30只，周龄为6-8周，雄性。按随机区组法分为Ctrl组和1μg剂量组，每组6只。Ctrl组给予肌肉注射0.1mL/只的溶剂(PBS溶液调整制剂mRNA浓度至为0.1mg/mL，同时调整制剂的渗透压至等渗)，间隔3周给药，连续免疫两次，末次给药免疫后的14天采血并分离血清，通过假病毒中和试验检测血清中分别针对Prototype、Beta、Gamma、Delta和Omicron假病毒的中和抗体滴度，结果如图8所示。结果发现，相较于SEQ ID NO：31、SEQ ID NO：32的mRNA，SEQ ID NO：34、SEQ ID NO：39的mRNA的假病毒中和抗体滴度更高。Methods: 30 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group and 1 μg dose group according to random block method, with 6 rats in each group. The Ctrl group was given intramuscular injection of 0.1 mL/rat of solvent (PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration Blood was collected 14 days after immunization and serum was separated, and the neutralizing antibody titers against Prototype, Beta, Gamma, Delta and Omicron pseudoviruses were detected in the serum by pseudovirus neutralization test, the results are shown in Figure 8. It was found that compared with the mRNAs of SEQ ID NO: 31 and SEQ ID NO: 32, the mRNAs of SEQ ID NO: 34 and SEQ ID NO: 39 had higher titers of pseudovirus neutralizing antibodies.
4、发明人在Gamma NTD_RBD(SEQ ID NO：12)的基础上加入D80A、Δ242-244和R246I三个Beta突变位点后得到BetaGamma NTD_RBD(SEQ ID NO：13)，并进一步考察了实施例4制备的Gamma NTD_RBD和BetaGamma NTD_RBD抗原的mRNA疫苗针对Gamma和Beta毒株的中和抗体水平。4. The inventor obtained BetaGamma NTD_RBD (SEQ ID NO: 13) after adding D80A, Δ242-244 and R246I three Beta mutation sites on the basis of Gamma NTD_RBD (SEQ ID NO: 12), and further investigated Example 4 The prepared Gamma NTD_RBD and BetaGamma NTD_RBD antigen mRNA vaccines are aimed at the neutralizing antibody levels of Gamma and Beta strains.
方法：取检疫及适应性观察合格的BALB/c小鼠18只，周龄为6-8周，雄性。按随机区组法分为Ctrl组和1μg剂量组，每组6只。Ctrl组给予肌肉注射0.1mL/只的溶剂(PBS溶液调整制剂mRNA浓度至为0.1mg/mL，同时调整制剂的渗透压至等渗)，间隔3周给药，连续免疫两次，末次给药免疫后的14天采血并分离血清，通过假病毒中和试验检测血清中分别针对Gamma、Beta和Delta假病毒的中和抗体滴度，结果如图9所示。结果发现，相较于SEQ ID NO：12的mRNA，SEQ ID NO：13的mRNA的假病毒中和抗体滴度更高，其假病毒中和抗体滴度为10 3左右。 Methods: 18 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group and 1 μg dose group according to random block method, with 6 rats in each group. The Ctrl group was given intramuscular injection of 0.1 mL/mouse of solvent (PBS solution to adjust the preparation mRNA concentration to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration Blood was collected 14 days after immunization and the serum was separated, and the neutralizing antibody titers against Gamma, Beta and Delta pseudoviruses in the serum were detected by the pseudovirus neutralization test, and the results were shown in Figure 9. As a result, it was found that compared with the mRNA of SEQ ID NO: 12, the mRNA of SEQ ID NO: 13 had a higher titer of pseudovirus neutralizing antibodies, and its titer of pseudovirus neutralizing antibodies was about 10 3 .
实施例6：mRNA疫苗抗病毒感染持久性能考察Example 6: Investigation of mRNA Vaccine Antiviral Infection Persistence Performance
发明人进一步考察了实施例3制得的含有Delta S-2P(SEQ ID NO：22)抗原的的mRNA脂质纳米粒抗新冠病毒感染持久性能。The inventors further investigated the persistent performance of the mRNA lipid nanoparticles containing the Delta S-2P (SEQ ID NO: 22) antigen prepared in Example 3 against novel coronavirus infection.
方法：取检疫及适应性观察合格的BALB/c小鼠12只，周龄为6-8周，雄性。按随机区组法分为Ctrl组和5μg剂量组，每组6只。Ctrl组给予肌肉注射0.1mL/只的溶剂(PBS溶液调整制剂mRNA浓度至为0.1mg/mL，同时调整制剂的渗透压至等渗)，间隔3周给药，连续免疫两次，末次给药免疫后的0.5、1、2、4、6、9、12个月采血并分离血清，通过ELISA检测各时间点的血清中分别针对Delta的S蛋白的结合抗体滴度，结果如图10所示。发明人惊喜的发现：本发明的mRNA疫苗连续肌肉注射免疫两次BALB/c小鼠后12个月内，针对Delta毒株的特异性结合抗体滴度可维持在到10 5～10 6之间(图10)，提示本发明的mRNA疫苗具有比较持久的抗病毒感染能力。 Methods: 12 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. They were divided into Ctrl group and 5μg dose group according to random block method, with 6 rats in each group. The Ctrl group was given intramuscular injection of 0.1 mL/mouse of solvent (PBS solution to adjust the preparation mRNA concentration to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration At 0.5, 1, 2, 4, 6, 9, and 12 months after immunization, blood was collected and serum was separated, and the titers of binding antibodies against Delta’s S protein in the serum at each time point were detected by ELISA, and the results are shown in Figure 10 . The inventors were surprised to find that the specific binding antibody titer against the Delta strain can be maintained between 10 5 and 10 6 within 12 months after two consecutive intramuscular injections of the mRNA vaccine of the present invention to immunize BALB/c mice twice ( FIG. 10 ), suggesting that the mRNA vaccine of the present invention has relatively durable antiviral infection ability.
