WO2022027702A1 - 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白多聚体纳米疫苗 - Google Patents

一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白多聚体纳米疫苗 Download PDF

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WO2022027702A1
WO2022027702A1 PCT/CN2020/108259 CN2020108259W WO2022027702A1 WO 2022027702 A1 WO2022027702 A1 WO 2022027702A1 CN 2020108259 W CN2020108259 W CN 2020108259W WO 2022027702 A1 WO2022027702 A1 WO 2022027702A1
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protein
rbd
hpf
tag
seq
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French (fr)
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张辉
邹帆
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广州千扬生物医药技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the technical field of biomedicine. More specifically, it relates to a novel coronavirus (SARS-CoV-2, also known as 2019-nCoV) S protein two-domain subunit nanovaccine based on Helicobacter pylori ferritin.
  • SARS-CoV-2 also known as 2019-nCoV
  • 2019-nCoV S protein two-domain subunit nanovaccine based on Helicobacter pylori ferritin.
  • the novel coronavirus SARS-CoV-2 (also known as 2019-nCoV), the novel coronavirus pneumonia caused by it, is spreading globally. Due to the unclear source and pathogenesis of the new coronavirus pneumonia, and the lack of specific antiviral drugs, it has brought great difficulties to clinical diagnosis, treatment and control of the epidemic, resulting in a serious social burden and crisis.
  • the prepared vaccine can significantly increase the level of the neutralizing antibody of the host against the virus, and the produced antibody has the ability to strongly block the virus from invading the target cell.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of 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 susceptible people.
  • the receptor binding domain (RBD) of the virus is used alone or together with the heptad repeat (HR) as a double antigen fragment, and is combined with Helicobacter pylori_Ferritin (HPF)
  • HR heptad repeat
  • HPF Helicobacter pylori_Ferritin
  • this program is:
  • heptad repeat sequence Heptad Repeat, HR
  • ST tag signal peptide
  • purification tag express through plasmid transfection in eukaryotic cell expression system (such as 293F or CHO cells), and obtain His-tag-ST-HR after purification protein.
  • Helicobacter pylori polymer protein (Helicobacter pylori_Ferritin, HPF) plus SC tag, signal peptide and purification tag, through plasmid transfection eukaryotic cell expression system (such as 293F or CHO cells) expression, purified to obtain His-tag- SC-HPF protein.
  • His-tag-SC-HPF protein can self-assemble into spherical tetrameric nanoparticles.
  • the His-tag-SC-HPF protein self-assembled into tetrameric nanoparticles was incubated with His-tag-ST-RBD protein, and the RBD-HPF subunit was formed through the action of ST-SC covalent bond. Tetrameric protein.
  • the His-tag-SC-HPF protein self-assembled into tetrameric nanoparticles was incubated with His-tag-ST-RBD protein and His-tag-ST-HR protein, the ST-SC covalent bond interacted with the protein. Forms the RBD-HR-HPF two-subunit tetrameric protein.
  • This tetrameric protein can display RBD protein alone or RBD/HR protein together on the surface of nanoparticles, overcoming the shortcomings of insufficient immunogenicity of RBD monomer, and can effectively induce stronger immune response and generate neutralization Antibodies against SARS-CoV-2 virus invading target cells.
  • the vaccine prepared by the scheme of the present invention can significantly increase the level of neutralizing antibodies of the host against SARS-CoV-2; and the vaccine preparation method of the present invention is simple, and the protein contains His tag and is easy to purify, which has been proved in the clinical trials registered by NIH Considering the safety of Ferritin antigen as a nano-vaccine carrier, the vaccine can be quickly applied to clinical trials.
  • the first object of the present invention is to provide a method for improving the immunogenicity of an antigen.
  • Another object of the present invention is to provide a novel coronavirus based on a twenty-tetramerized subunit constructed from a novel coronavirus (SARS-CoV-2) RBD subunit or RBD-HR double subunit and a bacterial polymer antigen.
  • SARS-CoV-2 novel coronavirus
  • 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-novel 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, a vector or a transgenic cell line that encodes and expresses the novel coronavirus antigen.
