WO2023070873A1 - Procédé de préparation de particules pseudo-virales de sars-cov-2 et utilisation de particules pseudo-virales de sars-cov-2 - Google Patents

Procédé de préparation de particules pseudo-virales de sars-cov-2 et utilisation de particules pseudo-virales de sars-cov-2 Download PDF

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WO2023070873A1
WO2023070873A1 PCT/CN2021/138037 CN2021138037W WO2023070873A1 WO 2023070873 A1 WO2023070873 A1 WO 2023070873A1 CN 2021138037 W CN2021138037 W CN 2021138037W WO 2023070873 A1 WO2023070873 A1 WO 2023070873A1
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cov
sars
plasmid
virus
protein
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戴俊彪
马英新
毛国斌
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • 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
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the technical field of molecular biology, in particular to a preparation method and application of SARS-CoV-2 virus-like particles.
  • VLP Virus-like particle
  • human VLP vaccines mainly include HBV vaccine, HPV vaccine and HEV vaccine, while veterinary VLP vaccine is used to prevent porcine circovirus type 2.
  • the human VLP vaccines under development also involve a variety of viruses, including influenza virus, HIV, Ebola virus, etc.
  • the veterinary VLP vaccines under development include foot-and-mouth disease virus, porcine parvovirus vaccine, and swine fever virus.
  • most of the current VLPs only contain part or all of the structural proteins, and the research on non-structural proteins and auxiliary proteins is limited, and the immune response induced by vaccines is limited.
  • SARS-CoV-2 The new coronavirus (SARS-CoV-2) is a single-stranded RNA virus with high virus mutation ability, and its mutant strains have higher virus infectivity. Therefore, the development of vaccines is particularly important.
  • SARS-CoV-2 vaccines based on a variety of technologies have entered the clinical stage, less research has been conducted on SARS-CoV-2 VLP-based vaccines.
  • One of the key reasons is the lack of a simple and efficient SARS-CoV-2 VLP synthesis and preparation platform.
  • the present invention provides a de novo artificial synthesis and preparation method of SARS-CoV-2 VLP, including splitting the SARS-CoV-2 genome into multiple fragments, synthesizing these fragments and assembling them in a vector Above, the packaging cells were transfected to obtain VLPs.
  • the first aspect of the present invention provides a composition for the preparation of SARS-CoV-2 virus-like particles, including the following (a), also including any one of the following (b) and (c) or Any two:
  • a first plasmid comprising a Stru ⁇ S fragment comprising a nucleic acid sequence encoding at least one structural protein of the SARS-CoV-2 virus or a mutant thereof and/or at least one accessory protein or a mutant thereof, and The ⁇ S fragment does not include a nucleic acid sequence encoding the S protein of SARS-CoV-2 virus or a mutant thereof;
  • the second plasmid that comprises S segment comprises the nucleic acid sequence of the S protein of coding SARS-CoV-2 virus or its mutant;
  • a third plasmid comprising the packaging signal segment of the SARS-CoV-2 virus, said packaging signal segment comprising the packaging signal sequence of the SARS-CoV-2 virus.
  • the mutation of the S protein relative to the S protein of SARS-CoV-2 virus includes N331Q, N501Y, D614G and/or P681H.
  • the Stru ⁇ S fragment comprises any of the following:
  • At least one of the structural protein and accessory protein has a mutation relative to the wild-type SARS-CoV-2 virus
  • the packaging signal sequence is nucleotides 19900-20000 of NCBI Serial No. NC_045512.2 or nucleotides 19773-20335 of NCBI Serial No. NC_045512.2.
  • the packaging signal fragment comprises ORF1ab of the SARS-CoV-2 viral genome.
  • Another aspect of the present invention provides a method for preparing the above composition, including preparing the first plasmid, the second plasmid and/or the third plasmid by the following methods:
  • step (2) Transfecting the short DNA fragment and the linearized plasmid vector obtained in step (1) into yeast cells, and performing homologous recombination to obtain the first plasmid, the second plasmid and/or the third plasmid respectively.
  • Another aspect of the present invention provides a method for preparing SARS-CoV-2 virus-like particles, comprising: transfecting packaging cells with any of the above compositions to obtain SARS-CoV-2 virus-like particles.
  • the preparation method also includes preparing the first plasmid, the second plasmid and/or the third plasmid by the following methods:
  • step (2) Transfecting the short DNA fragment and the linearized plasmid vector obtained in step (1) into yeast cells, and performing homologous recombination to obtain the first plasmid, the second plasmid and/or the third plasmid respectively.
  • the preparation method further comprises separately amplifying the first plasmid, the second plasmid and/or the third plasmid in Escherichia coli, and then transfecting the packaging cells.
  • Another aspect of the present invention provides SARS-CoV-2 virus-like particles prepared by the above preparation method.
  • Another aspect of the present invention provides the use of the SARS-CoV-2 virus-like particle prepared by the above composition or the above preparation method in the preparation of a vaccine for preventing or treating SARS-CoV-2 virus infection.
  • Another aspect of the present invention provides the use of the SARS-CoV-2 virus-like particles prepared by the above-mentioned composition or the above-mentioned preparation method in the research of SARS-CoV-2 virus-infected cells in vitro.
  • the research on the SARS-CoV-2 virus infected cells can be research on the interaction between VLP and cells, research on the process of virus infecting cells, and/or research on the effect of virus components, etc.
  • the present invention has the following advantages and effects:
  • the present invention uses a mammalian cell eukaryotic expression system to express structural proteins, nonstructural proteins and auxiliary proteins of SARS-CoV-2 to assemble into VLPs, which can be used for vaccines.
  • the vaccine preparation method does not involve live viruses. Compared with inactivated virus vaccines and live attenuated vaccines, this method has better safety; compared with polypeptide or nucleic acid vaccines, this method has better immunogenicity.
  • the present invention adopts the method of splitting the viral genome, first synthesizing small fragment genes, and then performing genome assembly through the yeast homologous recombination system, which is simple, fast and efficient.
  • the present invention utilizes the genome splitting strategy, and the VLP contains complete virion morphology such as structural proteins and non-structural proteins, which has strong immunogenicity and is expected to be developed into a high-efficiency vaccine with cross-protective efficacy.
  • Figure 1 shows the generation of SARS-CoV-2 pseudoviruses.
  • A Scheme of the full-length SARS-CoV-2 genome and disassembled parts, including the SARS-CoV-2 S, ORF1ab, and Stru ⁇ S genomes.
  • B The isolated SARS-CoV-2 genome was assembled in yeast to construct the plasmids SARS-CoV-2 S, ORF1ab, and Stru ⁇ S.
  • C SARS-CoV-2 pseudoviruses were obtained by co-transfection of plasmids SARS-CoV-2 S, ORF1ab, and Stru ⁇ S.
  • SARS-CoV-2 pseudovirus cannot produce progeny virus after infection of 293T/hACE2 cells.
  • Figure 2 shows the construction and characterization of the SARS-CoV-2 pseudovirus.
  • A Confocal microscopy images of 293T cells co-transfected with plasmids SARS-CoV-2 S, ORF1ab-mCherry, and Stru ⁇ S-EGFP 24 hours after transfection.
  • B Detection of SARS-CoV-2 S and N proteins in cell lysates (upper) and SARS-CoV-2 pseudoviruses (lower) by Western blot. GAPDH was used as a loading control for western blotting.
  • C Morphology and size of SARS-CoV-2 pseudoviruses and VLPs detected by TEM. Scale bar: 20 nm.
  • Figure 3 shows the analysis and validation of the packaging signal (PS).
  • PS packaging signal
  • C Detection of EGFP, SARS-CoV-2 S and N proteins in cell lysates (left) and VLP (EGFP-PS583) (right) by Western blot. GAPDH was used as a loading control for western blotting and RT-PCR.
  • FIG. 4 shows the functional validation of different SARS-CoV-2 proteins.
  • A Statistical analysis of DiO-labeled SARS-CoV-2 VLPs in the cytoplasm to test the effect of the SARS-CoV-2 S mutation on virus infectivity.
  • B Confocal microscopy image of 293T/hACE2 cells infected by VLP (EGFP-PS583), VLP ( ⁇ N-EGFP-PS583) or VLP (EGFP), and observe the green fluorescence in the cells 48 hours after infection.
  • C Detection of S and N proteins in different types of VLPs by Western blot. The morphology and size of VLPs were observed by TEM. Scale bar: 20 nm.
  • Figure 5 shows (A) the scheme of dual fluorescently labeled VLPs (QD-DiO).
  • B The morphology and size of VLPs (QD-DiO) were characterized by TEM, and the white arrows indicate the QDs. Scale bar: 20 nm.
  • C Co-localization of DiO and QD signals in VLP(QD-DiO).
  • D The dynamic process of VLP(QD-DiO) entering 293T/hACE2 cells.
