WO2023070873A1 - Method for preparing sars-cov-2 virus-like particles and use of sars-cov-2 virus-like particles - Google Patents

Method for preparing sars-cov-2 virus-like particles and use of sars-cov-2 virus-like particles Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
cov
sars
plasmid
virus
protein
Prior art date
Application number
PCT/CN2021/138037
Other languages
French (fr)
Chinese (zh)
Inventor
戴俊彪
马英新
毛国斌
Original Assignee
中国科学院深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院深圳先进技术研究院 filed Critical 中国科学院深圳先进技术研究院
Publication of WO2023070873A1 publication Critical patent/WO2023070873A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

A composition for preparing SARS-CoV-2 virus-like particles. The composition comprises a first plasmid containing a Stru ΔS fragment, and any one or two selected from a second plasmid containing an S fragment and a third plasmid containing a packaging signal fragment of the SARS-CoV-2 virus. The method for preparing the composition comprises: respectively splitting a Stru ΔS fragment, an S fragment and/or an ORF1ab packaging signal fragment into short DNA fragments having homologous sequences between adjacent fragments, and performing homologous recombination on the short DNA fragments and a linear plasmid vector in a yeast cell. The present invention further relates to a method for preparing SARS-CoV-2 virus-like particles using the composition, the obtained SARS-CoV-2 virus-like particles, and the use of the composition in the preparation of a vaccine for preventing or treating SARS-CoV-2 virus infections and in the in-vitro research on cells infected by the SARS-CoV-2 virus.

