WO2021174567A1 - 一种基于细菌复合物的新型冠状病毒s蛋白双区域亚单位纳米疫苗 - Google Patents

一种基于细菌复合物的新型冠状病毒s蛋白双区域亚单位纳米疫苗 Download PDF

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WO2021174567A1
WO2021174567A1 PCT/CN2020/078709 CN2020078709W WO2021174567A1 WO 2021174567 A1 WO2021174567 A1 WO 2021174567A1 CN 2020078709 W CN2020078709 W CN 2020078709W WO 2021174567 A1 WO2021174567 A1 WO 2021174567A1
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rbd
antigen
coronavirus
amino acid
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French (fr)
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张辉
马显才
邹帆
袁耀昌
李镕
张旭
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中山大学
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Priority claimed from CN202010144032.4A external-priority patent/CN111217919B/zh
Priority claimed from CN202010144031.XA external-priority patent/CN111217918B/zh
Application filed by 中山大学 filed Critical 中山大学
Priority to US17/908,916 priority Critical patent/US20230090422A1/en
Publication of WO2021174567A1 publication Critical patent/WO2021174567A1/zh

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Definitions

  • the invention belongs to the technical field of biomedicine. More specifically, it relates to a novel coronavirus (tentative name SARS-CoV-2, also known as 2019-nCoV) S protein double-region subunit nano-vaccine based on bacterial complex.
  • SARS-CoV-2 also known as 2019-nCoV
  • Coronavirus is a type of single positive-stranded RNA virus with an envelope, which can be widespread in humans, other mammals and birds, and cause respiratory, digestive, liver, and nervous system diseases.
  • coronaviruses that can cause human diseases.
  • four kinds of 229E, OC43, NL63 and HKU1 basically only cause common cold symptoms in people with immunodeficiency, and the other two are known as SARS-CoV and MERS-CoV, which can cause serious infectious diseases.
  • the length of the single-stranded positive RNA genome at the 5'end of the coronavirus is between 26.2 and 31.7 kb, which is the longest among all RNA viruses.
  • ORF open reading frames
  • the first ORF contains two thirds of the genome and encodes the replicase protein, while the last third contains a fixed sequence of structural protein genes: (HE)-S-E-M-N.
  • HE structural protein gene
  • ORFs encoding accessory proteins between these genes.
  • the genome is packaged into a spiral nucleocapsid, which is surrounded by a lipid bilayer derived from the host. This viral membrane contains at least three viral proteins, namely spike protein (S), membrane protein (M) and envelope protein (E).
  • the M and E proteins are mainly involved in the assembly of the virus, while the S protein mediates the binding of the virus to the receptor on the host cell membrane and fusion with the host cell membrane. Therefore, the S protein plays an important role in the tissue tropism, cell fusion and virulence of the virus, and is the main neutralizing antigen of the coronavirus.
  • the Receptor Binding Domain (RBD) of MERS-CoV and SARS-CoV S proteins is considered to be the most important antigen target region that induces the body to produce neutralizing antibodies.
  • RBD can focus the neutralizing antibodies produced by the body's stimulation more on the receptor binding to the virus, which can improve the immunogenicity and immune efficiency of the vaccine.
  • MERS-CoV invades cells through the binding of RBD to the host cell receptor (CD26, also known as DPP4).
  • SARS-CoV enters the cell through its RBD binding to the host cell receptor ACE2.
  • ACE2 host cell receptor
  • RBD monomer vaccines derived from MERS-CoV and SARS-CoV can only trigger lower levels of pseudovirus neutralizing antibodies after inoculation in animal models.
  • the technical problem to be solved by the present invention is to overcome the shortcomings of the existing novel coronavirus therapeutic drugs and vaccines, and to develop a safe and effective vaccine against SARS-CoV-2 as soon as possible to protect the susceptible population.
  • the receptor binding domain (RBD) of the virus and the fusion peptide (Fusion peptide, FP) are used together as the dual antigen fragments, and the antigen multimerization is realized based on the bacterial complex, and an RBD-FP is constructed and developed. Antigen multimer complex.
  • the receptor binding domain (RBD) of the virus and the fusion peptide (Fusion peptide, FP) are used as dual antigen fragments, and are combined with bacterial complexes (such as Pyrococcus furiosus_Ferritin, Ferritin ( PF)) or dioxytetrahydropteridine synthase polymer protein (Lumazine Synthase, LS)) together to form a fusion protein to achieve antigen multimerization.
  • bacterial complexes such as Pyrococcus furiosus_Ferritin, Ferritin ( PF)
  • LS dioxytetrahydropteridine synthase polymer protein
  • signal peptides and purification tags are added to transfect eukaryotic cells through plasmids Expression systems (such as 293F cells) express self-assembling fusion proteins, and fusion protein monomers can be assembled into spherical twenty-tetramer nanoparticles or spherical sixty-mer nanoparticles through self-assembly, and display them on nanoparticles On the surface, it overcomes the shortcomings of insufficient immunogenicity of RBD monomers, can effectively cause a stronger immune response, and produce antibodies that neutralize the SARS-CoV-2 pseudovirus invading target cells.
  • the vaccine of the present invention can significantly increase the level of neutralizing antibodies of the host against SARS-CoV-2; and the preparation method of the vaccine of the present invention is simple, and the protein contains His tag and is easy to purify.
  • the clinical trials registered by NIH have proved that Ferritin is derived from bacteria. With the safety of the nano-vaccine carrier produced by LS, the vaccine can be quickly applied in clinical trials.
  • the purpose of the present invention is to provide a method for improving the immunogenicity of antigens.
  • Another object of the present invention is to provide a coronavirus antigen with improved immunogenicity.
  • Another object of the present invention is to provide the application of the novel coronavirus antigen in the preparation of novel coronavirus vaccines and anti-new coronavirus drugs.
  • Another object of the present invention is to provide a method for preparing the novel coronavirus antigen.
  • Another object of the present invention is to provide a nucleotide sequence, vector or transgenic cell line encoding and expressing the novel coronavirus antigen.
  • the present invention first provides a method for improving the immunogenicity of an antigen.
  • the method is to use the receptor binding domain (RBD) of the virus and the fusion peptide (FP) together as a dual antigen, and further interact with bacteria.
  • the complex is fused to form a new fusion protein as an antigen; the bacterial complex is Pyrococcus furiosus_Ferritin (Ferritin (PF)) or 2,4-dioxotetrahydropteridine synthase polymer Protein (Lumazine Synthase, LS).
  • the fusion of the receptor binding domain (RBD), fusion peptide (FP) and Ferritin (PF) of the virus is composed of the fusion protein RBD-FP-PF_Ferritin.
  • the virus receptor binding domain (RBD), fusion peptide (FP) and LS are fused to form the fusion protein LS-RBD-FP.
  • Ferritin is a self-assembled globular protein.
  • the distance between the amino terminals of every two adjacent subunits on its surface is about 4.5-7.5nm, which is suitable for loading antigen on the outer surface.
  • PF_Ferritin a ferritin derived from Pyrococcus spontaneously, it can spontaneously form multimerization, and it can induce strong humoral and cellular immune responses after being loaded with antigen on the surface. It is a very ideal carrier and can increase a single immunity.
  • Dioxytetrahydropteridine synthase (Lumazine synthase, LS) is a widely used display platform in the research of self-assembled nanoparticle vaccines, which can self-assemble into icosahedral nanoparticles with an inner diameter of 9nm and an outer diameter of about 15nm.
  • LS nanoparticles have achieved good results in the treatment of AIDS, DC vaccines, ricin vaccines and other antigen display.
  • LS nanoparticles can increase the number of antigens that can be carried by a single immunization and greatly increase the amount of neutralizing antibodies. It solves the shortcomings of weaker immunity caused by RBD monomer vaccine.
  • the method for improving antigen immunogenicity of the present invention uses the receptor binding domain (RBD) of the virus and the fusion peptide (Fusion peptide, FP) as the dual antigen fragments, and is based on the Pyrococcus furiosus_Ferritin, Ferritin (PF)) or Aquifex aeolicus strain 2,4-Dioxytetrahydropteridine synthase polymer protein (Lumazine Synthase, LS) realizes antigen multimerization, which can overcome the shortcomings of insufficient immunogenicity of RBD monomers. It can effectively cause a stronger immune response and can significantly increase the host's level of neutralizing antibodies against SARS-CoV-2.
  • RBD and FP are the areas that binds to the receptor; 2FP is the area that fuses with the cell membrane of the receptor. "Combination” and “fusion” constitute the two most critical and early steps for the virus to invade cells. The construction of a fusion protein of two regions for immunization has not been reported in previous studies of single-segment vaccines. In addition, we also carry out the multimerization of Ferritin (PF) or LS on the antigen fragments.
  • PF Ferritin
  • the "dual antigen + multimer" strategy of the present invention can achieve a more effective, rapid and stable effect of stimulating the body to produce an effective immune response in terms of quality (RBD+FP dual antigen) and quantity (multimerization) .
  • the above-mentioned antigens of the present invention are preferably applicable to coronavirus antigens, and the receptor binding domain RBD and fusion peptide FP of the virus are the receptor binding domain RBD and fusion peptide FP of the coronavirus.
  • the novel coronavirus SARS-CoV-2 antigen is included, and the receptor binding domain RBD and the fusion peptide FP of the coronavirus are the receptor binding domain RBD and the fusion peptide FP of the new coronavirus SARS-CoV-2.
  • the novel coronavirus SARS-CoV-2 antigen is the surface spike protein (S protein) neutralizing antigen of the novel coronavirus SARS-CoV-2, the receptor binding domain RBD of the coronavirus and the fusion peptide FP It is the receptor binding domain RBD of the new coronavirus SARS-CoV-2 and the fusion peptide FP.
  • S protein surface spike protein
  • amino acid sequence of the RBD of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 1; the amino acid sequence of FP is shown in SEQ ID NO: 2.
  • SEQ ID NO: 1 and SEQ ID NO: 2 can be directly connected to obtain the fusion protein RBD-FP.
  • SEQ ID NO: 1 and SEQ ID NO: 2 are connected by a hinge region Linker to form a new fusion protein RBD-FP.