  • the present invention first provides a method for improving antigen immunogenicity, which is to express the receptor binding domain (RBD) of coronavirus, and the obtained protein and Helicobacter pylori polymer protein (Helicobacter pylori_Ferritin, HPF) pass through Covalently bind to form a new RBD-HPF subunit multimeric protein as an antigen.
  • RBD receptor binding domain
  • HPF Helicobacter pylori polymer protein
  • the described method for improving antigen immunogenicity is to express the receptor binding domain (Receptor binding domain, RBD) and heptad repeat region (Heptad Repeat, HR) of coronavirus respectively, and the obtained protein is then combined with Helicobacter pylori.
  • the multimeric protein (Helicobacter pylori_Ferritin, HPF) forms a new RBD-HR-HPF double subunit multimeric protein by covalent bonding as an antigen.
  • the method is to link the receptor binding domain (RBD) of the virus with the ST tag, signal peptide, and tag, and after fusion, the SP-His-tag-ST-RBD protein is formed;
  • the peptide repeat sequence (Heptad Repeat, HR) is linked with ST tags, signal peptides, and tags to form SP-His-tag-ST-HR protein after fusion;
  • the tag, signal peptide, and tag link are fused to form SP-His-tag-SC-HPF protein.
  • the protein containing the tag SC provided by the present invention can be combined with the protein containing the ST tag through covalent action.
  • the SP-His-tag-ST-RBD protein constructed by the method is incubated with the SP-His-tag-SC-HPF protein to form a new RBD-HPF subunit multimeric protein as an antigen.
  • the SP-His-tag-ST-RBD protein constructed by the method is mixed with the SP-His-tag-ST-HR protein in a certain ratio, and then incubated with the SP-His-tag-SC-HPF protein together to form a composition.
  • a novel RBD-HR-HPF two-subunit multimeric protein serves as an antigen.
  • Ferritin is a self-assembled globular protein, and the distance between the amino termini of every two adjacent subunits on the surface is about 4.5-7.5 nm, which is suitable for loading antigens on the outer surface.
  • HPF a ferritin derived from Helicobacter pylori, can spontaneously form multimerization, and after the surface is loaded with antigen, it can induce strong humoral and cellular immune responses.
  • the number of antigens that can be carried by the immunization solves the shortcomings of the weaker immunity caused by the RBD monomer vaccine.
  • RBD and HR are the reasons for choosing RBD and HR.
  • 1 RBD is the region that binds to receptors
  • 2 HR is the structural basis of S protein-mediated membrane fusion.
  • Binding" and "fusion” constitute the two most critical and earliest steps in the entry of viruses into cells. The immunization of two regions to construct a fusion protein has not been reported in previous studies of single-segment vaccines.
  • the antigen fragments with HPF we also multimerize the antigen fragments with HPF, and aggregate the double antigens together to form nanoparticles, which further increases the number of antigen-bearing antigens in a single immunization, so it can be more fully and stably contacted with immune cells in the human body to stimulate the production of antibody.
  • the "double antigen + multimer” strategy of the present invention can achieve the effect of stimulating the body to produce an effective immune response more effectively, rapidly and stably in terms of quality (RBD + HR double antigen) and quantity (multimerization). .
  • 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 the ST tag is shown in SEQ ID NO: 2.
  • SEQ ID NO: 1 and SEQ ID NO: 2 can be directly connected to obtain fusion protein ST-RBD.
  • SEQ ID NO: 2 is connected with SEQ ID NO: 1 to form a new fusion protein ST-RBD with the hinge region Linker.
  • the Linker can be GSG.
  • the amino acid sequence of ST-RBD of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 3.
  • the amino acid sequence of ST-RBD of the new coronavirus SARS-CoV-2 is shown in SEQ ID NO: 9.
  • amino acid sequence of HR of the new coronavirus SARS-CoV-2 is shown in SEQ ID NO:4; the amino acid sequence of ST is shown in SEQ ID NO:2.
  • SEQ ID NO:2 and SEQ ID NO:4 can be directly connected to obtain fusion protein ST-HR.
  • SEQ ID NO: 2 and SEQ ID NO: 4 are connected to form a new fusion protein ST-HR by hinge region Linker.
  • the Linker can be GSG.
  • the amino acid sequence of ST-HR of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 5.
  • the amino acid sequence of ST-HR of the new coronavirus SARS-CoV-2 is shown in SEQ ID NO: 10.