实施例7：异源RBD融合蛋白抗原多价mRNA疫苗抗原表达及免疫性能考察Example 7: Antigen expression and immune performance investigation of heterologous RBD fusion protein antigen multivalent mRNA vaccine
1、选择异源RBD融合蛋白抗原数量最多的B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon(SEQ ID NO：40，具体参见实施例1及其表1)mRNA免疫原，考察其在体外是否形成多聚体结构。1. Select B.1.617.2_RBD-Beta_RBD-Gamma_RBD-RBD-Foldon (SEQ ID NO: 40, see Example 1 and Table 1 for details) mRNA immunogen with the largest number of heterologous RBD fusion protein antigens, and investigate its in vitro Whether to form a multimeric structure.
方法：RBD免疫原整合到质粒(hCD2.4)的开放阅读框中，该质粒含有T7启动子和聚A尾、SP、LP和C末端Foldon(FD)三聚体标签(实验流程如图11A所示)。通过在还原条件下使用免疫印迹试验证实异源融合RBD蛋白在HEK293T细胞中的表达(实验结果图11B所示)。Methods: The RBD immunogen was integrated into the open reading frame of a plasmid (hCD2.4) containing a T7 promoter and poly A tail, SP, LP and C-terminal Foldon (FD) trimer tags (the experimental scheme is shown in Figure 11A shown). The expression of the heterologous fusion RBD protein in HEK293T cells was confirmed by using immunoblotting under reducing conditions (the experimental results are shown in FIG. 11B ).
为了证实SARS-CoV-2RBD十二聚体的形成，通过在非还原条件下使用免疫印迹试验检测其表达(图11C)。这些结果表明，异源RBD蛋白可以在293T细胞和上清液的细胞内表达(图11B)。通过凝胶电泳进一步确认异源RBD蛋白(HRBD)约为120kDa，并且确认含有C末端FD三聚体标签(HRBD-F)的融合RBD蛋白约为300kDa(图11C)。To confirm the formation of the SARS-CoV-2 RBD dodecamer, its expression was detected by immunoblotting under non-reducing conditions (Fig. 11C). These results indicated that heterologous RBD proteins could be expressed intracellularly in 293T cells and supernatants (FIG. 11B). The heterologous RBD protein (HRBD) was further confirmed by gel electrophoresis to be approximately 120 kDa, and the fusion RBD protein containing a C-terminal FD trimer tag (HRBD-F) was confirmed to be approximately 300 kDa (Fig. 11C).
以上结果表明，四种异源RBD融合蛋白在T4三聚作用下形成十二聚体结构。The above results indicated that the four heterologous RBD fusion proteins formed a dodecamer structure under the action of T4 trimerization.
2、根据实施例4的方法，发明人将融合RBD mRNA封装到可电离脂质纳米粒MC3-LNPs中，形成自组装SARS-CoV-2RBD十二聚体通用mRNA疫苗(HRBD十二聚体)，并考察了针对野生型(WT)、Beta、Delta和Omicron假病毒的中和抗体滴度。2. According to the method of Example 4, the inventor encapsulates fusion RBD mRNA into ionizable lipid nanoparticles MC3-LNPs to form a self-assembled SARS-CoV-2 RBD dodecamer universal mRNA vaccine (HRBD dodecamer) , and investigated the neutralizing antibody titers against wild-type (WT), Beta, Delta and Omicron pseudoviruses.
方法：取检疫及适应性观察合格的BALB/c小鼠30只，周龄6-8周，雄性。按随机区组法分为Ctrl组和5μg、10μg剂量组，每组6只。Ctrl组给予肌肉注射0.1mL/只的溶剂(PBS溶液调整制剂mRNA浓度至为0.1mg/mL，同时调整制剂的渗透压至等渗)，间隔3周给药，连续免疫两次，末次给药免疫后的0.5、1、2、4、6、9、12个月采血并分离血清，末次给药免疫后的35天采血并分离血清，通过假病毒中和试验检测血清中分别针对野生型(WT)、Beta、Delta和Omicron假病毒的中和抗体滴度，实验方案参见图13，结果如图12所示。Methods: 30 male BALB/c mice, aged 6-8 weeks, were selected for quarantine and adaptive observation. According to random block method, they were divided into Ctrl group and 5μg and 10μg dose groups, with 6 rats in each group. The Ctrl group was given intramuscular injection of 0.1 mL/rat of solvent (PBS solution to adjust the mRNA concentration of the preparation to 0.1 mg/mL, and at the same time adjust the osmotic pressure of the preparation to isotonicity), administered at intervals of 3 weeks, two consecutive immunizations, and the last administration Blood was collected and serum was collected at 0.5, 1, 2, 4, 6, 9, and 12 months after immunization, blood was collected and serum was separated 35 days after the last dose of immunization, and serum was tested for wild-type ( The neutralizing antibody titers of WT), Beta, Delta and Omicron pseudoviruses, the experimental scheme is shown in Figure 13, and the results are shown in Figure 12.
结果表明：本发明的自组装SARS-CoV-2RBD十二聚体诱导针对野生型(WT)、Beta、Delta和Omicron的中和抗体，可以作为一种通用的mRNA疫苗，够诱导强大的特异性RBD中和抗体，对抗进化突变引起的SARS-CoV-2免疫逃逸。而且，10μg剂量组，C端有Foldon结构的自组装SARS-CoV-2RBD表现出更高的针对Delta和Omicron的中和抗体 水平。The results show that: the self-assembled SARS-CoV-2RBD dodecamer of the present invention induces neutralizing antibodies against wild type (WT), Beta, Delta and Omicron, can be used as a general mRNA vaccine, and can induce strong specificity RBD neutralizing antibody against SARS-CoV-2 immune escape caused by evolutionary mutation. Moreover, in the 10 μg dose group, the self-assembled SARS-CoV-2 RBD with the Foldon structure at the C-terminus showed higher levels of neutralizing antibodies against Delta and Omicron.
3、为了进一步研究两次自组装SARS-CoV-2RBD十二聚体(C端有Foldon)免疫是否引发SARS-CoV-2特异性T细胞免疫反应，发明人通过流式细胞仪检测脾细胞中SARS-CoV-2RBD特异性CD4和CD8T细胞。3. In order to further study whether two self-assembled SARS-CoV-2 RBD dodecamers (with Foldon at the C-terminal) immunization can trigger a SARS-CoV-2 specific T cell immune response, the inventor detected splenocytes by flow cytometry SARS-CoV-2 RBD-specific CD4 and CD8 T cells.