  • the Linker can be GGSGGSGGSGGSGGG.
  • the amino acid sequence of RBD and FP of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 3.
  • the protein shown in SEQ ID NO: 3 fused with RBD and FP can be further constructed with Ferritin (PF) or LS to construct a multimerization fusion protein antigen.
  • PF Ferritin
  • the amino acid sequence of Ferritin (PF) is shown in SEQ ID NO: 4.
  • SEQ ID NO: 3 and SEQ ID NO: 4 can be directly connected to obtain a new fusion protein.
  • SEQ ID NO: 3 and SEQ ID NO: 4 are connected by a hinge region Linker to form a new fusion protein RBD-FP-PF_Ferritin.
  • the Linker may be GSG.
  • the amino acid sequence of the resulting fusion protein RBD-FP-PF_Ferritin is shown in SEQ ID NO: 5.
  • the method for improving the immunogenicity of the antigen of the present invention is to combine the receptor binding domain (RBD) of the virus and the fusion peptide FP with the Pyrococcus
  • the protein Helicobacter pylori_Ferritin, Ferritin (PF)
  • RBD-FP-PF_Ferritin the receptor binding domain of the virus
  • PF Ferritin
  • the signal peptide is a secreted signal peptide (Signal peptide, SP).
  • the purification tag is a His-tag. The signal peptide and purification tag are added to the amino acid N-terminus of RBD.
  • the amino acid sequence of the new coronavirus SARS-CoV-2 SP, His-tag, RBD and FP fusion is shown in SEQ ID NO: 6; the amino acid sequence of Ferritin (PF) is shown in SEQ ID NO: 4 is shown.
  • the SEQ ID NO: 6 and SEQ ID NO: 4 can be directly connected.
  • SEQ ID NO: 6 and SEQ ID NO: 4 are connected by a hinge region Linker to form a new fusion protein RBD-FP-PF_Ferritin.
  • the Linker may be GSG.
  • the amino acid sequence of the resulting fusion protein RBD-FP-PF_Ferritin is shown in SEQ ID NO: 7 (as shown in Figure 2).
  • the present invention provides a SARS-CoV-2 antigen with improved immunogenicity containing a signal peptide and a purification tag.
  • the antigen is self-assembled into a twenty-tetramerized protein protein RBD-FP by using Pyrococcus ferritin. -PF_Ferritin (as shown in Figure 1).
  • the Pyrococcus furiosus_Ferritin (Ferritin (PF)) is a bacterial complex ferritin.
  • the bacterial complex ferritin forms a globular protein that exists in bacteria, and it mainly acts to control polynuclear trioxide. The rate and location of iron formation are transported to and from the mineralized nucleus by hydrated iron ions and protons.
  • the globular form of ferritin is composed of a monomeric subunit protein (Ferritin), which is a polypeptide with a molecular weight of about 17-20 kD. The sequence of such a monomeric ferritin subunit is represented by SEQ ID NO: 4. These monomeric ferritin subunit proteins self-assemble into a globular ferritin protein containing 24 monomeric ferritin subunit proteins.
  • the fusion protein RBD-FP-PF_Ferritin can assemble RBD-FP-PF_Ferritin monomers into spherical twenty-tetrameric nanoparticles through the self-assembly of Ferritin (PF), and display the RBD-FP dual-domain antigen on the surface of the nanoparticles. It can effectively cause a stronger immune response in the receptor and produce antibodies that neutralize the SARS-CoV-2 pseudovirus invading target cells.
  • the twenty-tetramerized RBD-FP-PF_Ferritin of the present invention can overcome the shortcomings of insufficient immunogenicity of RBD monomers and significantly increase the neutralizing antibody titer.
  • the present invention also provides a coronavirus antigen with improved immunogenicity, specifically a new self-assembled and twenty-tetramerized fusion protein RBD-FP-PF_Ferritin constructed by the above method.
  • the amino acid sequence of the novel coronavirus SARS-CoV-2 antigen (a new fusion protein RBD-FP-PF_Ferritin) is shown in SEQ ID NO: 5 (through SEQ ID NO: 1 and SEQ ID NO: 2 with hinge region GGSGGSGGSGGSGGG is connected to obtain SEQ ID NO: 3, and SEQ ID NO: 3 is connected with SEQ ID NO: 4 to form a hinge region GSG); or the amino acid sequence formed after adding a signal peptide and a purification tag is as shown in SEQ ID NO: 7 Shown (through SEQ ID NO: 6 and SEQ ID NO: 4 are connected with the hinge region GSG).
  • the novel coronavirus SARS-CoV-2 antigen (a new fusion protein RBD-FP-PF_Ferritin) contains the signal peptide and purification tag disclosed herein, and the SARS-CoV-2
  • the RBD protein, the FP protein, and the self-assembling subunit protein Ferritin are sequentially connected, wherein the RBD-FP-PF_Ferritin protein can self-assemble into a nanoparticle, which displays the immunogenic part of the RBD-FP protein on the surface.
  • the RBD-FP-PF_Ferritin vaccine has the potential to protect people susceptible to SARS-CoV.
  • amino acid sequence of the LS is shown in SEQ ID NO: 8.
  • SEQ ID NO: 8 and SEQ ID NO: 3 can be directly connected to obtain a new fusion protein.
  • SEQ ID NO: 8 and SEQ ID NO: 3 are connected by a hinge region Linker to form a new fusion protein LS-RBD-FP.
  • the Linker may be GGSGGSGGSGGSGGSGGG.
  • the amino acid sequence of the resulting fusion protein LS-RBD-FP is shown in SEQ ID NO: 9.
  • the method for improving antigen immunogenicity of the present invention is to combine LS, the receptor binding domain (RBD) of the virus and the fusion peptide FP into a fusion protein LS- After RBD-FP, a signal peptide and purification tag are added to express the antigen through a eukaryotic expression system.
  • RBD receptor binding domain
  • the signal peptide is a secreted signal peptide (Signal peptide, SP).
  • the purification tag is a His-tag. The signal peptide and purification tag are added to the amino acid N-terminus of RBD.
  • the SP, LS, RBD, FP, His-tag fusion amino acid sequence of the new coronavirus SARS-CoV-2 nano vaccine is shown in SEQ ID NO: 11 (as shown in Figure 12).
  • the present invention provides a SARS-CoV-2 antigen with improved immunogenicity containing a signal peptide and a purification tag. Assembled into sixty-merized protein protein LS-RBD-FP (as shown in Figure 11).
  • the polymer protein is derived from the Aquifex aeolicus strain, and the self-assembling protein is 2,4-dioxotetrahydropteridine synthase (lumazine synthase, LS).
  • the monomeric LS subunit of the present invention is the full-length, single polypeptide, or any part of the LS protein capable of directing the self-assembly of the monomeric LS subunit into a nanoparticle.
  • the nano-vaccine formed by LS has a spherical form and contains a sixty-mer composed of 12 pentamer units.
  • the fusion protein LS-RBD-FP can assemble LS-RBD-FP monomers into spherical sixty-mer nanoparticles through LS self-assembly, and display them on the surface of the nanoparticles, which can effectively induce stronger immunity of the receptor. In response, an antibody that neutralizes the SARS-CoV-2 pseudovirus invading the target cell is produced.
  • the sixty-merized LS-RBD-FP of the present invention can overcome the shortcomings of insufficient immunogenicity of RBD monomers, and greatly improve the neutralizing antibody titer.
  • the invention also provides a coronavirus antigen with improved immunogenicity, specifically a new self-assembled and sixty-merized fusion protein LS-RBD-FP constructed by the above method.
  • the amino acid sequence of the novel coronavirus SARS-CoV-2 antigen (a new fusion protein LS-RBD-FP) is shown in SEQ ID NO: 9 (by SEQ ID NO: 1 and SEQ ID NO: 2 in the hinge region GGSGGSGGSGGSGGG is connected to obtain SEQ ID NO: 3, SEQ ID NO: 3 and SEQ ID NO: 8 are connected to form a hinge region GGSGGSGGSGGSGGSGGG); the amino acid sequence formed by adding the signal peptide is shown in SEQ ID NO: 10; or plus The amino acid sequence formed after adding the signal peptide and purification tag is shown in SEQ ID NO: 11.
  • the novel coronavirus SARS-CoV-2 antigen (a new fusion protein LS-RBD-FP) contains the signal peptide disclosed herein, the self-assembled LS protein, and SARS-CoV-
  • the RBD protein, FP protein and purification tag of 2 are sequentially connected, wherein the LS-RBD-FP protein can self-assemble into nanoparticles, which display the immunogenic part of the RBD-FP protein on the surface.
  • the LS-RBD-FP vaccine has the potential to protect SARS-CoV-2 susceptible populations.
  • the application of the coronavirus antigen provided by the present invention in the preparation of anti-coronavirus drugs is also within the protection scope of the present invention.
  • RBD-FP-PF_Ferritin protein can be used in combination with SAS adjuvant to prepare an anti-SARS-CoV-2 coronavirus vaccine.
  • the application also includes the preparation of a kit; the kit contains the protein antigen, or a DNA molecule encoding the antigen, or a recombinant vector/expression reagent for expressing the antigen Box/transgenic cell line/recombinant bacteria.
  • nucleotide sequence encoding/expressing the fusion protein antigen of the present invention should also be within the protection scope of the present invention.
  • the present invention also provides an alternative preparation method of the above antigen, which is specifically as follows:
  • the preparation of the fusion protein antigen RBD-FP-PF_Ferritin is the nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 3 and SEQ ID NO: 4 in direct series or hinge series, SEQ ID NO: 6 and SEQ ID NO: 4
  • the nucleotide sequence corresponding to the amino acid shown in direct tandem or hinged tandem, the nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 5, or the nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 7 is added at the 3'end
  • the translation stop codon was added and cloned into the eukaryotic expression vector (as shown in Figure 3, pcDNA3.1-Intron-WPRE), after digestion and sequencing were correct (as shown in Figure 4), the eukaryotic expression system was transiently transfected (For example, 293F cells) express the nano-antigen (shown in Figure 5).
  • the cell supernatant is collected and purified to obtain the new coronavirus SARS-CoV-2 antigen (twenty-tetramer RBD-FP-PF_Ferritin protein, Under non-reducing conditions (without DTT), the size is about 50Kd).