  • amino acid sequence of HPF is shown in SEQ ID NO:6
  • amino acid sequence of SC is shown in SEQ ID NO:7.
  • SEQ ID NO: 6 and SEQ ID NO: 7 can be directly connected to obtain fusion protein SC-HPF.
  • SEQ ID NO: 6 is connected with SEQ ID NO: 7 to form a new fusion protein SC-HPF with the hinge region Linker.
  • the Linker can be GSG.
  • the amino acid sequence of SC-HPF of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 8.
  • the amino acid sequence of SC-HPF of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 11.
  • SEQ ID NO: 9 protein and SEQ ID NO: 11 protein can be co-incubated to obtain a new RBD-HPF subunit multimer protein through covalent bond.
  • SEQ ID NO: 9 protein and SEQ ID NO: 10 protein are mixed in different ratios and then co-incubated with SEQ ID NO: 11 protein to obtain a new RBD-HR-HPF double subunit multimeric protein by covalent bond.
  • 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 tetramerized subunit protein RBD using Helicobacter pylori ferritin. - HPF or the tetramerized bisubunit protein RBD-HR-HPF (as shown in Figure 1).
  • Antigens will be displayed on the surface of nanoparticles, which can effectively elicit stronger immune responses of receptors.
  • Mice immunized with RBD-HPF protein and RBD-HR-HPF protein both produce antibodies that neutralize SARS-CoV-2 invading target cells .
  • the tetramerized RBD-HPF protein and the RBD-HR-HPF protein of the present invention can overcome the shortcoming of insufficient immunogenicity of the RBD monomer, and significantly improve the neutralizing antibody production of the receptor against SARS-CoV-2.
  • the novel coronavirus SARS-CoV-2 antigen (tetrameric RBD-HPF protein) comprises the SP-His-tag-ST-RBD protein and SP-His disclosed herein -tag-SC-HPF proteins are covalently linked, wherein the SP-His-tag-SC-HPF proteins are capable of self-assembly into nanoparticles that display on the surface the immunostaining of SP-His-tag-RBD-ST proteins original part.
  • the RBD-HPF subunit multimeric vaccine has the potential to protect SARS-CoV susceptible populations.
  • the novel coronavirus SARS-CoV-2 antigen (tetrameric RBD-HR-HPF protein) comprises the His-tag-ST-RBD protein disclosed herein and His- The tag-ST-HR protein is covalently linked to the His-tag-SC-HPF protein after mixing in a ratio of 7:3, wherein the His-tag-SC-HPF protein can self-assemble into nanoparticles, which are on the surface
  • the His-tag-ST-RBD protein and the immunogenic portion of the His-tag-ST-HR protein are shown above.
  • the RBD-HR-HPF subunit multimeric vaccine has the potential to protect SARS-CoV susceptible populations.
  • the obtained coronavirus antigen with improved immunogenicity specifically a new self-assembled and tetramerized RBD-HPF protein and RBD-HR-HPF protein constructed by the above method, And the coronavirus vaccine prepared with this antigen should also be within the protection scope of the present invention.
  • the application of the coronavirus antigen in the preparation of anti-coronavirus drugs is also within the protection scope of the present invention.
  • an anti-SARS-CoV-2 vaccine can be prepared by using RBD-HPF protein or RBD-HR-HPF protein in combination with SAS adjuvant.
  • the present invention also provides a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria expressing the above-mentioned antigen (tetrameric RBD-HPF protein or tetrameric RBD-HR-HPF protein).
  • the present invention also provides an optional preparation method of the above antigen, specifically in the nucleotide sequence corresponding to the amino acids shown in SEQ ID NO: 1 and SEQ ID NO: 2 in direct tandem or hinge tandem, SEQ ID NO: 2 Nucleotide sequences corresponding to amino acids shown in direct tandem or hinge tandem with SEQ ID NO:4, nucleotide sequences corresponding to amino acids shown in SEQ ID NO:6 and SEQ ID NO:7 in direct tandem or hinge tandem, SEQ ID NO: 7 : 3, SEQ ID NO: 5, nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 8, or nucleotide corresponding to the amino acid shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11
  • the 3' end of the sequence is added with a translation stop codon, cloned into a eukaryotic expression vector, and after digestion and sequencing is correct, transiently transfected into a eukaryotic expression system (such as 293F cells) for the
  • His-tag-ST-RBD protein (SEQ ID NO: 9) was incubated with His-tag-SC-HPF (SEQ ID NO: 11) protein alone to form RBD-HPF through covalent action twenty-four aggregate protein, or mix His-tag-ST-RBD protein and His-tag-ST-HR (SEQ ID NO: 10) protein in a certain ratio and then co-incubate with His-tag-SC-HPF protein. Covalently formed the RBD-HR-HPF twenty-tetrameric protein.