  • E Differential interference contrast (DIC) image of 293T/hACE2 cells. The black line indicates the trajectory of the virus.
  • F,G Analysis of the mean velocity (F) and MSD plot (G) of the virions shown in (D).
  • Figure 6 shows the construction and characterization of the SARS-CoV-2 S plasmid.
  • A RT-PCR analysis of the gene encoding the SARS-CoV-2 S protein in cell lysates. Red arrows indicate expected amplified sequences. nRT: Not reverse transcriptome.
  • B Detection of SARS-CoV-2 S protein in cell lysates by Western blotting, GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 7 shows the construction and characterization of the SARS-CoV-2 Stru ⁇ S-EGFP plasmid.
  • A Schematic representation of the Stru ⁇ S-EGFP construction.
  • B RT-PCR analysis of SARS-CoV-2 ORF3a and N protein-encoding genes in cell lysates, red arrows indicate expected amplified sequences, GAPDH is used as loading control, nRT: non-reverse transcriptome.
  • C Fluorescent imaging of 293T cells transfected with SARS-CoV-2 Stru ⁇ S-EGFP plasmid for 24 hours.
  • D Detection of N protein in cell lysates (left) and VLPs (right) by western blot, GAPDH was used as a loading control.
  • E Morphology and size of SARS-CoV-2 VLP (Stru ⁇ S) detected by transmission electron microscopy. Scale bar: 20 nm.
  • Figure 8 shows the construction and characterization of the SARS-CoV-2 ORF1ab-mCherry plasmid.
  • A Schematic representation of the ORF1ab-mCherry construct.
  • B RT-PCR analysis of SARS-CoV-2 NSP1 and NSP16 encoding genes in cell lysates, red arrows indicate expected amplified sequences, GAPDH is used as loading control, nRT: non-reverse transcriptome.
  • C Fluorescence imaging of 293T cells transfected with SARS-CoV-2 ORF1ab-mCherry plasmid for 24 hours.
  • Figure 9 shows the construction and characterization of SARS-CoV-2 VLP (Stru ⁇ S-S).
  • A Fluorescence imaging of 293T cells co-transfected with SARS-CoV-2 Stru ⁇ S-EGFP and S plasmid for 24 hours.
  • B Detection of N protein in cell lysates (top) and VLPs (Stru ⁇ S-S) (bottom) by Western blot, GAPDH was used as a loading control.
  • C Morphology and size of SARS-CoV-2 VLP (Stru ⁇ S-S) detected by transmission electron microscopy. Scale bar: 20 nm.
  • Figure 10 shows the construction and characterization of SARS-CoV-2 VLP (Stru ⁇ S-ORF1ab).
  • A Fluorescent imaging of 293T cells co-transfected with SARS-CoV-2 Stru ⁇ S-EGFP and ORF1ab-mCherry plasmids for 24 hours.
  • B Detection of N protein in cell lysates (top) and VLP (Stru ⁇ S-ORF1ab) (bottom) by Western blot, GAPDH was used as a loading control.
  • C Morphology and size of SARS-CoV-2 VLP (Stru ⁇ S-ORF1ab) detected by transmission electron microscopy. Scale bar: 20 nm.
  • Figure 11 shows the validation of the biosafety of the fake SARS-CoV-2 virus.
  • A Expression of hACE2 receptor on construct 293T/hACE2 cells detected by Western blot.
  • B Detection of S and N proteins in cell lysates (left) and supernatants (right) by Western blot.
  • C 293T/hACE2 cells were infected for 48 hours after co-transfection by VLP or plasmid SARS-CoV-2 S, ORF1ab-mCherry and Stru ⁇ S-EGFP, and GAPDH was used as loading control.
  • C Images of 293T/hACE2 cells 72 hours after infection with wild-type SARS-CoV-2 virus and SARS-CoV-2 VLP.
  • Figure 12 shows RNA structure predictions based on aligned consensus sequences. Bat SARS-like coronavirus PS (MG772933.1:19773-20355), SARS PS (NC-004718.3:19712-20294) and SARS-CoV-2 PS (NC-045512.2:19773- 20355) shared structure.
  • Figure 13 shows the analysis and validation of packaged signals.
  • PS101 gene in cell lysate was detected by RT-PCR, the red arrow indicated the expected amplified sequence, GAPDH was used as loading control, nRT: non-reverse transcribed group.
  • B Detection of S, N proteins and EGFP in cell lysates (left) and VLPs (right) by Western blot, GAPDH was used as a loading control.
  • C Fluorescence imaging of 293T/hACE2 cells infected with VLP(EGFP-PS101) and VLP(EGFP) for 48 hours.
  • Figure 14 shows the construction and characterization of SARS-CoV-2 S(N331Q).
  • A Schematic representation of the SARS-CoV-2 S(N331Q) construct.
  • B RT-PCR analysis of the gene encoding SARS-CoV-2 S(N331Q) in cell lysates, the red arrow indicates the expected amplified sequence, nRT: no reverse transcription.
  • C Detection of SARS-CoV-2 S(N331Q) protein in cell lysates by Western blotting, and GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 15 shows the construction and characterization of SARS-CoV-2 S(N501Y).
  • A Schematic representation of the SARS-CoV-2 S(N501Y) construct.
  • B RT-PCR analysis of the gene encoding SARS-CoV-2 S(N501Y) in cell lysates, red arrows indicate expected amplicons, nRT: non-reverse transcriptome.
  • C Detection of SARS-CoV-2 S(N501Y) protein in cell lysates by Western blotting, and GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 16 shows the construction and characterization of SARS-CoV-2 S(D614G).
  • A Schematic representation of the SARS-CoV-2 S(D614G) construct.
  • B RT-PCR analysis of the gene encoding SARS-CoV-2 S(D614G) in cell lysate, the red arrow indicates the expected amplified sequence, nRT: non-reverse transcriptome.
  • C Detection of SARS-CoV-2 S(D614G) protein in cell lysates by Western blotting, and GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 17 shows the construction and characterization of SARS-CoV-2 S(P681H).
  • A Schematic representation of the SARS-CoV-2 S(P681H) construction.
  • B RT-PCR analysis of the gene encoding SARS-CoV-2 S(P681H) in cell lysate, the red arrow indicates the expected amplified sequence, nRT: non-reverse transcriptome.
  • C Detection of SARS-CoV-2 S(P681H) protein in cell lysates by Western blotting, and GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 18 shows the construction and characterization of SARS-CoV-2 VLPs with S protein mutations. Detection of VLP(N331Q)(A), VLP(N501Y)(B), VLP(D614G)(B) and VLP(P681H)(D) by Western blot. The morphology and size of SARS-CoV-2 VLPs were examined by transmission electron microscopy. Scale bar: 20 nm.
  • Figure 19 shows the construction and characterization of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ N plasmid.
  • A Schematic diagram of the construction of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ N plasmid.
  • B RT-PCR analysis of SARS-CoV-2 ORF3a and N protein-encoding genes in cell lysates, red arrows indicate expected amplified sequences, nRT: non-reverse transcriptome.
  • C Detection of SARS-CoV-2 N protein in cell lysates by Western blotting, GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 20 shows the construction and characterization of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ E plasmid.
  • A Schematic diagram of the construction of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ E plasmid.
  • B RT-PCR analysis of SARS-CoV-2 ORF3a, E and N protein-encoding genes in cell lysates, red arrows indicate expected amplified sequences, nRT: non-reverse transcriptome.
  • C Detection of SARS-CoV-2 N protein in cell lysates by Western blotting, GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 21 shows the construction and characterization of SARS-CoV-2 VLP (VLP/ ⁇ E) with E protein deletion.
  • VLP/ ⁇ E SARS-CoV-2 VLP
  • A Morphology and size of VLP/ ⁇ E detected by transmission electron microscopy. Scale bar: 20 nm.
  • B Confocal microscopy images of 293T/hACE2 cells infected with VLP/ ⁇ E for 48 hours.
  • Figure 22 shows the construction and characterization of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ M plasmid.
  • A Schematic diagram of the construction of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ M plasmid.
  • B RT-PCR analysis of SARS-CoV-2 ORF3a, M and N protein-encoding genes in cell lysates, red arrows indicate expected amplified sequences, nRT: non-reverse transcriptome.
  • C Detection of SARS-CoV-2 N protein in cell lysates by Western blotting, GAPDH was used as a loading control for Western blotting and RT-PCR.
  • FIG. 23 shows the construction and characterization of M protein-deleted SARS-CoV-2 VLPs (VLP/ ⁇ E).
  • VLP/ ⁇ E M protein-deleted SARS-CoV-2 VLPs
  • A Morphology and size of VLP/ ⁇ M detected by transmission electron microscopy. Scale bar: 20 nm.
  • B Confocal microscopy images of 293T/hACE2 cells infected with VLP/ ⁇ M for 48 hours.