  • LS-RBD-FP The nucleotide sequence (SEQ ID NO: 9) and SEQ ID NO: 10 corresponding to the amino acid shown in SEQ ID NO: 8 and SEQ ID NO: 3 directly in series or in hinge series
  • the nucleotide sequence corresponding to the amino acid shown, or the nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 11 is added with a translation stop codon at the 3'end, and cloned into a eukaryotic expression vector (as shown in Figure 13, pcDNA3 .1-Intron-WPRE), after digestion and correct sequencing (as shown in Figure 14), transiently transfect eukaryotic expression system (such as 293F cells) to express the nanoantigen (as shown in Figure 15), and collect after expression
  • the cell supernatant is purified to obtain the new coronavirus SARS-CoV-2 antigen (the sixty-mer LS-RBD-FP protein, the size is about 50K
  • the eukaryotic expression system includes, but is not limited to, HEK293T cells, 293F cells, CHO cells, sf9 and other cell lines and cell lines that can be used to express eukaryotic proteins.
  • the schemes for introducing the corresponding protein into the eukaryotic expression system include, but are not limited to, various transfection, infection, and transposition schemes.
  • the purification method is to filter the cell supernatant expressing the antigen to remove cell debris, and pass it through a 10K ultrafiltration tube (Millipore) for preliminary purification, and then pass through a HisTrap HP nickel column (GE) , Lectin column (GE) captures the target protein, and finally purified by molecular sieve chromatography using Siperose6Increase10/300GL column (GE) to obtain high-purity target protein (as shown in Figure 6-7, Figure 16-17).
  • GE HisTrap HP nickel column
  • GE Lectin column
  • the buffer for ultrafiltration elution is: PBS buffer with pH 7.4.
  • the elution buffer for the nickel column is: pH 7.4 PBS containing 500 mM Imidazole.
  • the packing of the Lectin column is: Concanavalin A (Con A), Wheat germ agglutinin (WGA), and the elution machine for column elution is: methyl- ⁇ -D-mannopyranoside, GlcNAc.
  • the buffer for molecular sieve chromatography is: PBS buffer with pH 7.4.
  • the receptor binding domain (RBD) of the virus and the fusion peptide (Fusion peptide, FP) are used together as a dual antigen fragment, and are combined with a bacterial complex (such as Pyrococcus furiosus_Ferritin, Ferritin (Pyrococcus furiosus_Ferritin, Ferritin ( PF)) or derived from the Aquifex aeolicus strain dioxytetrahydropteridine synthase polymer protein (Lumazine Synthase, LS) to form a fusion protein to achieve antigen multimerization.
  • a bacterial complex such as Pyrococcus furiosus_Ferritin, Ferritin (Pyrococcus furiosus_Ferritin, Ferritin ( PF)
  • Aquifex aeolicus strain dioxytetrahydropteridine synthase polymer protein Lomazine Synthase, LS
  • the signal peptide and purification tag are added, and the plasmid is transfected Eukaryotic cell expression systems (such as 293F cells) express self-assembled fusion proteins, and RBD-FP can form twenty-tetramer nano-antigens or sixty-mer nano-antigens through Ferritin (PF) or LS self-assembly.
  • PF Ferritin
  • LS self-assembly
  • the present invention combines RBD-FP-PF_Ferritin nano antigen and LS-RBD-
  • the experiment of immunizing Balb/c mice with FP nano antigen has confirmed that the neutralizing antibody produced after 10 days of immunization has the ability to strongly block the SARS-CoV-2 pseudovirus from invading target cells.
  • the preparation method of the vaccine of the present invention is simple and easy to purify.
  • the safety of Ferritin and LS as nano vaccine carriers has been proved in clinical trials registered by NIH, and the vaccines can be quickly applied to clinical trials.
  • Figure 1 is a schematic diagram of RBD-FP-PF_Ferritin fusion protein self-assembled nanoparticles.
  • Figure 2 is a schematic diagram of the structure of the RBD-FP-PF_Ferritin fusion protein.
  • Figure 3 is a schematic diagram of the plasmid structure expressing RBD-FP-PF_Ferritin.
  • Figure 4 shows the verification of RBD-FP-PF_Ferritin fusion restriction digestion.
  • Figure 5 is an immunofluorescence image of 293F cells transfected with RBD-FP-PF_Ferritin fusion protein.
  • Figure 6 is a molecular sieve diagram of the purified RBD-FP-PF_Ferritin fusion protein.
  • Figure 7 is an SDS-PAGE image of purification of RBD-FP-PF_Ferritin fusion protein (about 50KD).
  • Figure 8 shows the immunization strategy of fusion protein nano-vaccine mice.
  • Figure 9 shows the detection strategy of neutralizing antibody titer in mouse serum.
  • FIG. 10 shows that mice immunized with RBD-FP-PF_Ferritin nano-vaccine produce neutralizing antibodies that block SARS-CoV-2 from invading target cells.
  • Figure 11 is a schematic diagram of self-assembled nanoparticles of LS-RBD-FP fusion protein.
  • Figure 12 is a schematic diagram of the structure of the LS-RBD-FP fusion protein.
  • Figure 13 is a schematic diagram of the structure of a plasmid expressing LS-RBD-FP.
  • Figure 14 shows the LS-RBD-FP fusion restriction digestion verification.
  • Figure 15 is an immunofluorescence image of 293F cells transfected with LS-RBD-FP fusion protein.
  • Figure 16 is a molecular sieve diagram of LS-RBD-FP fusion protein purification.
  • Figure 17 is an SDS-PAGE image of LS-RBD-FP fusion protein purification (approximately 50KD).
  • Figure 18 shows the neutralizing antibody produced by the mouse immunized LS-RBD-FP nano vaccine to prevent SARS-CoV-2 from invading target cells.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the construction and preparation method of the fusion protein RBD-FP-PF_Ferritin is as follows:
  • the 3'end of the RBD-FP-PF_Ferritin nucleotide sequence was added with a translation stop codon and cloned into the Xho I and Xba I digestion sites of the expression vector (pcDNA3.1-Intron-WPRE) added with I ntron and WPRE to enhance expression Between the points, construct the expression vector pcDNA3.1-Intron-WPRE-RBD-FP-Ferritin(PF)-IRE S-GFP (as shown in Figure 3).
  • the recombinant plasmid was transformed into DH5 ⁇ competent cells, cultured overnight at 37°C, and positive clones were identified by screening and PCR.
  • the endotoxin-free plasmid is extracted and used for the expression of nano antigen protein after restriction enzyme digestion and sequencing verification (as shown in Figure 4).
  • the plasmid was transfected into HEK293F cells through the liposome transfection protocol, and the cell supernatant was harvested by centrifugation after 3 days of transfection (the RBD-FP-PF_Ferritin protein transfected 293F cell immunofluorescence map is shown in Figure 5), and the target protein RBD was performed -FP-PF_Ferritin purification.
  • the cell supernatant expressing RBD-FP-PF_Ferritin was filtered through a 0.22 ⁇ m filter membrane to remove cell debris. After ultrafiltration through a 10K ultrafiltration tube, the filtered cell supernatant was combined with Histrap-excel at 4°C for 30 minutes, and a HisTrap excel nickel column was used for crude purification.
  • PBS pH 7.4 buffer and low-concentration imidazole buffer (PBS, 50 mM Imidazole, pH 7.4) to wash 50 ml respectively to remove impurities that flow through.
  • PBS pH 7.4 buffer and low-concentration imidazole buffer
  • the target protein was eluted with a buffer containing high imidazole (PBS, 500 mM Imidazole, pH 7.4).
  • the target protein was enriched using a Lectin Agarose column (GE) with Con A and WGA at a ratio of 1:1.
  • GE Lectin Agarose column
  • the RBD-FP-PF_Ferritin fusion protein obtained in Example 1 was diluted with physiological saline to 100 ⁇ g/ml according to Table 1, and was emulsified in groups with the equal volume of adjuvant SAS. Then Balb/C mice aged 6-8 weeks were immunized in groups.
  • the immunization strategy is shown in Figure 8, that is, by intraperitoneal injection, each mouse received 3 vaccine immunizations on day 0, week 3 (day 21), and week 14 (day 108), each with 200 ⁇ l of vaccine. Inoculation volume (10 ⁇ g).
  • the mouse serum was obtained by centrifugation at 4°C and 2800 rpm for 15 minutes after standing for a period of time until the serum was separated, and it was immediately used in the SARS-CoV-2 pseudovirus neutralization detection experiment.
  • the Spike protein of SARS-CoV-2 was synthesized and inserted into the pcDNA3.1 expression vector.
  • the expression vector of SARS-CoV-2Spike protein was co-transfected with pHIV-luciferase and psPAX2 plasmids into 293T cells. After 5 hours of transfection, the cells were washed twice with PBS and replaced with serum-free DMEM medium to continue the culture. After 48 hours, the supernatant was collected and centrifuged to remove cell debris. Then dissolve it with a small volume of serum-free DMEM to obtain HIV-luc/SARS-CoV-2-S pseudovirus.
  • the pseudovirus can effectively simulate the process of wild-type SARS-CoV-2 invading cells. When it infects the production cell or target cell, the expression of the luciferase reporter gene carried by the SARS-CoV-2 pseudovirus can accurately reflect the result of the virus infection, so that the result of the experimental system can be read accurately and quickly, which can be used as an excellent
  • the antibody neutralization titer monitoring system shown in Figure 9).
  • TCID 50 Dilute the virus solution collected in the previous step by a 5-fold ratio and add it to HEK293T cells in a 96-well plate. After 4 hours of infection, the virus solution was discarded, the cells were washed twice with PBS, and replaced with DMEM complete medium containing 10% serum. After 48 hours, discard the medium, wash twice with PBS, add cell lysate, and shake for 30 minutes. After freezing and thawing at -80°C, take 30 ⁇ l from each well and use GloMax 96 (Promega) to detect the luciferase activity value. Calculate TCID 50 by Reed-Muech method.
  • the purified antibody was diluted by a 2-fold ratio, mixed with TCID 50 final concentration pseudovirus, and incubated at 37°C for 1 hour.