然后通过流式细胞仪检测脾细胞中SARS-CoV-2RBD特异性CD4和CD8T细胞(图14A)。结果表明，用覆盖SARS-CoV-2RBD的肽池刺激后，接种自组装SARS-CoV-2RBD十二聚体的小鼠脾细胞中的特异性CD4和CD8效应T细胞比接种异源RBD的小鼠显著增加。此外，在接种了10μg自组装SARS-CoV-2RBD十二聚体mRNA的小鼠中通过ICS检测了CD4和CD8T细胞产生的SARS-CoV-2RBD特异性细胞因子IL-4、IL-2和IFN-γ(图14B)。并且进行ELISpot分析，以目视分析RBD特异性IFN-γ的表达(图14C)。SARS-CoV-2 RBD-specific CD4 and CD8 T cells in splenocytes were then detected by flow cytometry (Fig. 14A). The results showed that specific CD4 and CD8 effector T cells in splenocytes inoculated with self-assembled SARS-CoV-2 RBD dodecamers were smaller than those inoculated with heterologous RBD after stimulation with a peptide pool covering the SARS-CoV-2 RBD Rats increased significantly. Furthermore, the production of SARS-CoV-2 RBD-specific cytokines IL-4, IL-2, and IFN by CD4 and CD8 T cells was detected by ICS in mice inoculated with 10 μg of self-assembled SARS-CoV-2 RBD dodecamer mRNA -γ (FIG. 14B). And ELISpot analysis was performed to visually analyze the expression of RBD-specific IFN-γ ( FIG. 14C ).
上述实验结果表明，10μg自组装SARS-CoV-2RBD十二聚体mRNA免疫小鼠的脾细胞中分泌的RBD特异性细胞因子IL-2和IFN-γ显著高于4N4T异源RBD免疫小鼠。自组装SARS-CoV-2RBD十二聚体免疫动物和HRBD非十二聚体之间IL-4分泌没有显著差异，表明Th1状态良好，没有潜在的有害Th2免疫反应。结果表明，自组装SARS-CoV-2RBD十二聚体mRNA疫苗可成功诱导Th1偏向的特异性细胞免疫反应。The above experimental results showed that the secretion of RBD-specific cytokines IL-2 and IFN-γ in splenocytes of 10 μg self-assembled SARS-CoV-2 RBD dodecamer mRNA immunized mice was significantly higher than that of 4N4T heterologous RBD immunized mice. There was no significant difference in IL-4 secretion between animals immunized with self-assembled SARS-CoV-2 RBD dodecamers and HRBD non-dodecamers, suggesting a healthy Th1 status without potentially deleterious Th2 immune responses. The results showed that the self-assembled SARS-CoV-2 RBD dodecamer mRNA vaccine could successfully induce a Th1-biased specific cellular immune response.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (89)

  1. 一种mRNA，其特征在于，所述mRNA的模板DNA包含抗原编码区；An mRNA, characterized in that the template DNA of the mRNA comprises an antigen coding region;
    所述抗原编码区编码不含信号肽的SARS-CoV-2病毒Delta突变株S蛋白，所述不含信号肽的SARS-CoV-2病毒Delta突变株S蛋白具有K986P和V987P突变中的至少一个突变。The antigen coding region encodes the SARS-CoV-2 virus Delta mutant strain S protein without signal peptide, and the SARS-CoV-2 virus Delta mutant strain S protein without signal peptide has at least one of K986P and V987P mutations mutation.
  2. 根据权利要求1所述的mRNA，其特征在于，所述抗原编码区编码氨基酸序列如SEQ ID NO：7所示的蛋白。The mRNA according to claim 1, wherein the antigen coding region encodes a protein having an amino acid sequence as shown in SEQ ID NO:7.
  3. 根据权利要求1所述的mRNA，其特征在于，所述SARS-CoV-2病毒Delta突变株S蛋白进一步具有如下突变的至少之一：The mRNA according to claim 1, wherein the SARS-CoV-2 virus Delta mutant strain S protein further has at least one of the following mutations:
    F817P、A892P、A899P和A942P。F817P, A892P, A899P, and A942P.
  4. 根据权利要求3所述的mRNA，其特征在于，所述抗原编码区编码氨基酸序列如SEQ ID NO：8所示的蛋白。The mRNA according to claim 3, wherein the antigen coding region encodes a protein having an amino acid sequence as shown in SEQ ID NO: 8.
  5. 根据权利要求1～4任一项所述的mRNA，其特征在于，所述抗原编码区进一步编码具有突变的野生型SARS-CoV-2病毒的S蛋白的RBD结构域，所述突变为C538S。The mRNA according to any one of claims 1 to 4, wherein the antigen coding region further encodes the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with a mutation, and the mutation is C538S.
  6. 根据权利要求5所述的mRNA，其特征在于，所述具有突变的野生型SARS-CoV-2病毒的S蛋白的RBD结构域的氨基酸序列如SEQ ID NO：6所示。The mRNA according to claim 5, wherein the amino acid sequence of the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with mutation is as shown in SEQ ID NO:6.
  7. 根据权利要求5或6所述的mRNA，其特征在于，所述具有突变的野生型SARS-CoV-2病毒的S蛋白的RBD结构域的C端与所述不含信号肽的SARS-CoV-2病毒Delta突变株S蛋白的N端相连。The mRNA according to claim 5 or 6, characterized in that, the C-terminus of the RBD domain of the S protein of the wild-type SARS-CoV-2 virus with a mutation is related to the SARS-CoV- The N-terminus of the S protein of the 2 viral Delta mutant strains is connected.
  8. 根据权利要求1～7任一项所述的mRNA，其特征在于，所述mRNA的模板DNA进一步包含信号肽编码区。The mRNA according to any one of claims 1-7, wherein the template DNA of the mRNA further comprises a signal peptide coding region.
  9. 根据权利要求8所述的mRNA，其特征在于，所述信号肽编码区编码SARS-CoV-2病毒Delta突变株的信号肽。The mRNA according to claim 8, wherein the signal peptide coding region encodes the signal peptide of the SARS-CoV-2 virus Delta mutant strain.