  • Figure 24 shows the construction and characterization of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ ORF10 plasmid.
  • A Schematic diagram of the construction of the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ ORF10 plasmid.
  • B RT-PCR analysis of SARS-CoV-2 ORF3a, N and ORF10 protein-coding genes in cell lysates, red arrows indicate expected amplified sequences, nRT: non-reverse transcriptome.
  • C Detection of SARS-CoV-2 N protein in cell lysates by Western blotting, GAPDH was used as a loading control for Western blotting and RT-PCR.
  • Figure 25 shows the characterization of protein-deleted SARS-CoV-2 VLPs.
  • A Schematic diagram of plasmid construction for protein-deleted SARS-CoV-2 VLPs.
  • B Fluorescence colocalization results (DiO and QDs) of protein-deficient SARS-CoV-2 VLPs.
  • Numerical ranges described herein such as temperature ranges, time ranges, composition or concentration ranges, or other numerical ranges, etc., include end values, all intermediate ranges, and subranges (such as between an intermediate value and a certain ranges between end values), and all individual values, especially intermediate ranges, subranges, and individual integer values that end at integer values. Also, any intermediate ranges, subranges, and all individual values stated within a stated numerical range may be excluded from said numerical range.
  • nucleic acid sequence and “nucleotide sequence” are used interchangeably and refer to a sequence composed of bases, sugars and phosphate backbone connections.
  • the nucleic acid sequence of the present invention may be deoxyribonucleic acid sequence (DNA) or ribonucleic acid sequence (RNA), and may include natural bases or unnatural bases, which may be single-stranded or double-stranded, and may be coding sequences or non-coding sequence.
  • the "vector” when expressing a gene or a protein encoded by a gene, the "vector” may be a plasmid, and in some cases, the terms “vector” and “plasmid” are used interchangeably.
  • nucleic acid sequence is usually from 5' end to 3' end
  • amino acid sequence is usually from N-terminus to C-terminus
  • the methods of the invention can be performed in vitro or in vivo.
  • Reverse genetics is one of the main means of virus rescue (rescuing) or VLP preparation, and the acquisition of viral genome is the most important step.
  • Nuclease ligation is the main method to obtain the viral genome, but the reaction process of this method is complex and the efficiency is low.
  • the full-length viral genome is highly toxic when it is transformed into the large intestine.
  • the homologous recombination method in yeast is to use the highly efficient homologous recombination system in yeast cells to realize the assembly method of multiple homologous series of DNA fragments.
  • the present invention splits and assembles the genome of SARS-CoV-2 into three plasmids, and through cell transfection, a complete structure can be obtained SARS-CoV-2 VLP with high toxicity provides a good tool platform for further vaccine development.
  • the present invention provides three fragments of the genome of SARS-CoV-2, each fragment is assembled into a plasmid, and a total of three plasmids are obtained, and any one, any two, or Three co-transfected packaging cells can obtain the VLP of SARS-CoV-2.
  • Said SARS-CoV-2 can be wild-type SARS-CoV-2 or any mutant thereof.
  • the genome of wild-type SARS-CoV-2 can be determined by NCBI sequence number NC_045512.2.
  • the three fragments are the S fragment containing the coding sequence of the S protein of SARS-CoV-2, the Stru ⁇ S fragment containing the coding sequences of all structural proteins and all auxiliary proteins except the S protein, and the Stru ⁇ S fragment containing the packaging signal sequence. Wraps a signal fragment.
  • SARS-CoV-2 All structural proteins of SARS-CoV-2 include S protein (spike glycoprotein), E protein (small envelope protein), M protein (membrane glycoprotein) and N protein (nucleocapsid protein). All accessory proteins of SARS-CoV-2 include proteins encoded by ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, and ORF10.
  • the S protein (spike glycoprotein) encoded by the S fragment may be a wild-type S protein or a mutant thereof.
  • the S protein mutant has any one or more mutations selected from N331Q, N501Y, D614G, and P681H relative to the wild-type S protein.
  • the wild-type S protein sequence may be a protein sequence encoded by nucleotides 21563-25384 of NCBI sequence number NC_045512.2, or determined by NCBI sequence number YP_009724390.1.
  • the sequence encoding the S protein included in the S fragment is nucleotides 21563-25384 of NCBI sequence number NC_045512.2.
  • the Stru ⁇ S fragment encodes any one or more of structural proteins and auxiliary proteins, such as any of E protein (small envelope protein), M protein (membrane glycoprotein), and N protein (nucleocapsid protein)
  • E protein small envelope protein
  • M protein membrane glycoprotein
  • N protein nucleocapsid protein
  • the Stru ⁇ S fragment of the present invention encodes all structural proteins and auxiliary proteins except the S protein, in some embodiments of the present invention, compared to the most preferred solution, the Stru ⁇ S fragment encoded
  • the encoded protein can also further lack one or more structural proteins, and/or lack one or more auxiliary proteins, for example, the protein encoded by the Stru ⁇ S fragment can be without Contain any one, any two or three of E protein (small envelope protein), M protein (membrane glycoprotein), N protein (nucleocapsid protein), and/or may not contain any auxiliary protein one or more.
  • the wild-type structural protein and/or auxiliary protein refers to the structural protein and/or auxiliary protein of wild-type SARS-CoV-2.
  • the nucleic acid encoding the structural protein and the accessory protein contained in the Stru ⁇ S fragment is nucleotides 25393-29674 of NCBI sequence number NC_045512.2.
  • the packaging signal fragment at least includes the packaging signal sequence of SARS-CoV-2, and may also include other sequences in the ORF1ab sequence of SARS-CoV-2.
  • the packaging signal sequence is nucleotides 19900-20000 of NCBI Serial No. NC_045512.2 or nucleotides 19773-20335 of NCBI Serial No. NC_045512.2.
  • the packaging signal fragment may comprise the full length of ORF1ab or a fragment thereof, that is, the full length of ORF1ab of SARS-CoV-2 or a fragment thereof. Any one or any several ORFs in the full length or fragments of ORF1ab may be wild type or a mutant thereof.
  • the fragment of ORF1ab may lack one or more ORFs in the ORF1ab sequence, but at least comprise a packaging signal sequence.
  • the ORF in the ORF1ab sequence of the wild type refers to the ORF in the ORF1ab sequence of the wild type SARS-CoV-2.
  • ORF1ab is nucleotides 266-21555 of NCBI Serial No. NC_045512.2.
  • wild-type can be applied to a gene, protein or virus strain, and generally refers to isolated from a naturally occurring source.
  • the wild type is usually the gene, protein or virus strain most frequently observed in the population and is thus designated "wild type".
  • mutant refers to one or more nucleotide or amino acid insertions, replaced or missing.
  • a “mutant” usually still has the basic functional properties of the wild type, but the relative level of the functional properties may be changed compared with the wild type, for example, the activity of the protein or the protein encoded by the gene is increased or decreased.
  • mutants may also be naturally occurring, such as viruses that mutate naturally, resulting in naturally occurring mutants of the virus, and "wild type” may refer to the earliest naturally occurring variant of the same gene, protein, or virus strain. of those.
  • the genome of wild-type SARS-CoV-2 can be determined by NCBI sequence number NC_045512.2, and each ORF in each structural protein, auxiliary protein, and/or ORF1ab of the wild type can be determined by NCBI sequence number NC_045512.2 Encoded or determined by the genome sequence indicated.
  • the virus produced by SARS-CoV-2 through natural mutation during its existence and transmission in nature, as well as the genes contained in the virus genome and the proteins encoded by it, can all be called “mutants”.
  • the "mutants” in the present invention also include those viruses obtained by artificially transforming naturally occurring wild-type SARS-CoV-2 and naturally mutated SARS-CoV-2, their genomes, any of their genes, and any of their genes. A mutant protein.
  • each of S fragment, Stru ⁇ S fragment and ORF1ab fragment is assembled into a plasmid for expressing genes in these fragments or proteins encoded by them.
  • the plasmid comprising the Stru ⁇ S segment is referred to as the first plasmid
  • the plasmid comprising the S segment is referred to as the second plasmid
  • the plasmid comprising the packaging signal segment is referred to as the third plasmid.
  • the numbering of plasmids is for distinction only and does not imply a specific sequence structure implied in these plasmids.
  • the initial plasmid vectors used to prepare the first plasmid, the second plasmid and the third plasmid can be the same or different, for example, the same initial plasmid vector can be used for the three, or three different initial plasmid vectors can be used, Or it can be that two are the same but different from the other.
  • the initial plasmid vector used to prepare the first plasmid, the second plasmid or the third plasmid can be any suitable vector common in the art, for example, a common vector in the art that can shuttle between yeast and bacteria and can transfect packaging cells Any suitable vector, including but not limited to pEASY-T1, pRS415, pJS356, pEGFP-N1, etc.