  • the mixture was added to a 96-well plate in HEK293T cells that had a density of about 70%. After 48 hours, the culture medium was discarded, the cells were washed twice with PBS, and the cell lysate was added to detect the luciferase activity value.
  • results show that the combination of the RBD-FP-PF_Ferritin fusion protein of the present invention and the SAS adjuvant can stimulate mouse humoral immunity 10 days after one immunization, which is less than the neutralizing antibody titer stimulated by the parallel control group, and the difference is significant.
  • the construction and preparation method of the fusion protein LS-RBD-FP is as follows:
  • the 3'end of the LS-RBD-FP nucleotide sequence was added with a translation stop codon and cloned into the Xho I and Xba I restriction sites of the expression vector (pcDNA3.1-Intron-WPRE) that added Intron and WPRE to enhance expression
  • the expression vector pcDNA3.1-Intron-WPRE-LS-RBD-FP-IRES-GFP was constructed (as shown in Figure 13).
  • the recombinant plasmid was transformed into DH5 ⁇ competent cells, cultured overnight at 37°C, and positive clones were identified by screening and PCR.
  • the endotoxin-free plasmid was extracted and used for the expression of nano antigen protein after restriction enzyme digestion and sequencing verification (as shown in Figure 14).
  • the plasmid was transfected into HEK293F cells through the liposome transfection protocol, and the cell supernatant was harvested by centrifugation after 3 days of transfection (the immunofluorescence map of 293F cells transfected with LS-RBD-FP protein is shown in Figure 15), and the target protein LS was performed. -RBD-FP purification.
  • the cell supernatant expressing LS-RBD-FP was filtered through a 0.22 ⁇ m filter membrane to remove cell debris. After ultrafiltration through a 10K ultrafiltration tube, the filtered cell supernatant was combined with Histrap-excel at 4°C for 30 minutes, and a HisTrap excel nickel column was used for crude purification.
  • PBS pH 7.4 buffer and low-concentration imidazole buffer (PBS, 50 mM Imidazole, pH 7.4) to wash 50 ml respectively to remove impurities that flow through.
  • PBS pH 7.4 buffer and low-concentration imidazole buffer
  • the target protein was eluted with a buffer containing high imidazole (PBS, 500mM Imidazole, pH 7.4;).
  • PBS Lectin Agarose column
  • the buffer for molecular sieve chromatography is: PBS, pH 7.4. After the target protein is concentrated, it is divided into small aliquots, quickly frozen in liquid nitrogen, and stored at -80°C.
  • the LS-RBD-FP fusion protein obtained in Example 1 was diluted with physiological saline to 100 ⁇ g/ml according to Table 1, and was emulsified in groups with an equal volume of adjuvant SAS. Then Balb/C mice aged 6-8 weeks were immunized in groups.
  • the immunization strategy is shown in Figure 8, that is, by intraperitoneal injection, each mouse received 3 vaccine immunizations on day 0, week 3 (day 21), and week 14 (day 108), each with 200 ⁇ l of vaccine. Inoculation volume (10 ⁇ g).
  • the mouse serum was obtained by centrifugation at 4°C and 2800 rpm for 15 minutes after standing for a period of time until the serum was separated, and it was immediately used in the SARS-CoV-2 pseudovirus neutralization detection experiment.
  • the Spike protein of SARS-CoV-2 was synthesized and inserted into the pcDNA3.1 expression vector.
  • the expression vector of SARS-CoV-2Spike protein was co-transfected with pHIV-luciferase and psPAX2 plasmids into 293T cells. After 5 hours of transfection, the cells were washed twice with PBS and replaced with serum-free DMEM medium to continue the culture. After 48 hours, the supernatant was collected and centrifuged to remove cell debris. Then dissolve with a small volume of serum-free DMEM to obtain HIV-luc/SARS-CoV-2-S pseudovirus.
  • the pseudovirus can effectively simulate the process of wild-type SARS-CoV-2 invading cells. When it infects the production cell or target cell, the expression of the luciferase reporter gene carried by the SARS-CoV-2 pseudovirus can accurately reflect the result of the virus infection, so that the result of the experimental system can be read accurately and quickly, which can be used as an excellent
  • the antibody neutralization titer monitoring system shown in Figure 9).
  • TCID 50 Dilute the virus solution collected in the previous step by a 5-fold ratio and add it to HEK293T cells in a 96-well plate. After 4 hours of infection, the virus solution was discarded, the cells were washed twice with PBS, and replaced with DMEM complete medium containing 10% serum. After 48 hours, discard the culture medium, wash twice with PBS, add cell lysate, shake and lyse for 30 minutes. After freezing and thawing at -80°C, take 30 ⁇ l from each well and use GloMax 96 (Promega) to detect the luciferase activity value. Calculate TCID 50 by Reed-Muech method.