  10. 根据权利要求9所述的mRNA，其特征在于，所述信号肽的氨基酸序列如SEQ ID NO：2所示。The mRNA according to claim 9, wherein the amino acid sequence of the signal peptide is as shown in SEQ ID NO:2.
  11. 根据权利要求1～9任一项所述的mRNA，其特征在于，所述mRNA的模板DNA编码的氨基酸序列如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：29和SEQ ID NO：30中的至少一种所示。According to the mRNA according to any one of claims 1 to 9, it is characterized in that the amino acid sequence encoded by the template DNA of the mRNA is such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO : 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 30 At least one of the shown.
  12. 一种mRNA，其特征在于，所述mRNA的模板DNA包含抗原编码区；An mRNA, characterized in that the template DNA of the mRNA comprises an antigen coding region;
    所述抗原编码区编码SARS-CoV-2病毒Delta突变株的NTD_RBD结构域，所述SARS-CoV-2病毒Delta突变株的NTD_RBD结构域具有C538S突变。The antigen coding region encodes the NTD_RBD domain of the SARS-CoV-2 virus Delta mutant strain, and the NTD_RBD domain of the SARS-CoV-2 virus Delta mutant strain has a C538S mutation.
  13. 根据权利要求12所述的mRNA，其特征在于，所述SARS-CoV-2病毒Delta突变株的氨基酸序列如SEQ ID NO：9所示。The mRNA according to claim 12, wherein the amino acid sequence of the SARS-CoV-2 virus Delta mutant is as shown in SEQ ID NO: 9.
  14. 根据权利要求12或13所述的mRNA，其特征在于，所述抗原编码区进一步编码具有突变的野生型SARS-CoV-2的RBD结构域，所述突变为C538S。The mRNA according to claim 12 or 13, wherein the antigen coding region further encodes the RBD domain of wild-type SARS-CoV-2 with a mutation, the mutation being C538S.
  15. 根据权利要求14所述的mRNA，其特征在于，所述具有突变的野生型SARS-CoV-2的RBD结构域的氨基酸序列如SEQ ID NO：6所示。The mRNA according to claim 14, wherein the amino acid sequence of the RBD domain of the wild-type SARS-CoV-2 with mutation is as shown in SEQ ID NO:6.
  16. 根据权利要求12～15任一项所述的mRNA，其特征在于，所述抗原编码区进一步编码Foldon片段。The mRNA according to any one of claims 12-15, wherein the antigen coding region further encodes a Foldon fragment.
  17. 根据权利要求16所述的mRNA，其特征在于，所述Foldon片段的氨基酸序列如SEQ ID NO：11所示。The mRNA according to claim 16, wherein the amino acid sequence of the Foldon fragment is as shown in SEQ ID NO: 11.
  18. 根据权利要求12～17任一项所述的mRNA，其特征在于，所述抗原编码区进一步编码linker。The mRNA according to any one of claims 12-17, wherein the antigen coding region further encodes a linker.
  19. 根据权利要求18所述的mRNA，其特征在于，所述linker的氨基酸序列为GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10或(GSG) 10中的至少一种。 The mRNA according to claim 18, wherein the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 or (GSG) 10 .
  20. 根据权利要求12～19任一项所述的mRNA，其特征在于，所述模板DNA进一步包含信号肽编码区。The mRNA according to any one of claims 12-19, wherein the template DNA further comprises a signal peptide coding region.
  21. 根据权利要求20所述的mRNA，其特征在于，所述信号肽编码区编码SARS-CoV-2病毒Delta突变株的信号肽。The mRNA according to claim 20, wherein the signal peptide coding region encodes the signal peptide of the SARS-CoV-2 virus Delta mutant strain.
  22. 根据权利要求21所述的mRNA，其特征在于，所述信号肽的氨基酸序列如SEQ ID NO：2所示。The mRNA according to claim 21, wherein the amino acid sequence of the signal peptide is as shown in SEQ ID NO:2.
  23. 根据权利要求12～22任一项所述的mRNA，其特征在于，所述mRNA的模板DNA编码的氨基酸序列如SEQ ID NO：9、SEQ ID NO：18、或SEQ ID NO：19、SEQ ID NO：27、SEQ ID NO：31或SEQ ID NO：32中的至少一种所示。The mRNA according to any one of claims 12 to 22, wherein the amino acid sequence encoded by the template DNA of the mRNA is such as SEQ ID NO: 9, SEQ ID NO: 18, or SEQ ID NO: 19, SEQ ID At least one of NO: 27, SEQ ID NO: 31 or SEQ ID NO: 32.
  24. 一种mRNA，其特征在于，所述mRNA的模板DNA包含抗原编码区；An mRNA, characterized in that the template DNA of the mRNA comprises an antigen coding region;
    所述抗原编码区编码SARS-CoV-2病毒Delta突变株的RBD结构域和SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域中的至少之一；The antigen coding region encodes at least one of the RBD domain of the SARS-CoV-2 virus Delta mutant and the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant;
    其中，所述SARS-CoV-2病毒Delta突变株的RBD结构域具有C538S突变；Wherein, the RBD domain of the SARS-CoV-2 virus Delta mutant has a C538S mutation;
    所述SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域具有D80A突变、R246I突变和C538S突变，以及增加Beta突变株的Δ242-244。The NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant strain has D80A mutation, R246I mutation and C538S mutation, and increases the Δ242-244 of the Beta mutant strain.
  25. 根据权利要求24所述的mRNA，其特征在于，所述SARS-CoV-2病毒Delta突变株的RBD结构域的氨基酸序 列如SEQ ID NO：14或SEQ ID NO：28所示。The mRNA according to claim 24, wherein the amino acid sequence of the RBD domain of the SARS-CoV-2 virus Delta mutant is as shown in SEQ ID NO: 14 or SEQ ID NO: 28.
  26. 根据权利要求24～25任意一项所述的mRNA，其特征在于，所述SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域的氨基酸序列如SEQ ID NO：13所示。The mRNA according to any one of claims 24 to 25, wherein the amino acid sequence of the NTD_RBD domain of the SARS-CoV-2 virus Gamma mutant is as shown in SEQ ID NO: 13.