  • the sequence encoding structural protein and/or auxiliary protein contained in the first plasmid, the sequence encoding S protein in the second plasmid and/or the packaging signal sequence in the third plasmid or the full length of ORF1ab or its fragments can be combined with the expression regulation sequence Operably connected.
  • the expression regulatory sequence is a sequence that regulates the expression and/or translation of the fragments contained in the plasmid, including but not limited to 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), Promoters, enhancers, terminators, selectable marker genes, post-transcriptional regulatory elements, internal ribosome entry sites (IRES), cleavable sequences and/or polyadenylation signals (polyA), etc.
  • Usable promoters include, but are not limited to, promoters derived from viruses or mammals (including humans) and the like. Promoters can be constitutive or inducible. Examples of viral promoters include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, RSV promoter, chicken ⁇ -actin (CBA) promoter, CMV early enhancer/chicken ⁇ -actin (CAG) promoter, simian virus 40 (SV40) promoter, etc.
  • CMV cytomegalovirus
  • RSV RSV promoter
  • CBA CMV early enhancer/chicken ⁇ -actin
  • SV40 simian virus 40
  • mammalian promoters include, but are not limited to, the human elongation factor 1a (EF1a) promoter, the human ubiquitin C (UCB) promoter, the mouse phosphoglycerate kinase (PGK) promoter, the RNA polymerase type III promoter (such as U6 and H1) etc.
  • constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, ⁇ -actin Promoter and phosphoglycerol kinase (PGK) promoter and EF1a promoter.
  • inducible promoters include, but are not limited to, the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, and the like.
  • MT zinc-inducible sheep metallothionein
  • Dex dexamethasone-inducible mouse mammary tumor virus
  • T7 polymerase promoter system and the like.
  • the usable post-transcriptional regulatory element may be, for example, a viral post-transcriptional regulatory element such as woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), hepatitis B virus post-transcriptional regulatory element (HBVPRE) or RNA transport element (RTE).
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • HBVPRE hepatitis B virus post-transcriptional regulatory element
  • RTE RNA transport element
  • Selectable markers that can be used include, but are not limited to, eg, drug resistance selectable markers. Such selectable marker genes may encode factors necessary for the survival or growth of cells grown in selective media. Host cells not transformed with a vector containing a selection gene will not survive in culture. Proteins that confer resistance to antibiotics or other toxins, examples of which include, but are not limited to, ampicillin, hygrocylin, Neomycin, methotrexate, kanamycin, gentamicin, Zeocin, or tetracycline.
  • the selectable marker can also be a fluorescent protein, such as mCherry, GFP, BFP, EGFP, etc.
  • a cleavable sequence may be any sequence that, after expression, is capable of self-cleaving or being cleaved by other means (eg, by a specific enzyme).
  • a cleavable sequence may be a sequence capable of self-cleavage after expression.
  • Such sequences are well known to those skilled in the art, for example, ribozyme sequences with self-cleavage function, such as splicing ribozyme sequences.
  • Splicing ribozymes can catalyze the self-cleavage of their own RNA at specific sites. Splicing ribozymes include, but are not limited to, hammerhead ribozymes, hairpin ribozymes, HDV ribozymes, or RNaseP.
  • a cleavable sequence can also be a sequence that can be cleaved by other means, such as by specific enzymes. Such sequences are well known to those skilled in the art, for example tRNA sequences.
  • the tRNA sequence can be cut off the 5' end additional sequence of the tRNA under the action of tRNA 5' maturation enzyme (RZaseP), or the sequence at the 3' end of the tRNA can be cut off under the action of the 3' endonuclease RZase F.
  • the cleavable sequence can be a tRNA sequence that can be cut by RZaseP to the 5' end sequence, or can be a tRNA sequence that can be cut by RZase F to the 3' end sequence. Any tRNA that is capable of being cleaved upon maturity can be used in the present invention.
  • a polyadenylation signal sequence may be located in a transcription termination region, such as the 3' untranslated region, examples of which include, but are not limited to, bovine growth hormone (bGH) poly(A), SV40 polyA, thymidine kinase (TK) poly(A) sequence etc.
  • bGH bovine growth hormone
  • SV40 SV40 polyA
  • TK thymidine kinase
  • 5' untranslated region (5'UTR), 3' untranslated region (3'UTR) can use any suitable virus 5'UTR and 3'UTR, such as 5'UTR and 3'UTR of SARS-CoV-2.
  • the nucleic acid encoding the structural protein and/or accessory protein in the first plasmid is operably with the promoter, 5'UTR at the 5' end, and the 3'UTR at the 3' end, polyA and optional cleavable sequence connect.
  • the nucleic acid encoding the S protein in the second plasmid is operably linked to a promoter at the 5' end and polyA at the 3' end.
  • the sequence of ORF1ab from SARS-CoV-2 in the third plasmid is operable with the promoter, 5'UTR at the 5' end, and the 3'UTR at the 3' end, polyA and optional cleavable sequence connect.
  • the Stru ⁇ S fragment, S fragment or ORF1ab fragment can be assembled into the first plasmid, the second plasmid or the third plasmid by transformation-associated recombination (Transformation-associated recombination, TAR) cloning method.
  • TAR cloning uses yeast in vivo recombination system to splice and synthesize large fragments of target DNA.
  • the specific method is: co-transfect multiple fragments of target DNA with homologous ends and a linearized TAR vector into yeast, and use yeast
  • the highly efficient recombination system in cells enables homologous recombination between the vector and the homologous sequences of the target DNA fragments and the homologous sequences of different target DNA fragments to produce vectors with the target DNA.
  • S fragment, Stru ⁇ S fragment or ORF1ab fragment can be used as target DNA for TAR cloning.
  • the TAR vector may be a plasmid vector.
  • the target DNA is split into multiple DNA fragments.
  • homologous sequences also called homologous arms, overlapping sequences or overlapping segments
  • the DNA fragments located at both ends of the target DNA are respectively aligned with the linear
  • the two ends of the linearized vector have homologous sequences, and these DNA fragments and the linearized vector are co-transferred into yeast, so that they undergo homologous recombination in yeast to assemble into a vector containing the target DNA.
  • the "adjacent" means that the positions of the two DNA fragments on the target DNA are adjacent.
  • each DNA fragment split is usually 2-5kb, for example the lower limit and the upper limit of the length range of each DNA fragment split can be respectively about 2kb, about 2.1kb, about 2.2kb, about 2.3kb, about 2.4kb, about 2.5kb, about 2.6kb, about 2.7kb, about 2.8kb, about 2.9kb, about 3kb, about 3.1kb, about 3.2kb, about 3.3kb, about 3.4kb, about 3.5kb, about 3.6kb, About 3.7kb, about 3.8kb, about 3.9kb, about 4kb, about 4.1kb, about 4.2kb, about 4.3kb, about 4.4kb, about 4.5kb, about 4.6kb, about 4.7kb, about 4.8kb, about 4.9kb , about 5 kb, preferably 3-4 kb, more preferably about 3 kb.
  • the length of the homologous sequence is usually 50-300 bp, preferably 100
  • the Stru ⁇ S fragment can be resolved into 3 DNA fragments.
  • the S segment can be split into 2 DNA segments.
  • the ORF1ab fragment can be split into 2 DNA fragments.
  • the split DNA fragments can be synthesized by methods known in the art, such as in vitro synthesis, and the synthesized DNA fragments can be single-stranded or double-stranded fragments.
  • Stru ⁇ S fragment, S fragment or ORF1ab fragment described in the present invention may only contain the gene of SARS-CoV-2 to be expressed, without other sequences (such as coding sequence or expression control sequence), and may also contain the gene of SARS-CoV-2 to be expressed.
  • the expression control sequence can be added to the vector by any means, for example, the expression control sequence can be included in the Stru ⁇ S fragment, the S fragment and/or the ORF1ab fragment, together with the gene of SARS-CoV-2 expressed in these fragments Split into multiple DNA fragments containing homology arms and assembled into plasmids by yeast homologous recombination (TAR).
  • the aforementioned expression control sequence can also be added to the plasmid vector in advance, and the DNA fragment with homologous arms split with the Stru ⁇ S fragment, S fragment or ORF1ab fragment is subjected to homologous recombination in yeast.
  • the Stru ⁇ S fragment, S fragment Or the ORF1ab fragment only contains the gene of SARS-CoV-2 to be expressed, but does not include the expression control sequence.
  • some regulatory sequences are included in the Stru ⁇ S fragment, the S fragment and/or the ORF1ab fragment, and together with the genes of SARS-CoV-2 expressed in these fragments, they are split into multiple sequences containing homology arms.
  • a DNA fragment was assembled into a plasmid by yeast homologous recombination (TAR), and other expression regulatory sequences were added to the plasmid vector in advance.