  • the purified antibody was diluted by a 2-fold ratio, mixed with TCID 50 final concentration pseudovirus, and incubated at 37°C for 1 hour.
  • the mixture was added to a 96-well plate in HEK293T cells that had a density of about 70%. After 48 hours, the culture medium was discarded, the cells were washed twice with PBS, and the cell lysate was added to detect the luciferase activity value.

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Abstract

本发明公开了一种基于细菌复合物的新型冠状病毒S蛋白双区域亚单位纳米疫苗。本发明以病毒的受体结合域(Receptor binding domain，RBD)和融合肽(Fusion peptide，FP)共同作为双抗原，并与细菌复合物(如PF_Ferritin或Lumazine Synthase(LS)连接组成融和蛋白，实现抗原多聚化；再利用真核细胞表达系统表达，可通过自组装作用形成二十四聚体纳米抗原或六十聚体纳米抗原。该方案可克服RBD单体免疫原性不足的缺点，所得疫苗能显著的提高宿主针对病毒的中和抗体的水平，产生的抗体具有强力阻挡病毒入侵靶细胞的能力。而且本发明疫苗制备方法简单、易于纯化，安全性高，疫苗可较快的应用于临床试验。

Description

一种基于细菌复合物的新型冠状病毒S蛋白双区域亚单位纳米疫苗 技术领域
本发明属于生物医药技术领域。更具体地，涉及一种基于细菌复合物的新型冠状病毒(暂定名称SARS-CoV-2，又名2019-nCoV)S蛋白双区域亚单位纳米疫苗。
背景技术
自2019年12月以来，在中国湖北武汉出现一系列不明原因肺炎病例，临床表现与病毒性肺炎极为相似；主要临床表现为发热、疲乏、干咳等，严重者可发生休克、脓毒血症、呼吸衰竭而死亡。利用9例下呼吸道样本的深度测序分析表明，存在一种新型冠状病毒，暂定命名为SARS-CoV-2(又名2019-nCoV)。截至到2月19日，国内已有7万余例患者确诊，仍有5000余例的疑似病例，造成超过1600人的死亡，日本、泰国、韩国、美国、和欧洲多国也已确诊上百例病例，有在中国乃至世界蔓延的势头。由于目前新型冠状病毒肺炎的病毒来源、发病机制等尚不明确，并且缺乏特效抗病毒药物，为临床诊治和控制疫情带来极大困难，造成严重的社会负担和危机。
目前，人类仍缺乏有效的抗SARS-CoV-2的疫苗，在这种严峻的形势下，尽快开发安全、有效的针对SARS-CoV-2的疫苗用以保护易感人群，对于我国的人民健康与国家安全具有重要意义。
对于疫苗的研发，就必须先要了解病毒的结构。冠状病毒是一类具有包膜的单正链RNA病毒，能够在人和其他哺乳动物以及鸟类中广泛存在，并导致呼吸、消化、肝脏和神经系统等类型的疾病。在本次疫情发生以前，目前已知有6种冠状病毒可以引发人类疾病。其中，四种229E，OC43，NL63和HKU1基本上只会导致免疫缺陷的人引起普通感冒症状，而另外两种就是我们熟知的SARS-CoV和MERS-CoV，会引发严重的传染性疾病。冠状病毒5'端的单链阳性RNA基因组的长度介于26.2和31.7kb之间，是所有RNA病毒中最长的。其基因组有六到十个开放的读码框(ORF)。第一个ORF包含基因组的三分之二，并编码复制酶蛋白质，而最后三分之一含有固定顺序的结构蛋白基因：(HE)-S-E-M-N。在这些基因之间存在着编码辅助蛋白的多个ORF。基因组被包装成螺旋状的核衣 壳，核衣壳被宿主来源的脂质双层所包围。这个病毒膜至少含有三种病毒蛋白，即刺突蛋白(S)和膜蛋白(M)以及包膜蛋白(E)。
其中，M和E蛋白主要参与病毒的组装，而S蛋白介导了病毒与宿主细胞膜上的受体结合并与宿主细胞膜相融合。因此，S蛋白在病毒的组织嗜性、细胞融合和毒力等方面起重要作用，是冠状病毒的主要中和抗原。MERS-CoV、SARS-CoV S蛋白的受体结合区(Receptor Binding Domain,RBD)被认为是诱导机体产生中和抗体的最主要的抗原靶区域。RBD作为疫苗能够将机体刺激产生的中和抗体更加聚焦在针对病毒的受体结合，可以提高疫苗的免疫原性和免疫效率。MERS-CoV通过RBD与宿主细胞的受体(CD26，又名DPP4)结合而侵入细胞，SARS-CoV通过其RBD与宿主细胞受体ACE2结合而进入细胞，其作为疫苗的核心能够将机体刺激产生的中和抗体更加聚焦在针对病毒的受体结合，进而提高疫苗的免疫原性和中和效率。然而在前期的研究中，来源于MERS-CoV以及SARS-CoV的RBD单体疫苗接种动物模型后仅能引发较低的假病毒中和抗体水平。
因此，目前针对新型冠状病毒SARS-CoV-2开展高免疫原性和中和效率的疫苗的研发工作迫在眉睫。
发明内容
本发明要解决的技术问题是克服现有新型冠状病毒治疗药物以及疫苗的不足，为尽快开发安全、有效的针对SARS-CoV-2的疫苗用以保护易感人群。本发明以病毒的受体结合域(Receptor binding domain,RBD)及融合肽(Fusion peptide,FP)共同作为双抗原片段，并基于细菌复合物实现抗原多聚化，构建开发了一种RBD-FP抗原多聚体复合物。具体是以病毒的受体结合域(Receptor binding domain,RBD)及融合肽(Fusion peptide,FP)共同作为双抗原片段，并与细菌复合物(如火球菌多聚物蛋白(Pyrococcus furiosus_Ferritin,Ferritin(PF))或二氧四氢喋啶合酶多聚物蛋白(Lumazine Synthase,LS))共同组成融和蛋白，实现抗原多聚化，同时加上信号肽及纯化标签，通过质粒转染真核细胞表达系统(如293F细胞)表达可自组装的融合蛋白，可通过自行组装将融合蛋白单聚体组装成球状二十四聚体纳米颗粒或球状六十聚体纳米颗粒，将其展示在纳米颗粒表面，克服了RBD单体免疫原性不足的缺点，能够有效地引起更强的免疫反应，产生中和SARS-CoV-2假病毒入侵靶细胞的抗体。本发明的疫苗能显著的提高宿 主针对SARS-CoV-2的中和抗体的水平；而且本发明疫苗制备方法简单、蛋白含有His标签易于纯化，NIH登记的临床试验中已证明了细菌来源的Ferritin和LS制作纳米疫苗载体的安全性，疫苗可较快的应用于临床试验。
本发明的目的是提供一种提高抗原免疫原性的方法。
本发明另一目的是提供一种免疫原性提高的冠状病毒抗原。
本发明另一目的是提供所述新型冠状病毒抗原在制备新型冠状病毒疫苗及抗新型冠状病毒药物中的应用。
本发明再一目的是提供所述新型冠状病毒抗原的制备方法。
本发明再一目的是提供编码表达所述新型冠状病毒抗原的核苷酸序列、载体或转基因细胞系。
本发明上述目的通过以下技术方案实现：
本发明首先提供一种提高抗原免疫原性的方法，所述方法是将病毒的受体结合域(Receptor binding domain,RBD)和融合肽(Fusion peptide,FP)共同作为双抗原，并进一步与细菌复合物融合组成一个新的融和蛋白后作为抗原；所述细菌复合物为火球菌多聚物蛋白(Pyrococcus furiosus_Ferritin,Ferritin(PF))或2,4-二氧四氢喋啶合酶多聚物蛋白(Lumazine Synthase,LS)。
病毒的受体结合域(RBD)、融合肽(FP)与Ferritin(PF)融合组成的为融和蛋白RBD-FP-PF_Ferritin。
病毒的受体结合域(RBD)、融合肽(FP)与LS融合组成的为融和蛋白LS-RBD-FP。
铁蛋白(Ferritin)作为一种可自组装的球形蛋白，其表面每相邻两个亚单位的氨基端间距约为4.5-7.5nm，适合在外表面负载抗原。利用PF_Ferritin这样一种来源于火球菌的铁蛋白能够自发形成多聚化的特性，且表面负载抗原后能诱发很强的体液免疫反应及细胞免疫反应，是非常理想的载体，可以增加单次免疫所能够承载的抗原的数量，大大提高了中和抗体滴度，解决RBD单体疫苗引发较弱免疫的缺点。
二氧四氢喋啶合酶(Lumazine synthase，LS)是自组装纳米颗粒疫苗研究中广泛使用的展示平台,能够自组装成内径9nm、外径15nm左右的二十面体纳米颗粒。LS纳米颗粒在AIDS的治疗、DC疫苗、蓖麻毒素疫苗等抗原展示方面均取得了较好的效果，LS纳米颗粒可以增加单次免疫所能够承载的抗原的数量，大 大提高了中和抗体滴度，解决RBD单体疫苗引发较弱免疫的缺点。
本发明提高抗原免疫原性的方案以病毒的受体结合域(Receptor binding domain,RBD)及融合肽(Fusion peptide,FP)共同作为双抗原片段，并基于火球菌多聚物蛋白(Pyrococcus furiosus_Ferritin,Ferritin(PF))或Aquifex aeolicus菌株2,4-二氧四氢喋啶合酶多聚物蛋白(Lumazine Synthase,LS)实现抗原多聚化，能够克服RBD单体免疫原性不足的缺点，能够有效地引起更强的免疫反应，能显著的提高宿主针对SARS-CoV-2的中和抗体的水平。
在以往抗原研究中，尤其是SARS的研究，仅仅关注某一个区段的免疫原性，比如RBD区域，但目前相关疫苗的研发均宣告失败，因此我们考虑采用双区段进行抗原免疫。选择RBD和FP的原因在于：①RBD是和受体结合的区域；②FP是和受体细胞膜融合的区域。“结合”和“融合”构成了病毒侵入细胞的最关键最早期的两个步骤。将两个区域构建融合蛋白进行免疫在以往单区段疫苗研究中是不曾报道的。另外我们还对抗原片段进行Ferritin(PF)的多聚化或LS的多聚化，利用Ferritin(PF)或LS能够自发形成多聚化的特性，将双抗原聚集在一起形成纳米颗粒，进一步增加单次免疫承载抗原的数量，因此可以更加充分而稳定的和人体内免疫细胞进行接触而刺激产生抗体。本发明的这种“双抗原+多聚体”的策略，可从质量(RBD+FP双抗原)和数量(多聚化)上达到更加有效、快速、稳定地刺激机体产生有效免疫反应的效果。
优选地，本发明上述抗原优选适用于冠状病毒抗原，所述病毒的受体结合域RBD和融合肽FP为冠状病毒的受体结合域RBD和融合肽FP。