  • 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 protocols for introducing corresponding proteins into eukaryotic expression systems include, but are not limited to, various transfection, infection, site-directed knock-in, transposition protocols, and the like.
  • the purification method is to filter the cell supernatant expressing the antigen to remove cell debris, and pass through a 10K ultrafiltration tube (Millipore) for preliminary purification, and then pass through a HisTrap HP nickel column (GE) , Lectin column (GE) to capture the target protein (His-tag-ST-RBD protein, His-tag-ST-HR protein, His-tag-SC-HPF protein), and co-incubate the RBD- HPF and RBD-HR-HPF tetramer fusion protein were purified by molecular sieve chromatography using Superose 6Increase 10/300GL column (GE) to obtain high-purity target protein (as shown in Figure 3-6).
  • GE HisTrap HP nickel column
  • GE Lectin column
  • RBD- HPF and RBD-HR-HPF tetramer fusion protein were purified by molecular sieve chromatography using Superose 6Increase 10/300GL column (GE) to obtain high-pur
  • the ultrafiltration elution buffer is: Tris buffer pH 7.4.
  • the nickel column elution buffer is: Tris buffer, pH 7.4, containing 500 mM Imidazole.
  • the packing material of Lectin column is: Concanavalin A (Con A), Wheat germ agglutinin (WGA), and the eluent for column elution is: methyl- ⁇ -D-mannopyranoside, GlcNAc.
  • the buffer of the molecular sieve chromatography is: Tris buffer of pH 7.4.
  • the nanovaccine obtained in the present invention is a purified tetramer RBD-HPF protein or a tetramer protein of RBD-HR-HPF; the monomer size of the RBD-HPF protein is about 65kD.
  • the monomeric size of the RBD-HR-HPF protein was 65 kD (RBD-HPF monomeric protein) and 80 kD (HR-HPF monomeric protein).
  • nucleotide sequences encoding the above-mentioned antigens of the present invention should also fall within the protection scope of the present invention.
  • the invention provides a method for improving the immunogenicity of an antigen, and prepares a coronavirus antigen with improved immunogenicity, specifically co-incubating the His-tag-ST-RBD protein with the His-tag-SC-HPF protein alone, through Covalently form RBD-HPF tetramer protein, this RBD-HPF tetramer protein is used as an antigen for preparing vaccine; or His-tag-ST-RBD protein and His-tag-ST-HR protein are combined After mixing in a certain proportion, it was incubated with His-tag-SC-HPF protein to form RBD-HR-HPF tetramer protein through covalent action, and this RBD-HR-HPF tetramer protein was used as antigen.
  • the vaccine preparation method of the present invention is simple, the protein contains His tag and is easy to purify, and the safety of Ferritin antigen as a nano-vaccine carrier has been proved in the clinical trial registered by NIH, and the vaccine can be quickly applied to the clinical trial.
  • Fig. 1 is an electron microscope counterstaining image (left: RBD-HPF, right: RBD-HR-HPF).
  • Figure 2 is a schematic diagram of the structure (top: SP-His-tag-ST-RBD, middle: SP-His-tag-ST-HR, bottom: SP-His-tag-SC-HPF).
  • Figure 3 shows the Coomassie brilliant blue staining bands of the tetrameric protein of RBD-HPF after purification.
  • Figure 4 shows the Coomassie brilliant blue staining band of the tetrameric protein of RBD-HR-HPF after purification.
  • Figure 5 is a molecular sieve diagram of purified RBD-HPF tetrameric protein after purification.
  • Figure 6 is a molecular sieve diagram of purified RBD-HR-HPF tetrameric protein after purification.