  27. 根据权利要求24～26任一项所述的mRNA，其特征在于，所述抗原编码区进一步编码具有突变的野生型SARS-CoV-2病毒的RBD结构域，所述突变为C538S。The mRNA according to any one of claims 24-26, wherein the antigen coding region further encodes the RBD domain of the wild-type SARS-CoV-2 virus with a mutation, the mutation being C538S.
  28. 根据权利要求27所述的mRNA，其特征在于，所述具有突变的野生型SARS-CoV-2病毒的RBD结构域的氨基酸序列如SEQ ID NO：6所示。The mRNA according to claim 27, wherein the amino acid sequence of the RBD domain of the wild-type SARS-CoV-2 virus with mutation is as shown in SEQ ID NO: 6.
  29. 根据权利要求24～28所述的mRNA，其特征在于，所述抗原编码区编码SARS-CoV-2病毒Delta突变株的RBD结构域、SARS-CoV-2病毒Gamma突变株的NTD_RBD结构域和具有突变的野生型SARS-CoV-2病毒的RBD结构域。According to the described mRNA of claim 24～28, it is characterized in that, the RBD domain of described antigen coding region coding SARS-CoV-2 virus Delta mutant strain, the NTD_RBD domain of SARS-CoV-2 virus Gamma mutant strain and have The RBD domain of the mutated wild-type SARS-CoV-2 virus.
  30. 根据权利要求24～29任一项的mRNA，其特征在于，所述抗原编码区进一步编码Foldon片段。The mRNA according to any one of claims 24-29, wherein the antigen coding region further encodes a Foldon fragment.
  31. 根据权利要求30的mRNA，其特征在于，所述Foldon片段的氨基酸序列如SEQ ID NO：11所示。According to the mRNA of claim 30, it is characterized in that, the aminoacid sequence of described Foldon fragment is as shown in SEQ ID NO:11.
  32. 根据权利要求24～31任一项所述的mRNA，其特征在于，所述抗原编码区进一步编码linker。The mRNA according to any one of claims 24-31, wherein the antigen coding region further encodes a linker.
  33. 根据权利要求32所述的mRNA，其特征在于，所述linker的氨基酸序列如GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10和(GSG) 10中的至少一种所示。 The mRNA according to claim 32, wherein the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
  34. 根据权利要求24～33任一项所述的mRNA，其特征在于，所述mRNA的模板DNA进一步含有信号肽编码区。The mRNA according to any one of claims 24-33, wherein the template DNA of the mRNA further contains a signal peptide coding region.
  35. 根据权利要求34所述的mRNA，其特征在于，所述信号肽编码区编码SARS-CoV-2病毒Beta突变株的信号肽。The mRNA according to claim 34, wherein the signal peptide coding region encodes the signal peptide of the SARS-CoV-2 virus Beta mutant strain.
  36. 根据权利要求35所述的mRNA，其特征在于，所述SARS-CoV-2病毒Beta突变株的信号肽的氨基酸序列如SEQ ID NO：3所示。The mRNA according to claim 35, wherein the amino acid sequence of the signal peptide of the SARS-CoV-2 virus Beta mutant strain is as shown in SEQ ID NO:3.
  37. 根据权利要求24～36任一项所述的mRNA，其特征在于，所述抗原编码区编码的氨基酸序列如SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：28、SEQ ID NO：34、SEQ ID NO：33中的至少一种所示。The mRNA according to any one of claims 24 to 36, wherein the amino acid sequence encoded by the antigen coding region is such as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 21. At least one of SEQ ID NO: 28, SEQ ID NO: 34, and SEQ ID NO: 33.
  38. 一种mRNA，其特征在于，所述mRNA的模板DNA包含抗原编码区；An mRNA, characterized in that the template DNA of the mRNA comprises an antigen coding region;
    所述抗原编码区编码SARS-CoV-2病毒Delta突变株的S蛋白的RBD结构域、SARS-CoV-2病毒Beta突变株S蛋白的RBD结构域、SARS-CoV-2病毒Gamma突变株S蛋白的RBD结构域和野生型SARS-CoV-2病毒的S蛋白的RBD结构域。The antigen coding region encodes the RBD domain of the S protein of the SARS-CoV-2 virus Delta mutant strain, the RBD domain of the SARS-CoV-2 virus Beta mutant strain S protein, and the SARS-CoV-2 virus Gamma mutant strain S protein RBD domain and the RBD domain of the S protein of the wild-type SARS-CoV-2 virus.
  39. 根据权利要求38所述的mRNA，其特征在于，所述抗原编码区进一步编码Foldon片段。The mRNA according to claim 38, wherein the antigen coding region further encodes a Foldon fragment.
  40. 根据权利要求39所述的mRNA，其特征在于，所述Foldon片段的氨基酸序列如SEQ ID NO：11所示。The mRNA according to claim 39, wherein the amino acid sequence of the Foldon fragment is as shown in SEQ ID NO: 11.
  41. 根据权利要求38～40任一项所述的mRNA，其特征在于，所述抗原编码区进一步编码linker。The mRNA according to any one of claims 38-40, wherein the antigen coding region further encodes a linker.
  42. 根据权利要求41所述的mRNA，其特征在于，所述linker的氨基酸序列如GGGGS、(GGGGS) 3、(GGGGS) 6、(GGS) 10和(GSG) 10中的至少一种所示。 The mRNA according to claim 41, wherein the amino acid sequence of the linker is at least one of GGGGS, (GGGGS) 3 , (GGGGS) 6 , (GGS) 10 and (GSG) 10 .
  43. 根据权利要求38～42任一项所述的mRNA，其特征在于，所述模板DNA进一步包含信号肽编码区。The mRNA according to any one of claims 38-42, wherein the template DNA further comprises a signal peptide coding region.
  44. 根据权利要求43所述的mRNA，其特征在于，所述信号肽编码区编码SARS-CoV-2病毒Beta突变株的信号肽。The mRNA according to claim 43, wherein the signal peptide coding region encodes the signal peptide of the SARS-CoV-2 virus Beta mutant strain.