Description

SARS-CoV-2病毒样颗粒的制备方法及其应用Preparation method and application of SARS-CoV-2 virus-like particles 技术领域technical field
本发明涉及分子生物学技术领域，特别是涉及SARS-CoV-2病毒样颗粒的制备方法及其应用。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.
背景技术Background technique
病毒样颗粒(VLP)是一种由病毒单一或多个结构蛋白自行装配而成的高度结构化的蛋白颗粒。VLP在宿主内无法实现复制繁殖，但是其包含部分或全部蛋白，仍然可以很好地引起宿主的强免疫响应。因此，VLP可以作为一种潜在的高效疫苗制剂。一般而言，所有的VLP均可形成独特的结构，其表面布满了重复的蛋白结构，其较强的免疫原性使其可作为疫苗。VLP在疫苗研究中倍受关注，目前上市或在研的VLP疫苗中，人用VLP疫苗主要有HBV疫苗、HPV疫苗以及HEV疫苗，而兽用VLP疫苗为用于预防猪圆环病毒2型。在研的人用VLP疫苗还涉及了多种病毒，包括流感病毒、艾滋病病毒、埃博拉病毒等，在研兽用VLP疫苗则还有口蹄疫病毒、猪细小病毒疫苗、猪瘟病毒等。然而，目前大部分的VLP中只含有部分或者全部的结构蛋白，在对非结构蛋白、辅助蛋白等方面的研究受限，且作为疫苗引起的免疫反应有限。Virus-like particle (VLP) is a highly structured protein particle self-assembled from a single or multiple structural proteins of the virus. VLP cannot achieve replication and reproduction in the host, but it contains part or all of the protein and can still elicit a strong immune response from the host. Therefore, VLP can be used as a potential high-efficiency vaccine formulation. Generally speaking, all VLPs can form a unique structure, and its surface is covered with repeated protein structures. Its strong immunogenicity makes it suitable for vaccines. VLP has attracted much attention in vaccine research. Among the VLP vaccines currently on the market or under research, 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., and the veterinary VLP vaccines under development include foot-and-mouth disease virus, porcine parvovirus vaccine, and swine fever virus. However, 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.
新型冠状病毒(SARS-CoV-2)是一种单链RNA病毒，具有较高的病毒变异能力，其变异毒株具有更高的病毒传染力，因此，疫苗的开发尤为重要。尽管基于多种技术的SARS-CoV-2疫苗已经进入临床阶段，基于SARS-CoV-2 VLP疫苗研究较少。其中一个关键的原因是缺乏一个简单，高效的SARS-CoV-2 VLP合成与制备平台。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. Although 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.
发明内容Contents of the invention
鉴于目前存在的技术问题，本发明提供了一种SARS-CoV-2 VLP从头人工合成及制备方法，包括将SARS-CoV-2基因组拆分为多个片段，合成这些片段并将其组装在载体上，转染包装细胞获得VLP。In view of the current technical problems, 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.
本发明的第一方面提供一种用于制备SARS-CoV-2病毒样颗粒的组合物，包括下述(a)，还包括选自下述(b)和(c)中的任一种或任两种：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)包含StruΔS片段的第一质粒，所述ΔS片段包含编码SARS-CoV-2病毒的至少一种结构蛋白或其突变体和/或至少一种辅助蛋白或其突变体的核酸序列，且所述ΔS片段不包含编码SARS-CoV-2病毒的S蛋白或其突变体的核酸序列；(a) a first plasmid comprising a StruΔS 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 ΔS fragment does not include a nucleic acid sequence encoding the S protein of SARS-CoV-2 virus or a mutant thereof;
(b)包含S片段的第二质粒，所述S片段包含编码SARS-CoV-2病毒的S蛋白或其突变体的核酸序列；(b) the second plasmid that comprises S segment, and described S segment comprises the nucleic acid sequence of the S protein of coding SARS-CoV-2 virus or its mutant;
(c)包含SARS-CoV-2病毒的包装信号片段的第三质粒，所述包装信号片段包含SARS-CoV-2病毒的包装信号序列。(c) 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.
在一些实施方案中，所述的S蛋白的突变体相对于SARS-CoV-2病毒的S蛋白的突变包括N331Q、N501Y、D614G和/或P681H。In some embodiments, the mutation of the S protein relative to the S protein of SARS-CoV-2 virus includes N331Q, N501Y, D614G and/or P681H.
在一些实施方案中，所述StruΔS片段包含下述任一种：In some embodiments, the StruΔS fragment comprises any of the following:
(1)编码SARS-CoV-2病毒的除S蛋白之外的全部结构蛋白和全部辅助蛋白的核酸序列；(1) Nucleic acid sequences encoding all structural proteins and all auxiliary proteins of the SARS-CoV-2 virus except the S protein;
(2)与(1)相比，所述结构蛋白和辅助蛋白中的至少一种相对于野生型SARS-CoV-2病毒具有突变；和(2) Compared with (1), at least one of the structural protein and accessory protein has a mutation relative to the wild-type SARS-CoV-2 virus; and
(3)与(1)相比，缺失所述结构蛋白和辅助蛋白中的任一种。(3) Compared with (1), any one of the structural protein and auxiliary protein is deleted.
在一些实施方案中，所述包装信号序列是NCBI序列号NC_045512.2的第19900-20000位核苷酸或NCBI序列号NC_045512.2的第19773-20335位核苷酸。In some embodiments, 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.
在一些实施方案中，所述包装信号片段包含SARS-CoV-2病毒基因组的ORF1ab。In some embodiments, the packaging signal fragment comprises ORF1ab of the SARS-CoV-2 viral genome.
本发明的另一方面提供了上述组合物的制备方法，包括分别通过下述方法制备第一质粒、第二质粒和/或第三质粒：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:
(1)将StruΔS片段、S片段和/或ORF1ab包装信号片段分别拆分为在相邻片段之间具有同源序列的DNA短片段；(1) Separate the StruΔS fragment, the S fragment and/or the ORF1ab packaging signal fragment into short DNA fragments with homologous sequences between adjacent fragments;
(2)将由步骤(1)获得的DNA短片段和线性化质粒载体转入酵母细胞中，进行同源重组，分别获得第一质粒、第二质粒和/或第三质粒。(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.
本发明的另一方面提供了SARS-CoV-2病毒样颗粒的制备方法，包括：用上述任一种的组合物转染包装细胞，获得SARS-CoV-2病毒样颗粒。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.
在一些实施方案中，该制备方法，还包括分别通过下述方法制备第一质粒、第二质粒和/或第三质粒：In some embodiments, the preparation method also includes preparing the first plasmid, the second plasmid and/or the third plasmid by the following methods:
(1)将StruΔS片段、S片段和/或包装信号片段分别拆分为在相邻片段之间具有同源序列的DNA短片段；(1) Separate the StruΔS fragment, the S fragment and/or the packaging signal fragment into short DNA fragments with homologous sequences between adjacent fragments;
(2)将由步骤(1)获得的DNA短片段和线性化质粒载体转入酵母细胞中，进行同源重组，分别获得第一质粒、第二质粒和/或第三质粒。(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.
在一些实施方案中，所述制备方法，进一步包括将第一质粒、第二质粒和/或第三质粒分别在大肠杆菌中进行扩增，然后转染包装细胞。In some embodiments, 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.
本发明的另一方面提供由上述制备方法制备获得的SARS-CoV-2病毒样颗粒。Another aspect of the present invention provides SARS-CoV-2 virus-like particles prepared by the above preparation method.
本发明的另一方面提供由上述组合物组合物或由上述制备方法制备获得的SARS-CoV-2病毒样颗粒在制备用于预防或治疗SARS-CoV-2病毒感染的疫苗中的用途。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.
本发明的另一方面提供由上述组合物组合物或由上述制备方法制备获得的SARS-CoV-2病毒样颗粒在体外用于SARS-CoV-2病毒感染细胞的研究中的用途。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.
所述SARS-CoV-2病毒感染细胞的研究可以是研究VLP与细胞的相互作用、研究病毒对细胞的侵染过程和/或研究病毒成分的作用等。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.
本发明与现有技术相比，具有以下优点和效果：Compared with the prior art, the present invention has the following advantages and effects:
A、本发明利用哺乳细胞真核表达系统表达SARS-CoV-2的结构蛋白、非结构蛋白及辅助蛋白等以组装成VLP，可用于疫苗，该疫苗制备方法不涉及活病毒。相比于灭活病毒疫苗和减毒活疫苗，该方法安全性更好；相比于多肽或核酸疫苗，该方法免疫原性更好。A. 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.
B、本发明采用病毒基因组拆分的方法，先合成小片段基因，再通过酵母同源重组系统进行基因组组装，该方法简便、快速且效率高。B. 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.
C、本发明利用基因组拆分策略，VLP中含有结构蛋白与非结构蛋白等完整的病毒粒子形貌，免疫原性强，有望发展成为具有交叉保护效力的高效疫苗。C. 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.
附图说明Description of drawings
通过以下详细的描述并结合附图将更充分地理解本发明，其中相似的元件以相似的方式编号，其中：The present invention will be more fully understood from the following detailed description when taken in conjunction with the accompanying drawings, in which like elements are numbered in a like manner, in which:
图1显示了SARS-CoV-2假病毒的生成。(A)全长SARS-CoV-2基因组和拆分部分的方案，包括SARS-CoV-2 S、ORF1ab和StruΔS基因组。(B)分离的SARS-CoV-2基 因组在酵母中组装以构建质粒SARS-CoV-2 S、ORF1ab和StruΔS。(C)SARS-CoV-2假病毒是通过共转染质粒SARS-CoV-2 S、ORF1ab和StruΔS获得的。SARS-CoV-2假病毒感染293T/hACE2细胞后不能产生子代病毒。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 StruΔS genomes. (B) The isolated SARS-CoV-2 genome was assembled in yeast to construct the plasmids SARS-CoV-2 S, ORF1ab, and StruΔS. (C) SARS-CoV-2 pseudoviruses were obtained by co-transfection of plasmids SARS-CoV-2 S, ORF1ab, and StruΔS. SARS-CoV-2 pseudovirus cannot produce progeny virus after infection of 293T/hACE2 cells.
图2显示了SARS-CoV-2假病毒的构建和表征。(A)转染后24小时，共转染质粒SARS-CoV-2 S、ORF1ab-mCherry和StruΔS-EGFP的293T细胞的共聚焦显微镜图像。(B)通过蛋白质印迹检测细胞裂解液(上)和SARS-CoV-2假病毒(下)中的SARS-CoV-2 S和N蛋白。GAPDH用作蛋白质印迹的上样对照。(C)TEM检测到的SARS-CoV-2假病毒和VLPs的形态和大小。比例尺：20nm。通过共转染质粒SARS-CoV-2 S、ORF1ab-mCherry和StruΔS-EGFP获得的SARS-CoV-2假病毒用于293T/hACE2细胞侵染的共聚焦显微镜图像(D)和实验方案(E)。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 StruΔS-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. Confocal microscope image (D) and experimental protocol (E) of SARS-CoV-2 pseudovirus obtained by co-transfection of plasmids SARS-CoV-2 S, ORF1ab-mCherry and StruΔS-EGFP for 293T/hACE2 cell infection .
图3显示了包装信号(PS)的分析和验证。(A)使用Biopython软件分析SARS-CoV-2、SARS-CoV和类蝙蝠SARS-CoV的ORF1b区域的序列(nt 19500-20400)。这三种SARS相关病毒中相同位置的二级结构由RNAstructure Web Server使用默认参数预测，并由Vienna RNA Web Server进行分析。两个相同的RNA茎环(SL1和SL2)用矩形框指示。(B)由质粒EGFP-PS583转染的细胞裂解液中PS583基因的RT-PCR分析。红色箭头指示预期的扩增序列。nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液(左)和VLP(EGFP-PS583)(右)中的EGFP、SARS-CoV-2 S和N蛋白。GAPDH用作蛋白质印迹和RT-PCR的上样对照。用VLP(EGFP-PS583)侵染293T/hACE2细胞48小时后的共聚焦显微镜成像(D)和实验方案(E)。用VLP(EGFP-PS583)侵染293T细胞和VLP(EGFP)侵染293T/hACE2细胞作为对照。。Figure 3 shows the analysis and validation of the packaging signal (PS). (A) The sequences of the ORF1b region (nt 19500-20400) of SARS-CoV-2, SARS-CoV and bat-like SARS-CoV were analyzed using Biopython software. The secondary structures at the same positions in these three SARS-associated viruses were predicted by RNAstructure Web Server using default parameters and analyzed by Vienna RNA Web Server. Two identical RNA stem-loops (SL1 and SL2) are indicated by rectangular boxes. (B) RT-PCR analysis of PS583 gene in lysates of cells transfected with plasmid EGFP-PS583. Red arrows indicate expected amplified sequences. nRT: Not reverse transcriptome. (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. Confocal microscopy image (D) and experimental scheme (E) of 293T/hACE2 cells infected with VLP (EGFP-PS583) for 48 hours. 293T cells were infected with VLP (EGFP-PS583) and 293T/hACE2 cells were infected with VLP (EGFP) as controls. .
图4显示了不同SARS-CoV-2蛋白的功能验证。(A)细胞质中DiO标记的SARS-CoV-2 VLP的统计分析，以测试SARS-CoV-2 S突变对病毒侵染能力的影响。(B)VLP(EGFP-PS583)、VLP(ΔN-EGFP-PS583)或VLP(EGFP)侵染293T/hACE2细胞后的共聚焦显微镜成像图，观察侵染后48小时后细胞内的绿色荧光。(C)通过蛋白质印迹检测不同类型VLP中的S和N蛋白。通过TEM观察VLP的形态和大小。比例尺：20nm。Figure 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 (ΔN-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.
图5显示了(A)双荧光标记VLP(QD-DiO)的方案。(B)通过TEM表征VLP(QD-DiO)的形态和大小，白色箭头指示QD。比例尺：20nm。(C)VLP(QD-DiO)中DiO和QD信号的共定位。(D)VLP(QD-DiO)进入293T/hACE2细胞的动态过程。(E)293T/hACE2细胞的微分干涉对比(DIC)图像。黑线指示了病毒的轨迹。(F,G)分析(D)中显示的病毒粒子的平均速度(F)和MSD图(G)。(H)VLP(QD-DiO)的RNA-QD和Env-DiO在293T/hACE2细胞中实时解离的动态过程。(I-K)RNA-QD和Env-DiO的轨迹(I)、平均速度(J)和MSD图(K)。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). (H) Dynamic process of RNA-QD and Env-DiO dissociation of VLP(QD-DiO) in real time in 293T/hACE2 cells. (I-K) Trajectories (I), average velocity (J) and MSD plots (K) of RNA-QD and Env-DiO.
图6显示了SARS-CoV-2 S质粒的构建和表征。(A)细胞裂解液中SARS-CoV-2 S蛋白编码基因的RT-PCR分析。红色箭头指示预期的扩增序列。nRT：未逆转录组。(B)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 S蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。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.
图7显示了SARS-CoV-2 StruΔS-EGFP质粒的构建和表征。(A)StruΔS-EGFP构建的示意图。(B)细胞裂解液中SARS-CoV-2 ORF3a和N蛋白编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，GAPDH用作上样对照，nRT：未逆转录组。(C)SARS-CoV-2 StruΔS-EGFP质粒转染293T细胞24小时后的荧光成像。(D)通过蛋白质印迹检测细胞裂解液(左)和VLP(右)的N蛋白，GAPDH用作上样对照。(E)通过透射电子显微镜检测SARS-CoV-2 VLP(StruΔS)的形态和大小。比例尺：20nm。Figure 7 shows the construction and characterization of the SARS-CoV-2 StruΔS-EGFP plasmid. (A) Schematic representation of the StruΔS-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 StruΔS-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 (StruΔS) detected by transmission electron microscopy. Scale bar: 20 nm.
图8显示了SARS-CoV-2 ORF1ab-mCherry质粒的构建和表征。(A)ORF1ab-mCherry构建的示意图。(B)细胞裂解物中SARS-CoV-2 NSP1和NSP16编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，GAPDH用作上样对照，nRT：未逆转录组。(C)SARS-CoV-2 ORF1ab-mCherry质粒转染293T细胞24小时后的荧光成像。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.
图9显示了SARS-CoV-2 VLP(StruΔS-S)的构建和表征。(A)SARS-CoV-2 StruΔS-EGFP和S质粒共转染293T细胞24小时后的荧光成像。(B)通过蛋白质印迹检测细胞裂解液(上)和VLP(StruΔS-S)(下)的N蛋白，GAPDH用作上样对照。(C)通过透射电子显微镜检测SARS-CoV-2 VLP(StruΔS-S)的形态和大小。比例尺：20nm。Figure 9 shows the construction and characterization of SARS-CoV-2 VLP (StruΔS-S). (A) Fluorescence imaging of 293T cells co-transfected with SARS-CoV-2 StruΔS-EGFP and S plasmid for 24 hours. (B) Detection of N protein in cell lysates (top) and VLPs (StruΔS-S) (bottom) by Western blot, GAPDH was used as a loading control. (C) Morphology and size of SARS-CoV-2 VLP (StruΔS-S) detected by transmission electron microscopy. Scale bar: 20 nm.
图10显示了SARS-CoV-2 VLP(StruΔS-ORF1ab)的构建和表征。(A)SARS-CoV-2 StruΔS-EGFP和ORF1ab-mCherry质粒共转染293T细胞24小时后的荧光成像。(B)通过蛋白质印迹检测细胞裂解液(上)和VLP(StruΔS-ORF1ab)(下)的N蛋白，GAPDH用作上样对照。(C)通过透射电子显微镜检测SARS-CoV-2 VLP(StruΔS-ORF1ab)的形态和大小。比例尺：20nm。Figure 10 shows the construction and characterization of SARS-CoV-2 VLP (StruΔS-ORF1ab). (A) Fluorescent imaging of 293T cells co-transfected with SARS-CoV-2 StruΔS-EGFP and ORF1ab-mCherry plasmids for 24 hours. (B) Detection of N protein in cell lysates (top) and VLP (StruΔS-ORF1ab) (bottom) by Western blot, GAPDH was used as a loading control. (C) Morphology and size of SARS-CoV-2 VLP (StruΔS-ORF1ab) detected by transmission electron microscopy. Scale bar: 20 nm.
图11显示了假SARS-CoV-2病毒生物安全性的验证。(A)通过蛋白质印迹检测构建293T/hACE2细胞上hACE2受体的表达。(B)通过蛋白质印迹检测细胞裂解液(左)和上清(右)的S和N蛋白。(C)通过VLP或质粒SARS-CoV-2 S，ORF1ab-mCherry和StruΔS-EGFP共转染后感染293T/hACE2细胞48小时，GAPDH用作上样对照。(C)感染野生型SARS-CoV-2病毒和SARS-CoV-2 VLP后72小时后的293T/hACE2细胞图像。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 StruΔS-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.
图12显示了基于比对共有序列的RNA结构预测。多重比对LocARNA使用默认参数预测的蝙蝠SARS样冠状病毒PS(MG772933.1:19773-20355)，SARS PS(NC-004718.3:19712-20294)和SARS-CoV-2 PS(NC-045512.2:19773-20355)的共有结构。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.
图13显示了包装信号的分析和验证。(A)通过RT-PCR检测细胞裂解液中PS101基因，红色箭头指示预期的扩增序列，GAPDH用作上样对照，nRT：未逆转录组。(B)通过蛋白质印迹检测细胞裂解液(左)和VLP(右)的S，N蛋白和EGFP，GAPDH用作上样对照。(C)VLP(EGFP-PS101)和VLP(EGFP)感染293T/hACE2细胞48小时后的荧光成像。Figure 13 shows the analysis and validation of packaged signals. (A) 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.
图14显示了SARS-CoV-2 S(N331Q)构建和表征。(A)SARS-CoV-2 S(N331Q)构建的示意图。(B)细胞裂解液中SARS-CoV-2 S(N331Q)编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，nRT：没有逆转录。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 S(N331Q)蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。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.
图15显示了SARS-CoV-2 S(N501Y)构建和表征。(A)SARS-CoV-2 S(N501Y)构建的示意图。(B)细胞裂解物中SARS-CoV-2 S(N501Y)编码基因的RT-PCR分析，红色箭头表示预期的扩增子，nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 S(N501Y)蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。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.
图16显示了SARS-CoV-2 S(D614G)构建和表征。(A)SARS-CoV-2 S(D614G)构建的示意图。(B)细胞裂解液中SARS-CoV-2 S(D614G)编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 S(D614G)蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。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.
图17显示了SARS-CoV-2 S(P681H)构建和表征。(A)SARS-CoV-2 S(P681H)构建的示意图。(B)细胞裂解液中SARS-CoV-2 S(P681H)编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 S(P681H)蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。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.
图18显示了具有S蛋白突变的SARS-CoV-2 VLP的构建和表征。通过蛋白质印迹检测VLP(N331Q)(A)，VLP(N501Y)(B),VLP(D614G)(B)和VLP(P681H)(D)。通过透射电子显微镜检测SARS-CoV-2 VLP的形态和大小。比例尺：20nm。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.
图19显示了SARS-CoV-2 StruΔS-EGFP/ΔN质粒的构建和表征。(A)SARS-CoV-2 StruΔS-EGFP/ΔN质粒构建示意图。(B)细胞裂解液中SARS-CoV-2 ORF3a和N蛋白编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 N蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。Figure 19 shows the construction and characterization of the SARS-CoV-2 StruΔS-EGFP/ΔN plasmid. (A) Schematic diagram of the construction of the SARS-CoV-2 StruΔS-EGFP/ΔN 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.
图20显示了SARS-CoV-2 StruΔS-EGFP/ΔE质粒的构建和表征。(A)SARS-CoV-2 StruΔS-EGFP/ΔE质粒构建示意图。(B)细胞裂解液中SARS-CoV-2 ORF3a，E和N蛋白编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 N蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。Figure 20 shows the construction and characterization of the SARS-CoV-2 StruΔS-EGFP/ΔE plasmid. (A) Schematic diagram of the construction of the SARS-CoV-2 StruΔS-EGFP/ΔE 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.
图21显示了E蛋白缺失的SARS-CoV-2 VLP(VLP/ΔE)的构建和表征。(A)通过透射电子显微镜检测VLP/ΔE的形态和大小。比例尺：20nm。(B)VLP/ΔE感染293T/hACE2细胞48小时后的共聚焦显微镜成像图。Figure 21 shows the construction and characterization of SARS-CoV-2 VLP (VLP/ΔE) with E protein deletion. (A) Morphology and size of VLP/ΔE detected by transmission electron microscopy. Scale bar: 20 nm. (B) Confocal microscopy images of 293T/hACE2 cells infected with VLP/ΔE for 48 hours.
图22显示了SARS-CoV-2 StruΔS-EGFP/ΔM质粒的构建和表征。(A)SARS-CoV-2 StruΔS-EGFP/ΔM质粒构建示意图。(B)细胞裂解液中SARS-CoV-2 ORF3a，M和N蛋白编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 N蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。Figure 22 shows the construction and characterization of the SARS-CoV-2 StruΔS-EGFP/ΔM plasmid. (A) Schematic diagram of the construction of the SARS-CoV-2 StruΔS-EGFP/ΔM 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.
图23显示了M蛋白缺失的SARS-CoV-2 VLP(VLP/ΔE)的构建和表征。(A)通过透射电子显微镜检测VLP/ΔM的形态和大小。比例尺：20nm。(B)VLP/ΔM感染293T/hACE2细胞48小时后的共聚焦显微镜成像图。Figure 23 shows the construction and characterization of M protein-deleted SARS-CoV-2 VLPs (VLP/ΔE). (A) Morphology and size of VLP/ΔM detected by transmission electron microscopy. Scale bar: 20 nm. (B) Confocal microscopy images of 293T/hACE2 cells infected with VLP/ΔM for 48 hours.