  • YPD Peptone glucose medium
  • the vector containing the target DNA assembled in yeast can be extracted from yeast by conventional plasmid extraction methods to obtain the first plasmid, second plasmid or third plasmid, and then optionally transferred to E. coli for amplification; or
  • the total yeast DNA can be directly extracted, transformed into Escherichia coli with the total yeast DNA and amplified to obtain the first plasmid, the second plasmid or the third plasmid.
  • Methods for obtaining and amplifying recombinant plasmids from yeast are well known in the art.
  • the total yeast DNA can be transferred into Escherichia coli, a single clone can be selected, and after verification, a large number of plasmids can be amplified and extracted, and the plasmids can be stored for future use.
  • the program is processed by junction PCR; then select the correct bacteria to extract the plasmid, and perform enzyme digestion verification.
  • any two or three of the first plasmid, the second plasmid and the third plasmid are used to transfect the packaging cells, and they are expressed and assembled in the packaging cells, so that the SARS-CoV-2 can be obtained virus-like particles.
  • the plasmids that can be used to transfect packaging cells include the first plasmid and any one or both of the second plasmid and the third plasmid.
  • any kind of plasmid can be transfected into mammalian cells (such as 293T cells) alone, and the intracellular RNA and protein can be extracted after lysing the cells and detected separately. Detection by RT-PCR and Western blot, and verification of plasmid expression can also be achieved by selective markers, such as fluorescent protein imaging.
  • virus-like particle VLP
  • pseudovirus recombinant virus
  • rVP recombinant viral particle
  • the obtained viral particles wherein at least one component contained in the viral genome is expressed and assembled by genetic engineering methods.
  • the "virus-like particles” of the present invention may not contain viral genomes, or contain genes that do not encode viral proteins, and therefore are non-replicative and non-infectious.
  • the "recombinant virus particles" in the present invention refer to the use of the first plasmid, the second Virus particles obtained by transfecting packaging cells with any two or three of the second plasmid and the third plasmid, the virus particles may or may not contain nucleic acid substances.
  • the plasmids used to transfect packaging cells include plasmids with packaging signals
  • the obtained virus particles contain nucleic acid material;
  • the plasmids used to transfect packaging cells do not include plasmids with packaging signals, the obtained Virus particles do not contain nucleic acid material.
  • rSAR2-CoV-2 and SAR2-CoV-2 VLP can be used interchangeably.
  • Transfection in the present invention refers to the transfer of polynucleotides, such as nucleic acid molecules, plasmids, etc., from the outside of the cells into the cells, so that the polynucleotides have functions in the cells. Transfection methods are well known to those skilled in the art, such as calcium phosphate or liposome-mediated transfection. As known to those skilled in the art, liposomes that can be used include lipofectamine 8000 and the like.
  • the transfection method comprises making 293T grow to a density of about 80%, transfecting 293T cells after mixing the plasmid with lipofectamine 8000, and the medium is DMEM supplemented with 10% FBS; after 6 hours, the medium is replaced with DMEM, collected the supernatant solution after 48h.
  • Packaging cells for the production of recombinant virus particles are well known to those skilled in the art.
  • packaging cells that can be used include, but are not limited to, mammalian (including human) cells, insect cells, plant cells, microorganisms or yeast, such as HEK293 A series of cells (such as HEK293A, HEK293T or HEK293FT), A549 cells or Vero cells, etc.
  • the medium and culture methods used to produce the recombinant virus particles are known to those skilled in the art, and commercially available or custom-made medium can be used, or one or more cell culture components known in the art can be added and supplemented, Including but not limited to glucose, vitamins, amino acids and or growth factors to increase the titer of recombinant virus particles in the production culture.
  • Recombinant viral vector production cultures can be grown under conditions appropriate to the particular host cell (ie, packaging cell) used.
  • viral particles can be purified, if desired, from cell lysates or viral supernatants using a variety of conventional methods, including ultrafiltration, e.g., using nitrocellulose filters; adsorption, e.g., using calcium phosphate Or ion exchange is well known to adsorb viruses or impurities, followed by elution with salt solution; chromatography, such as Sephadex chromatography, ion exchange chromatography, affinity chromatography, etc.; centrifugation, such as differential centrifugation, CsCl or Sucrose density gradient centrifugation; precipitation methods, such as polyethylene glycol precipitation (such as using PEG2000), isoelectric point precipitation or neutral salt precipitation, etc.
  • ultrafiltration e.g., using nitrocellulose filters
  • adsorption e.g., using calcium phosphate Or ion exchange is well known to adsorb viruses or impurities, followed by elution with salt solution
  • chromatography such as Sephadex
  • the above purification methods may also be used in combination.
  • This method can be used for experiments such as VLP infection; as another example of the purification method, the collected virus culture supernatant was washed with 20% (w/v) The sucrose solution was subjected to ultracentrifugation at 30,000rpm for 4h to preliminarily purify VLP.
  • This VLP purification method can be used for the characterization of RNA, protein, and VLP morphology.
  • the recombinant viral particles of the present invention can be used as active ingredients in pharmaceutical compositions or vaccines to treat diseases caused by SARS-CoV-2.
  • the term "vaccine” refers to a preparation comprising said recombinant viral particles.
  • the dose of recombinant virus particles contained in the vaccine can be adjusted by those skilled in the art, for example, according to the disease condition, subject and treatment schedule.
  • Vaccines generally contain a therapeutically effective amount of recombinant viral particles.
  • Treatment in the present invention refers to preventing or alleviating (eg, reducing, alleviating or curing) at least one symptom associated with a disease state.
  • the "therapeutically effective amount” in the present invention is an amount sufficient to prevent or alleviate (eg, reduce, relieve or cure) at least one symptom associated with a disease state.
  • the dosage of the pharmaceutical composition or vaccine can be conveniently determined by one skilled in the art, for example by first identifying a dose effective to elicit a prophylactic or therapeutic immune response, for example by measuring serum titers of virus-specific immunoglobulins or by Measure the inhibition ratio of antibodies in serum samples or urine samples or mucosal secretions.
  • the pharmaceutical composition or vaccine of the present invention may comprise the recombinant virus particle of the present invention and a pharmaceutically acceptable carrier or excipient.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
  • the pharmaceutical compositions or vaccines of the present invention may contain adjuvants, which are well known to those skilled in the art. Exemplary adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, BCG, and the like.
  • the pharmaceutical composition or vaccine of the invention should be suitable for administration to a subject, eg be sterile, non-particulate and/or non-pyrogenic.
  • the pharmaceutical composition or vaccine can be formulated in a solid form, such as lyophilized powder, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder that can be used to prepare injections.
  • compositions or vaccines of the invention include, but are not limited to, parenteral (e.g., intradermal, intramuscular, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or via suppositories). Administration can be systemic or local.
  • parenteral e.g., intradermal, intramuscular, intravenous, and subcutaneous
  • epidural e.g., epidural and mucosal
  • mucosal e.g., intranasal and oral or pulmonary routes or via suppositories.
  • Administration can be systemic or local.
  • the recombinant viral particles of the present invention can also be used in in vitro studies of SARS-CoV-2 virus-infected cells, for example, can be used for in vitro drug screening, such as in vitro screening of drugs that can inhibit SARS-CoV-2 virus-infected cells; develop antibodies,
  • the recombinant virus particle can be used as an immunogen to prepare antibodies or detect antibody neutralization activity; it can also be used to develop virus vaccines, for example, it can be aimed at emerging virus mutants, especially newly emerging virus mutants with mutant S proteins, convenient Rapidly develop vaccines.
  • the principle of genome splitting, synthesis and assembly is shown in FIG. 1 .
  • the genome ORF1ab was split into 6 fragments with a size of 3-5kb, the S protein gene was split into 2 fragments, and other structural proteins or auxiliary protein genes were split into 3 fragments; then, each fragment was synthesized in vitro and Three fragments of ORF1ab, S and Stru ⁇ S were assembled by yeast homologous recombination technology; then the three fragments were constructed on the corresponding vectors, and transformed into E. coli to obtain plasmids; finally, the plasmids were transfected into mammalian cells to obtain VLPs. Characterization of various VLPs is shown in Figures 3-4.
  • the present invention provides a de novo artificial synthesis and preparation method of SARS-CoV-2 VLP, specifically comprising:
  • Step 1 SARS-CoV-2 genome disassembly, synthesis and assembly
  • 5'UTR, 3'UTR and PolyA tail are added to both ends of ORF1ab and Stru ⁇ S coding sequences, and CMV enhancer is added to the 5' of each sequence; PolyA tail is added to the 3' end of S protein coding sequence ; and add CAG enhancer at 5';
  • YPD yeast extract powder peptone glucose medium
  • the specific operation steps are as follows: first, pick EPI300 single clone and inoculate it in 5mL LB liquid medium, cultivate it overnight, transfect the total yeast DNA by electroporation, apply the transferred bacteria solution on the resistance plate to pick the single clone, and follow the PCR method.