  45. 根据权利要求44所述的mRNA，其特征在于，所述SARS-CoV-2病毒Beta突变株的信号肽的氨基酸序列如SEQ ID NO：3所示。The mRNA according to claim 44, wherein the amino acid sequence of the signal peptide of the SARS-CoV-2 virus Beta mutant strain is as shown in SEQ ID NO:3.
  46. 根据权利要求38～45任一项所述的mRNA，其特征在于，所述模板DNA编码的氨基酸序列如SEQ ID NO：39和SEQ ID NO：40中的至少一种所示。The mRNA according to any one of claims 38 to 45, wherein the amino acid sequence encoded by the template DNA is at least one of SEQ ID NO: 39 and SEQ ID NO: 40.
  47. 根据权利要求1～46任一项所述的mRNA，其特征在于，所述模板DNA的启动子为T7或SP6启动子。The mRNA according to any one of claims 1-46, wherein the promoter of the template DNA is a T7 or SP6 promoter.
  48. 根据权利要求1～46任一项所述的mRNA，其特征在于，所述模板DNA进一步包含有5’端非翻译区。The mRNA according to any one of claims 1 to 46, wherein the template DNA further comprises a 5' untranslated region.
  49. 根据权利要求48所述的mRNA，其特征在于，所述5’端非翻译区的核苷酸序列如SEQ ID NO：1所示。The mRNA according to claim 48, wherein the nucleotide sequence of the 5' untranslated region is as shown in SEQ ID NO: 1.
  50. 根据权利要求1～46任一项所述的mRNA，其特征在于，所述模板DNA进一步包含3’端非翻译区。The mRNA according to any one of claims 1-46, wherein the template DNA further comprises a 3' untranslated region.
  51. 根据权利要求50所述的mRNA，其特征在于，所述的3’端非翻译区的核苷酸序列如SEQ ID NO：41所示。The mRNA according to claim 50, wherein the nucleotide sequence of the 3' untranslated region is as shown in SEQ ID NO: 41.
  52. 根据权利要求1～46任一项所述的mRNA，其特征在于，所述模板DNA的3’端进一步连接polyA。The mRNA according to any one of claims 1 to 46, wherein the 3' end of the template DNA is further linked to polyA.
  53. 根据权利要求52所述的mRNA，其特征在于，所述polyA的核苷酸序列如SEQ ID NO：42所示。The mRNA according to claim 52, wherein the nucleotide sequence of said polyA is as shown in SEQ ID NO: 42.
  54. 根据权利要求1～46任一项所述的mRNA，其特征在于，所述模板DNA由所述启动子、5’端非翻译区、信号肽编码区、抗原编码区、3’端非翻译区和polyA连接组成。The mRNA according to any one of claims 1 to 46, wherein the template DNA consists of the promoter, the 5' end untranslated region, the signal peptide coding region, the antigen coding region, and the 3' end untranslated region Concatenated with polyA.
  55. 根据权利要求1～54任一项所述的mRNA，其特征在于，所述SARS-CoV-2病毒Delta突变株为B.1.617.1突变株或B.1.617.2突变株。The mRNA according to any one of claims 1 to 54, wherein the SARS-CoV-2 virus Delta mutant is a B.1.617.1 mutant or a B.1.617.2 mutant.
  56. 一种mRNA疫苗，其特征在于，包含：An mRNA vaccine, characterized in that it comprises:
    权利要求1～55任一项所述的mRNA，以及任选地药学上可接受的辅料或者辅助性成分。The mRNA according to any one of claims 1-55, and optionally pharmaceutically acceptable adjuvants or auxiliary components.
  57. 根据权利要求56所述的mRNA疫苗，其特征在于，所述辅助性成分为运载所述mRNA的纳米载体；和/或所述辅料包括选自注射剂缓冲介质、冻干或冷冻保护剂中的至少之一。The mRNA vaccine according to claim 56, wherein the auxiliary component is a nanocarrier carrying the mRNA; and/or the auxiliary material includes at least one selected from injection buffer media, lyophilization or cryoprotectants one.
  58. 根据权利要求57所述的mRNA疫苗，其特征在于，所述纳米载体包括选自脂质体、纳米粒、微球和脂质纳米载体中的至少之一。The mRNA vaccine according to claim 57, wherein the nanocarrier comprises at least one selected from liposomes, nanoparticles, microspheres and lipid nanocarriers.
  59. 根据权利要求57所述的mRNA疫苗，其特征在于，所述纳米载体是采用以下至少一种脂质材料制备而成：The mRNA vaccine according to claim 57, wherein the nanocarrier is prepared from at least one of the following lipid materials:
    DOTAP、DOTMA、DOTIM、DDA、DC-Chol、CCS、diC14-脒、DOTPA、DOSPA、DTAB、TTAB、CTAB、DORI、DORIE及其衍生物、DPRIE、DSRIE、DMRIE、DOGS、DOSC、LPLL、DODMA、DDAB、Dlin-MC3-DMA、CKK-E12、C12-200、DSPC、DMG-PEG、DOPE、磷脂酰乙醇胺、磷脂酰胆碱与胆固醇。DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and its derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, phosphatidylethanolamine, phosphatidylcholine and cholesterol.
  60. 根据权利要求59所述的mRNA疫苗，其特征在于，所述脂质材料：mRNA的质量比为(0.5～50):1，优选为(2～10):1。The mRNA vaccine according to claim 59, characterized in that the lipid material:mRNA mass ratio is (0.5-50):1, preferably (2-10):1.
  61. 根据权利要求59或60所述的mRNA疫苗，其特征在于，所述mRNA疫苗是采用微流控设备将所述mRNA和脂质材料自组装形成；或者The mRNA vaccine according to claim 59 or 60, wherein the mRNA vaccine is formed by self-assembly of the mRNA and lipid materials using a microfluidic device; or
    所述mRNA疫苗是通过所述纳米载体与mRNA进行孵育形成的。The mRNA vaccine is formed by incubating the nanocarrier with mRNA.
  62. 权利要求56～61任一项所述mRNA疫苗的制备方法，其特征在于，包括：The preparation method of the mRNA vaccine according to any one of claims 56-61, characterized in that it comprises:
    将所述纳米载体和mRNA在溶液进行混合处理，以便获得所述mRNA疫苗。The nanocarrier and mRNA are mixed in a solution so as to obtain the mRNA vaccine.