图24显示了SARS-CoV-2 StruΔS-EGFP/ΔORF10质粒的构建和表征。(A)SARS-CoV-2 StruΔS-EGFP/ΔORF10质粒构建示意图。(B)细胞裂解物中SARS-CoV-2 ORF3a，N和ORF10蛋白编码基因的RT-PCR分析，红色箭头指示预期的扩增序列，nRT：未逆转录组。(C)通过蛋白质印迹检测细胞裂解液中SARS-CoV-2 N蛋白，GAPDH用作蛋白质印迹和RT-PCR的上样对照。Figure 24 shows the construction and characterization of the SARS-CoV-2 StruΔS-EGFP/ΔORF10 plasmid. (A) Schematic diagram of the construction of the SARS-CoV-2 StruΔS-EGFP/ΔORF10 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.
图25显示了蛋白缺失的SARS-CoV-2 VLP的表征。(A)蛋白缺失SARS-CoV-2 VLP的质粒构建示意图。(B)蛋白缺失SARS-CoV-2 VLP的荧光共定位结果(DiO与QDs)。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.
具体实施方式Detailed ways
除非另外指出，本发明中所用的术语具有本领域通常所理解的含义，可以通过参考本领域技术人员已知的标准教科书，参考书、文献加以理解。本文所提供的定义和解释用于明确它们在本发明范围内的特定用途。本发明所引用的参考文献均通过引用将其全文并入本文。Unless otherwise indicated, the terms used in the present invention have meanings commonly understood in the art, which can be understood by referring to standard textbooks, reference books, and literature known to those skilled in the art. Definitions and explanations are provided herein to clarify their specific use within the scope of the present invention. All references cited herein are hereby incorporated by reference in their entirety.
不希望受到任何特定理论的束缚，本发明中对于相关理论或机理的解释仅仅是用于帮助理解发明，不应视为对本发明所保护的方案的限制。Not wishing to be bound by any particular theory, the explanation of the relevant theories or mechanisms in the present invention is only for helping to understand the invention, and should not be regarded as a limitation on the protection scheme of the present invention.
本发明中所使用的术语“包含”，或与其具有类似含义的其他术语形式“包括”、“含有”或“具有”等，应当理解为包括所列出的要素，但不排除其他要素的存在；这些术语也包括仅由所列出的要素组成的情况。术语“由……组成”是指仅由所列举的要素组成。术语“基本上由……组成”表示不排除对所涉及的方案没有显著影响的要素。The term "comprises" used in the present invention, or other term forms "comprising", "containing" or "having" with similar meanings, should be understood as including the listed elements, but not excluding the existence of other elements ; these terms also include the case where it consists only of the listed elements. The term "consisting of" means consisting only of the recited elements. The term "consisting essentially of" indicates that elements that do not have a significant effect on the formulation involved are not excluded.
除非另外特别说明，本文中的“a”、“an”或“the”包括单数形式和复数形式。术语“至少一个”或“至少一种”或其它类似表述的含义等同于“一个或更多个”或“一种或更多种”的含义。Unless specifically stated otherwise, "a", "an" or "the" herein includes both singular and plural forms. The term "at least one" or "at least one" or other similar expressions has the same meaning as "one or more" or "one or more".
本文中所述的数值范围，例如温度范围，时间范围，组成或浓度范围，或其他数值范围等，包括该范围内的端值、所有中间范围、子范围(例如在某个中间值和某个端值之间的范围)以及所有单个数值，特别是以整数数值为端值的中间范围、子范围和单个的整数数值。并且，所述数值范围中所描述的任何中间范围、子范围以及所有单个数值可以从所述的数值范围中排除。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.
本文中所述的“约”、“大约”的意思为所述数值的±20％、±18％、±15％、±12％、±10％、±9％、±8％、±7％、±6％、±5％、±4％、±3％、±2％、±1％或±0.5％的范围，所述范围包扩所述范围的端点及所述范围内的任一数值。"About" and "approximately" mentioned herein mean ±20%, ±18%, ±15%, ±12%, ±10%, ±9%, ±8%, ±7% of the stated value , ±6%, ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5%, the range includes the endpoints of the range and any value within the range .
本文中所述的术语“和/或”应当理解为由该术语连接的多个要素中的任何一个要素、任意两个要素、任意三个要素或任意的更多个要素组成的组合。The term "and/or" described herein should be understood as a combination of any one element, any two elements, any three elements or any more elements among the elements connected by the term.
本发明中，术语“核酸序列”和“核苷酸序列”可以互换使用，是指由碱基、糖和磷酸骨架连接构成的序列。本发明的核酸序列可以是脱氧核糖核酸序列(DNA)或核糖核酸序列(RNA)，且可包括天然碱基或非天然碱基，其可以是单链或双链，可以是编码序列或非编码序列。In the present invention, the terms "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.
本发明中，当表达基因或基因所编码的蛋白时，“载体”可以是质粒，在一些情况下，术语“载体”和“质粒”可以互换使用。In the present invention, 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.
本发明中，除非另有说明，核酸序列的描述通常是从5’端至3’端，氨基酸序列的描述通常是从N端至C端。In the present invention, unless otherwise specified, the description of nucleic acid sequence is usually from 5' end to 3' end, and the description of amino acid sequence is usually from N-terminus to C-terminus.
本发明的方法可以是在体外或体内进行。The methods of the invention can be performed in vitro or in vivo.
反向遗传学是病毒拯救(rescuing)或VLP制备的主要手段之一，病毒基因组的获得是其中最为重要的一步。核酸酶连接是获取病毒基因组的主要方法，但是该方法反应过程复杂且效率较低，同时，病毒全长基因组在转化大肠时存在较大的毒性。酵母体内同源重组法是利用酵母细胞内高效的同源重组系统来实现多个相互存在同源系列DNA片段的组装方法。本发明通过独特的基因组拆分设计，以及基因组的合成与酵母体内同源重组组装技术，将SARS-CoV-2的基因组拆分并组装成三个质粒，通过细胞转染，即可获得结构完整性高的SARS-CoV-2 VLP，为进一步的疫苗开发提供了很好的工具平台。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. At the same time, 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. Through the unique genome splitting design, as well as genome synthesis and yeast in vivo homologous recombination assembly technology, 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.
具体而言，本发明提供了SARS-CoV-2的基因组的三个片段，每一个片段被组装到一个质粒中，共获得三个质粒，使用这三个质粒中的任一个、任两个或三个共转染包装细胞，可以获得SARS-CoV-2的VLP。Specifically, 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.
所述的SARS-CoV-2可以是野生型的SARS-CoV-2或者是其任何突变体。野生型SARS-CoV-2的基因组可由NCBI序列号NC_045512.2确定。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.
所述三个片段分别是包含SARS-CoV-2的S蛋白的编码序列的S片段、包含除S蛋白之外的所有结构蛋白和所有辅助蛋白的编码序列的StruΔS片段、以及包含包装信号序列的包装信号片段。The three fragments are the S fragment containing the coding sequence of the S protein of SARS-CoV-2, the StruΔS fragment containing the coding sequences of all structural proteins and all auxiliary proteins except the S protein, and the StruΔS fragment containing the packaging signal sequence. Wraps a signal fragment.
SARS-CoV-2的全部结构蛋白包括S蛋白(刺突糖蛋白)、E蛋白(小包膜蛋白)、M蛋白(膜糖蛋白)和N蛋白(核衣壳蛋白)。SARS-CoV-2的全部辅助蛋白包括ORF3a、ORF3b、ORF6、ORF7a、ORF7b、ORF8和ORF10编码的蛋白。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.
所述S片段编码的S蛋白(刺突糖蛋白)可以是野生型S蛋白或其突变体。在一些实施方案中，S蛋白突变体相对于野生型S蛋白具有选自N331Q、N501Y、D614G及P681H的任一个或更多个的突变。野生型S蛋白序列可以是由NCBI序列号NC_045512.2的21563-25384位核苷酸编码的蛋白质序列，或者由NCBI序列号YP_009724390.1确定。在一些实施方案中，S片段中包含的编码S蛋白的序列是NCBI序列号NC_045512.2的第21563-25384位核苷酸。The S protein (spike glycoprotein) encoded by the S fragment may be a wild-type S protein or a mutant thereof. In some embodiments, 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. In some embodiments, the sequence encoding the S protein included in the S fragment is nucleotides 21563-25384 of NCBI sequence number NC_045512.2.
所述StruΔS片段编码结构蛋白和辅助蛋白中的任一种或任几种，例如E蛋白(小包膜蛋白)、M蛋白(膜糖蛋白)、N蛋白(核衣壳蛋白)中的任一种、任两种或三种，和/或辅助蛋白中的任一种或任几种，这些蛋白独立地可以是野生型或其突变体。虽然最优选地，本发明的StruΔS片段编码除S蛋白之外的所有结构蛋白和辅助蛋白，但在本发明的一些实施例中，相比于该最优选的方案而言，所述StruΔS片段所编码的蛋白也可以进一步缺少一种或更多种结构蛋白，和/或缺少一种或更多种辅助蛋白，例如，所述StruΔS片段所编码的蛋白相比于该最优选的方案，可以不包含E蛋白(小包膜蛋白)、M蛋白(膜糖蛋白)、N蛋白(核衣壳蛋白)中的任一种、任两种或三种，和/或可以不包含辅助蛋白中的任一种或更多种。野生型的结构蛋白和/或辅助蛋白是指野生型SARS-CoV-2的结构蛋白和/或辅助蛋白。在一些实施方案中，StruΔS片段中包含的编码结构蛋白和辅助蛋白的核酸是NCBI序列号NC_045512.2的第25393-29674位核苷酸。The StruΔS 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) One, any two or three, and/or any one or any several of auxiliary proteins, these proteins can be wild type or mutants thereof independently. Although most preferably, the StruΔS 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 StruΔS 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 StruΔS 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. In some embodiments, the nucleic acid encoding the structural protein and the accessory protein contained in the StruΔS fragment is nucleotides 25393-29674 of NCBI sequence number NC_045512.2.
所述包装信号片段至少包含SARS-CoV-2的包装信号序列，还可以包含SARS-CoV-2的ORF1ab序列中的其他序列。在一些实施方案中，所述包装信号序列为NCBI序列号NC_045512.2的第19900-20000位核苷酸或NCBI序列号NC_045512.2的第19773-20335位核苷酸。在一些实施方案中，所述包装信号片段可以包含ORF1ab的全长或其片段，即SARS-CoV-2的ORF1ab全长或其片段。ORF1ab的全长或片段中所任一种或任几种ORF可以是野生型或其突变体。在本发明的一些实施方案中，所述ORF1ab的片段可以缺少ORF1ab序列中的一种或更多种ORF，但至少包含包装信号序列。野生型的ORF1ab序列中的ORF是指野生型SARS-CoV-2的ORF1ab序列中的ORF。在一些实施方案中，ORF1ab是NCBI序列号NC_045512.2的第266-21555位核苷酸。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. In some embodiments, 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. In some embodiments, 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. In some embodiments of the invention, 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. In some embodiments, ORF1ab is nucleotides 266-21555 of NCBI Serial No. NC_045512.2.
如本文所使用的，术语“野生型”可用于基因、蛋白质或病毒株，通常是指从天然存在的来源所分离的。野生型通常是在种群中最频繁观察到的基因、蛋白质或病毒株，因而指定为“野生型”。术语“突变体”是指当与野生型基因、蛋白质或病毒株相比，在基因的核酸序列、蛋白质的氨基酸序列或病毒株的基因组序列具有一个或更多个核苷酸或氨基酸的插入、取代或缺失。“突变体”通常仍具有野生型的基本功能特性，但其功能特性的相对水平与野生型相比可能发生改变，例如蛋白质或基因编码的蛋白质的活性有所提高或降低。应当理解，“突变体”也可能是天然存在的，例如病毒在会发生自然变异，导致产生天然存在的病毒突变体，“野生型”可以指同一基因、蛋白质或病毒株中最早出现的天然存在的那些。本发明中，野生型SARS-CoV-2的基因组可由NCBI序列号NC_045512.2确定，野生型的各个结构蛋白、辅助蛋白、和/或ORF1ab中的各个ORF可以是由NCBI序列号NC_045512.2所示的基因组序列编码或确定的。SARS-CoV- 2在自然界存在和传播过程中通过自然变异而产生病毒以及该病毒基因组中包含的基因及其编码的蛋白质，均可以被称为是“突变体”。本发明中的“突变体”也包括那些对天然存在的野生型SARS-CoV-2和自然变异的SARS-CoV-2进行人工改造而得到的病毒、其基因组、其任一种基因和其任一种蛋白质的突变体。As used herein, the term "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". The term "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. It should be understood that "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. In the present invention, 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.
本发明中，S片段、StruΔS片段和ORF1ab片段中的每一个被组装到一个质粒中，所述质粒用于表达这些片段中的基因或其编码的蛋白。在一些实施方案中，包含StruΔS片段的质粒被称为第一质粒，包含S片段的质粒被称为第二质粒，包含包装信号片段的质粒被称为第三质粒。对质粒的编号仅仅是为了进行区分，并非意味着这些质粒中隐含着特定的序列结构。In the present invention, each of S fragment, StruΔS fragment and ORF1ab fragment is assembled into a plasmid for expressing genes in these fragments or proteins encoded by them. In some embodiments, the plasmid comprising the StruΔS segment is referred to as the first plasmid, the plasmid comprising the S segment is referred to as the second plasmid, and 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.
用于制备第一质粒、第二质粒和第三质粒的初始质粒载体可以是相同的或不同的，例如三者可以用相同的一种初始质粒载体，或者可以使用三种不同的初始质粒载体，或者可以是其中二者相同，但与另一者不同。用于制备第一质粒、第二质粒或第三质粒的初始质粒载体可以是本领域常见的任何适当的载体，例如本领域常见的可以在酵母和细菌之间穿梭，并可以转染包装细胞的任何适当的载体，包括但不限于pEASY-T1、pRS415、pJS356、pEGFP-N1等。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.
第一质粒中包含的编码结构蛋白和/或辅助蛋白的序列、第二质粒中编码S蛋白的序列和/或第三质粒中的包装信号序列或ORF1ab的全长或其片段可以与表达调节序列可操作地连接。所述表达调节序列是质粒中所含的片段的表达和/或翻译具有调控作用的序列，包括但不限于5’非翻译区(5’UTR)、3’非翻译区(3’UTR)、启动子、增强子、终止子、选择标记基因、转录后调节元件、内部核糖体进入位点(IRES)、可切割序列和/或多腺苷酸化信号(polyA)等。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.
可使用的启动子包括但不限于来自病毒或哺乳动物(包括人)等的启动子等。启动子可以是组成型的或诱导型的。病毒启动子的示例包括但不限于巨细胞病毒(CMV)立即早期启动子、RSV启动子、鸡β-肌动蛋白(CBA)启动子、CMV早期增强子/鸡β-肌动蛋白(CAG)启动子、猿猴病毒40(SV40)启动子等。哺乳动物启动子的示例包括但不限于人延伸因子1a(EF1a)启动子、人泛素C(UCB)启动子、小鼠磷酸甘油酸激酶(PGK)启动子、RNA聚合酶III型启动子(如U6和H1)等。组成型启动子的示例包括但不限于逆转录病毒劳氏肉瘤病毒(RSV)LTR启动子、巨细胞病毒(CMV)启动子、SV40启动子、二氢叶酸还原酶启动子、β-肌动蛋白启动子和磷酸甘油激酶(PGK)启动子和EF1a启动子。诱导型启动子的示例包括但不限于锌诱导的羊金属硫蛋白(MT)启动子、地塞米松(Dex)诱导的小鼠乳腺肿瘤病毒(MMTV)启动子、T7聚合酶启动子系统等。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 β-actin (CBA) promoter, CMV early enhancer/chicken β-actin (CAG) promoter, simian virus 40 (SV40) promoter, etc. Examples of 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. Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin Promoter and phosphoglycerol kinase (PGK) promoter and EF1a promoter. Examples of 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.
可使用的转录后调节元件例如可以是病毒转录后调节元件如土拨鼠肝炎病毒转录后调节元件(WPRE)、乙型肝炎病毒转录后调节元件(HBVPRE)或RNA转运元件(RTE)等。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).
可使用的选择标记包括但不限于，例如药物抗性选择标记。这种选择标记基因可编码对于生长在选择培养基中的细胞的存活或生长必要的因子。未用包含选择基因的载体转化的宿主细胞将不会在培养基中存活。可以选择给予对抗生素或其他毒素的抗性、补充营养缺陷型的缺陷、或供给从培养基中扣留的关键营养元素的蛋白作为选择标记，抗生素或其他毒素的示例包括但不限于氨苄青霉素、潮霉素、新霉素、甲氨喋呤、卡那霉素、庆大霉素、Zeocin、或四环素。选择标记还可以是荧光蛋白，例如mCherry、GFP、BFP、EGFP等。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.
可切割序列可以是任何在表达后能自切割或能通过其它方式被切割(例如被特定的酶切割)的序列。可切割序列可以是在表达后能够自切割的序列。这样的序列是本领域 技术人员熟知的，例如具有自切割功能的核酶序列，如剪切型核酶序列。剪切型核酶可以催化自身RNA在特定的位点自我剪切。剪切型核酶包括但不限于锤头核酶、发夹核酶、HDV核酶或RNaseP。可切割序列还可以是能通过其它方式被切割，例如被特定的酶切割的序列。这样的序列是本领域技术人员熟知的，例如tRNA序列。tRNA序列能在tRNA 5’成熟酶(RZaseP)作用下被切掉tRNA的5’端附加序列，或者可以在3’内切核酸酶RZase F的作用下被切掉tRNA 3’端的序列。因此，所述可切割序列可以是可被RZaseP切割5’端附加序列的tRNA序列，或者可以是可被RZase F切割3’端序列的tRNA序列。任何在成熟时能够被切割的tRNA都可以用于本发明。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. Therefore, 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.
多腺苷酸化信号序列可以位于转录终止区，例如3’非翻译区中，其示例包括但不限于牛生长激素(bGH)poly(A)、SV40 polyA、胸苷激酶(TK)poly(A)序列等。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.
5’非翻译区(5’UTR)、3’非翻译区(3’UTR)可以使用任何适合的病毒5’UTR和3’UTR，例如SARS-CoV-2的5’UTR和3’UTR。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.
在一些实施方案中，第一质粒中编码结构蛋白和/或辅助蛋白的核酸与5’端的启动子、5’UTR，以及3’端的3’UTR、polyA和任选的可切割序列可操作地连接。在一些实施方案中，第二质粒中编码S蛋白的核酸与5’端的启动子和3’端的polyA可操作地连接。在一些实施方案中，第三质粒中来自SARS-CoV-2的ORF1ab的序列与5’端的启动子、5’UTR，以及3’端的3’UTR、polyA和任选的可切割序列可操作地连接。In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
在本发明中，StruΔS片段、S片段或ORF1ab片段可以通过转化介导的重组(Transformation-associated recombination,TAR)克隆方法被组装到第一质粒、第二质粒或第三质粒中。TAR克隆的是利用酵母体内重组系统对大片段的目的DNA进行拼接合成，具体方法是：将具有同源末端的目的DNA的多个片段和线性化的TAR载体共同转染到酵母中，利用酵母细胞中高效的重组系统，使载体与目的DNA片段的同源序列以及不同目的DNA片段的同源序列间进行同源重组，即可产生带有目的DNA的载体。本发明中可以以S片段、StruΔS片段或ORF1ab片段为目的DNA进行TAR克隆。所述TAR载体可以是质粒载体。具体而言，目的DNA被拆分为多个DNA片段，相邻DNA片段之间具有同源序列(也可以叫同源臂、重叠序列或重叠片段)，位于目的DNA两端的DNA片段分别与线性化的载体两端具有同源序列，将这些DNA片段和线性化的载体共同转入酵母中，使得它们在酵母体内发生同源重组，以组装成含有目的DNA的载体。所述“相邻”是指两个DNA片段在目的DNA上的位置是相邻的。拆分的每个DNA片段的长度通常为2-5kb，例如拆分的每个DNA片段的长度范围的下限和上限可以可以分别是约2kb、约2.1kb、约2.2kb、约2.3kb、约2.4kb、约2.5kb、约2.6kb、约2.7kb、约2.8kb、约2.9kb、约3kb、约3.1kb、约3.2kb、约3.3kb、约3.4kb、约3.5kb、约3.6kb、约3.7kb、约3.8kb、约3.9kb、约4kb、约4.1kb、约4.2kb、约4.3kb、约4.4kb、约4.5kb，约4.6kb、约4.7kb、约4.8kb、约4.9kb、约5kb，优选为3-4kb，更优选为约3kb。同源序列的长度通常为50-300bp，优选为100-250bp，更优选为150-200bp。In the present invention, the StruΔS 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. In the present invention, S fragment, StruΔS fragment or ORF1ab fragment can be used as target DNA for TAR cloning. The TAR vector may be a plasmid vector. Specifically, the target DNA is split into multiple DNA fragments. There are homologous sequences (also called homologous arms, overlapping sequences or overlapping segments) between adjacent DNA fragments. 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. The length of 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 100-250 bp, more preferably 150-200 bp.
在一些实施方案中，StruΔS片段可以被拆分为3个DNA片段。在一些实施方案中，S片段可以被拆分为2个DNA片段。在一些实施方案中，ORF1ab片段可以被拆分为2个DNA片段。In some embodiments, the StruΔS fragment can be resolved into 3 DNA fragments. In some embodiments, the S segment can be split into 2 DNA segments. In some embodiments, the ORF1ab fragment can be split into 2 DNA fragments.
拆分后的DNA片段可以通过本领域已知的方法合成，如体外合成，所合成的DNA片段可以是单链或双链片段。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.
应当理解，本发明所述的StruΔS片段、S片段或ORF1ab片段可以仅含有所要表达的SARS-CoV-2的基因，不含有其他序列(例如编码序列或表达调控序列)，也可以包含所要表达的SARS-CoV-2的基因和前述的一种、两种或更多种表达调控序列。表达调 控序列可以通过任何方式被添加到载体中，例如，表达调控序列可以被包含在StruΔS片段、S片段和/或ORF1ab片段中，与这些片段中所表达的SARS-CoV-2的基因一同被拆分为包含同源臂的多个DNA片段，并通过酵母同源重组(TAR)被组装到质粒中。前述的表达调控序列也可以被事先添加到质粒载体中，与StruΔS片段、S片段或ORF1ab片段拆分的带有同源臂的DNA片段在酵母中进行同源重组，此时StruΔS片段、S片段或ORF1ab片段仅含有所要表达的SARS-CoV-2的基因，而不包含表达调控序列。在另一些实施方案中，一些调控序列被包含在StruΔS片段、S片段和/或ORF1ab片段中，与这些片段中所表达的SARS-CoV-2的基因一同被拆分为包含同源臂的多个DNA片段，并通过酵母同源重组(TAR)被组装到质粒中，另一些表达调控序列则被事先添加到质粒载体中。It should be understood that the StruΔS 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 gene of SARS-CoV-2 and the aforementioned one, two or more expression control sequences. The expression control sequence can be added to the vector by any means, for example, the expression control sequence can be included in the StruΔS 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 StruΔS fragment, S fragment or ORF1ab fragment is subjected to homologous recombination in yeast. At this time, the StruΔS 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. In other embodiments, some regulatory sequences are included in the StruΔS 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.
DNA片段和线性化的载体在酵母体内同源重组的方法是本领域技术人员熟知的，例如，一个具体的实例是：首先制备酵母感受态，从平板上挑取酵母菌株并接种到5mL酵母浸出粉胨葡萄糖培养基(YPD)，培养过夜；从扩摇的菌液中取出适量再次接种到5mL YPD，在30℃，200rpm条件下培养至OD 600＝0.4～0.6。清洗菌体，取待连接的DNA片段150ng、线性化的载体100ng，混合均匀，并加入感受态酵母细胞；随后，从同源臂的两端约100bp设计引物扩增，挑取酵母单克隆过夜培养，按照PCR程序处理进行junction PCR；验证正确后，从平板上挑取菌体，接种于5mL液体SC-LEU培养基，培养过夜，收集菌体；最后，重悬菌体并提取酵母总DNA备用。 The method for homologous recombination of DNA fragments and linearized vectors in yeast is well known to those skilled in the art. For example, a specific example is: first prepare yeast competent, pick yeast strains from the plate and inoculate into 5mL yeast extract Peptone glucose medium (YPD), cultured overnight; take an appropriate amount from the expanded bacterial solution and inoculate it into 5 mL of YPD again, and culture at 30°C and 200 rpm until OD 600 =0.4-0.6. Wash the cells, take 150ng of the DNA fragment to be ligated and 100ng of the linearized carrier, mix them evenly, and add them into competent yeast cells; then, design primers for amplification from about 100bp at both ends of the homology arm, and pick a single yeast clone overnight Cultivate and perform junction PCR according to the PCR procedure; after the verification is correct, pick the bacteria from the plate, inoculate in 5mL liquid SC-LEU medium, culture overnight, and collect the bacteria; finally, resuspend the bacteria and extract the total yeast DNA spare.