  • the program is processed by junction PCR; then select the correct bacteria to extract the plasmid, and perform enzyme digestion verification. After the enzyme digestion verification is correct, perform sequencing verification; after the verification is correct, a large number of E. coli are amplified, and the plasmid is extracted, and the plasmid is saved for future use.
  • the second step SARS-CoV-2 VLP expression and preparation method
  • the three plasmids constructed by splitting the SARS-CoV-2 genome were verified separately. Firstly, the three plasmids were separately transfected into 293T cells, and then the cells were lysed, and the RNA and protein in the cells were extracted respectively, and verified by RT-PCR and western blot respectively; at the same time, the EGFP in SARS-CoV-2 Stru ⁇ S and the The method of fluorescence imaging of mCherry in SARS-CoV-2 ORF1ab realizes the verification of plasmid expression.
  • (2) 293T grows to a density of about 80%, respectively transfected with SARS-CoV-2 Stru ⁇ S, SARS-CoV-2 Stru ⁇ S and SARS-CoV-2 S, SARS-CoV-2 Stru ⁇ S and SARS-CoV-2 ORF1ab, and SARS-CoV-2 Stru ⁇ S, SARS-CoV-2 S, and SARS-CoV-2 ORF1ab, the medium was opti-MEM; after 6 hours, the medium was replaced with DMEM, and the supernatant solution was collected after 48 hours.
  • plasmid co-transfection to prepare VLP and its verification include any of the following steps:
  • Stru ⁇ S, S and ORF1ab plasmids were co-transfected into 293T cells to prepare Stru ⁇ S+S+ORF1ab VLP
  • Stru ⁇ S, ORF1ab and S mutant plasmids were co-transfected into 293T cells to prepare Stru ⁇ S+S mutation +ORF1ab VLP
  • S protein plasmid that is, SARS-CoV-2 S
  • N331Q, N501Y, D614G, and P681H are taken as examples; each S mutant plasmid is transfected into 293T cells, lysed Cells, the expression of the S mutant plasmid was verified by RT-PCR.
  • S mutant VLP for each S protein mutation, spread 293T cells on a 100mm cell culture dish, and when the cell density is about 90%, transfect 15 ⁇ g SARS- CoV-2 Stru ⁇ S, 15 ⁇ g SARS-CoV-2 S mutation and 15 ⁇ g SARS-CoV-2 ORF1ab plasmid, change the medium after 6h; collect the supernatant after 48h, which is the VLP of Stru ⁇ S+S mutation +ORF1ab; The expression of the plasmid was verified by imaging method, the intracellular expression and the S protein and N protein in VLP were analyzed by western blot, and the size and shape of VLP were verified by transmission electron microscopy.
  • nucleotide numbers of the SARS-CoV-2 genome and its parts described in the present invention and the following examples are all subject to NCBI sequence number NC_045512.2, and the amino acids of the S protein and its mutants
  • the position numbers are all based on the amino acid sequence of the S protein, that is, the first amino acid of the S protein is numbered 1.
  • Plasmids Three plasmids of SARS-CoV-2 S, ORF1ab-mCherry and Stru ⁇ S-EGFP were constructed based on the TAR system in yeast to prepare virus particles of SARS-CoV-2. .
  • a CAG promoter is added at the 5' end of the codon-optimized cDNA sequence encoding the SARS-CoV-2 S glycoprotein, and a bGH poly(A) signal is added at its 3' end, which will contain the promoter and poly(A) ) signal was split into two fragments (SEQ ID NO:1, SEQ ID NO:2) with overlapping sequences, and the SARS-
  • the cDNA sequence of the CoV-2 S glycoprotein was assembled and cloned into the pEASY-T1 vector to form the plasmid pEASY-T-S (also referred to as SARS-CoV-2 S plasmid or S plasmid in the following examples).
  • the CMV promoter, 5'UTR, and 3'UTR, HDV, SV40 polyA and mCherry encoding genes were added at the 5' end of ORF1ab of SARS-CoV-2, and the resulting sequence was split into six fragments with overlapping sequences (SEQ ID NO:3-8), these overlapping fragments were assembled in yeast and cloned into the pJS356 vector (the pJS356 vector was added on the basis of the pR415 plasmid to induce high expression of the low-copy plasmid
  • the ori2-oriV element and the SopA, SopB, RepE and SopC functional elements obtained for high expression in Escherichia coli) form the plasmid pJS356-ORF1ab-mCherry (also known as SARS-CoV-2 in the following examples ORF1ab plasmid, SARS-CoV-2 ORF1ab-mCherry plasmid, ORF1ab-m
  • CMV promoter, T7 promoter, 5 'UTR, and 3'UTR, HDV, SV40 polyA and EGFP coding genes were added at the 3' end, and the resulting sequence was split into three fragments with overlapping sequences (SEQ ID NO:9-11), in These overlapping fragments were assembled in yeast and cloned into the pJS356 vector to form the plasmid pJS356-Stru ⁇ S-EGFP (also referred to as SARS-CoV-2 Stru ⁇ S plasmid, SARS-CoV-2 Stru ⁇ S-EGFP plasmid, Stru ⁇ S-EGFP in the following examples) EGFP plasmid or Stru ⁇ S plasmid).
  • the obtained plasmids were verified by digestion and sequencing.
  • Plasmid pEGFP-N1 was treated with Not I restriction endonuclease, and the PS-containing fragment was inserted to generate pEGFP-N1-PS101 or pEGFP-N1-PS583.
  • Site-directed mutagenesis use the SARS-CoV-2 S plasmid as a template to construct S protein gene mutants, including SARS-CoV-2 S(N331Q), SARS-CoV-2 S(N501Y), SARS-CoV-2 S(D614G) and SARS-CoV-2 S(P681H) a total of four plasmids. Select 15-20 bases near the target mutation site as the forward primer, and select the complementary sequence as the reverse primer as the reverse primer, which are listed in Table 1. After site-directed mutagenesis PCR, the template strand was digested with the restriction endonuclease DpnI. The product was directly transformed into Escherichia coli DH5 ⁇ competent cells, and a single clone was selected and sequenced.
  • ORF1b RNA secondary structure Sequences in the ORF1b region (nt 19500-20400) of SARS-CoV-2, SARS-CoV and bat-like SARS-CoV were selected from the reference genome using Biopython. Secondary structure was predicted by the RNA structure web server with default parameters and visualized by the Vienna RNA web server.
  • 293T cells stably expressing human ACE2 (293T/hACE2) were constructed. 293T cells were transfected with hACE2 plasmid to produce recipient hACE2, and selected under 2 ⁇ g/mL puromycin. Expression of hACE2 cells was detected by Western blotting. 293T cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycin, and cultured at 37°C in a 5% CO2 incubator. 293T/hACE2 cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycin, and 2 ⁇ g/mL puromycin, and cultured at 37°C in a 5% CO2 incubator.
  • VLPs Construction of VLPs: In order to obtain VLPs, use lipofectamine 8000 to mix with SARS-CoV-2 S (10 ⁇ g), ORF1ab-mCherry (10 ⁇ g) and Stru ⁇ S-EGFP (10 ⁇ g) plasmids, and then transfect them into 293T cells, and in 10% Culture in DMEM medium with FBS for 48 hours.
  • VLP EGFP-PS101
  • VLP VLP (EGFP-PS583) assembled with a packaging signal
  • pEGFP-N1-PS101 1 ⁇ g
  • pEGFP-N1-PS583 1 ⁇ g
  • SARS-CoV-2 S 10 ⁇ g
  • Stru ⁇ S (10 ⁇ g) plasmids were co-transfected into 293T cells for 48 hours.
  • VLP purification 48 hours after transfection, the culture supernatant was collected and filtered with a 0.45 ⁇ m filter. The medium supernatant was added to a 20% sucrose pad and ultracentrifuged at 30000 rpm for 3 hours using a SW32 rotor. Purified VLPs were used for transmission electron microscopy. For western blot analysis, the culture supernatant was added on top of a 20% to 40% density gradient sucrose and ultracentrifuged at 30000 rpm for 4 h using a SW41 rotor. VLPs were collected using PEG20000 enrichment for fluorescence imaging.
  • RNA extraction and RT-PCR Pass the total RNA from the supernatant of the V2 cell culture medium of the FastPure Cell/Tissue Total RNA Isolation Kit, and transcribe it into cDNA within 48 hours according to the kit instructions.
  • cDNA design the specific primers listed in Table 1 to amplify the coding region of SARS-CoV-2 S, N, NSP1, NSP16 or ORF3a, and verify the PCR products by electrophoresis on 1% agarose gel.
  • VLPs were adhered to carbon-coated copper grids for 10 min and stained with 2% (w/v) phosphotungstic acid (pH 7.1) for 1 min, and the samples were observed with a 200 kV transmission electron microscope.