  63. 根据权利要求62所述的制备方法，其特征在于，所述纳米载体的脂质材料为阳离子脂质材料。The preparation method according to claim 62, characterized in that the lipid material of the nanocarrier is a cationic lipid material.
  64. 一种蛋白，其特征在于，所述蛋白是由权利要求1～55任一项所述mRNA的模板DNA编码形成的。A protein, characterized in that the protein is coded by the template DNA of the mRNA according to any one of claims 1-55.
  65. 一种蛋白，其特征在于，所述蛋白具有如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：9、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：6、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：39和SEQ ID NO：40中的至少一种所示的氨基酸序列；或者，A kind of protein, it is characterized in that, described protein has such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 17, SEQ ID NO: ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO : 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , the amino acid sequence of at least one of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 39, and SEQ ID NO: 40; or,
    所述蛋白具有如与SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：9、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：6、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：39和SEQ ID NO：40中的至少一种具有至少80％同一性的氨基酸序列。Said protein has such properties as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23. SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, Amino acid sequences having at least 80% identity to at least one of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 39 and SEQ ID NO: 40.
  66. 一种抗体，其特征在于，所述抗体具有权利要求64或65所述的蛋白结合活性。An antibody, characterized in that the antibody has the protein-binding activity of claim 64 or 65.
  67. 根据权利要求66所述的抗体，其特征在于，所述抗体为多克隆抗体或单克隆抗体。The antibody according to claim 66, wherein the antibody is a polyclonal antibody or a monoclonal antibody.
  68. 一种偶联物，其特征在于，包含：A conjugate, characterized in that it comprises:
    权利要求66～67任一项所述的抗体；以及the antibody of any one of claims 66-67; and
    偶联部分，所述偶联部分与所述抗体相连。a coupling moiety attached to the antibody.
  69. 根据权利要求68所述的偶联物，其特征在于，所述偶联部分包括选自为载体、药物、毒素、细胞因子、蛋白标签、修饰物、成像分子和化疗剂中的至少之一。The conjugate according to claim 68, wherein the coupling moiety comprises at least one selected from the group consisting of carriers, drugs, toxins, cytokines, protein tags, modifiers, imaging molecules and chemotherapeutic agents.
  70. 一种蛋白或多肽疫苗，其特征在于，包含：A protein or polypeptide vaccine, characterized in that it comprises:
    权利要求64或65所述的蛋白作为抗原成分。The protein according to claim 64 or 65 as an antigenic component.
  71. 根据权利要求70所述的蛋白或多肽疫苗，其特征在于，进一步包括药学上可接受的辅料或者辅助性成分。The protein or polypeptide vaccine according to claim 70, further comprising pharmaceutically acceptable adjuvants or auxiliary components.
  72. 根据权利要求71所述的蛋白或多肽疫苗，其特征在于，进一步包含免疫佐剂。The protein or polypeptide vaccine according to claim 71, further comprising an immune adjuvant.
  73. 根据权利要求72所述的蛋白或多肽疫苗，其特征在于，所述免疫佐剂包括选自弗氏不完全佐剂、完全弗氏佐剂、氢氧化铝佐剂、磷酸铝佐剂、乳佐剂、脂质体佐剂和微生物佐剂中的至少之一。The protein or polypeptide vaccine according to claim 72, wherein the immune adjuvant comprises Freund's incomplete adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, milk adjuvant at least one of liposome adjuvant and microbial adjuvant.
  74. 一种核酸分子，其特征在于，编码权利要求64～65任一项所述的蛋白、权利要求66～67任一项所述的抗体或权利要求68～69任一项所述的偶联物。A nucleic acid molecule, characterized in that it encodes the protein according to any one of claims 64-65, the antibody according to any one of claims 66-67 or the conjugate according to any one of claims 68-69 .
  75. 一种载体，其特征在于，携带权利要求74所述的核酸分子。A carrier, characterized in that it carries the nucleic acid molecule of claim 74.
  76. 根据权利要求75所述的载体，其特征在于，所述载体为真核载体或原核载体。The vector according to claim 75, wherein the vector is a eukaryotic vector or a prokaryotic vector.
  77. 根据权利要求75或76所述的载体，其特征在于，所述载体包括选自质粒载体、腺病毒载体、慢病毒载体和腺相关病毒载体中的至少之一。The vector according to claim 75 or 76, wherein the vector comprises at least one selected from the group consisting of plasmid vectors, adenovirus vectors, lentivirus vectors and adeno-associated virus vectors.
  78. 载体疫苗，其特征在于，包括活性成分；The carrier vaccine is characterized in that it comprises an active ingredient;
    所述活性成分是通过将权利要求1～55任一项所述mRNA的模板DNA的抗原编码区，或者权利要求74所述的核酸 分子装载到权利要求75～77任一项所述的载体得到的。The active ingredient is obtained by loading the antigen coding region of the mRNA template DNA according to any one of claims 1 to 55, or the nucleic acid molecule according to claim 74, onto the carrier according to any one of claims 75 to 77 of.
  79. 根据权利要求78所述的载体疫苗，其特征在于，所述腺病毒载体为复制缺陷型腺病毒载体。The vector vaccine according to claim 78, wherein the adenoviral vector is a replication-defective adenoviral vector.
  80. 一种药物组合物，其特征在于，包括：A pharmaceutical composition, characterized in that, comprising:
    权利要求1～55任一项所述mRNA、权利要求56～61任一项所述mRNA疫苗、依据权利要求62～63任一项所述的方法制备的mRNA疫苗、权利要求64～65任一项所述的蛋白、权利要求68～69任一项所述的偶联物、权利要求70～73任一项所述的蛋白或多肽疫苗或权利要求78～79任一项所述的载体疫苗。The mRNA of any one of claims 1-55, the mRNA vaccine of any one of claims 56-61, the mRNA vaccine prepared according to the method of any one of claims 62-63, any one of claims 64-65 The protein described in item 1, the conjugate described in any one of claims 68-69, the protein or polypeptide vaccine described in any one of claims 70-73, or the vector vaccine described in any one of claims 78-79 .