在酵母中组装好的含有目的DNA的载体，可以通过常规的质粒提取方法从酵母中提取出来，获得第一质粒、第二质粒或第三质粒，然后可选择地转入大肠杆菌扩增；或可以直接提取酵母总DNA，用酵母总DNA转化大肠杆菌并扩增，获得第一质粒、第二质粒或第三质粒。本领域熟知从酵母中获得并扩增重组质粒的方法，例如可以将酵母总DNA转入大肠杆菌，挑选单克隆，验证正确后，大量扩增并提取质粒，保存质粒以备用。作为一个具体实例，首先挑取EPI300单克隆并接种于5mL LB液体培养基，过夜培养，采用电转的方式转染酵母总DNA，将转移菌液涂在抗性平板上挑取单克隆，按照PCR程序处理进行junction PCR；随后选择验证正确的菌提取质粒，并进行酶切验证，酶切验证正确后进行测序验证；验证正确后，大量扩增大肠杆菌，并提取质粒，保存质粒以备用。本发明中，使用选自第一质粒、第二质粒和第三质粒中的任意两种或三种转染包装细胞，使他们在包装细胞中表达并组装，即可获得SARS-CoV-2的病毒样颗粒。在一些实施方案中，可以用于转染包装细胞的质粒包括第一质粒和选自第二质粒和第三质粒中的任一种或两种。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. For example, 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. As a specific example, first pick a single clone of EPI300 and inoculate it in 5mL LB liquid medium, cultivate it overnight, and transfect the total yeast DNA by electroporation, apply the transferred bacteria solution on a resistant plate to pick a single clone, and perform PCR 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. In the present invention, 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. In some embodiments, 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.
在进行转染之前，可以先对质粒的表达进行验证，例如可以将任一种质粒单独转染哺乳动物细胞(如293T细胞)，裂解细胞后提取胞内RNA及蛋白并分别进行检测，例如可以通过RT-PCR和Western blot进行检测，还可以通过选择性标记，例如荧光蛋白成像的方法实现对质粒表达的验证。Before transfection, the expression of the plasmid can be verified first. For example, 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.
本发明中，术语“病毒样颗粒(VLP)”、“假病毒”、“重组病毒”和“重组病毒颗粒(rVP)”可以互换使用，是指通过包含至少一种重组DNA技术步骤的方法获得的病毒颗粒，其中病毒基因组中包含的至少一种组分通过基因工程方法进行表达和组装。本发明的“病毒样颗粒”可以不含病毒基因组，或者含有不编码病毒蛋白的基因，因此是非复制性和非感染性的，所述“重组病毒颗粒”在本发明中指使用第一质粒、第二质粒和第三质粒中的任意两种或三种转染包装细胞获得的病毒颗粒，所述病毒颗粒中可以包含或不包含核酸物质。当用于转染包装细胞的质粒中包括具有包装信号的质粒时，所获得的病毒颗粒中包含核酸物质，当用于转染包装细胞的质粒中不包括具有包装 信号的质粒时，所获得的病毒颗粒中不包含核酸物质。例如，本发明中，rSAR2-CoV-2与SAR2-CoV-2 VLP可以互换使用。In the present invention, the terms "virus-like particle (VLP)", "pseudovirus", "recombinant virus" and "recombinant viral particle (rVP)" are used interchangeably and refer to a process comprising at least one step of recombinant DNA technology 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. When the plasmids used to transfect packaging cells include plasmids with packaging signals, the obtained virus particles contain nucleic acid material; when the plasmids used to transfect packaging cells do not include plasmids with packaging signals, the obtained Virus particles do not contain nucleic acid material. For example, in the present invention, rSAR2-CoV-2 and SAR2-CoV-2 VLP can be used interchangeably.
本发明所述的“转染”是指使多核苷酸，如核酸分子、质粒等，从细胞外转移到细胞内，使得该多核苷酸在所述细胞内具有功能。转染方法是本领域技术人员熟知的，例如磷酸钙或脂质体介导的转染。如本领域技术人员所知，可使用的脂质体包括lipofectamine 8000等。在一些实施方案中，转染方法包括使293T长至密度约为80％，用质粒与lipofectamine 8000混合后转染293T细胞，培养基为添加了10％FBS的DMEM；6h后，更换培养基为DMEM，48h后收集上清溶液。"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. In some embodiments, 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.
用于生产重组病毒颗粒的包装细胞是本领域技术人员熟知的，例如，可使用的包装细胞包括，但不限于，哺乳动物(包括人)细胞、昆虫细胞、植物细胞、微生物或酵母，例如HEK293系列的细胞(如HEK293A、HEK293T或HEK293FT)、A549细胞或Vero细胞等。Packaging cells for the production of recombinant virus particles are well known to those skilled in the art. For example, 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.
用于生产重组病毒颗粒的培养基和培养方法是本领域技术人员已知的，可以采用商购的或定制的培养基，或者进一步添加补充本领域已知的一个或多个细胞培养组分，包括但不限于葡萄糖、维生素、氨基酸和或生长因子，以增加生产培养物中重组病毒颗粒的滴度。重组病毒载体生产培养物可以在适合于所使用的特定宿主细胞(即包装细胞)的条件下生长。在产生重组病毒载体之后，如果需要，可使用多种常规方法从细胞裂解物或病毒上清液中纯化病毒颗粒，包括超滤法，例如使用硝酸纤维素滤膜；吸附法，例如使用磷酸钙或离子交换熟知吸附病毒或杂质，随后用盐溶液洗脱；层析法，例如葡聚糖凝胶层析、离子交换层析、亲和层析等；离心法，例如差速离心、CsCl或蔗糖密度梯度离心；沉淀法，例如聚乙二醇沉淀(如使用PEG2000)，等电点沉淀或中性盐沉淀等。上述纯化方法也可以组合使用。在一些实施方案中，作为纯化方法的一个示例，配制浓度为10％(w/v)的PEG20000溶液，以体积比1:1混合PEG20000与培养的病毒上清溶液，4℃放置16h进行浓缩，离心收集沉淀物，然后用opti-MEM重悬以备用，该方法可用于VLP的侵染等实验；作为纯化方法的另一个示例，将收集的病毒培养上清过20％(w/v)的蔗糖溶液，以30000rpm超速离心4h对VLP进行初步纯化，此VLP纯化方法可用于RNA、蛋白及VLP形貌等的表征。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. Following production of recombinant viral vectors, 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. The above purification methods may also be used in combination. In some embodiments, as an example of the purification method, prepare a PEG20000 solution with a concentration of 10% (w/v), mix PEG20000 and the cultured virus supernatant solution at a volume ratio of 1:1, place at 4°C for 16 hours to concentrate, The precipitate was collected by centrifugation, and then resuspended with opti-MEM for future use. 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.
本发明的重组病毒颗粒可以作为活性成分用在药物组合物或疫苗中，以治疗由SARS-CoV-2引起的疾病。术语“疫苗”指包含所述重组病毒颗粒的制剂。疫苗中所包含的重组病毒颗粒的剂量可由本领域技术人员例如根据疾病状况、受试者和治疗时间表进行调整。疫苗中通常包含治疗有效量的重组病毒颗粒。本发明所述的“治疗”是指预防或缓解(例如，降低、减轻或治愈)至少一种与疾病状态相关的症状。本发明所述的“治疗有效量”即足以预防或缓解(例如，降低、减轻或治愈)至少一种与疾病状态相关的症状的量。所述药物组合物或疫苗的剂量可方便地由本领域技术人员来确定，例如通过首先鉴定有效引发预防性或治疗性免疫应答的剂量，例如通过测量病毒特异性免疫球蛋白的血清效价或通过测量血清样品或尿样或粘膜分泌物中抗体的抑制比。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.
本发明的药物组合物或疫苗可以包含本发明的重组病毒颗粒和药学上可接受的载体或赋形剂。药学上可接受的载体包括但不限于盐水、缓冲盐水、右旋糖、水、甘油、无菌等张水性缓冲液及其组合。本发明的药物组合物或疫苗可以包含佐剂，佐剂对于本领域技术人员而言是公知的。示例性的佐剂包括完全弗氏佐剂、不完全弗氏佐剂、氢氧化铝佐剂、BCG等。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.
本发明的药物组合物或疫苗应当适合于施用给受试者，例如是是无菌的、非颗粒的和/或非致热原性的。所述药物组合物或疫苗可被配制为固体形式，如可用于配制注射 液的冻干粉、液体溶液、悬液、乳液、片剂、丸剂、胶囊剂、持续释放配制物或散剂等。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.
本发明的药物组合物或疫苗的施用方法包括但不限于肠胃外给药(例如皮内、肌肉内、静脉内和皮下)、硬膜外和粘膜(例如鼻内和经口或肺部途径或通过栓剂)。给药可为系统性的或局部的。Methods of administration of the pharmaceutical 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.
本发明的重组病毒颗粒还可以用在SARS-CoV-2病毒感染细胞的体外研究中，例如可用于体外药物筛选，如在体外筛选能够抑制SARS-CoV-2病毒感染细胞的药物；开发抗体，例如可以该重组病毒颗粒作为免疫原制备抗体或检测抗体中和活性；还可用于开发病毒疫苗，例如可以针对新出现的病毒突变体，特别是新出现的具有突变S蛋白的病毒突变体，方便快捷地开发疫苗。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, For example, 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.
在一些实施方案中，基因组的拆分、合成及组装原理如图1所示。首先，将基因组ORF1ab拆分成6个大小为3-5kb的片段、S蛋白基因拆分成2个片段、其他结构蛋白或辅助蛋白基因拆分为3个片段；然后，体外合成每个片段并通过酵母同源重组技术组装成ORF1ab、S及StruΔS三个片段；再将三个片段构建在相应载体上，转入大肠杆菌，获得质粒；最后将质粒转染哺乳动物细胞，获得VLP。各种VLP的表征如图3-4所示。In some embodiments, the principle of genome splitting, synthesis and assembly is shown in FIG. 1 . First, 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 StruΔS 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.
在一些实施方案中，本发明提供了一种SARS-CoV-2 VLP从头人工合成及制备方法，具体包括：In some embodiments, the present invention provides a de novo artificial synthesis and preparation method of SARS-CoV-2 VLP, specifically comprising:
第一步：SARS-CoV-2基因组拆分，合成及组装Step 1: SARS-CoV-2 genome disassembly, synthesis and assembly
(1)将SARS-CoV-2基因组拆分成ORF1ab、StruΔS和S三个部分，其中ORF1ab序列从266到21555，包含所有非结构蛋白的序列；StruΔS序列从25393到29674，包含除S蛋白外所有的结构蛋白与辅助蛋白的序列；S序列从21563到25384，包含S蛋白的序列(具体序列见附件)；(1) Split the SARS-CoV-2 genome into three parts: ORF1ab, StruΔS and S, where the ORF1ab sequence ranges from 266 to 21555, including the sequences of all non-structural proteins; the StruΔS sequence ranges from 25393 to 29674, including the S protein Sequences of all structural proteins and auxiliary proteins; S sequence from 21563 to 25384, including the sequence of S protein (see the attachment for the specific sequence);
(2)将ORF1ab拆分成6个约3kb的片段，将StruΔS拆分成3个约3kb的片段，将S拆分成2个3kb的片段，每个片段均采用体外合成的方法，并在相邻片段上设计overlap片段。(2) Split ORF1ab into 6 fragments of about 3kb, split StruΔS into 3 fragments of about 3kb, and split S into 2 fragments of 3kb, each fragment is synthesized in vitro, and in Design overlap fragments on adjacent fragments.
(3)将上一步骤拆分的片段利用酵母体内平台进行酵母同源重组，利用相邻片段间的overlap通过同源重组的方法组装成为ORF1ab、StruΔS及S三个完整的片段，同时分别组装到质粒载体pJS356和pEASY-T-CAG上得到三个质粒SARS-CoV-2 ORF1ab、SARS-CoV-2 StruΔS和SARS-CoV-2 S。其中在ORF1ab、StruΔS编码序列两端分别加上5’UTR及3’UTR与PolyA尾巴，同时每条序列的5’加上CMV的增强子；在S蛋白编码序列的3’端加上PolyA尾巴；并在5’加上CAG的增强子；(3) Use the yeast in vivo platform to carry out yeast homologous recombination on the fragments split in the previous step, and use the overlap between adjacent fragments to assemble into three complete fragments of ORF1ab, StruΔS and S through homologous recombination, and assemble them separately Three plasmids SARS-CoV-2 ORF1ab, SARS-CoV-2 StruΔS and SARS-CoV-2 S were obtained on the plasmid vectors pJS356 and pEASY-T-CAG. Among them, 5'UTR, 3'UTR and PolyA tail are added to both ends of ORF1ab and StruΔS 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';
酵母同源重组的具体操作步骤如下：首先制备酵母感受态，从平板上挑取酵母菌株并接种到5mL酵母浸出粉胨葡萄糖培养基(YPD)，培养过夜；从扩摇的菌液中取出适量再次接种到5mL YPD，在30℃，200rpm条件下培养至OD 600＝0.4～0.6。清洗菌体，取待连接的DNA片段(约3kb)150ng、线性化的载体100ng，混合均匀，并加入感受态酵母细胞；随后，从同源臂的两端约100bp设计引物扩增，挑取酵母单克隆过夜培养，按照PCR程序处理进行junction PCR；验证正确后，从平板上挑取菌体，接种于5mL液体SC-LEU培养基，培养过夜，收集菌体；最后，重悬菌体并提取酵母总DNA备用； The specific operation steps of yeast homologous recombination are as follows: first prepare the yeast competent state, pick the yeast strain from the plate and inoculate it into 5mL yeast extract powder peptone glucose medium (YPD), and cultivate it overnight; Inoculate into 5mL YPD again, and culture at 30°C and 200rpm until OD 600 =0.4-0.6. Wash the bacteria, take 150ng of the DNA fragment (about 3kb) to be ligated, and 100ng of the linearized carrier, mix them evenly, and add them into competent yeast cells; then, design primers to amplify from about 100bp at both ends of the homology arm, pick Yeast monoclonal culture was carried out overnight, and junction PCR was carried out according to the PCR procedure; after the verification was correct, the bacteria were picked from the plate, inoculated in 5mL liquid SC-LEU medium, cultured overnight, and the bacteria were collected; finally, the bacteria were resuspended and collected. Extract the total yeast DNA for later use;
(4)将酵母总DNA转入大肠杆菌，挑选单克隆，验证正确后，大量扩增并提取质粒，保存质粒以备用；(4) Transfer the total yeast DNA into Escherichia coli, select a single clone, and after verification is correct, amplify and extract the plasmid in large quantities, and save the plasmid for future use;
具体操作步骤如下：首先挑取EPI300单克隆并接种于5mL LB液体培养基，过夜培养，采用电转的方式转染酵母总DNA，将转移菌液涂在抗性平板上挑取单克隆，按 照PCR程序处理进行junction PCR；随后选择验证正确的菌提取质粒，并进行酶切验证，酶切验证正确后进行测序验证；验证正确后，大量扩增大肠杆菌，并提取质粒，保存质粒以备用。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.
第二步：SARS-CoV-2 VLP表达及制备方法The second step: SARS-CoV-2 VLP expression and preparation method
(1)对SARS-CoV-2基因组拆分成而构建的三个质粒分别进行验证。首先分别将三个质粒单独转染293T细胞，然后裂解细胞，分别提取细胞内的RNA及蛋白，并分别进行RT-PCR及western blot等的验证；同时通过SARS-CoV-2 StruΔS中的EGFP和SARS-CoV-2 ORF1ab中的mCherry的荧光成像的方法实现对质粒表达的验证。(1) 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 StruΔS and the The method of fluorescence imaging of mCherry in SARS-CoV-2 ORF1ab realizes the verification of plasmid expression.
(2)293T长至密度约为80％，分别转染SARS-CoV-2 StruΔS，SARS-CoV-2 StruΔS与SARS-CoV-2 S，SARS-CoV-2 StruΔS与SARS-CoV-2 ORF1ab，及SARS-CoV-2 StruΔS、SARS-CoV-2 S及SARS-CoV-2 ORF1ab，培养基为opti-MEM；6h后，更换培养基为DMEM，48h后收集上清溶液。(2) 293T grows to a density of about 80%, respectively transfected with SARS-CoV-2 StruΔS, SARS-CoV-2 StruΔS and SARS-CoV-2 S, SARS-CoV-2 StruΔS and SARS-CoV-2 ORF1ab, and SARS-CoV-2 StruΔS, 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.
(3)配置浓度为10％(w/v)的PEG20000溶液，以体积比1:1混合PEG20000与病毒上清溶液，4度放置16h进行浓缩，离心收集沉淀物，然后用opti-MEM重悬以备用，以上方法可用于VLP的侵染等实验。将收集的上清过20％(w/v)的蔗糖溶液，以30000rpm超速离心4h对VLP进行初步纯化，此VLP纯化方法主要用于RNA、蛋白及VLP形貌等的表征。(3) Prepare a PEG20000 solution with a concentration of 10% (w/v), mix PEG20000 and the virus supernatant solution at a volume ratio of 1:1, place at 4 degrees for 16 hours to concentrate, collect the precipitate by centrifugation, and then resuspend with opti-MEM As a spare, the above method can be used for experiments such as VLP infection. The collected supernatant was passed through 20% (w/v) sucrose solution and ultracentrifuged at 30000rpm for 4h to preliminarily purify VLP. This VLP purification method is mainly used for the characterization of RNA, protein and VLP morphology.
在一些实施方案中，质粒共转制备VLP及其验证，包括下述任一种步骤：In some embodiments, plasmid co-transfection to prepare VLP and its verification include any of the following steps:
(1)将StruΔS的质粒单转入293T细胞制备StruΔS的VLP(1) Transform the plasmid of StruΔS into 293T cells to prepare the VLP of StruΔS
将293T铺在100mm的细胞培养皿，至细胞密度约为90％时，用转染试剂lipo8000在每个培养皿中转染15μg SARS-CoV-2 StruΔS，6h后更换培养基。48h后收集上清容液，即为VLP。将培养皿中细胞裂解进行RT-PCR与western blot以验证质粒在293T细胞中的表达；采用荧光成像的方法验证质粒的表达，采用western blot分析VLP中的N蛋白，采用透射电镜表征的方法验证VLP的大小与形貌； Spread 293T on a 100mm cell culture dish, and when the cell density is about 90%, transfect 15 μg of SARS-CoV-2 StruΔS in each dish with transfection reagent lipo8000, and replace the medium after 6 hours. After 48 hours, the supernatant was collected, which was VLP. The cells in the culture dish were lysed for RT-PCR and western blot to verify the expression of the plasmid in 293T cells; the expression of the plasmid was verified by fluorescence imaging, the N protein in VLP was analyzed by western blot, and the expression of the plasmid was verified by transmission electron microscopy The size and shape of the VLP;
(2)将StruΔS与S的质粒共转入293T细胞制备StruΔS+S的VLP(2) Co-transfer the plasmids of StruΔS and S into 293T cells to prepare the VLP of StruΔS+S
将293T铺在100mm的细胞培养皿，至细胞密度约为90％时，用转染试剂lipo8000在每个培养皿中转染15μg SARS-CoV-2 StruΔS与15μg SARS-CoV-2 S，6h后更换培养基；72h后收集上清容液，即为StruΔS+S的VLP。采用荧光成像的方法验证质粒的表达，采用western blot分析胞内表达及VLP中的S蛋白和N蛋白；采用透射电镜表征的方法验证VLP的大小与形貌； Spread 293T on a 100mm cell culture dish until the cell density is about 90%, transfect 15 μg SARS-CoV-2 StruΔS and 15 μg SARS-CoV-2 S in each culture dish with transfection reagent lipo8000, after 6 hours Replace the medium; collect the supernatant after 72 hours, which is the VLP of StruΔS+S. Fluorescence imaging was used to verify the expression of the plasmid, and western blot was used to analyze the intracellular expression and the S protein and N protein in the VLP; the size and shape of the VLP were verified by transmission electron microscopy;
(3)将StruΔS与ORF1ab的质粒共转入293T细胞制备StruΔS+ORF1ab的VLP(3) Co-transfer the plasmids of StruΔS and ORF1ab into 293T cells to prepare the VLP of StruΔS+ORF1ab
将293T铺在100mm的细胞培养皿，至细胞密度约为90％时，用转染试剂lipo8000在每个培养皿中转染15μg SARS-CoV-2 StruΔS与15μg SARS-CoV-2 ORF1ab，6h后更换培养基。48h后收集上清容液，即为StruΔS+ORF1ab的VLP。采用荧光成像的方法验证质粒的表达，采用western blot分析胞内表达及VLP中的N蛋白，采用透射电镜表征的方法验证VLP的大小与形貌； Spread 293T on a 100mm cell culture dish until the cell density is about 90%, transfect 15 μg SARS-CoV-2 StruΔS and 15 μg SARS-CoV-2 ORF1ab in each culture dish with transfection reagent lipo8000, after 6 hours Replace medium. After 48 hours, the supernatant was collected, which was the VLP of StruΔS+ORF1ab. The expression of the plasmid was verified by fluorescence imaging, the intracellular expression and the N protein in VLP were analyzed by western blot, and the size and shape of VLP were verified by transmission electron microscopy;
(4)将StruΔS，S与ORF1ab的质粒共转入293T细胞制备StruΔS+S+ORF1ab的VLP(4) StruΔS, S and ORF1ab plasmids were co-transfected into 293T cells to prepare StruΔS+S+ORF1ab VLP
将293T铺在100mm的细胞培养皿，至细胞密度约为90％时，用转染试剂lipo8000在每个培养皿中转染15μg SARS-CoV-2 StruΔS，15μg SARS-CoV-2 S与15μg SARS-CoV-2 ORF1ab，6h后更换培养基；48h后收集上清溶液，即为StruΔS+S+ORF1ab的VLP；采用荧光成像的方法验证质粒的表达，采用western blot分析胞内表达及VLP中的S蛋白和N蛋白，采用透射电镜表征的方法验证VLP的大小与形貌； Spread 293T on a 100mm cell culture dish, and when the cell density is about 90%, transfect 15 μg SARS-CoV-2 StruΔS, 15 μg SARS-CoV-2 S and 15 μg SARS in each culture dish with transfection reagent lipo8000 -CoV-2 ORF1ab, replace the medium after 6 hours; collect the supernatant solution after 48 hours, which is the VLP of StruΔS+S+ORF1ab; use fluorescence imaging to verify the expression of the plasmid, and use western blot to analyze intracellular expression and VLP For S protein and N protein, the size and shape of VLP were verified by transmission electron microscopy;
(5)将StruΔS，ORF1ab与S 突变的质粒共转入293T细胞制备StruΔS+S 突变+ORF1ab的VLP (5) StruΔS, ORF1ab and S mutant plasmids were co-transfected into 293T cells to prepare StruΔS+S mutation +ORF1ab VLP
首先，通过PCR的方法对S蛋白质粒(即SARS-CoV-2 S)进行单位点的突变，本案例以N331Q、N501Y、D614G及P681H为例；将每个S突变质粒转染293T细胞，裂解细胞，采用RT-PCR的方法验证S突变质粒的表达。然后，制备S突变VLP，对于每种S蛋白突变，将293T细胞铺在100mm的细胞培养皿，至细胞密度约为90％时，用转染试剂lipo8000在每个培养皿中转染15μg SARS-CoV-2 StruΔS，15μg SARS-CoV-2 S 突变与15μg SARS-CoV-2 ORF1ab的质粒，6h后更换培养基；48h后收集上清溶液，即为StruΔS+S 突变+ORF1ab的VLP；采用荧光成像的方法验证质粒的表达，采用western blot分析胞内表达及VLP中的S蛋白和N蛋白，采用透射电镜表征的方法验证VLP的大小与形貌。 First, carry out a single-point mutation on the S protein plasmid (that is, SARS-CoV-2 S) by the PCR method. In this case, 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. Then, prepare 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 μg SARS- CoV-2 StruΔS, 15μg SARS-CoV-2 S mutation and 15μg SARS-CoV-2 ORF1ab plasmid, change the medium after 6h; collect the supernatant after 48h, which is the VLP of StruΔS+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.
下面通过实施例，并结合附图，对本发明的技术方案作进一步详细的说明，但本发明不限于下面的实施例。除非另有说明，本发明中以及下述实施例中所述的SARS-CoV-2基因组及其部分的核苷酸编号均以NCBI序列号NC_045512.2为准，S蛋白及其突变体的氨基酸位置编号均以S蛋白的氨基酸序列为准，即以S蛋白的第1位氨基酸为编号1。The technical solution of the present invention will be further described in detail through the following examples in conjunction with the accompanying drawings, but the present invention is not limited to the following examples. Unless otherwise specified, the 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.
实施例1：材料和方法Example 1: Materials and methods