  • FIG. 7 shows that mice immunized with RBD-HPF nanovaccine and RBD-HR-HPF nanovaccine produced neutralizing antibodies that blocked the invasion of SARS-CoV-2 wild virus into target cells.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • RBD-HPF subunit multimeric protein is as follows:
  • the 3' end of the nucleotide sequence corresponding to SP-His-tag-ST-RBD was added with a translation stop codon and cloned into an expression vector (pcDNA3.1-Intron-WPRE) added with Intron and WPRE for enhanced expression to construct an expression vector .
  • the recombinant plasmids were transformed into DH5 ⁇ competent cells, cultured at 37°C overnight, and positive clones were identified by screening and PCR.
  • the endotoxin-depleted plasmid was extracted and verified by enzyme digestion and sequencing for the expression of nano-antigen protein.
  • the plasmid was transfected into HEK293F cells by the lipofection protocol. After 3 days of transfection, the cell supernatant was harvested by centrifugation. The cell supernatant expressing His-tag-ST-RBD was filtered through a 0.22 ⁇ m filter to remove cell debris. .
  • 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.
  • 50 ml of Tris (pH 7.4) buffer and low-concentration imidazole buffer (PBS, 50 mM Imidazole, pH 7.4) were used to wash 50 ml respectively to remove the flow-through impurity protein.
  • PBS 50 mM Imidazole, pH 7.4
  • the target protein was eluted by a high imidazole-containing buffer (Tris-HCl, 500 mM Imidazole, pH 7.4;).
  • Tris-HCl 500 mM Imidazole, pH 7.4;
  • the target protein was enriched using a Lectin Agarose column (GE) packed with Con A and WGA at a ratio of 1:1.
  • the 3' end of the nucleotide sequence corresponding to SP-His-tag-ST-HR was added with a translation stop codon and cloned into an expression vector (pcDNA3.1-Intron-WPRE) added with Intron and WPRE for enhanced expression to construct an expression vector .
  • the recombinant plasmids were transformed into DH5 ⁇ competent cells, cultured at 37°C overnight, and positive clones were identified by screening and PCR.
  • the endotoxin-depleted plasmid was extracted and verified by enzyme digestion and sequencing for the expression of nano-antigen protein.
  • the plasmid was transfected into HEK293F cells by the lipofection protocol. After 3 days of transfection, the cell supernatant was harvested by centrifugation, and the cell supernatant expressing His-tag-ST-HR was filtered through a 0.22 ⁇ m filter to remove cell debris. .
  • 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.
  • 50 ml of Tris (pH 7.4) buffer and low-concentration imidazole buffer (Tris-HCl, 50 mM Imidazole, pH 7.4) were used to wash 50 ml respectively to remove the flow-through impurity proteins.
  • the target protein was eluted by a high imidazole-containing buffer (Tris-HCl, 500 mM Imidazole, pH 7.4;).
  • the target protein was enriched using a Lectin Agarose column (GE) packed with Con A and WGA at a ratio of 1:1.
  • GE Lectin Agarose column
  • the 3' end of the nucleotide sequence corresponding to SP-His-tag-SC-HPF was added with a translation stop codon and cloned into an expression vector (pcDNA3.1-Intron-WPRE) added with Intron and WPRE to enhance expression to construct an expression vector .
  • the recombinant plasmid was transformed into DH5 ⁇ competent cells, cultured at 37°C overnight, and positive clones were identified by screening and PCR.
  • the endotoxin-depleted plasmid was extracted and verified by enzyme digestion and sequencing for the expression of nano-antigen protein.
  • the plasmid was transfected into HEK293F cells by the lipofection protocol.
  • the cell supernatant was harvested by centrifugation.
  • the cell supernatant expressing His-tag-SC-HPF was filtered through a 0.22 ⁇ m filter to remove cell debris. .
  • 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.
  • 50 ml of Tris (pH 7.4) buffer and low-concentration imidazole buffer (Tris-HCl, 50 mM Imidazole, pH 7.4) were used to wash 50 ml respectively to remove the flow-through impurity proteins.
  • the target protein was eluted by a high imidazole-containing buffer (Tris-HCl, 500 mM Imidazole, pH 7.4;). Subsequently, the target protein was enriched using a Lectin Agarose column (GE) packed with Con A and WGA at a ratio of 1:1.