  • quantum dot nanobeacons tellurium powder (20 mg) and sodium borohydride (11 mg) were added to a round-bottomed flask containing 1 mL of ultrapure water, and the mixture was stirred for 5 h under anaerobic and ice-bath conditions to obtain sodium telluride hydride .
  • cadmium chloride, zinc chloride, and N-acetyl-L-cysteine were mixed in a 1:1:4 ratio and the pH was adjusted to 9.0 by NaOH.
  • BHQ2 and phosphorothioate co-modified DNA, cadmium precursor, and sodium hydride telluride were mixed and transferred into a tetrafluoroethylene-lined stainless steel autoclave.
  • the mixture was heated to 200°C for 36 min to obtain BHQ2-DNA functionalized CdTe:Zn 2+ QDs, and the quantum dots were purified by centrifugation at 8000 rpm for 10 min with an Amicon Ultra-4 centrifugal filter device (50 kDa).
  • the BHQ2-DNA functionalized CdTe:Zn 2+ QDs were annealed at 95°C for 10 min and at room temperature for 30 min to form a stem-loop structure.
  • Fluorescent labeling of VLPs To construct fluorescently labeled VLPs, 40 ⁇ L of annealed quantum dot nanobeacons (10 ⁇ M) were added to the culture medium 6 hours after transfection of the SARS-CoV-2 plasmid to label the viral genome, and the virus self-assembled Nanobeacons were tagged in the VLP during the period. Add 0.5 ⁇ L DiO (1 mM) to 2 mL of the collected virus liquid and incubate at 37 °C for 1 hour to label the viral envelope.
  • Virus infection and fluorescence imaging 293T/hACE2 cells were seeded in confocal culture dishes and cultured in DMEM containing 10% FBS, 1% penicillin and streptomycin, and 2 ⁇ g/mL puromycin. Fluorescently labeled VLPs were incubated with host cells 293T/hACE2 for 30 minutes at 4°C, and the medium was replaced with fresh medium to remove unbound particles. The confocal dish was sealed with parafilm and then transferred to a 37 degree 5% CO 2 incubator. Imaging was performed by an UltraView Vox confocal laser scanning system at 488 nm and 561 nm excitation.
  • SARS-CoV-2 ORF1ab nucleotide position: 266-21555
  • SARS-CoV-2 CoV-2 S nucleotide position: 21563-25384
  • SARS-CoV-2 Stru ⁇ S nucleotide position: 25393-29674
  • SARS-CoV-2 ORF1ab SARS-CoV-2 S and SARS-CoV-2 Stru ⁇ S respectively
  • the constructed plasmids were co-transfected into 293T cells to produce SARS-CoV-2 VLPs (Fig. 1B).
  • SARS-CoV-2 ORF1ab RNA containing packaging signal (PS) sequence assembles with structural proteins to form SARS-CoV-2 VLPs; infectivity of SARS-CoV-2 VLPs It is mediated by the binding of the spike protein to the hACE2 receptor on the host cell surface.
  • PS packaging signal
  • SARS-CoV-2 VLP construction strategy is expected to provide a safe and general platform for virology research.
  • the SARS-CoV-2 spike glycoprotein (S) acts as a receptor binding site that can mediate membrane fusion and virus entry.
  • S S protein coding sequence
  • the SARS-CoV-2 S plasmid was transfected into 293T cells, and the results of S gene analysis by RT-PCR are shown in Figure 6A.
  • the expressed SARS-CoV-2 S protein was verified by Western blotting, which showed two major bands, a full-length protein of 180 kDa and a cleaved protein of 110 kDa (Fig. 6B).
  • SARS-CoV-2 Stru ⁇ S gene sequence was fused with EGFP to construct a plasmid named SARS-CoV-2 Stru ⁇ S-EGFP (Fig. 7A); at the same time, we constructed a structural protein (E, envelope; M, membrane; N, nuclear capsid) and accessory protein expression vectors.
  • accessory protein expression vectors To verify the activity of the SARS-CoV-2 Stru ⁇ S-EGFP plasmid, the ORF3a and N genes were analyzed by RT-PCR in 293T cells (Fig. 7B); EGFP expression was observed 24 hours after plasmid transfection (Fig. 7C), and The expression of SARS-CoV-2 N protein was determined by western blotting (Fig. 7D).
  • VLP The assembly and secretion of VLP (Stru ⁇ S-S) depend on the co-expression of SARS-CoV-2 S plasmid and Stru ⁇ S-EGFP plasmid; 24h after transfection, the fluorescence of EGFP was detected by confocal microscope, and SARS-CoV was detected by Western blot -2 S and N proteins, which indicated that SARS-CoV-2 S and Stru ⁇ S-EGFP RNA could be expressed in transfected cells ( Figure 9A and 9B). In order to characterize its shape and size, VLP (Stru ⁇ S-S) was negatively stained and observed under TEM under TEM (Fig.
  • VLP Stru ⁇ S-S
  • FIG. 9B we also performed Western blot analysis on the structural proteins of the assembled VLP (Stru ⁇ S-S), and bands with expected molecular weights were also obtained ( FIG. 9B ).
  • the rescued VLPs were tested for safety in a BSL-3 laboratory.
  • the 293T/hACE2 cell line we constructed is susceptible to SARS-CoV-2 infection; the hACE2 receptor expressed by the cells can be verified by western blotting ( Figure 11A).
  • 293T/hACE2 cells were de-infected with SARS-CoV-2 VLPs to test whether the infected cells produced progeny virus. Whether the viral structural proteins are expressed in the cells and assembled into progeny viruses is detected by Western blot, and the results show that no corresponding bands can be observed. The results show that SARS-CoV-2 VLP cannot replicate, express structural proteins, and cannot assemble in host cells to produce progeny viruses.
  • SARS-CoV-2 VLPs Representative images of 293T/hACE2 cells infected with SARS-CoV-2 VLPs and wild-type SARS-CoV-2 virus showed significant differences in virus titers due to the inability of SARS-CoV-2 VLPs to replicate (Fig. 11C). Therefore, the SARS-CoV-2 VLP we constructed can be used as a safe and suitable model to study the life cycle and infection mechanism of the SARS-CoV-2 virus, and it is expected to further understand the life cycle of the virus, which can be used for antiviral drugs and vaccines. provide a theoretical basis for the development of
  • VLP mCherry was assembled by co-transfection of SARS-CoV-2 S plasmid, Stru ⁇ S-EGFP plasmid and mCherry plasmid (obtained by directly inserting mCherry into pEGFP-N1 vector).
  • the nucleocapsid of coronavirus interacts with viral RNA by recognizing a specific packaging signal (PS) sequence to promote the assembly of genomic RNA into virus particles.
  • PS packaging signal
  • Bioinformatics analysis was used to predict the PS position of SARS-CoV-2, and the prediction results showed that the PS was near the 3' end of the ORF1b region.
  • RNA stem-loops are highly conserved, especially SL1 and SL2 (Fig. 12). These results suggest that the stable stem-loop structure may have PS function in SARS-CoV-2.
  • PS101, nt 19900-20000 a conserved stem-loop structure
  • PS583, nt 19773-20335 additional flanks A longer region of the sequence
  • the predicted PS sequence was fused with the 3' non-coding region of the EGFP reporter gene, and the plasmids pEGFP-N1-PS101 and pEGFP-N1-PS583 were constructed and transfected into 293T cells respectively; EGFP-PS101 and EGFP-PS583 RNA The expression of was determined by RT-PCR (Fig. S8A and 3B). To confirm the assembly of the predicted PS RNA, VLPs (EGFP-PS101) and VLPs (EGFP-PS583) were collected and used to infect 293T/hACE2 cells, as shown in Figure 3E.
  • SARS-CoV-2 S plasmid, Stru ⁇ S plasmid and pEGFP-N1-PS101/pEGFP-N1-PS583 plasmid were co-transfected into 293T cells to produce VLP (EGFP-PS101) or VLP (EGFP-PS583), respectively.
  • VLP EGFP-PS101
  • EGFP-PS583 VLP
  • S protein, N protein, and EGFP were expressed and assembled into VLPs, and several proteins were analyzed by western blot (Fig. 13B and 3C). Structural proteins were detected in VLPs, whereas EGFP could not be detected.
  • VLP (EGFP) was obtained by co-transfection of SARS-CoV-2 S plasmid, Stru ⁇ S plasmid and pEGFP-N1 plasmid, and as a control group, EGFP mRNA without PS sequence on the surface could not be packaged into VLP (green fluorescent protein) . These results confirm that the predicted sequence has a PS function in SARS-CoV-2, which is important for the assembly of viral genomic RNA into VLPs.
  • VLP S (mutation) was assembled after co-transfection of cells with SARS-CoV-2 S (mutation) plasmid, Stru ⁇ S plasmid and ORF1ab plasmid.
  • SARS-CoV-2 S and N proteins in the virions were identified by Western blot, and the expected bands are shown in Figure 18.