质粒：基于酵母中的TAR系统构建SARS-CoV-2 S、ORF1ab-mCherry和StruΔS-EGFP三个质粒，以制备SARS-CoV-2的病毒颗粒。。在密码子优化过的编码SARS-CoV-2 S糖蛋白的cDNA序列的5’端添加CAG启动子，在其3’端添加bGH poly(A)signal，将该带有启动子和poly(A)signal的序列拆分为两个带有重叠序列的片段(SEQ ID NO:1,SEQ ID NO:2)，通过酵母中的同源重组将带有启动子和poly(A)signal的SARS-CoV-2 S糖蛋白的cDNA序列组装并克隆到pEASY-T1载体上，形成质粒pEASY-T-S(在以下实施例中也被称为SARS-CoV-2 S质粒或S质粒)。在SARS-CoV-2的ORF1ab的5’端添加CMV启动子、5’UTR，并在3’端添加3’UTR、HDV、SV40 polyA和mCherry编码基因，并将由此获得的序列拆分为六个带有重叠序列的片段(SEQ ID NO:3-8)，在酵母中将这些重叠片段组装并克隆到pJS356载体(pJS356载体是在pR415质粒的基础上加入用于诱导低拷贝质粒的高表达的ori2-oriV元件和用于在大肠杆菌中高效表达的SopA、SopB、RepE及SopC功能元件获得的)中，形成质粒pJS356-ORF1ab-mCherry(在以下实施例中又可称SARS-CoV-2 ORF1ab质粒、SARS-CoV-2 ORF1ab-mCherry质粒、ORF1ab-mCherry质粒或ORF1ab质粒)。在编码全部结构蛋白(刺突蛋白除外)和全部辅助蛋白(辅助蛋白包括ORF3a，ORF3b，ORF6，ORF7a，ORF7b，ORF8，ORF10)的DNA序列的3’端添加CMV启动子、T7启动子、5’UTR，并在3’端添加3’UTR、HDV、SV40 polyA和EGFP编码基因，并将由此获得的序列拆分为三个带有重叠序列的片段(SEQ ID NO:9-11)，在酵母中将这些重叠片段组装，克隆到pJS356载体中，形成质粒pJS356-StruΔS-EGFP(在以下实施例中又可称SARS-CoV-2 StruΔS质粒、SARS-CoV-2 StruΔS-EGFP质粒、StruΔS-EGFP质粒或StruΔS质粒)。通过酶切和测序验证获得的质粒。Plasmids: Three plasmids of SARS-CoV-2 S, ORF1ab-mCherry and StruΔS-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-mCherry plasmid or ORF1ab plasmid). 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-StruΔS-EGFP (also referred to as SARS-CoV-2 StruΔS plasmid, SARS-CoV-2 StruΔS-EGFP plasmid, StruΔS-EGFP in the following examples) EGFP plasmid or StruΔS plasmid). The obtained plasmids were verified by digestion and sequencing.
将通过生信分析得到的SARS-CoV-2基因组RNA(nt 19900-20000或19773-20335)的包装信号(packaging signal，简称PS)的DNA序列插入pEGFP-N1载体中EGFP基因的3'非编码区，构建质粒pEGFP-N1-PS101或pEGFP-N1-PS583。从质粒pJS356-ORF1ab-mCherry扩增得到PS101(nt 19900-20000)或PS583(nt 19773-20335)。用 Not I限制性核酸内切酶处理质粒pEGFP-N1，插入含PS的片段以产生pEGFP-N1-PS101或pEGFP-N1-PS583。Insert the DNA sequence of the packaging signal (PS for short) of SARS-CoV-2 genomic RNA (nt 19900-20000 or 19773-20335) obtained through bioinformatics analysis into the 3' non-coding of the EGFP gene in the pEGFP-N1 vector Region, construct plasmid pEGFP-N1-PS101 or pEGFP-N1-PS583. PS101 (nt 19900-20000) or PS583 (nt 19773-20335) were amplified from plasmid pJS356-ORF1ab-mCherry. 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.
定点突变：利用SARS-CoV-2 S质粒作为模板构建S蛋白基因突变体，包括SARS-CoV-2 S(N331Q)，SARS-CoV-2 S(N501Y)，SARS-CoV-2 S(D614G)和SARS-CoV-2 S(P681H)共四种质粒。选择目标突变位点附近15-20碱基作为正向引物，反向引物选择互补序列作为反向引物，其列于表1。定点突变PCR后，用限制性内切酶DpnI消化模板链。产物直接转化至大肠杆菌DH5α感受态细胞，选择单克隆并测序。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 DH5α competent cells, and a single clone was selected and sequenced.
ORF1b RNA二级结构的预测：使用Biopython从参考基因组中选取SARS-CoV-2，SARS-CoV和类蝙蝠SARS-CoV的ORF1b区域中的序列(nt 19500-20400)。二级结构由具有默认参数的RNA结构web服务器预测，并由Vienna RNA web服务器进行可视化分析。Prediction of 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.
相似结构的多序列比对和预测：我们预测并比较了ORF1b 3'末端的保守RNA茎环，这是SARS相关病毒中预测的包装信号。使用LocARNA web服务器对SARS-CoV-2(NC-045512.2:19773-20355)，SARS-CoV(NC-004718.3:19712-20294)和类蝙蝠SARS-CoV(MG772933.1:19773-20355)进行比对。Multiple sequence alignment and prediction of similar structures: We predicted and compared a conserved RNA stem-loop at the 3' end of ORF1b, a predicted packaging signal in SARS-related viruses. Comparison of SARS-CoV-2 (NC-045512.2:19773-20355), SARS-CoV (NC-004718.3:19712-20294) and bat-like SARS-CoV (MG772933.1:19773-20355) using the LocARNA web server .
细胞构建和培养：本研究构建了稳定表达人ACE2的293T细胞(293T/hACE2)。用hACE2质粒转染293T细胞产生受体hACE2，并在2μg/mL嘌呤霉素下进行筛选。通过蛋白质印记来检测hACE2细胞的表达。293T细胞在添加10％FBS、1％青霉素和链霉素的DMEM培养基中培养，并将其培养在37℃，5％CO 2培养箱中。293T/hACE2细胞在添加10％FBS、1％青霉素和链霉素以及2μg/mL嘌呤霉素的DMEM培养基中培养，并将其培养在37℃，5％CO 2培养箱中。 Cell construction and culture: In this study, 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 μg/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 μg/mL puromycin, and cultured at 37°C in a 5% CO2 incubator.
VLPs的构建：为了获得VLPs，使用lipofectamine 8000与SARS-CoV-2 S(10μg),ORF1ab-mCherry(10μg)和StruΔS-EGFP(10μg)质粒混合后转染到293T细胞中，并在含有10％FBS的DMEM培养基中培养48小时。为了形成组装有包装信号的VLP(EGFP-PS101)或者VLP(EGFP-PS583)，将pEGFP-N1-PS101(1μg)或pEGFP-N1-PS583(1μg)与SARS-CoV-2 S(10μg)和StruΔS(10μg)质粒一起共转染到293T细胞48小时。Construction of VLPs: In order to obtain VLPs, use lipofectamine 8000 to mix with SARS-CoV-2 S (10 μg), ORF1ab-mCherry (10 μg) and StruΔS-EGFP (10 μg) plasmids, and then transfect them into 293T cells, and in 10% Culture in DMEM medium with FBS for 48 hours. To form a VLP (EGFP-PS101) or VLP (EGFP-PS583) assembled with a packaging signal, pEGFP-N1-PS101 (1 μg) or pEGFP-N1-PS583 (1 μg) was mixed with SARS-CoV-2 S (10 μg) and StruΔS (10 μg) plasmids were co-transfected into 293T cells for 48 hours.
VLP纯化：转染后48小时后收集培养基上清并用0.45μm滤膜进行过滤。将培养基上清加到20％蔗糖垫上，并使用SW32转子以30000rpm超速离心3小时。纯化后的VLP用于拍摄透射电子显微镜。对于蛋白质免疫印迹分析，将培养基上清加到20％至40％密度梯度蔗糖的顶部，并使用SW41转子以30000rpm超速离心4小时。采用PEG20000浓缩的方法收集VLP用于荧光成像。VLP purification: 48 hours after transfection, the culture supernatant was collected and filtered with a 0.45 μm 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提取和RT-PCR：通过FastPure细胞/组织总RNA分离试剂盒V2细胞培养基上清的总RNA，并根据试剂盒说明书在48小时转录成cDNA。使用cDNA作为模板，设计表1中列出的特异性引物用以扩增SARS-CoV-2 S，N，NSP1，NSP16或ORF3a的编码区，通过在1％琼脂糖凝胶电泳验证PCR产物。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. Using cDNA as a template, 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.
蛋白质免疫印迹分析：用含有蛋白酶抑制剂和PMSF的RIPA缓冲液在冰上裂解细胞或VLP五分钟，并以13000g离心5分钟除去大的细胞碎片。蛋白质裂解物在100度下煮沸10分钟，在4-20％丙烯酰胺凝胶上分离，并转移到硝酸纤维素膜上。用含1％Tween-20和5％牛奶的TBST缓冲封闭硝酸纤维素膜，4度过夜，然后与IRDye 680RD山羊抗小鼠抗体或IRDye 800CW山羊抗兔抗体一起温育。通过ChemiDoc MP成像系统来获得分析结果。Western blot analysis: Cells or VLPs were lysed with RIPA buffer containing protease inhibitors and PMSF for five minutes on ice and centrifuged at 13000 g for 5 minutes to remove large cell debris. Protein lysates were boiled at 100°C for 10 min, separated on 4-20% acrylamide gels, and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked with TBST buffer containing 1% Tween-20 and 5% milk, overnight at 4, and then incubated with IRDye 680RD goat anti-mouse antibody or IRDye 800CW goat anti-rabbit antibody. Analytical results were obtained with the ChemiDoc MP imaging system.
透射电子显微镜：将VLP粘附到碳涂覆的铜网上10分钟，并用2％(w/v)磷钨酸(pH 7.1)染色1分钟，用200kV透射电子显微镜观察样品。Transmission electron microscopy: 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.
量子点纳米信标的制备：将碲粉(20mg)和硼氢化钠(11mg)加入含1mL超纯水的圆底烧瓶中，将混合物在无氧和冰浴条件下搅拌5小时以获得碲氢化钠。为了制备镉前体，将氯化镉，氯化锌和N-乙酰基-L-半胱氨酸按照1:1:4的混合并通过NaOH将pH调节至9.0。将BHQ2和硫代磷酸酯共修饰的DNA，镉前体和碲氢化钠混合并转移到四聚氟乙烯内衬的不锈钢高压反应釜中。将混合物加热至200度36分钟以获得BHQ2-DNA功能化的CdTe:Zn 2+QDs，通过用Amicon Ultra-4离心过滤装置(50kDa)以8000rpm离心10分钟来纯化量子点。对BHQ2-DNA功能化的CdTe:Zn 2+QDs进行95度退火处理保持10分钟并在室温下保持30分钟以形成茎环结构。 Preparation of 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 . To prepare the cadmium precursor, 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.
VLP的荧光标记：为了构建带荧光标记VLP，在转染SARS-CoV-2质粒后6小时加入40μL退火的量子点纳米信标(10μM)到培养基中以标记病毒基因组，并且在病毒自组装期过程中将纳米信标标记在VLP中。在2mL收集的病毒液中加入0.5μL DiO(1mM)，在37℃下孵育1小时以标记病毒囊膜。Fluorescent labeling of VLPs: To construct fluorescently labeled VLPs, 40 μL of annealed quantum dot nanobeacons (10 μM) 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 μL DiO (1 mM) to 2 mL of the collected virus liquid and incubate at 37 °C for 1 hour to label the viral envelope.
病毒感染和荧光成像：将293T/hACE2细胞接种于共聚焦培养皿中，在含10％FBS、1％青霉素和链霉素和2μg/mL嘌呤霉素的DMEM中培养。荧光标记的VLP与宿主细胞293T/hACE2在4℃孵育保持30分钟，并用新鲜培养基替换培养基以除去未结合的颗粒。共聚焦培养皿用封口膜密封，然后转移到37度5％CO 2培养箱中。通过UltraView Vox共聚焦激光扫描系统在488nm和561nm激发下进行成像。 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 μg/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.
实施例2 结果和讨论Example 2 Results and Discussion
结果result
SARS-CoV-2 VLP的构建和表征Construction and Characterization of SARS-CoV-2 VLPs
为组装具侵染能力的SARS-CoV-2 VLP，我们将SARS-CoV-2基因组的cDNA全长分为三部分：SARS-CoV-2 ORF1ab(核苷酸位置：266-21555)、SARS-CoV-2 S(核苷酸位置：21563-25384)和SARS-CoV-2 StruΔS(核苷酸位置：25393-29674)，且都包含组成型启动子，具体如图1A所示。使用化学合成的病毒基因组DNA片段，分别在酵母中进行组装构建成分别含有SARS-CoV-2 ORF1ab、SARS-CoV-2 S和SARS-CoV-2 StruΔS的质粒；构建好的质粒共转染到293T细胞中以产生SARS-CoV-2 VLP(图1B)。在293T细胞中，DNA片段被翻译并表达病毒蛋白；包含包装信号(PS)序列的SARS-CoV-2 ORF1ab RNA与结构蛋白组装形成SARS-CoV-2 VLP；SARS-CoV-2 VLP的感染性是由刺突蛋白与宿主细胞表面hACE2受体的结合所介导。在病毒粒子中仅组装包包装序列基因，不组装所有结构蛋白基因的情况下，已受SARS-CoV-2 VLP感染的细胞不会产生子代病毒(图1C)。因此，我们的SARS-CoV-2 VLP构建策略有望为病毒学研究提供一个安全且通用的平台。In order to assemble SARS-CoV-2 VLP with infective ability, we divided the full-length cDNA of SARS-CoV-2 genome into three parts: SARS-CoV-2 ORF1ab (nucleotide position: 266-21555), SARS-CoV-2 CoV-2 S (nucleotide position: 21563-25384) and SARS-CoV-2 StruΔS (nucleotide position: 25393-29674), and both contain constitutive promoters, as shown in Figure 1A. Using chemically synthesized viral genome DNA fragments, they were assembled in yeast to construct plasmids containing SARS-CoV-2 ORF1ab, SARS-CoV-2 S and SARS-CoV-2 StruΔS respectively; the constructed plasmids were co-transfected into 293T cells to produce SARS-CoV-2 VLPs (Fig. 1B). In 293T cells, DNA fragments are translated and viral proteins are expressed; 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. Cells infected with SARS-CoV-2 VLPs did not produce progeny viruses when only the packaging sequence genes were assembled in the virions, but not all the structural protein genes (Fig. 1C). Therefore, our SARS-CoV-2 VLP construction strategy is expected to provide a safe and general platform for virology research.
SARS-CoV-2刺突糖蛋白(S)作为受体结合位点，可以介导膜融合和病毒进入。我们组装并优化了S蛋白编码序列，并在哺乳动物细胞中表达S蛋白。将SARS-CoV-2 S质粒转染293T细胞，通过RT-PCR分析S基因的结果如图6A所示。表达的SARS-CoV-2 S蛋白通过蛋白免疫印迹验证，结果显示有两个主要条带，180kDa的全长蛋白和110kDa的裂解蛋白(图6B)。The SARS-CoV-2 spike glycoprotein (S) acts as a receptor binding site that can mediate membrane fusion and virus entry. We assembled and optimized the S protein coding sequence and expressed the S protein in mammalian cells. 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 StruΔS基因序列与EGFP融合构建成质粒，命名为SARS-CoV-2 StruΔS-EGFP(图7A)；同时，我们构建了结构蛋白(E，包膜；M，膜；N，核衣壳)和辅助蛋白的表达载体。为了验证SARS-CoV-2 StruΔS-EGFP质粒的活性，在293T细胞中通过RT-PCR分析了ORF3a和N基因(图7B)；在质粒转染24小时后观察到EGFP表达(图7C)，并通过蛋白免疫印迹确定SARS-CoV-2 N蛋白的表达(图7D)。结构蛋白可以组装成病毒样颗粒VLP(StruΔS)；我们在转染后48小时收集培 养基，转移至超离管中(底部含20％蔗糖垫)以分离VLP。通过蛋白免疫印迹法验证VLP(StruΔS)中确实含有SARS-CoV-2 N蛋白(图7D)；VLP(StruΔS)的形态大小则通过TEM进行观察，我们发现不含刺突蛋白的球形颗粒的估计直径为75±14nm(n＝30，图7E)。The SARS-CoV-2 StruΔS gene sequence was fused with EGFP to construct a plasmid named SARS-CoV-2 StruΔS-EGFP (Fig. 7A); at the same time, we constructed a structural protein (E, envelope; M, membrane; N, nuclear capsid) and accessory protein expression vectors. To verify the activity of the SARS-CoV-2 StruΔS-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). Structural proteins can be assembled into virus-like particles VLPs (StruΔS); we collected the medium 48 hours after transfection and transferred to ultracentrifuge tubes (with 20% sucrose pad at the bottom) to isolate VLPs. It was verified by Western blotting that the VLP (StruΔS) does contain the SARS-CoV-2 N protein (Fig. 7D); the shape and size of the VLP (StruΔS) was observed by TEM, and we found that the estimated size of the spherical particles without the spike protein The diameter was 75±14 nm (n=30, FIG. 7E ).
我们将SARS-CoV-2 ORF1ab基因序列与mCherry序列融合构建成质粒，质粒表达的多聚蛋白1a(pp1a)和pp1ab可以被病毒蛋白酶自切割成16个非结构蛋白(NSP)(图8A)。通过RT-PCR评估SARS-CoV-2 ORF1ab-mCherry质粒的活性，同时使用共聚焦显微镜可在转染后48小时内观察到mCherry信号(图8B和8C)。We fused the SARS-CoV-2 ORF1ab gene sequence with the mCherry sequence to construct a plasmid, and the polyprotein 1a (pp1a) and pp1ab expressed by the plasmid could be self-cleaved into 16 nonstructural proteins (NSP) by viral proteases (Figure 8A). The activity of the SARS-CoV-2 ORF1ab-mCherry plasmid was assessed by RT-PCR, while the mCherry signal could be observed within 48 hours after transfection using confocal microscopy (Figure 8B and 8C).
VLP(StruΔS-S)的组装和分泌依赖于SARS-CoV-2 S质粒和StruΔS-EGFP质粒的共表达；转染后24h，共聚焦显微镜检测到EGFP的荧光，蛋白免疫印迹检测到SARS-CoV-2 S和N蛋白，这表明SARS-CoV-2 S和StruΔS-EGFP RNA可以在转染的细胞中表达(图9A和9B)。为表征其形貌和尺寸，对VLP(StruΔS-S)负染色后在TEM下进行透射电镜观察(图9C)；观察结果表明VLP(StruΔS-S)是带有刺突蛋白的球形颗粒，平均大小估计为74±16nm(n＝30，不包含刺突)。同时我们也对组装VLP(StruΔS-S)的结构蛋白进行蛋白免疫印迹分析，也能得到预期分子量的条带(图9B)。The assembly and secretion of VLP (StruΔS-S) depend on the co-expression of SARS-CoV-2 S plasmid and StruΔS-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 StruΔS-EGFP RNA could be expressed in transfected cells (Figure 9A and 9B). In order to characterize its shape and size, VLP (StruΔS-S) was negatively stained and observed under TEM under TEM (Fig. 9C); the observation results showed that VLP (StruΔS-S) was a spherical particle with a spike protein, with an average The size was estimated to be 74±16 nm (n=30, excluding spikes). At the same time, we also performed Western blot analysis on the structural proteins of the assembled VLP (StruΔS-S), and bands with expected molecular weights were also obtained ( FIG. 9B ).
VLP(StruΔS-ORF1ab)是由SARS-CoV-2 StruΔS-EGFP质粒和ORF1ab-mCherry质粒共转染293T细胞而产生的；在转染后24小时对细胞进行成像，可以观察到明显的荧光(图10A)；同时，通过蛋白免疫印迹检测到SARS-CoV-2 N蛋白在转染细胞中的表达(图10B)。接着，进行透射电镜观察以分析VLP(StruΔS-ORF1ab)的形态特征。不含刺突蛋白的球形颗粒的直径约为79±13nm(n＝30，图10C)。VLP(StruΔS-ORF1ab)的SARS-CoV-2 N蛋白通过蛋白免疫印迹法测定(图10B)。VLP (StruΔS-ORF1ab) was produced by co-transfecting 293T cells with SARS-CoV-2 StruΔS-EGFP plasmid and ORF1ab-mCherry plasmid; the cells were imaged 24 hours after transfection, and obvious fluorescence could be observed (Fig. 10A); meanwhile, the expression of SARS-CoV-2 N protein in transfected cells was detected by western blot (Fig. 10B). Next, TEM observation was performed to analyze the morphological characteristics of VLP (StruΔS-ORF1ab). The diameter of spherical particles without Spike protein was approximately 79±13 nm (n=30, FIG. 10C ). The SARS-CoV-2 N protein of VLP (StruΔS-ORF1ab) was determined by western blotting (Fig. 10B).
要制备SARS-CoV-2 VLP，需要三质粒共转染到239T细胞中。转染后24小时内，能观察到EGFP和mCherry信号，表明在细胞中质粒进行了复制和转录(图2A)。为了检测SARS-CoV-2 S和N蛋白的表达，在转染后48小时，收集细胞裂解产液进行蛋白免疫印迹分析。同时，为了分析病毒颗粒的形态表征，进行了透射电镜观察；透射电镜图像显示圆形病毒颗粒且具有典型尖峰冠状，且颗粒的直径约为76±12nm(n＝30，不包含刺突)。SARS-CoV-2 VLP和其他VLPs似乎具有相似的形状和尺寸(图2C)。病毒粒子中的SARS-CoV-2 S和N蛋白，通过蛋白免疫印迹进行分析可得到预期的条带(图2B)。To prepare SARS-CoV-2 VLPs, three plasmids were co-transfected into 239T cells. Within 24 hours after transfection, EGFP and mCherry signals could be observed, indicating replication and transcription of the plasmid in the cells (Fig. 2A). In order to detect the expression of SARS-CoV-2 S and N proteins, 48 hours after transfection, the cell lysates were collected for western blot analysis. At the same time, in order to analyze the morphological characterization of virus particles, transmission electron microscope observations were carried out; transmission electron microscope images showed round virus particles with typical peaked crowns, and the diameter of the particles was about 76±12nm (n=30, excluding spikes). SARS-CoV-2 VLPs and other VLPs appear to have similar shapes and sizes (Fig. 2C). Analysis of the SARS-CoV-2 S and N proteins in the virion by western blot yielded the expected bands (Fig. 2B).
为了避免潜在的生物安全问题，将拯救的病毒样颗粒在BSL-3实验室进行了安全测试。我们构建的293T/hACE2细胞株对SARS-CoV-2感染具有易感性；细胞表达的hACE2受体，可以通过蛋白免疫印迹进行验证(图11A)。用SARS-CoV-2 VLP去感染293T/hACE2细胞，以测试受感染的细胞是否产生了子代病毒。病毒结构蛋白是否在细胞中进行了表达并组装成子代病毒，用蛋白免疫印迹进行检测，结果显示没能观察到对应的条带。结果表明，SARS-CoV-2 VLP不能进行复制、表达结构蛋白，进而不能在宿主细胞中组装产生子代病毒。由于SARS-CoV-2 VLP无法复制，感染SARS-CoV-2 VLP和野生型SARS-CoV-2病毒的293T/hACE2细胞的代表性图像显示病毒存在显着的滴度差异(图11C)。因此，我们构建的SARS-CoV-2 VLP，可以作为一种安全、适宜的模型来研究SARS-CoV-2病毒的生命周期和感染机制，有望进一步理解病毒的生命周期，为抗病毒药物及疫苗的开发提供一定理论基础。To avoid potential biosafety issues, 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. 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
为了进一步探究病毒基因组RNA的组装，我们利用SARS-CoV-2 VLP感染293T/hACE2细胞(图2E)。在病毒颗粒感染48小时后，通过共聚焦显微镜成像可以观察到mCherry信号，但可能由于ORF1ab-mCherry mRNA的低拷贝数，受感染细胞中的红色荧光非常微弱(图2D)。VLP(mCherry)由SARS-CoV-2 S质粒、StruΔS-EGFP 质粒和mCherry质粒(直接将mCherry插入pEGFP-N1载体获得)三质粒共转染组装而成。在VLP(mCherry)感染的细胞中未观察到荧光信号，表明没有包装信号(PS)序列的mCherry mRNA无法包装成颗粒。这说明位于ORF1ab片段中的SARS-CoV-2包装信号，对于病毒RNA组装形成颗粒具有重要作用。To further explore the assembly of viral genomic RNA, we infected 293T/hACE2 cells with SARS-CoV-2 VLPs (Fig. 2E). 48 hours after virus particle infection, mCherry signal could be observed by confocal microscopy imaging, but the red fluorescence in infected cells was very weak, possibly due to the low copy number of ORF1ab-mCherry mRNA (Fig. 2D). VLP (mCherry) was assembled by co-transfection of SARS-CoV-2 S plasmid, StruΔS-EGFP plasmid and mCherry plasmid (obtained by directly inserting mCherry into pEGFP-N1 vector). No fluorescent signal was observed in VLP(mCherry)-infected cells, indicating that mCherry mRNA without a packaging signal (PS) sequence cannot be packaged into particles. This shows that the SARS-CoV-2 packaging signal located in the ORF1ab fragment plays an important role in the assembly of viral RNA to form particles.
SARS-CoV-2中包装信号的预测和验证Prediction and validation of packaging signals in SARS-CoV-2
根据之前的报道，冠状病毒的核衣壳通过识别特定的包装信号(PS)序列与病毒RNA相互作用，以促进基因组RNA组装成病毒颗粒。用生物信息学分析进行预测SARS-CoV-2的PS位置，预测结果表明PS在靠近ORF1b区域的3'末端。同时，我们预测了SARS-CoV-2、SARS-CoV和类蝙蝠SARS-CoV的RNA二级结构；预测结果表明，三种类型的病毒的RNA二级结构中有都含有两个稳定的茎环(SL1和SL2)(图3A)。PS共有结构的多重比对进一步证实了RNA茎环具有高度保守性，尤其是SL1和SL2(图12)。这些结果表明，稳定的茎环结构可能在SARS-CoV-2中具备PS功能。为了进一步探究，根据包装序列的生信预测结果，我们选择了两个区域来测试包装活性，一个是包含保守茎环结构的短区域(PS101,nt 19900-20000)，另外一个是带有额外侧翼序列的较长区域(PS583,nt 19773-20335)。According to previous reports, 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. 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. At the same time, we predicted the RNA secondary structures of SARS-CoV-2, SARS-CoV and bat-like SARS-CoV; the prediction results showed that the RNA secondary structures of the three types of viruses all contained two stable stem-loops (SL1 and SL2) (Fig. 3A). Multiple alignments of PS consensus structures further confirmed that 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. To further explore, according to the bioinformatics prediction results of the packaging sequence, we selected two regions to test packaging activity, one is a short region containing a conserved stem-loop structure (PS101, nt 19900-20000), and the other is with additional flanks A longer region of the sequence (PS583, nt 19773-20335).