  • a high imidazole-containing buffer Tris-HCl, 500 mM Imidazole, pH 7.4;.
  • the target protein was enriched using a Lectin Agarose column (GE) packed with Con A and WGA at a ratio of 1:1.
  • the prepared His-tag-ST-RBD protein and His-tag-SC-HPF protein were mixed at room temperature in a molar ratio of 1:1. Incubate for 12 hours, and use Superose 6 Increase10/300GL column (GE) for purification by molecular sieve chromatography to obtain a tetrameric RBD-HPF protein with a purity greater than 99%, that is, the RBD-HPF subunit multimeric protein (as shown in the figure). 3, 5), the buffer of molecular sieve chromatography is: Tris-HCl, pH 7.4. After the target protein was concentrated, it was divided into small portions, quickly frozen in liquid nitrogen and stored at -80°C.
  • the RBD-HPF subunit multimeric protein and RBD-HR-HPF subunit multimeric protein obtained in Example 1 were diluted to 100 ⁇ g/ml with physiological saline according to Table 1, and emulsified in groups with equal volume of adjuvant SAS.
  • Group immunizations were then performed on 6-8 week old BALB/c mice. By intraperitoneal injection, each mouse received 3 times of vaccine immunization on day 0, week 3 (21 days), and week 14 (day 108), each with an inoculation volume of 200 ⁇ l (10 ⁇ g). On days 10, 31, and 108, the mice were bled from the orbit. The mouse serum was left standing for a period of time until the serum was precipitated, and then obtained by centrifugation at 4°C and 2800 rpm for 15 minutes, and immediately used for the SARS-CoV-2 virus neutralization detection experiment.
  • Antigen/Control antigen content adjuvant Number of animals (only) RBD-HPF protein 10 ⁇ g SAS 4 RBD-HR-HPF protein 10 ⁇ g SAS 4 RBD protein 10 ⁇ g SAS 4
  • Novel coronavirus SARS-CoV-2 wild virus source isolated from fecal samples of new crown patients provided by the Eighth People's Hospital of Guangzhou.
  • Vero E6 cells were seeded into 96-well plates at a density of 2 x 104 cells per well.
  • Dilute serum Serum is inactivated at 56°C for 30 minutes (this inactivation step is generally performed immediately after obtaining the serum), and the serum is diluted 5-fold or 10-fold according to the experimental requirements.
  • two dilution gradients are selected, namely 100-fold and 1000-fold. times.
  • Table 2 For example, to obtain a system with a final concentration of 10-fold, 100-fold and 100-fold diluted serum, dilute as shown in Table 2 below:
  • Fixation Remove the supernatant, add 200ul of 4% paraformaldehyde to each well, fix it overnight at 4°C and transfer it to P2 to continue the experiment. The fixative was removed and each well was washed 3 times with 200ul PBS.
  • Dry plate remove the color developing solution, wash each well with 200ul ddH 2 O for 3 minutes each time, and wash twice in total. And pat dry, count with ELISPOT.