优选包括新型冠状病毒SARS-CoV-2抗原，所述冠状病毒的受体结合域RBD和融合肽FP为新型冠状病毒SARS-CoV-2的受体结合域RBD和融合肽FP。
更具体优选是指新型冠状病毒SARS-CoV-2抗原为新型冠状病毒SARS-CoV-2的表面刺突蛋白(S蛋白)中和抗原，所述冠状病毒的受体结合域RBD和融合肽FP为新型冠状病毒SARS-CoV-2的受体结合域RBD和融合肽FP。
具体地，新型冠状病毒SARS-CoV-2的RBD的氨基酸序列如SEQ ID NO:1所示；FP的氨基酸序列如SEQ ID NO:2所示。
SEQ ID NO:1与SEQ ID NO:2可以直接连接得到融合蛋白RBD-FP。
或SEQ ID NO:1与SEQ ID NO:2以铰链区Linker相连构成一个新的融和蛋白RBD-FP。作为一种可选择的优选方案，所述Linker可以为 GGSGGSGGSGGSGGG。当所述Linker为GGSGGSGGSGGSGGG时，新型冠状病毒SARS-CoV-2的RBD与FP的氨基酸序列如SEQ ID NO:3所示。
另外，基于此可进一步将RBD与FP融合的SEQ ID NO:3所示蛋白与Ferritin(PF)或LS构建多聚化融合蛋白抗原。
与Ferritin(PF)融合的方案如下：
所述Ferritin(PF)的氨基酸序列如SEQ ID NO:4所示。
SEQ ID NO:3与SEQ ID NO:4可以直接连接得到新的融和蛋白。
或SEQ ID NO:3与SEQ ID NO:4以铰链区Linker相连构成一个新的融和蛋白RBD-FP-PF_Ferritin。作为一种可选择的优选方案，所述Linker可以为GSG。当所述Linker为GSG时，所得融和蛋白RBD-FP-PF_Ferritin的氨基酸序列如SEQ ID NO:5所示。
进一步优选地，作为一种可选择的实施方案，本发明所述提高抗原免疫原性的方法是将病毒的受体结合域(Receptor binding domain,RBD)和融合肽FP，与火球菌多聚物蛋白(Helicobacter pylori_Ferritin,Ferritin(PF))组成融和蛋白RBD-FP-PF_Ferritin后，再加上信号肽及纯化标签，通过真核表达系统表达出抗原。
优选地，所述信号肽为分泌型信号肽(Signal peptide,SP)。优选地，所述纯化标签为His标签(His-tag)。所述信号肽及纯化标签是加在RBD的氨基酸N端。
加上信号肽及纯化标签后，新型冠状病毒SARS-CoV-2的SP、His-tag、RBD与FP融合的氨基酸序列如SEQ ID NO:6所示；所述Ferritin(PF)的氨基酸序列如SEQ ID NO:4所示。
所述SEQ ID NO:6与SEQ ID NO:4可以直接相连。
或SEQ ID NO:6与SEQ ID NO:4以铰链区Linker相连构成一个新的融和蛋白RBD-FP-PF_Ferritin。作为一种可选择的优选方案，所述Linker可以为GSG。
当所述Linker为GSG时，所得融和蛋白RBD-FP-PF_Ferritin的氨基酸序列如SEQ ID NO:7所示(如图2所示)。
即本发明提供了一种含有信号肽及纯化标签的免疫原性提高的SARS-CoV-2抗原，所述抗原是利用火球菌铁蛋白自行组装为二十四聚体化的蛋白蛋白RBD-FP-PF_Ferritin(如图1所示)。
所述火球菌多聚物蛋白(Pyrococcus furiosus_Ferritin,Ferritin(PF))为一种细菌复合物铁蛋白，所述细菌复合物铁蛋白形成存在于细菌的球状蛋白，其主要作用于控制多核三氧化二铁形成的速率和位置，通过水合铁离子和质子运输到矿化核和从矿化核运输。铁蛋白的球状形式是由单体亚基蛋白(Ferritin)构成的，其为具有分子量约17-20kD的多肽。一个这样的单体铁蛋白亚基的序列的如SEQ ID NO:4表示。这些单体铁蛋白亚基蛋白自组装为包含24个单体铁蛋白亚基蛋白的球状铁蛋白蛋白质。
该融和蛋白RBD-FP-PF_Ferritin可通过Ferritin(PF)自行组装作用将RBD-FP-PF_Ferritin单聚体组装成球状二十四聚体纳米颗粒，将RBD-FP双区域抗原展示在纳米颗粒表面，能够有效地引起受体更强的免疫反应，产生中和SARS-CoV-2假病毒入侵靶细胞的抗体。本发明的二十四聚体化的RBD-FP-PF_Ferritin可克服RBD单体免疫原性不足的缺点，显著提高了中和抗体滴度。
本发明还提供一种免疫原性提高的冠状病毒抗原，具体是由上述方法构建得到的一个新的可自行组装并二十四聚体化的融合蛋白RBD-FP-PF_Ferritin。
所述新型冠状病毒SARS-CoV-2抗原(一个新的融合蛋白RBD-FP-PF_Ferritin)的氨基酸序列如SEQ ID NO:5所示(通过SEQ ID NO:1和SEQ ID NO:2以铰链区GGSGGSGGSGGSGGG相连得到SEQ ID NO:3，SEQ ID NO:3再与SEQ ID NO:4以铰链区GSG相连构成)；或再加上信号肽及纯化标签后形成的氨基酸序列如SEQ ID NO:7所示(通过SEQ ID NO:6与SEQ ID NO:4以铰链区GSG相连构成)。
即作为本发明可选择的一种优选实施方案，新型冠状病毒SARS-CoV-2抗原(一个新的融合蛋白RBD-FP-PF_Ferritin)包含本文公开的信号肽及纯化标签、SARS-CoV-2的RBD蛋白与FP蛋白与自组装亚基蛋白Ferritin依次连接，其中所述RBD-FP-PF_Ferritin蛋白能够自组装为纳米颗粒，其在表面上展示RBD-FP蛋白的免疫原性部分。经进一步动物模型安全性与有效性研究后，RBD-FP-PF_Ferritin疫苗具备保护SARS-CoV易感人群的潜力。
与LS融合的方案如下：
所述LS的氨基酸序列如SEQ ID NO:8所示。
SEQ ID NO:8与SEQ ID NO:3可以直接连接得到新的融和蛋白。
或SEQ ID NO:8与SEQ ID NO:3以铰链区Linker相连构成一个新的融和蛋白LS-RBD-FP。作为一种可选择的优选方案，所述Linker可以为GGSGGSGGSGGSGGSGGG。当所述Linker为GGSGGSGGSGGSGGSGGG时，所得融和蛋白LS-RBD-FP的氨基酸序列如SEQ ID NO:9所示。
进一步优选地，作为一种可选择的实施方案，本发明所述提高抗原免疫原性的方法是将LS、病毒的受体结合域(Receptor binding domain,RBD)和融合肽FP组成融和蛋白LS-RBD-FP后，再加上信号肽及纯化标签，通过真核表达系统表达出抗原。
优选地，所述信号肽为分泌型信号肽(Signal peptide,SP)。优选地，所述纯化标签为His标签(His-tag)。所述信号肽及纯化标签是加在RBD的氨基酸N端。
加上信号肽后，新型冠状病毒SARS-CoV-2纳米疫苗的SP、LS、RBD与FP融合的氨基酸序列如SEQ ID NO:10所示。
加上His-tag标签后，新型冠状病毒SARS-CoV-2纳米疫苗的SP、LS、RBD、FP、His-tag融合的氨基酸序列如SEQ ID NO:11所示(如图12所示)。
即本发明提供了一种含有信号肽及纯化标签的免疫原性提高的SARS-CoV-2抗原，所述抗原是利用2,4-二氧四氢蝶啶合酶(lumazine synthase,LS)自组装为六十聚体化的蛋白蛋白LS-RBD-FP(如图11所示)。
所述多聚物蛋白是来源于Aquifex aeolicus菌株，自组装蛋白是2,4-二氧四氢蝶啶合酶(lumazine synthase,LS)。据本发明，本发明的单体LS亚基是能够指导单体LS亚基自组装成纳米颗粒的LS蛋白的全长，单个多肽，或其任何部分。LS形成的纳米疫苗为球状形式，包含12个五聚体单位组成的六十聚体。
该融和蛋白LS-RBD-FP可通过LS自行组装将LS-RBD-FP单聚体组装成球状六十聚体纳米颗粒，将其展示在纳米颗粒表面，能够有效地引起受体更强的免疫反应，产生中和SARS-CoV-2假病毒入侵靶细胞的抗体。本发明的六十聚体化的LS-RBD-FP可克服RBD单体免疫原性不足的缺点，大大提高了中和抗体滴度。
本发明还提供一种免疫原性提高的冠状病毒抗原，具体是由上述方法构建得到的一个新的可自行组装并六十聚体化的融合蛋白LS-RBD-FP。
所述新型冠状病毒SARS-CoV-2抗原(一个新的融合蛋白LS-RBD-FP)的氨基酸序列如SEQ ID NO:9所示(通过SEQ ID NO:1和SEQ ID NO:2以铰链区 GGSGGSGGSGGSGGG相连得到SEQ ID NO:3，SEQ ID NO:3再与SEQ ID NO:8以铰链区GGSGGSGGSGGSGGSGGG相连构成)；再加上信号肽形成的氨基酸序列如SEQ ID NO:10所示；或再加上信号肽及纯化标签后形成的氨基酸序列如SEQ ID NO:11所示。
即作为本发明可选择的一种优选实施方案，新型冠状病毒SARS-CoV-2抗原(一个新的融合蛋白LS-RBD-FP)包含本文公开的信号肽、自组装LS蛋白、SARS-CoV-2的RBD蛋白、FP蛋白及纯化标签依次连接，其中所述LS-RBD-FP蛋白能够自组装为纳米颗粒，其在表面上展示RBD-FP蛋白的免疫原性部分。经进一步动物模型安全性与有效性研究后，LS-RBD-FP疫苗具备保护SARS-CoV-2易感人群的潜力。
因此，本发明所提供的冠状病毒抗原在制备抗冠状病毒药物方面的应用，具体包括制备抗新型冠状病毒SARS-CoV-2药物的应用，也在本发明的保护范围之内。
作为一种可选择的实施方式，可以利用RBD-FP-PF_Ferritin蛋白与SAS佐剂合用制备抗SARS-CoV-2冠状病毒疫苗。
另外作为可选择的实施方式，所述应用还包括用于制备试剂盒；所述试剂盒中含有所述蛋白抗原，或者编码所述抗原的DNA分子，或者表达所述抗原的重组载体/表达试剂盒/转基因细胞系/重组菌。
另外，编码/表达本发明融合蛋白抗原的核苷酸序列、含有该核苷酸序列的重组载体、表达盒、转基因细胞系或重组菌，也应在本发明的保护范围之内。
本发明还提供上述抗原的一种可选择的制备方法，具体如下：
融合蛋白抗原RBD-FP-PF_Ferritin的制备：是在SEQ ID NO:3与SEQ ID NO:4直接串联或铰链串联所示氨基酸对应的核苷酸序列、SEQ ID NO:6与SEQ ID NO:4直接串联或铰链串联所示氨基酸对应的核苷酸序列、SEQ ID NO:5所示氨基酸对应的核苷酸序列、或SEQ ID NO:7所示氨基酸对应的核苷酸序列的3’端加上翻译终止密码子，克隆进真核表达载体(如图3所示，pcDNA3.1-Intron-WPRE)，经酶切以及测序正确后(如图4所示)，瞬时转染真核表达系统(如293F细胞)进行纳米抗原的表达(图5所示)，表达后收集细胞上清，纯化，即得到新型冠状病毒SARS-CoV-2抗原(二十四聚体RBD-FP-PF_Ferritin蛋白，非还原条件下(不加DTT)的情况下大小约为50Kd)。