将预测的PS序列与EGFP报告基因的3'非编码区融合表达，构建了质粒pEGFP-N1-PS101和质粒pEGFP-N1-PS583并分别转染到293T细胞中；EGFP-PS101和EGFP-PS583 RNA的表达通过RT-PCR来确定(图S8A和3B)。为了证实预测的PS RNA的组装，收集VLP(EGFP-PS101)和VLP(EGFP-PS583)并用于感染293T/hACE2细胞，如图3E所示。SARS-CoV-2 S质粒、StruΔS质粒和pEGFP-N1-PS101/pEGFP-N1-PS583质粒共转染到293T细胞中，分别产生VLP(EGFP-PS101)或VLP(EGFP-PS583)。转染细胞中，S蛋白、N蛋白和EGFP进行表达并组装成VLP，通过蛋白免疫印迹对几个蛋白进行分析(图13B和3C)。在VLP中检测到结构蛋白，而EGFP未能被检测到。为了验证EGFP-PS101或EGFP-PS583 RNA是否被包装到VLP中，用VLP感染293T/hACE2细胞并检查受感染细胞中的EGFP表达(图3E)。在VLP(EGFP-PS101)或VLP(EGFP-PS583)感染细胞48小时后，观察到EGFP的绿色荧光，并且具有较高的荧光强度，这说明其具有更好的包装活性。如图13C和3D所示，VLP感染细胞中的低荧光信号可能是由于包装到VLP中的EGFP-PS RNA拷贝数较低。VLP(EGFP)是通过SARS-CoV-2 S质粒、StruΔS质粒和pEGFP-N1质粒共转染得到，并作为对照组，表面不含PS序列的EGFP mRNA不能被包装到VLP中(绿色荧光蛋白)。这些结果证实预测的序列在SARS-CoV-2中具有PS功能，对于将病毒基因组RNA组装成VLP非常重要。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, StruΔS 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. In transfected cells, 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. To verify whether EGFP-PS101 or EGFP-PS583 RNA was packaged into VLPs, 293T/hACE2 cells were infected with VLPs and EGFP expression in infected cells was examined (Figure 3E). 48 hours after VLP (EGFP-PS101) or VLP (EGFP-PS583) infected cells, the green fluorescence of EGFP was observed with higher fluorescence intensity, which indicated that it had better packaging activity. As shown in Figure 13C and 3D, the low fluorescent signal in VLP-infected cells may be due to the low copy number of EGFP-PS RNA packaged into VLP. VLP (EGFP) was obtained by co-transfection of SARS-CoV-2 S plasmid, StruΔS 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.
结构蛋白在病毒感染和组装中的作用评估Assessment of the role of structural proteins in viral infection and assembly
此前的报道证实，SARS-CoV-2 S蛋白关联受体结合和膜融合，S蛋白的突变将极大地影响对宿主的感染能力。我们在SARS-CoV-2 S质粒的基础上分别构建了4个不同区域的突变，包括RBD区域22553位点A到C置换和RBD区域22555位点C到G置换的N331Q突变、23063位点A-T置换的N501Y；RBD区附近23403位点的A到G置换的D614G；以及弗林蛋白酶区附近23604位点C到A置换的P681H突变；S突变体的构建和表征如图14-17所示。VLP S(突变)由SARS-CoV-2 S(突变)质粒、StruΔS质粒和ORF1ab质粒的共转染细胞后组装完成。病毒粒子中的SARS-CoV-2 S和N蛋白通过蛋白质印迹进行鉴定，预期条带如图18所示。VLP S(突变)的形态和大小利用透射电镜进行分析，结果表明突变体与具有野生型S蛋白的VLP相似(图18)。为了检 测S突变病毒颗粒的感染性，用DiO标记脂质包膜产生荧光VLP S(突变)去感染293T/hACE2细胞。将细胞在感染后1小时或2小时进行固定，接着随机选择500个细胞在共聚焦显微镜下成像，统计有色颗粒用于分析。与野生型S的VLP相比，VLP(N501Y)、VLP(D614G)和VLP(P681H)具有更高的传染性，而VLP(N331Q)的传染性则明显降低(图4A)。结果与之前的报告一致，表明我们构建的系统可以用于评估S突变对SARS-CoV-2组装和感染性影响的能力。Previous reports have confirmed that the SARS-CoV-2 S protein is associated with receptor binding and membrane fusion, and mutations in the S protein will greatly affect the ability to infect the host. We constructed mutations in four different regions on the basis of the SARS-CoV-2 S plasmid, including the N331Q mutation of the RBD region 22553 site A to C substitution and the RBD region 22555 site C to G substitution, and the 23063 site A-T Substitution of N501Y; D614G of A to G substitution at site 23403 near the RBD region; and P681H mutation of C to A substitution at site 23604 near the furin region; construction and characterization of the S mutant are shown in Figures 14-17. VLP S (mutation) was assembled after co-transfection of cells with SARS-CoV-2 S (mutation) plasmid, StruΔS plasmid and ORF1ab plasmid. The 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). In order to detect the infectivity of S mutant virus particles, 293T/hACE2 cells were infected with DiO-labeled lipid envelope to produce fluorescent VLP S(mutant). Cells were fixed 1 hour or 2 hours after infection, and then 500 cells were randomly selected and imaged under a confocal microscope to count colored particles for analysis. Compared with the VLP of wild-type S, 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.
为了研究核衣壳(N)在病毒RNA包装中的必要性，我们在质粒SARS-CoV-2 StruΔS上删除了N蛋白的编码区，从而构建了质粒SARS-CoV-2 StruΔS-EGFP/ΔN((又可称SARS-CoV-2 StruΔS-EGFP/ΔN质粒、StruΔS-EGFP/ΔN质粒或StruΔS/ΔN质粒)图19A)。SARS-CoV-2 StruΔS-EGFP/ΔN质粒的活性和表达分别通过RT-PCR和蛋白免疫印迹进行检测(图19B和19C)。VLP(ΔN)可以通过SARS-CoV-2 S质粒、StruΔS-EGFP/ΔN质粒和ORF1ab三质粒共转染细胞后得到。VLP(ΔN)呈球形、具有冠状结构，直径为81±17nm(n＝30，不包括刺突)，如TEM图像所示。产生的VLP，我们用蛋白免疫印迹检测进行检测，检测到S蛋白，未能检测到N蛋白，这与预期的结果一致(图4C)。病毒RNA组装过程中是否依赖核衣壳，是通过将VLP(ΔN-EGFP-PS583)感染293T/hACE2细胞确定的；VLP(ΔN-EGFP-PS583)是通过与质粒SARS-CoV-2 S质粒、StruΔS/ΔN质粒以及包装信号质粒pEGFP-N1-PS583共转染产生。在VLP(ΔN-EGFP-PS583)感染的细胞中观察到EGFP的绿色荧光很少，如图4B所示；这与VLP(EGFP-PS583)感染的细胞存在显着差异。结果表明，N蛋白对于VLP的形成不是必需的，但它在病毒RNA包装中发挥重要作用。To investigate the necessity of nucleocapsid (N) in viral RNA packaging, we deleted the coding region of N protein on the plasmid SARS-CoV-2 StruΔS, thereby constructing the plasmid SARS-CoV-2 StruΔS-EGFP/ΔN( (It can also be called SARS-CoV-2 StruΔS-EGFP/ΔN plasmid, StruΔS-EGFP/ΔN plasmid or StruΔS/ΔN plasmid) Figure 19A). The activity and expression of the SARS-CoV-2 StruΔS-EGFP/ΔN plasmid were detected by RT-PCR and Western blotting, respectively (Figures 19B and 19C). VLP(ΔN) can be obtained by co-transfecting cells with SARS-CoV-2 S plasmid, StruΔS-EGFP/ΔN plasmid and ORF1ab three plasmids. The VLP (ΔN) was spherical with a coronal structure and a diameter of 81 ± 17 nm (n = 30, excluding spikes), as shown in TEM images. 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 (ΔN-EGFP-PS583); StruΔS/ΔN plasmid and packaging signal plasmid pEGFP-N1-PS583 co-transfected to produce. Little green fluorescence of EGFP was observed in VLP(ΔN-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.
关于冠状病毒的早期报道中，膜(M)和包膜(E)蛋白作为主要的功能成分，在病毒颗粒的产生中发挥了重要作用；本研究也探究了M和E蛋白对VLP制备的影响。分别删除M或E蛋白编码序列，得到SARS-CoV-2 StruΔS-EGFP/ΔM质粒和SARS-CoV-2 StruΔS-EGFP/ΔE质粒，并通过RT-PCR和蛋白免疫印迹进行验证(图20和22)。将StruΔS突变体质粒，即SARS-CoV-2 StruΔS-EGFP/ΔM质粒或SARS-CoV-2 StruΔS-EGFP/ΔE质粒与质粒SARS-CoV-2 S和ORF1ab-mCherry共转染到293T细胞中，分别产生StruΔS突变体病毒颗粒rVP(ΔM)或rVP(ΔE)。TEM图像和蛋白质印迹分析表明，缺乏M或E蛋白的病毒颗粒在转染细胞中仍然可获得(图4C、21A和23A)。StruΔS突变体病毒颗粒进一步用于感染293T/hACE2细胞，在感染48小时后没能观察到荧光信号(图21B和23B)，这与SARS-CoV-2 VLP感染后宿主细胞的表现相反，表明即使缺失M或E基因，转染细胞也能产生突变病毒粒子，但病毒粒子的功能受到了影响。与N缺失的突变体不同，M或E蛋白的缺失不影响病毒RNA的包装。In the early reports on coronaviruses, the membrane (M) and envelope (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 StruΔS-EGFP/ΔM plasmid and the SARS-CoV-2 StruΔS-EGFP/ΔE plasmid, which were verified by RT-PCR and Western blotting (Figures 20 and 22 ). The StruΔS mutant plasmid, that is, the SARS-CoV-2 StruΔS-EGFP/ΔM plasmid or the SARS-CoV-2 StruΔS-EGFP/ΔE plasmid was co-transfected with the plasmids SARS-CoV-2 S and ORF1ab-mCherry into 293T cells, StruΔS mutant virions rVP(ΔM) or rVP(ΔE) 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). StruΔS 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.
SARS-CoV-2基因组中开放阅读框10(ORF10)区域位于N基因下游，在之前的报道中ORF10蛋白似乎没有发挥重大作用。我们构建了敲除ORF10编码序列的SARS-CoV-2 StruΔS-EGFP/ΔORF10质粒，并进行表征(图24)；该质粒与质粒SARS-CoV-2 S、ORF1ab共转染细胞，从而产生VLP(ΔORF10)。VLP(ΔORF10)表现出与我们构建的SARS-CoV-2 VLP具有相似的形态和功能(图4C)，表明ORF10基因的缺失对病毒颗粒的组装没有影响。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. We constructed and characterized the SARS-CoV-2 StruΔS-EGFP/ΔORF10 plasmid that knocked out the ORF10 coding sequence (Figure 24); the plasmid was co-transfected with the plasmid SARS-CoV-2 S and ORF1ab to produce VLP ΔORF10). The VLP (ΔORF10) 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.
为了进一步确认上述结构蛋白/辅助蛋白对VLP组装的影响，我们对VLP中的基因组和囊膜分别进行了荧光标记，并通过荧光共定位效率初步探究了VLP的组装效率(图25)，结论与上述验证结果基本一致。In order to further confirm the influence of the above-mentioned structural proteins/accessory proteins on VLP assembly, we carried out fluorescent labeling on the genome and envelope of VLP, and preliminarily explored the assembly efficiency of VLP through the fluorescence colocalization efficiency (Figure 25). The conclusion is consistent with that of The above verification results are basically consistent.
rSARS-CoV-2病毒进入293T/hACE2细胞的实时成像。Live imaging of rSARS-CoV-2 virus entry into 293T/hACE2 cells.
我们制备了双荧光标记的SARS-CoV-2 VLP(QD-DiO)，以深入了解病毒进入过程，如图5A所示。病毒RNA与靶核酸序列杂交形成复合物，用量子点(QD)纳米信标标记后带有红色荧光，复合物最终被病毒颗粒包裹。在QD标记病毒内部观察到暗电子致密核心(图5B)。为了获得双荧光颗粒，用DiO标记脂质包膜。RNA-QD和Env-DiO的荧光共定位证实了成功构建双色rSARS-CoV-2(QD-DiO)(图5C)，其中rSARS-CoV-2也即SARS-CoV-2 VLP。We prepared double fluorescently labeled SARS-CoV-2 VLPs (QD-DiO) to gain insight into the virus entry process, as shown in Figure 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.
为了显示病毒进入的动态过程，通过共聚焦显微镜成像在293T/hACE2细胞中跟踪SARS-CoV-2 VLP(QD-DiO)颗粒。在实时成像期间，只有包含QD和DiO信号共定位，才当作单个病毒粒子。在293T/hACE2细胞的膜上观察到双色颗粒，并以主动方式转运到宿主细胞中(图5D)。该病毒颗粒的轨迹如图5E所示。本研究中，我们对活细胞中的2000多个单病毒颗粒的轨迹进行了跟踪和分析。结果表明，47.5％病毒颗粒通过内吞作用进入293T/hACE2细胞中，而其他颗粒仅附着在细胞膜表面，没有转运到细胞质中。To visualize the dynamic process of viral entry, 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.
病毒核心在胞吞进入宿主细胞过程中从包膜中的释放也被实时成像捕获。对活细胞中rSARS-CoV-2(QD-DiO)颗粒的跟踪显示，病毒核心释放到细胞质中。具有QD和DiO共定位信号(黄色)的病毒粒子在细胞质中成像，并且在病毒粒子的动态运动期间观察到红点与绿点的分离(图5F)。RNA-QD和Env-DiO的轨迹在分离行为后有所不同，如图5G所示。结果表明病毒核心从内体中成功逃逸，这是产生感染的必要过程。本研究中，我们在293T/hACE2细胞中分析了1000个单个粒子，并捕获了64个类似事件。较低的比例可能是由于在观察过程中被跟踪的粒子移出焦平面或荧光信号的光漂白。The release of the viral core from the envelope during endocytosis into the host cell was also captured by live imaging. Tracking of rSARS-CoV-2 (QD-DiO) particles in living cells revealed release of the viral core into the cytoplasm. Virions with QD and DiO colocalization signals (yellow) 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. The results indicated successful escape of the viral core from the endosome, a necessary process for generating infection. In this study, we analyzed 1000 individual particles in 293T/hACE2 cells and captured 64 similar events. The lower ratio could be due to the movement of tracked particles out of the focal plane or photobleaching of the fluorescent signal during observation.
讨论discuss
为了研究SARS-CoV-2，我们构建了病毒基因组拆分系统(split-virus-genom，SVG)，并进行了对应的表征分析。利用该系统产生了具有单轮感染的SARS-CoV-2 VLP，有望用于生物学探索和疫苗开发。包含包装信号的SARS-CoV-2 ORF1ab RNA被组装到rSARS-CoV-2中，而结构基因不在病毒粒子中，从而确保了系统的安全性。我们设计了一系列带有病毒基因的突变或缺失的rSARS-CoV-2突变体，以确定病毒成分的功能；探究了四种结构蛋白和ORF10蛋白在SARS-CoV-2 VLP感染和组装中的作用。同时，我们引入纳米信标和亲脂性染料，通过实时成像显示SARS-CoV-2 VLP通过内吞作用进入宿主细胞的动态过程。根据研究需要，可以在系统中设计各种荧光探针，从而可视化病毒生命周期的其他步骤。另外，在BSL-2实验室进行病毒学实验的通用平台对于研究SARS-CoV-2非常重要。基于我们构建的系统，可运用于目前几种类型的SARS-CoV-2研究：(1)SARS-CoV-2的功能分析可以通过设计rSARS-CoV-2突变体来进行每个成分的功能分析。(2)特定序列SARS-CoV-2包装信号的预测和验证，为研究参与病毒RNA组装的分子机制提供了可靠的途径。(3)基于多色标记SARS-CoV-2 VLP的单一病毒颗粒追踪，能够对活宿主细胞中病毒颗粒的侵染过程进行实时和精确地成像。(4)组装的具有不同成分的VLP促进了载体和疫苗的开发。结合这些优势，SVG系统可发展为研究SARS-CoV-2和其他冠状病毒的宝贵平台。In order to study SARS-CoV-2, we constructed a 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. We designed a series of rSARS-CoV-2 mutants with mutations or deletions of viral genes to determine the functions of viral components; explored the role of four structural proteins and ORF10 protein in SARS-CoV-2 VLP infection and assembly effect. At the same time, we introduced nanobeacons and lipophilic dyes to show the dynamic process of SARS-CoV-2 VLPs entering host cells through endocytosis through real-time imaging. Depending on research needs, various fluorescent probes can be designed into the system to visualize other steps of the virus life cycle. In addition, a common platform for virology experiments in the BSL-2 laboratory is very important for the study of SARS-CoV-2. Based on the system we built, it can be applied to several types of current SARS-CoV-2 research: (1) Functional analysis of SARS-CoV-2 can be performed by designing rSARS-CoV-2 mutants for functional analysis of each component . (2) The prediction and verification of the specific sequence SARS-CoV-2 packaging signal provides a reliable way to study the molecular mechanism involved in viral RNA assembly. (3) 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. (4) 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.
我们的实验结果证实SVG系统具备安全性。SARS-CoV-2的全基因组分为三个部分，病毒RNA组装所必需的包装信号位于ORF1ab片段中。我们的系统缺乏多个基因片段，不会生产野生型病毒。与包含部分功能结构的假病毒不同，我们生产的病毒体几乎具有SARS-CoV-2的所有成分。然而，由于病毒包装的RNA中缺乏结构基因，SARS-CoV-2 VLP无法复制和组装子代病毒。这些单轮感染性病毒粒子无法对宿主细胞 进行多轮感染，阻断了病毒的传播。不需要构建特定的稳定细胞系来生产可以在BSL-2实验室中安全操作的病毒颗粒，例如反式互补系统。根据病毒研究的需要，可以进一步从三个片段中删除一些ORF基因，产生安全性更高的rSARS-CoV-2突变体。Our experimental results confirm that the SVG system is secure. 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. There is no need to construct a specific stable cell line to produce virus particles that can be safely manipulated in a BSL-2 laboratory, such as a trans-complementation system. According to the needs of virus research, some ORF genes can be further deleted from the three fragments to produce rSARS-CoV-2 mutants with higher safety.
在SVG系统中，我们惊讶地发现编码M蛋白的基因的缺失并不影响病毒颗粒的形成，而在VLP(ΔM)感染的细胞中却未观察到病毒RNA表达的mCherry信号。这可能是由于突变病毒粒子中缺乏M蛋白干扰了病毒粒子的功能。因此，SARS-CoV-2 M蛋白在病毒功能中起主要作用，但对于病毒颗粒的形成并不是必需的。它不同于SARS-CoV，在SARS-CoV中M蛋白是病毒组装的关键因素。我们开发的系统也有一些限制。病毒组装效率有待提高，这对系统的进一步提升和疫苗开发具有重要意义。病毒体包装的RNA片段的优化可以促进SARS-CoV-2感染和复制的研究。本发明的SVG系统、组合物和方法可用作促进中和试验和抗病毒测试的强大工具，利用该病毒基因组拆分系统可获得多种假病毒颗粒，可用于病毒抗体的中和抗体试验、抗病毒药物的筛选、病毒疫苗的制备等。总之，我们为SARS-CoV-2的研究开发了一个SVG系统，可以在BSL-2实验室进行操作。产生的病毒粒子rSARS-CoV-2很容易进行工程改造，用于分析病毒生命过程和每个组件的功能。In the SVG system, we were surprised to find that deletion of the gene encoding the M protein did not affect viral particle formation, whereas no mCherry signal expressed by viral RNA was observed in VLP(ΔM)-infected cells. This may be due to the lack of M protein in the mutant virion interfering with the function of the virion. Thus, 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. Optimization of virion-packaged RNA fragments can facilitate studies of SARS-CoV-2 infection and replication. 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. In summary, we have developed an SVG system for SARS-CoV-2 research that can be operated in a BSL-2 laboratory. The resulting virion rSARS-CoV-2 was easily engineered for analysis of the viral life process and the function of each component.
表1Table 1
Figure PCTCN2021138037-appb-000001
Figure PCTCN2021138037-appb-000001
下面的序列中，带有下划线的序列为同源臂In the following sequences, the underlined sequences are homology arms
S蛋白表达序列拆分片段(包含密码子优化的S蛋白基因组3822bp)：Split fragment of S protein expression sequence (3822bp including codon-optimized S protein genome):
S-片段1(SEQ ID NO：1)：S-fragment 1 (SEQ ID NO: 1):
Figure PCTCN2021138037-appb-000002
Figure PCTCN2021138037-appb-000002
S-片段2(SEQ ID NO：2)：S-fragment 2 (SEQ ID NO: 2):
Figure PCTCN2021138037-appb-000003
Figure PCTCN2021138037-appb-000003
Figure PCTCN2021138037-appb-000004
Figure PCTCN2021138037-appb-000004
ORF1ab序列拆分片段：ORF1ab sequence split fragments:
ORF1ab-片段1(SEQ ID NO：3)：ORF1ab-fragment 1 (SEQ ID NO: 3):
Figure PCTCN2021138037-appb-000005
Figure PCTCN2021138037-appb-000005
Figure PCTCN2021138037-appb-000006
Figure PCTCN2021138037-appb-000006
Figure PCTCN2021138037-appb-000007
Figure PCTCN2021138037-appb-000007
ORF1ab-片段2(SEQ ID NO：4)：ORF1ab-fragment 2 (SEQ ID NO: 4):
Figure PCTCN2021138037-appb-000008
Figure PCTCN2021138037-appb-000008
Figure PCTCN2021138037-appb-000009
Figure PCTCN2021138037-appb-000009
ORF1ab-片段3(SEQ ID NO：5)：ORF1ab-fragment 3 (SEQ ID NO: 5):
Figure PCTCN2021138037-appb-000010
Figure PCTCN2021138037-appb-000010
Figure PCTCN2021138037-appb-000011
Figure PCTCN2021138037-appb-000011
Figure PCTCN2021138037-appb-000012
Figure PCTCN2021138037-appb-000012
ORF1ab-片段4(SEQ ID NO：6)：ORF1ab-fragment 4 (SEQ ID NO: 6):
Figure PCTCN2021138037-appb-000013
Figure PCTCN2021138037-appb-000013
Figure PCTCN2021138037-appb-000014
Figure PCTCN2021138037-appb-000014
ORF1ab-片段5(SEQ ID NO：7)：ORF1ab-fragment 5 (SEQ ID NO: 7):
Figure PCTCN2021138037-appb-000015
Figure PCTCN2021138037-appb-000015
Figure PCTCN2021138037-appb-000016
Figure PCTCN2021138037-appb-000016
ORF1ab-片段6(SEQ ID NO：8)：ORF1ab-fragment 6 (SEQ ID NO: 8):
Figure PCTCN2021138037-appb-000017
Figure PCTCN2021138037-appb-000017
Figure PCTCN2021138037-appb-000018
Figure PCTCN2021138037-appb-000018
StruΔS蛋白表达序列拆分片段：StruΔS protein expression sequence split fragments:
StruΔS-片段1(SEQ ID NO：9)：StruΔS-fragment 1 (SEQ ID NO: 9):
Figure PCTCN2021138037-appb-000019
Figure PCTCN2021138037-appb-000019
Figure PCTCN2021138037-appb-000020
Figure PCTCN2021138037-appb-000020
StruΔS-片段2(SEQ ID NO：10)：StruΔS-fragment 2 (SEQ ID NO: 10):
Figure PCTCN2021138037-appb-000021
Figure PCTCN2021138037-appb-000021
Figure PCTCN2021138037-appb-000022
Figure PCTCN2021138037-appb-000022
StruΔS-片段3(SEQ ID NO：11)：StruΔS-fragment 3 (SEQ ID NO: 11):
Figure PCTCN2021138037-appb-000023
Figure PCTCN2021138037-appb-000023
Figure PCTCN2021138037-appb-000024
Figure PCTCN2021138037-appb-000024
实施例1中使用的5’UTR(SEQ ID NO：12)5'UTR (SEQ ID NO: 12) used in Example 1
Figure PCTCN2021138037-appb-000025
Figure PCTCN2021138037-appb-000025
实施例1中使用的3’UTR(包含polyA尾巴，SEQ ID NO：13)3'UTR used in Example 1 (comprising polyA tail, SEQ ID NO: 13)
Figure PCTCN2021138037-appb-000026
Figure PCTCN2021138037-appb-000026
本发明的实施方式并不限于上述实施例所述，在不偏离本发明的精神和范围的情况下，本领域普通技术人员可以在形式和细节上对本发明做出各种改变和改进，而这些均被认为落入了本发明的保护范围。The embodiments of the present invention are not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements to the present invention in form and details, and these All are considered to fall into the protection scope of the present invention.