  81. 根据权利要求80所述的药物组合物，其特征在于，进一步包括药学上可接受的辅料。The pharmaceutical composition according to claim 80, further comprising pharmaceutically acceptable excipients.
  82. 权利要求1～55任一项所述mRNA、权利要求56～61任一项所述mRNA疫苗或依据权利要求62～63任一项所述的方法制备的mRNA疫苗、权利要求64～65任一项所述的蛋白、权利要求68～69任一项所述的偶联物、权利要求70～73任一项所述的蛋白或多肽疫苗、权利要求78～79任一项所述的载体疫苗或权利要求80～81任一项所述的药物组合物在制备预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病的药物中的用途。The mRNA of any one of claims 1-55, the mRNA vaccine of any one of claims 56-61, or the mRNA vaccine prepared according to the method of any one of claims 62-63, any of claims 64-65 The protein described in item 1, the conjugate described in any one of claims 68-69, the protein or polypeptide vaccine described in any one of claims 70-73, the vector vaccine described in any one of claims 78-79 Or the use of the pharmaceutical composition described in any one of claims 80 to 81 in the preparation of medicines for preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection.
  83. 权利要求1～55任一项所述mRNA、权利要求56～61任一项所述mRNA疫苗或依据权利要求62～63任一项所述的方法制备的mRNA疫苗、权利要求64～65任一项所述的蛋白、权利要求68～69任一项所述的偶联物、权利要求70～73任一项所述的蛋白或多肽疫苗、权利要求78～79任一项所述的载体疫苗或权利要求80～81任一项所述的药物组合物在预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病中的用途。The mRNA of any one of claims 1-55, the mRNA vaccine of any one of claims 56-61, or the mRNA vaccine prepared according to the method of any one of claims 62-63, any of claims 64-65 The protein described in item 1, the conjugate described in any one of claims 68-69, the protein or polypeptide vaccine described in any one of claims 70-73, the vector vaccine described in any one of claims 78-79 Or the use of the pharmaceutical composition described in any one of claims 80 to 81 in preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection.
  84. 权利要求1～55任一项所述mRNA、权利要求56～61任一项所述mRNA疫苗或依据权利要求62～63任一项所述的方法制备的mRNA疫苗、权利要求64～65任一项所述的蛋白、权利要求68～69任一项所述的偶联物、权利要求70～73任一项所述的蛋白或多肽疫苗、权利要求78～79任一项所述的载体疫苗或权利要求80～81任一项所述的药物组合物，用于预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病。The mRNA of any one of claims 1-55, the mRNA vaccine of any one of claims 56-61, or the mRNA vaccine prepared according to the method of any one of claims 62-63, any of claims 64-65 The protein described in item 1, the conjugate described in any one of claims 68-69, the protein or polypeptide vaccine described in any one of claims 70-73, the vector vaccine described in any one of claims 78-79 Or the pharmaceutical composition according to any one of claims 80 to 81, which is used to prevent SARS-CoV-2 infection and/or prevent and/or treat related diseases caused by SARS-CoV-2 infection.
  85. 一种预防SARS-CoV-2感染和/或预防和/或治疗SARS-CoV-2感染引起的相关疾病的方法，其特征在于，包括：A method for preventing SARS-CoV-2 infection and/or preventing and/or treating related diseases caused by SARS-CoV-2 infection, characterized in that it comprises:
    向受试者使用药学上可接受量的利要求1～55任一项所述mRNA、权利要求56～61任一项所述mRNA疫苗或依据权利要求62～63任一项所述的方法制备的mRNA疫苗、权利要求64～65任一项所述的蛋白、权利要求68～69任一项所述的偶联物、权利要求70～73任一项所述的蛋白或多肽疫苗、权利要求78～79任一项所述的载体疫苗或权利要求80～81任一项所述的药物组合物。Using a pharmaceutically acceptable amount of the mRNA described in any one of claims 1 to 55, the mRNA vaccine described in any one of claims 56 to 61, or prepared according to the method described in any one of claims 62 to 63 The mRNA vaccine of any one of claims 64-65, the conjugate of any one of claims 68-69, the protein or polypeptide vaccine of any one of claims 70-73, the protein or polypeptide vaccine of any one of claims 64-65, The vector vaccine according to any one of claims 78-79 or the pharmaceutical composition according to any one of claims 80-81.
  86. 根据权利要求85所述的方法，其特征在于，所述方法的给药途径包括选自注射、滴鼻或吸入。The method according to claim 85, characterized in that the administration route of the method is selected from injection, nasal drip or inhalation.
  87. 根据权利要求86所述的方法，其特征在于，所述注射的施用方式包括选自肌肉注射、皮下注射和静脉注射中的至少一种；或者The method according to claim 86, wherein the administration of the injection comprises at least one selected from intramuscular injection, subcutaneous injection and intravenous injection; or
    所述吸入的施用方式选自粉末吸入和雾化吸入中的至少一种。The administration mode of the inhalation is at least one selected from powder inhalation and nebulized inhalation.
  88. 根据权利要求85～87任一项所述的方法，其特征在于，所述施用的次数为1～20次，优选为1～10次，更优选为1、2、3、4、5或6次。The method according to any one of claims 85-87, characterized in that, the number of administrations is 1-20 times, preferably 1-10 times, more preferably 1, 2, 3, 4, 5 or 6 Second-rate.
  89. 根据权利要求85～87任一项所述的方法，其特征在于，进一步包含：The method according to any one of claims 85-87, further comprising:
    SARS-CoV-2感染者或者SARS-CoV-2暴露风险者施用所述mRNA、mRNA疫苗、蛋白或多肽疫苗、载体疫苗或药物组合物后，检测所述SARS-CoV-2感染者或者SARS-CoV-2暴露风险者抗体滴度。After administration of the mRNA, mRNA vaccine, protein or polypeptide vaccine, vector vaccine or pharmaceutical composition to a person infected with SARS-CoV-2 or a person at risk of exposure to SARS-CoV-2, detect the person infected with SARS-CoV-2 or a person at risk of exposure to SARS-CoV-2. Antibody titers in persons at risk of CoV-2 exposure.
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