融合蛋白抗原LS-RBD-FP的制备：在SEQ ID NO:8与SEQ ID NO:3直接串联或铰链串联所示氨基酸对应的核苷酸序列(SEQ ID NO:9)、SEQ ID NO:10所示氨基酸对应的核苷酸序列、或SEQ ID NO:11所示氨基酸对应的核苷酸序列的3’端加上翻译终止密码子，克隆进真核表达载体(如图13所示，pcDNA3.1-Intron-WPRE)，经酶切以及测序正确后(如图14所示)，瞬时转染真核表达系统(如293F细胞)进行纳米抗原的表达(图15所示)，表达后收集细胞上清，纯化，即得到新型冠状病毒SARS-CoV-2抗原(六十聚体LS-RBD-FP蛋白，非还原条件下(不加DTT)的情况下大小约为50Kd)。作为可选择的实施方案，所述真核表达系统包括但不限于HEK293T细胞、293F细胞、CHO细胞、sf9等可用于表达真核蛋白的细胞株、细胞系。相应蛋白导入真核表达系统的方案包括但不限于各种转染、感染、转座方案等。
作为可选择的实施方案，所述纯化方法是将表达所述抗原的细胞上清液过滤除去细胞碎片，并通10K超滤管(Millipore)进行初步的提纯，随即通过HisTrap HP镍柱(GE)、Lectin柱(GE)进行目的蛋白的捕获，最后通过使用Siperose6Increase10/300GL柱子(GE)进行分子筛层析进行纯化，获取高纯度的目的蛋白(如图6-7所示，图16-17)。
作为可选择的实施方案，超滤洗脱的缓冲液是：pH 7.4的PBS缓冲液。
作为可选择的实施方案，镍柱洗脱的缓冲液是：pH 7.4的PBS，含有500mM Imidazole。
作为可选择的实施方案，Lectin柱(GE)的填料为：Concanavalin A(Con A)，Wheat germ agglutinin(WGA)，柱洗脱的洗脱机是：methyl-α-D-mannopyranoside，GlcNAc。
作为可选择的实施方案，所述分子筛层析的缓冲液是：pH 7.4的PBS缓冲液。
本发明具有以下有益效果：
本发明将病毒的受体结合域(Receptor binding domain,RBD)及融合肽(Fusion peptide,FP)共同作为双抗原片段，并与细菌复合物(如火球菌多聚物蛋白(Pyrococcus furiosus_Ferritin,Ferritin(PF))或来源于Aquifex aeolicus菌株二氧四氢喋啶合酶多聚物蛋白(Lumazine Synthase,LS)组成融和蛋白，实现抗原多聚化，同时加上信号肽及纯化标签，通过质粒转染真核细胞表达系统(如 293F细胞)表达可自组装的融合蛋白，RBD-FP可通过Ferritin(PF)或LS自组装作用形成二十四聚体纳米抗原或六十聚体纳米抗原。该方案可克服RBD-FP单体免疫原性不足的缺点，所得疫苗能显著的提高宿主针对SARS-CoV-2的中和抗体的水平。本发明通过将RBD-FP-PF_Ferritin纳米抗原和LS-RBD-FP纳米抗原免疫Balb/c小鼠的实验已证实免疫10天后产生的中和抗体具有可强力阻挡SARS-CoV-2假病毒入侵靶细胞的能力。
而且本发明疫苗制备方法简单、易于纯化，NIH登记的临床试验中已证明了Ferritin和LS作为纳米疫苗载体的安全性，疫苗可较快的应用于临床试验。
附图说明
图1为RBD-FP-PF_Ferritin融合蛋白自行组装纳米颗粒示意图。
图2为RBD-FP-PF_Ferritin融合蛋白结构示意图。
图3为表达RBD-FP-PF_Ferritin的质粒结构示意图。
图4为RBD-FP-PF_Ferritin融合酶切验证。
图5为RBD-FP-PF_Ferritin融合蛋白转染293F细胞免疫荧光图。
图6为RBD-FP-PF_Ferritin融合蛋白纯化分子筛图。
图7为RBD-FP-PF_Ferritin融合蛋白纯化SDS-PAGE图(约50KD)。
图8为融合蛋白纳米疫苗小鼠免疫策略。
图9为小鼠血清中和抗体效价检测策略。
图10为小鼠免疫RBD-FP-PF_Ferritin纳米疫苗产生阻挡SARS-CoV-2入侵靶细胞的中和抗体。
图11为LS-RBD-FP融合蛋白自行组装纳米颗粒示意图。
图12为LS-RBD-FP融合蛋白结构示意图。
图13为表达LS-RBD-FP的质粒结构示意图。
图14为LS-RBD-FP融合酶切验证。
图15为LS-RBD-FP融合蛋白转染293F细胞免疫荧光图。
图16为LS-RBD-FP融合蛋白纯化分子筛图。
图17为LS-RBD-FP融合蛋白纯化SDS-PAGE图(约50KD)。
图18为小鼠免疫LS-RBD-FP纳米疫苗产生阻挡SARS-CoV-2入侵靶细胞的中和抗体。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。
除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 构建新型冠状病毒SARS-CoV-2抗原(融和蛋白RBD-FP-PF_Ferritin)
融和蛋白RBD-FP-PF_Ferritin自行组装纳米颗粒示意图以及结构示意图分别如图1和图2所示。
具体地，融和蛋白RBD-FP-PF_Ferritin的构建制备方法如下：
1、表达RBD-FP-PF_Ferritin融合蛋白的载体的制备
将RBD-FP-PF_Ferritin核苷酸序列3’端加上翻译终止密码子后克隆到添加I ntron以及WPRE增强表达的表达载体(pcDNA3.1-Intron-WPRE)的Xho I和Xba I酶切位点之间，构建表达载体pcDNA3.1-Intron-WPRE-RBD-FP-Ferritin(PF)-IRE S-GFP(如图3所示)。
重组质粒转化DH5α感受态细胞，37℃过夜培养，筛选和PCR鉴定出阳性克隆。提取去内毒素的质粒，经酶切以及测序验证后用于纳米抗原蛋白的表达(如图4所示)。将质粒通过脂质体转染的方案转染HEK293F细胞，转染3天后经离心收获细胞上清(RBD-FP-PF_Ferritin蛋白转染293F细胞免疫荧光图如图5所示)，进行目的蛋白RBD-FP-PF_Ferritin纯化。
2、RBD-FP-PF_Ferritin纳米抗原纯化
将表达RBD-FP-PF_Ferritin的细胞上清通过0.22μm的滤膜过滤，除去细胞碎片。经10K超滤管超滤后，将过滤后的细胞上清液与Histrap-excel于4℃结合30分钟，使用HisTrap excel镍柱进行粗纯。
之后，首先使用PBS(pH 7.4)缓冲液和低浓度咪唑缓冲液(PBS，50mM Imidazole，pH 7.4)分别进行洗涤50ml，去除流穿的杂蛋白。其后，通过含高咪唑缓冲液(PBS，500mM Imidazole，pH 7.4)进行目的蛋白洗脱。随后，目的蛋白使用Con A与WGA以1：1比例填料的Lectin Agarose柱(GE)进行目的蛋白的富集。
收集合并RBD-FP-PF_Ferritin二十四聚体的洗脱峰，最后通过使用Siperose6 Increase10/300GL柱子(GE)进行分子筛层析进行纯化，获得纯度大于99％的二 十四聚体RBD-FP-PF_Ferritin蛋白(如图6-7所示)，分子筛层析的缓冲液是：PBS，pH 7.4。目的蛋白浓缩后，分装成小份，用液氮迅速冷冻后于-80℃保存。
实施例2 小鼠免疫实验
将实施例1得到的RBD-FP-PF_Ferritin融合蛋白按照表1用生理盐水稀释至100μg/ml，并与等体积佐剂SAS进行分组乳化。然后对6-8周龄的Balb/C小鼠进行分组免疫。免疫策略如图8所示，即通过腹腔注射的方式，每只小鼠分别在第0天，第3周(21天)，第14周(108天)接受3次疫苗免疫，每次200μl的接种体积(10μg)。第10、31、108天，对小鼠进行眼眶取血。小鼠血清在静置一段时间待血清析出后，通过4℃，2800rpm离心15分钟获得，立刻用于SARS-CoV-2假病毒中和检测实验。
表1
抗原/对照 抗原含量 佐剂 动物数量(只)
RBD-FP-PF_Ferritin 10μg SAS 4
PBS 0 SAS 4
实施例3 假病毒中和试验
1、假病毒的制备：
根据NCBI公布序列，合成SARS-CoV-2的Spike蛋白，并将其插入pcDNA3.1表达载体。将SARS-CoV-2Spike蛋白的表达载体与pHIV-luciferase和psPAX2质粒共同转染293T细胞，转染5小时后，PBS洗涤细胞2次，换为无血清DMEM培养基继续培养。48小时后收取上清，离心去除细胞碎片。后用小体积无血清DMEM溶解获得HIV-luc/SARS-CoV-2-S假病毒。
该假病毒可以有效模拟野生型SARS-CoV-2入侵细胞的过程。当其感染生产细胞或靶细胞后，SARS-CoV-2假病毒所携带的荧光素酶报告基因的表达能够准确反映病毒感染结果，使得实验系统的结果能够被精准快捷地读取，可以作为优秀的抗体中和效价监测系统(如图9所示)。
2、假病毒TCID 50测定
将上一步收取的病毒液按5倍比稀释，加入到96孔板中的HEK293T细胞中。感染4小时后，弃掉病毒液，PBS洗涤细胞2次，换为含10％血清的DMEM完全培养基。48小时后，弃掉培养基，PBS洗涤2次，加入细胞裂解液，震荡 裂解30分钟。-80℃冻融一次后，每孔取30μl利用GloMax 96(Promega)检测荧光素酶活性值。通过Reed-Muech法计算TCID 50。
3、中和试验
将纯化的抗体2倍倍比稀释，与TCID 50终浓度假病毒混合，37℃共孵育1小时。将混合液加入到已密度为70％左右的HEK293T细胞中的96孔板中。48小时后，弃掉培养液，PBS洗涤细胞2次，加入细胞裂解液，检测荧光素酶活性值。
4、结果分析
结果如图10。将RBD-FP-PF_Ferritin纳米抗原免疫Balb/c小鼠后10天血清即检测到对SARS-CoV-2假病毒的中和活性，t检验显示实验组与对照组组间存在差异显著性。在显著性水平为0.05的情况下，双尾概率水平小于0.05。
结果表明，本发明RBD-FP-PF_Ferritin融合蛋白与SAS佐剂合用，经一次免疫后10天即可激发小鼠体液免疫，小于平行对照组所激发的中和抗体效价，且差异显著。
实施例4 构建新型冠状病毒SARS-CoV-2抗原(融和蛋白LS-RBD-FP)
融和蛋白LS-RBD-FP自行组装纳米颗粒示意图以及结构示意图分别如图11和图12所示。
具体地，融和蛋白LS-RBD-FP的构建制备方法如下：
1、表达LS-RBD-FP融合蛋白的载体的制备
将LS-RBD-FP核苷酸序列3’端加上翻译终止密码子后克隆到添加Intron以及WPRE增强表达的表达载体(pcDNA3.1-Intron-WPRE)的Xho I和Xba I酶切位点之间，构建表达载体pcDNA3.1-Intron-WPRE-LS-RBD-FP-IRES-GFP(如图13所示)。