Claims (12)

  1. 用于制备SARS-CoV-2病毒样颗粒的组合物，包括下述(a)，还包括选自下述Compositions for the preparation of SARS-CoV-2 virus-like particles, including the following (a), also include selected from the following
    (b)和(c)中的任一种或任两种：Either or both of (b) and (c):
    (a)包含StruΔS片段的第一质粒，所述ΔS片段包含编码SARS-CoV-2病毒的至少一种结构蛋白或其突变体和/或至少一种辅助蛋白或其突变体的核酸序列，且所述ΔS片段不包含编码SARS-CoV-2病毒的S蛋白或其突变体的核酸序列；(a) a first plasmid comprising a StruΔS 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 ΔS fragment does not include a nucleic acid sequence encoding the S protein of SARS-CoV-2 virus or a mutant thereof;
    (b)包含S片段的第二质粒，所述S片段包含编码SARS-CoV-2病毒的S蛋白或其突变体的核酸序列；(b) the second plasmid that comprises S segment, and described S segment comprises the nucleic acid sequence of the S protein of coding SARS-CoV-2 virus or its mutant;
    (c)包含SARS-CoV-2病毒的包装信号片段的第三质粒，所述包装信号片段包含SARS-CoV-2病毒的包装信号序列。(c) 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.
  2. 根据权利要求1所述的组合物，其中所述的S蛋白的突变体相对于SARS-CoV-2病毒的S蛋白的突变包括N331Q、N501Y、D614G和/或P681H。The composition according to claim 1, wherein the mutant of the S protein comprises N331Q, N501Y, D614G and/or P681H relative to the mutation of the S protein of the SARS-CoV-2 virus.
  3. 根据权利要求1或2所述的组合物，其中所述StruΔS片段包含下述任一种：The composition according to claim 1 or 2, wherein the StruΔS fragment comprises any of the following:
    (1)编码SARS-CoV-2病毒的除S蛋白之外的全部结构蛋白和全部辅助蛋白的核酸序列；(1) Nucleic acid sequences encoding all structural proteins and all auxiliary proteins of the SARS-CoV-2 virus except the S protein;
    (2)与(1)相比，所述结构蛋白和辅助蛋白中的至少一种相对于野生型SARS-CoV-2病毒具有突变；和(2) Compared with (1), at least one of the structural protein and accessory protein has a mutation relative to the wild-type SARS-CoV-2 virus; and
    (3)与(1)相比，缺失所述结构蛋白和辅助蛋白中的任一种。(3) Compared with (1), any one of the structural protein and auxiliary protein is deleted.
  4. 根据权利要求1-3任一项所述的组合物，其中所述包装信号序列是NCBI序列号NC_045512.2的第19900-20000位核苷酸或NCBI序列号NC_045512.2的第19773-20335位核苷酸。The composition according to any one of claims 1-3, wherein the packaging signal sequence is 19900-20000 nucleotides of NCBI sequence number NC_045512.2 or 19773-20335 of NCBI sequence number NC_045512.2 Nucleotides.
  5. 根据权利要求1-4任一项所述的组合物，其中所述包装信号片段包含SARS-CoV-2病毒基因组的ORF1ab。The composition according to any one of claims 1-4, wherein the packaging signal fragment comprises ORF1ab of the SARS-CoV-2 viral genome.
  6. 权利要求1-5任一项所述的组合物的制备方法，包括分别通过下述方法制备第一质粒、第二质粒和/或第三质粒：The method for preparing the composition according to any one of claims 1-5, comprising preparing the first plasmid, the second plasmid and/or the third plasmid by the following method respectively:
    (1)将StruΔS片段、S片段和/或ORF1ab包装信号片段分别拆分为在相邻片段之间具有同源序列的DNA短片段；(1) Separate the StruΔS fragment, the S fragment and/or the ORF1ab packaging signal fragment into short DNA fragments with homologous sequences between adjacent fragments;
    (2)将由步骤(1)获得的DNA短片段和线性化质粒载体转入酵母细胞中，进行同源重组，分别获得第一质粒、第二质粒和/或第三质粒。(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.
  7. SARS-CoV-2病毒样颗粒的制备方法，包括：A method for preparing SARS-CoV-2 virus-like particles, comprising:
    用权利要求1-5任一项的组合物转染包装细胞，获得SARS-CoV-2病毒样颗粒。Transfect packaging cells with the composition of any one of claims 1-5 to obtain SARS-CoV-2 virus-like particles.
  8. 根据权利要求7所述的制备方法，还包括The preparation method according to claim 7, further comprising
    分别通过下述方法制备第一质粒、第二质粒和/或第三质粒：Prepare the first plasmid, the second plasmid and/or the third plasmid respectively by the following methods:
    (1)将StruΔS片段、S片段和/或包装信号片段分别拆分为在相邻片段之间具有同源序列的DNA短片段；(1) Separate the StruΔS fragment, the S fragment and/or the packaging signal fragment into short DNA fragments with homologous sequences between adjacent fragments;
    (2)将由步骤(1)获得的DNA短片段和线性化质粒载体转入酵母细胞中，进行同源重组，分别获得第一质粒、第二质粒和/或第三质粒。(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.
  9. 根据权利要求8所述的制备方法，进一步包括将第一质粒、第二质粒和/或第三质粒分别在大肠杆菌中进行扩增，然后转染包装细胞。The preparation method according to claim 8, further comprising amplifying the first plasmid, the second plasmid and/or the third plasmid respectively in Escherichia coli, and then transfecting the packaging cells.
  10. 由权利要求7-9任一项所述的制备方法制备获得的SARS-CoV-2病毒样颗粒。The SARS-CoV-2 virus-like particle prepared by the preparation method described in any one of claims 7-9.
  11. 根据权利要求1-5任一项的组合物或根据权利要求10所述的SARS-CoV-2病毒样颗粒在制备用于预防或治疗SARS-CoV-2病毒感染的疫苗中的用途。Use of the composition according to any one of claims 1-5 or the SARS-CoV-2 virus-like particle according to claim 10 in the preparation of a vaccine for preventing or treating SARS-CoV-2 virus infection.
  12. 根据权利要求1-5任一项的组合物或根据权利要求10所述的SARS-CoV-2病毒样颗粒在体外用于SARS-CoV-2病毒感染细胞的研究中的用途。Use of the composition according to any one of claims 1-5 or the SARS-CoV-2 virus-like particle according to claim 10 for the research of SARS-CoV-2 virus infected cells in vitro.
PCT/CN2021/138037 2021-10-29 2021-12-14 Method for preparing sars-cov-2 virus-like particles and use of sars-cov-2 virus-like particles WO2023070873A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111271508.1 2021-10-29
CN202111271508.1A CN114395017A (en) 2021-10-29 2021-10-29 Preparation method and application of SARS-CoV-2 virus-like particle