  • the morphology and size of VLP S (mutant) were analyzed by transmission electron microscopy, and the results showed that the mutant was similar to the VLP with wild-type S protein ( Figure 18).
  • 293T/hACE2 cells were infected with DiO-labeled lipid envelope to produce fluorescent VLP S(mutant).
  • VLP(N501Y), VLP(D614G) and VLP(P681H) had higher infectivity, while the infectivity of VLP(N331Q) was significantly lower (Fig. 4A). The results are consistent with previous reports, suggesting that the system we constructed can be used to assess the ability of S mutations to affect SARS-CoV-2 assembly and infectivity.
  • VLP( ⁇ N) can be obtained by co-transfecting cells with SARS-CoV-2 S plasmid, Stru ⁇ S-EGFP/ ⁇ N plasmid and ORF1ab three plasmids.
  • the generated VLPs were detected by western blotting, and the S protein was detected, but the N protein was not detected, which was consistent with the expected results (Fig. 4C).
  • Whether the viral RNA assembly depends on nucleocapsid was determined by infecting 293T/hACE2 cells with VLP ( ⁇ N-EGFP-PS583); Stru ⁇ S/ ⁇ N plasmid and packaging signal plasmid pEGFP-N1-PS583 co-transfected to produce. Little green fluorescence of EGFP was observed in VLP( ⁇ N-EGFP-PS583)-infected cells, as shown in Figure 4B; this was significantly different from VLP(EGFP-PS583)-infected cells. The results showed that the N protein is not essential for VLP formation, but it plays an important role in viral RNA packaging.
  • M and E proteins as the main functional components, played an important role in the production of virus particles; this study also explored the influence of M and E proteins on VLP production .
  • the M or E protein coding sequences were deleted, respectively, to obtain the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ M plasmid and the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ E plasmid, which were verified by RT-PCR and Western blotting ( Figures 20 and 22 ).
  • the Stru ⁇ S mutant plasmid that is, the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ M plasmid or the SARS-CoV-2 Stru ⁇ S-EGFP/ ⁇ E plasmid was co-transfected with the plasmids SARS-CoV-2 S and ORF1ab-mCherry into 293T cells, Stru ⁇ S mutant virions rVP( ⁇ M) or rVP( ⁇ E) were produced, respectively.
  • TEM images and Western blot analysis showed that virus particles lacking M or E protein were still available in transfected cells (Fig. 4C, 21A and 23A).
  • Stru ⁇ S mutant virus particles were further used to infect 293T/hACE2 cells, and no fluorescent signal could be observed after 48 hours of infection (Fig. In the absence of M or E genes, transfected cells can also produce mutant virions, but the function of virions is affected. Unlike N-deleted mutants, deletion of M or E proteins did not affect viral RNA packaging.
  • the open reading frame 10 (ORF10) region of the SARS-CoV-2 genome is located downstream of the N gene, and the ORF10 protein did not seem to play a significant role in previous reports.
  • ORF10 open reading frame 10
  • the VLP ( ⁇ ORF10) exhibited similar morphology and function to our constructed SARS-CoV-2 VLP (Fig. 4C), indicating that deletion of the ORF10 gene had no effect on viral particle assembly.
  • RNA-QD-DiO double fluorescently labeled SARS-CoV-2 VLPs
  • FIG. 5A The viral RNA hybridizes with the target nucleic acid sequence to form a complex, which is labeled with a quantum dot (QD) nano-beacon with red fluorescence, and the complex is finally wrapped by the virus particle. A dark electron-dense core was observed inside the QD-labeled virus (Fig. 5B). To obtain dual fluorescent particles, the lipid envelope was labeled with DiO.
  • the fluorescent colocalization of RNA-QD and Env-DiO confirmed the successful construction of two-color rSARS-CoV-2 (QD-DiO) (Fig. 5C), where rSARS-CoV-2 is also known as SARS-CoV-2 VLP.
  • SARS-CoV-2 VLP(QD-DiO) particles were tracked in 293T/hACE2 cells by confocal microscopy imaging. During live imaging, only those containing QD and DiO signals co-localized were considered as single virions. Bicolor granules were observed on the membrane of 293T/hACE2 cells and were actively transported into the host cells (Fig. 5D). The trajectory of this virus particle is shown in Figure 5E. In this study, we tracked and analyzed the trajectories of more than 2000 single virus particles in living cells. The results showed that 47.5% of virus particles entered into 293T/hACE2 cells through endocytosis, while other particles only attached to the cell membrane surface and were not transported into the cytoplasm.
  • RNA-QD and Env-DiO were imaged in the cytoplasm, and separation of red and green dots was observed during the dynamic motion of the virion (Fig. 5F).
  • the trajectories of RNA-QD and Env-DiO were different after the separation behavior, as shown in Fig. 5G.
  • SARS-CoV-2 virus genome split system (split-virus-genom, SVG) and performed corresponding characterization analysis. This system was used to generate SARS-CoV-2 VLPs with a single round of infection, which is expected to be used for biological exploration and vaccine development.
  • the SARS-CoV-2 ORF1ab RNA containing the packaging signal was assembled into rSARS-CoV-2 without the structural gene in the virion, thus ensuring the safety of the system.
  • SARS-CoV-2 packaging signal provides a reliable way to study the molecular mechanism involved in viral RNA assembly.
  • Single virus particle tracking based on multicolor labeled SARS-CoV-2 VLPs enables real-time and precise imaging of virus particle infection process in living host cells.
  • the assembled VLPs with different components facilitate the development of vectors and vaccines. Combining these advantages, the SVG system could develop into a valuable platform for studying SARS-CoV-2 and other coronaviruses.
  • SVG-CoV-2 The full genome of SARS-CoV-2 is divided into three parts, and the packaging signal necessary for viral RNA assembly is located in the ORF1ab fragment. Our system lacks multiple gene segments and does not produce wild-type virus. Unlike pseudoviruses that contain partially functional structures, our virions have almost all components of SARS-CoV-2. However, due to the lack of structural genes in the virally packaged RNA, SARS-CoV-2 VLPs are unable to replicate and assemble progeny viruses. These single rounds of infectious virions are unable to infect host cells for multiple rounds, blocking viral transmission.
  • the SARS-CoV-2 M protein plays a major role in viral function but is not essential for viral particle formation. It differs from SARS-CoV, where the M protein is a key factor in virus assembly.
  • the system we developed also has some limitations. The efficiency of virus assembly needs to be improved, which is of great significance for the further improvement of the system and the development of vaccines.
  • the SVG system, composition and method of the present invention can be used as a powerful tool to promote neutralization tests and anti-virus tests, and a variety of pseudovirus particles can be obtained by using the virus genome splitting system, which can be used for neutralizing antibody tests of virus antibodies, Screening of antiviral drugs, preparation of virus vaccines, etc.
  • virus genome splitting system which can be used for neutralizing antibody tests of virus antibodies, Screening of antiviral drugs, preparation of virus vaccines, etc.
  • S-fragment 1 (SEQ ID NO: 1):
  • S-fragment 2 (SEQ ID NO: 2):
  • ORF1ab-fragment 1 (SEQ ID NO: 3):
  • ORF1ab-fragment 2 (SEQ ID NO: 4):
  • ORF1ab-fragment 3 (SEQ ID NO: 5):
  • ORF1ab-fragment 4 (SEQ ID NO: 6):
  • ORF1ab-fragment 5 (SEQ ID NO: 7):
  • ORF1ab-fragment 6 (SEQ ID NO: 8):
  • Stru ⁇ S-fragment 1 (SEQ ID NO: 9):
  • Stru ⁇ S-fragment 2 (SEQ ID NO: 10):
  • Stru ⁇ S-fragment 3 (SEQ ID NO: 11):

Abstract

L'invention concerne une composition pour préparer des particules pseudo-virales de SARS-CoV-2. La composition comprend un premier plasmide contenant un fragment Stru ΔS, et l'un quelconque ou deux parmi un deuxième plasmide contenant un fragment S et un troisième plasmide contenant un fragment de signal d'encapsidation virale de SARS-CoV-2. Le procédé de préparation de la composition comprend les étapes consistant à : diviser respectivement un fragment Stru ΔS, un fragment S et/ou un fragment de signal d'encapsidation ORF1ab en fragments d'ADN courts ayant des séquences homologues entre des fragments adjacents, et réaliser une recombinaison homologue sur les fragments d'ADN courts et un vecteur plasmidique linéaire dans une cellule de levure. La présente invention concerne en outre un procédé de préparation de particules pseudo-virales de SARS-CoV-2 à l'aide de la composition, les particules pseudo-virales de SARS-CoV -2 obtenues, et l'utilisation de la composition dans la préparation d'un vaccin pour la prévention ou le traitement d'infections par le virus de SARS-CoV-2 et dans la recherche in vitro sur des cellules infectées par le virus de SARS-CoV-2.
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