重组质粒转化DH5α感受态细胞，37℃过夜培养，筛选和PCR鉴定出阳性克隆。提取去内毒素的质粒，经酶切以及测序验证后用于纳米抗原蛋白的表达(如图14所示)。将质粒通过脂质体转染的方案转染HEK293F细胞，转染3天后经离心收获细胞上清(LS-RBD-FP蛋白转染293F细胞免疫荧光图如图15所示)，进行目的蛋白LS-RBD-FP纯化。
2、LS-RBD-FP纳米抗原纯化
将表达LS-RBD-FP的细胞上清通过0.22μm的滤膜过滤，除去细胞碎片。经 10K超滤管超滤后，将过滤后的细胞上清液与Histrap-excel于4℃结合30分钟，使用HisTrap excel镍柱进行粗纯。
之后，首先使用PBS(pH 7.4)缓冲液和低浓度咪唑缓冲液(PBS，50mM Imidazole，pH 7.4)分别进行洗涤50ml，去除流穿的杂蛋白。其后，通过含高咪唑缓冲液(PBS，500mM Imidazole，pH 7.4；)进行目的蛋白洗脱。随后，目的蛋白使用Con A与WGA以1：1比例填料的Lectin Agarose柱(GE)进行目的蛋白的富集。
收集合并LS-RBD-FP六十聚体的洗脱峰，最后通过使用Siperose6Increase10/300GL柱子(GE)进行分子筛层析进行纯化，获得纯度大于99％的六十聚体LS-RBD-FP蛋白(如图16-17所示)，分子筛层析的缓冲液是：PBS，pH7.4。目的蛋白浓缩后，分装成小份，用液氮迅速冷冻后于-80℃保存。
实施例5 小鼠免疫实验
将实施例1得到的LS-RBD-FP融合蛋白按照表1用生理盐水稀释至100μg/ml，并与等体积佐剂SAS进行分组乳化。然后对6-8周龄的Balb/C小鼠进行分组免疫。免疫策略如图8所示，即通过腹腔注射的方式，每只小鼠分别在第0天，第3周(21天)，第14周(108天)接受3次疫苗免疫，每次200μl的接种体积(10μg)。第10、31、108天，对小鼠进行眼眶取血。小鼠血清在静置一段时间待血清析出后，通过4℃，2800rpm离心15分钟获得，立刻用于SARS-CoV-2假病毒中和检测实验。
表1
抗原/对照 抗原含量 佐剂 动物数量(只)
LS-RBD-FP 10μg SAS 4
PBS 0 SAS 4
实施例6 假病毒中和试验
1、假病毒的制备：
根据NCBI公布序列，合成SARS-CoV-2的Spike蛋白，并将其插入pcDNA3.1表达载体。将SARS-CoV-2Spike蛋白的表达载体与pHIV-luciferase和psPAX2质粒共同转染293T细胞，转染5小时后，PBS洗涤细胞2次，换为无血清DMEM培养基继续培养。48小时后收取上清，离心去除细胞碎片。后用小体积无血清 DMEM溶解获得HIV-luc/SARS-CoV-2-S假病毒。
该假病毒可以有效模拟野生型SARS-CoV-2入侵细胞的过程。当其感染生产细胞或靶细胞后，SARS-CoV-2假病毒所携带的荧光素酶报告基因的表达能够准确反映病毒感染结果，使得实验系统的结果能够被精准快捷地读取，可以作为优秀的抗体中和效价监测系统(如图9所示)。
2、假病毒TCID 50测定
将上一步收取的病毒液按5倍比稀释，加入到96孔板中的HEK293T细胞中。感染4小时后，弃掉病毒液，PBS洗涤细胞2次，换为含10％血清的DMEM完全培养基。48小时后，弃掉培养基，PBS洗涤2次，加入细胞裂解液，震荡裂解30分钟。-80℃冻融一次后，每孔取30μl利用GloMax 96(Promega)检测荧光素酶活性值。通过Reed-Muech法计算TCID 50。
3、中和试验
将纯化的抗体2倍倍比稀释，与TCID 50终浓度假病毒混合，37℃共孵育1小时。将混合液加入到已密度为70％左右的HEK293T细胞中的96孔板中。48小时后，弃掉培养液，PBS洗涤细胞2次，加入细胞裂解液，检测荧光素酶活性值。
4、结果分析
结果如图18。将LS-RBD-FP纳米抗原免疫Balb/c小鼠后10天血清即检测到对SARS-CoV-2假病毒的中和活性，t检验显示实验组与对照组组间存在差异显著性。在显著性水平为0.05的情况下，双尾概率水平小于0.05。
结果表明，本发明LS-RBD-FP与SAS佐剂合用，经一次免疫后10天即可激发小鼠体液免疫，小于平行对照组所激发的中和抗体效价，且差异显著。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (21)

  1. 一种提高抗原免疫原性的方法，其特征在于，所述方法是将病毒的受体结合域(Receptor binding domain,RBD)和融合肽(Fusion peptide,FP)共同作为双抗原，并与细菌复合物融合组成一个新的融和蛋白后作为抗原；所述细菌复合物为火球菌多聚物蛋白(Pyrococcus furiosus_Ferritin,Ferritin(PF))或2,4-二氧四氢喋啶合酶多聚物蛋白(Lumazine Synthase,LS)。
  2. 根据权利要求1所述方法，其特征在于，所述抗原为冠状病毒抗原，所述病毒的受体结合域RBD和融合肽FP为冠状病毒的受体结合域RBD和融合肽FP。
  3. 根据权利要求2所述方法，其特征在于，所述冠状病毒抗原为新型冠状病毒SARS-CoV-2抗原，所述冠状病毒的受体结合域RBD和融合肽FP为新型冠状病毒SARS-CoV-2的受体结合域RBD和融合肽FP。
  4. 根据权利要求3所述方法，其特征在于，所述新型冠状病毒SARS-CoV-2抗原为新型冠状病毒SARS-CoV-2的表面刺突蛋白(S蛋白)抗原。
  5. 根据权利要求4所述方法，其特征在于，新型冠状病毒SARS-CoV-2的RBD的序列如SEQ ID NO:1所示，FP的氨基酸序列如SEQ ID NO:2所示，SEQ ID NO:1与SEQ ID NO:2可以直接连接，或两者以铰链区Linker相连构成一个新的融和蛋白RBD-FP；优选地，当所述Linker为GGSGGSGGSGGSGGG时所得融和蛋白RBD-FP的氨基酸序列如SEQ ID NO:3所示。
  6. 根据权利要求5所述方法，其特征在于，所述Ferritin(PF)的氨基酸序列如SEQ ID NO:4所示；SEQ ID NO:3与SEQ ID NO:4可以直接连接，或两者以铰链区Linker相连构成一个新的融和蛋白RBD-FP-PF_Ferritin；优选地，当所述Linker为GSG时所得融和蛋白RBD-FP-PF_Ferritin的氨基酸序列如SEQ ID NO:5所示。
  7. 根据权利要求1-6任一所述方法，其特征在于，融和蛋白再加上信号肽及纯化标签后，利用真核表达系统表达出抗原；优选地，所述信号肽为分泌型信号肽(Signal peptide,SP)；优选地，所述纯化标签为His标签(His-tag)；优选地，新型冠状病毒SARS-CoV-2的SP、His-tag、RBD与FP融合的氨基酸序列如SEQ ID NO:6所示。
  8. 根据权利要求7所述方法，其特征在于，所述SEQ ID NO:4与SEQ ID  NO:6所示序列可以直接连接，或两者以铰链区Linker相连构成一个新的融和蛋白RBD-FP-PF_Ferritin；优选地，当所述Linker为GSG时所得融和蛋白RBD-FP-PF_Ferritin的氨基酸序列如SEQ ID NO:7所示。
  9. 根据权利要求7所述方法，其特征在于，所述Lumazine Synthase(LS)的氨基酸序列如SEQ ID NO:8所示；SEQ ID NO:8与SEQ ID NO:3可以直接连接，或两者以铰链区Linker相连构成一个新的融和蛋白LS-RBD-FP；优选地，当所述Linker为GGSGGSGGSGGSGGSGGG时所得融和蛋白LS-RBD-FP的氨基酸序列如SEQ ID NO:9所示。
  10. 根据权利要求1-5、9任一所述方法，其特征在于，融和蛋白再加上信号肽后，利用真核表达系统表达出抗原；优选地，所述信号肽为分泌型信号肽(Signal peptide,SP)；优选地，新型冠状病毒SARS-CoV-2的SP、LS、RBD与FP融合的氨基酸序列如SEQ ID NO:10所示。
  11. 根据权利要求10所述方法，其特征在于，在SEQ ID NO:10融和蛋白加上纯化标签后，可用于融合蛋白的纯化；优选地，所述纯化标签为His标签(His-tag)；新型冠状病毒SARS-CoV-2纳米疫苗的SP、LS、RBD、FP与His-tag融合的氨基酸序列如SEQ ID NO:11所示。
  12. 一种免疫原性提高的冠状病毒抗原，其特征在于，依据权利要求1-8任一所述方法构建得到的新的融合蛋白RBD-FP-PF_Ferritin。
  13. 根据权利要求9所述冠状病毒抗原，其特征在于，新型冠状病毒SARS-CoV-2抗原(融合蛋白RBD-FP-PF_Ferritin)的氨基酸序列如SEQ ID NO:5或SEQ ID NO:7所示。
  14. 一种免疫原性提高的冠状病毒抗原，其特征在于，依据权利要求1-5、9-11任一所述方法构建得到的新的融合蛋白LS-RBD-FP。
  15. 根据权利要求14所述冠状病毒抗原，其特征在于，新型冠状病毒SARS-CoV-2抗原(融合蛋白LS-RBD-FP)的氨基酸序列如SEQ ID NO:9或SEQ ID NO:10或SEQ ID NO:11所示。
  16. 权利要求12-15任一所述冠状病毒抗原在制备抗冠状病毒药物方面的应用。
  17. 根据权利要求16所述的应用，其特征在于，所述应用是将所述冠状病毒抗原与SAS佐剂合用。
  18. 根据权利要求16或17所述的应用，其特征在于，所述应用是用于制备试剂盒；所述试剂盒中含有所述抗原，或者编码所述抗原的DNA分子，或者表达所述抗原的重组载体/表达盒/转基因细胞系/重组菌。
  19. 一种表达权利要求12-15任一所述抗原的核苷酸序列，含有该核苷酸序列的重组载体、表达盒、转基因细胞系或重组菌。
  20. 一种冠状病毒疫苗，其特征在于，以权利要求12-15任一所述冠状病毒抗原为抗原制备而成。
  21. 权利要求12-15任一所述抗原的制备方法，其特征在于，在SEQ ID NO:3与SEQ ID NO:4直接串联或铰链串联所示氨基酸对应的核苷酸序列、SEQ ID NO:6与SEQ ID NO:4直接串联或铰链串联所示氨基酸对应的核苷酸序列、SEQ ID NO:5所示氨基酸对应的核苷酸序列、或SEQ ID NO:7所示氨基酸对应的核苷酸序列的3’端加上翻译终止密码子，进行克隆表达，筛选正确的重组子，然后转染真核表达系统进行表达，表达后收集细胞上清，纯化，即得到冠状病毒纳米抗原RBD-FP-PF_Ferritin；
    或在SEQ ID NO:8与SEQ ID NO:3直接串联或铰链串联所示氨基酸对应的核苷酸序列(SEQ ID NO:9)、SEQ ID NO:10所示氨基酸对应的核苷酸序列、或SEQ ID NO:11所示氨基酸对应的核苷酸序列的3’端加上翻译终止密码子，进行克隆表达，筛选正确的重组子，然后转染真核表达系统进行表达，表达后收集细胞上清，纯化，即得到冠状病毒纳米抗原LS-RBD-FP。
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