Publications (1)

Publication Number Publication Date
WO2023070873A1 true WO2023070873A1 (en) 2023-05-04

Family

ID=81225768

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/138037 WO2023070873A1 (en) 2021-10-29 2021-12-14 Method for preparing sars-cov-2 virus-like particles and use of sars-cov-2 virus-like particles

Country Status (2)

Country Link
CN (1) CN114395017A (en)
WO (1) WO2023070873A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114395017A (en) * 2021-10-29 2022-04-26 中国科学院深圳先进技术研究院 Preparation method and application of SARS-CoV-2 virus-like particle

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111647053A (en) * 2020-04-16 2020-09-11 军事科学院军事医学研究院生命组学研究所 Polypeptide and application thereof in novel coronavirus detection and antibody or vaccine screening
CN113248579A (en) * 2020-02-12 2021-08-13 重庆医科大学 Novel coronavirus (2019-ncov) epitope, antibody and application thereof
WO2021176397A2 (en) * 2020-03-04 2021-09-10 Premas Biotech Pvt. Ltd Expression of sars-cov proteins, nucleic acid constructs, virus like proteins (vlps) and methods relevant thereto
US20210299245A1 (en) * 2020-03-31 2021-09-30 Sementis Limited Attenuated poxvirus vector based vaccine for protection against covid-19
WO2021211749A1 (en) * 2020-04-14 2021-10-21 The Regents Of The University Of California Multi-epitope pan-coronavirus vaccine compositions
CN114395017A (en) * 2021-10-29 2022-04-26 中国科学院深圳先进技术研究院 Preparation method and application of SARS-CoV-2 virus-like particle

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7556957B2 (en) * 2001-05-17 2009-07-07 Stichting Voor De Technische Wetenschappen Coronavirus-like particles comprising functionally deleted genomes
WO2006135413A2 (en) * 2004-09-02 2006-12-21 Qun Chen Encapsidation system for production of recombinant virus-like particles
US20230149536A1 (en) * 2020-04-17 2023-05-18 Vyriad, Inc. Compositions for treating and/or preventing coronavirus infections
CN112746059A (en) * 2020-09-15 2021-05-04 清华大学 Coronavirus cell model based on virus structural protein genetic complementation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113248579A (en) * 2020-02-12 2021-08-13 重庆医科大学 Novel coronavirus (2019-ncov) epitope, antibody and application thereof
WO2021176397A2 (en) * 2020-03-04 2021-09-10 Premas Biotech Pvt. Ltd Expression of sars-cov proteins, nucleic acid constructs, virus like proteins (vlps) and methods relevant thereto
US20210299245A1 (en) * 2020-03-31 2021-09-30 Sementis Limited Attenuated poxvirus vector based vaccine for protection against covid-19
WO2021211749A1 (en) * 2020-04-14 2021-10-21 The Regents Of The University Of California Multi-epitope pan-coronavirus vaccine compositions
CN111647053A (en) * 2020-04-16 2020-09-11 军事科学院军事医学研究院生命组学研究所 Polypeptide and application thereof in novel coronavirus detection and antibody or vaccine screening
CN114395017A (en) * 2021-10-29 2022-04-26 中国科学院深圳先进技术研究院 Preparation method and application of SARS-CoV-2 virus-like particle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
THOMAS SUNIL: "Mapping the Nonstructural Transmembrane Proteins of Severe Acute Respiratory Syndrome Coronavirus 2", JOURNAL OF COMPUTATIONAL BIOLOGY., MARY ANN LIEBERT INC., US, vol. 28, no. 9, 1 September 2021 (2021-09-01), US , pages 909 - 921, XP093060539, ISSN: 1066-5277, DOI: 10.1089/cmb.2020.0627 *
刘彬 等 (LIU, BIN ET AL.): "新型冠状病毒基因组结构与蛋白功能 (Genomic Structure and Protein Profiles of 2019 Novel Coronavirus)", 微生物与感染 (JOURNAL OF MICROBES AND INFECTIONS), vol. 15, no. 1, 25 February 2020 (2020-02-25), pages 52 - 57, XP055851290, DOI: 10.3969/j.issn.1673-6184.2020.01.010 *

Also Published As

Publication number Publication date
CN114395017A (en) 2022-04-26

Similar Documents

Publication Publication Date Title
CN112618707B (en) SARS-CoV-2 coronavirus vaccine and its preparation method
JP7239948B2 (en) RECOMBINANT RSV HAVING SILENT MUTATIONS, VACCINES, AND RELATED METHODS
AU2005279303B2 (en) Nucleic acid sequences encoding proteins capable of associating into a virus-like particle
CN110951699B (en) Recombinant rabies virus for expressing canine distemper virus structural protein and application thereof
WO2016086576A1 (en) Vector expressing poliomyelitis virus-like granule protein and method for preparing poliomyelitis virus-like granules
JPWO2019013258A1 (en) Photoregulated viral protein, its gene, and viral vector containing the gene
CN112458064A (en) Gatasavir full-length infectious clone, replicon system, preparation and application thereof
WO2023070873A1 (en) Method for preparing sars-cov-2 virus-like particles and use of sars-cov-2 virus-like particles
JP2023551982A (en) Multicistronic RNA vaccines and their uses
EP2460878B1 (en) Orf7 deficient varicella virus, vaccine comprising the virus and use thereof
JP2021502822A (en) Non-human papillomavirus for gene delivery in vitro and in vivo
JP2021500880A (en) Saladenovirus vector with two expression cassettes
KR20210126075A (en) Chimeric RSV and HMPV F proteins, immunogenic compositions, and methods of use
Wang et al. A novel viral vaccine platform based on engineered transfer RNA
CN113817753B (en) Expression of SARS-CoV-2 fiber protein or its variant S Δ21 Construction and use of pseudotyped VSV viruses
CN112079905B (en) Avian influenza virus-like particle antigen, vaccine, preparation method and application thereof
CN114292823A (en) Recombinant LaSota vaccine strain carrying genes VII type Newcastle disease virus F and HN genes and construction method and application thereof
CN111235114A (en) EV71 replication-defective virus and preparation method and application thereof
US20230203536A1 (en) Coronavirus rna replicons and use thereof as vaccines
Elfayres et al. Mammalian cells-based platforms for the generation of SARS-CoV-2 virus-like particles
CN114807232A (en) Construction and application of west nile virus infectious clone
WO2024015510A1 (en) Sars-cov-2 lacking the envelope protein as an attenuated vaccine virus against covid-19
EP1650308A1 (en) Nucleic acid sequences encoding proteins capable of associating into a virus-like particle
CN117860880A (en) RdRp-dependent African swine fever taRNA vaccine and construction method and application thereof
JP2005224237A (en) DEFECTIVE INTERFERING PARTICLE, POLIOVIRUS DEFICIENT VARIANT RNA, cDNA, AND PLASMID

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21962216

Country of ref document: EP

Kind code of ref document: A1