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Abstract

提供一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒S蛋白亚单位纳米疫苗。以新型冠状病毒的受体结合域(Receptor binding domain,RBD)表达蛋白与幽门螺旋杆菌多聚物蛋白(HP_Ferritin，HPF)的N端通过SpyTag/SpyCatcher(ST-SC)系统中分子间异肽键作用连接组成亚单位多聚体蛋白，实现抗原多聚化；或将RBD表达蛋白与七肽重复区(Heptad Repeat,HR)表达蛋白，共同与HPF蛋白以ST-SC共价结合组成双亚单位多聚体蛋白，实现抗原多聚化。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。同时基于序列分析，该方案有望研发出针对多种冠状病毒有效的疫苗。而且所述疫苗制备方法简单、易于纯化，安全性高，疫苗可较快的应用于临床试验。

Description

一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒S蛋白多聚体纳米疫苗 技术领域
本发明属于生物医药技术领域。更具体地，涉及一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒(SARS-CoV-2，又名2019-nCoV)S蛋白双区域亚单位纳米疫苗。
背景技术
新型冠状病毒SARS-CoV-2(又名2019-nCoV)，其引起的新型冠状病毒肺炎疫情在全球蔓延。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，对于人民健康与国家安全具有重要意义。因此，目前针对冠状病毒，尤其是新型冠状病毒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。
对于冠状病毒疫苗的研发，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。中国专利2020101440324(公开号CN111217919A)提供了一种基于火球菌铁蛋白的新型冠状病毒S蛋白双区域亚单位纳米疫苗，方案中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，并与火球菌多聚物蛋白(Pyrococcus furiosus_Ferritin,Ferritin(PF))融合组成一个新的融和蛋白RBD-FP-PF_Ferritin后作为抗原，提高了抗原免疫原性，所制得的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。
同时，我们对更多、更优的疫苗制备方案进行不断地探索研究。
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)单独或者与七肽重复序 列(Heptad Repeat,HR)共同作为双抗原片段，并与幽门螺旋杆菌多聚物蛋白(Helicobacter pylori_Ferritin,HPF)通过ST-SC共价作用实现抗原的多聚化，构建开发了抗原多聚体复合物。
此方案具体是：
以病毒的受体结合域(Receptor binding domain,RBD)加上ST标签、信号肽及纯化标签，通过质粒转染真核细胞表达系统(如293F或者CHO细胞)表达，经纯化获得His-tag-ST-RBD蛋白。
以七肽重复序列(Heptad Repeat,HR)加上ST标签、信号肽及纯化标签，通过质粒转染真核细胞表达系统(如293F或者CHO细胞)表达，经纯化获得His-tag-ST-HR蛋白。
以幽门螺旋杆菌多聚物蛋白(Helicobacter pylori_Ferritin,HPF)加上SC标签、信号肽及纯化标签，通过质粒转染真核细胞表达系统(如293F或者CHO细胞)表达，经纯化获得His-tag-SC-HPF蛋白。His-tag-SC-HPF蛋白可自行组装成球状二十四聚体纳米颗粒。
将自组装成二十四聚体纳米颗粒的His-tag-SC-HPF蛋白与His-tag-ST-RBD蛋白共同孵育，则通过ST-SC共价键的作用形成RBD-HPF亚单位二十四聚体蛋白。将自组装成二十四聚体纳米颗粒的His-tag-SC-HPF蛋白与His-tag-ST-RBD蛋白以及His-tag-ST-HR蛋白共同孵育，则通过ST-SC共价键作用形成RBD-HR-HPF双亚单位二十四聚体蛋白。
这种二十四聚体蛋白可将RBD蛋白单独或者RBD/HR蛋白共同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病毒入侵靶细胞的抗体。
本发明的方案所制得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Ferritin抗原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。
因此，本发明第一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基于新型冠状病毒(SARS-CoV-2)RBD亚单位或者RBD-HR双亚单位与细菌多聚物构建的二十四聚体化的亚单位的新型冠状病毒抗原。
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，是将冠状病毒的受体结合域(Receptor binding domain,RBD)进行表达，所得蛋白与幽门螺旋杆菌多聚物蛋白(Helicobacter pylori_Ferritin,HPF)通过共价结合的方式组成一个新的RBD-HPF亚单位多聚体蛋白后作为抗原。
或，所述提高抗原免疫原性的方法是分别将冠状病毒的受体结合域(Receptor binding domain,RBD)与七肽重复区(Heptad Repeat,HR)进行表达，所得蛋白再共同与幽门螺旋杆菌多聚物蛋白(Helicobacter pylori_Ferritin,HPF)通过共价结合的方式组成一个新的RBD-HR-HPF双亚单位多聚体蛋白后作为抗原。
具体操作方案中，所述方法是将病毒的受体结合域(Receptor binding domain,RBD)和ST标签、信号肽、标签链接，经过融合后形成SP-His-tag-ST-RBD蛋白；将七肽重复序列(Heptad Repeat,HR)和ST标签、信号肽、标签链接，经过融合后形成SP-His-tag-ST-HR蛋白；将幽门螺旋杆菌多聚物蛋白(Helicobacter pylori_Ferritin,HPF)和SC标签、信号肽、标签链接，经过融合后形成SP-His-tag-SC-HPF蛋白。本发明提供的含有标签SC的蛋白可与含有ST标签的蛋白通过共价作用结合。
进一步优选地，所述方法构建的SP-His-tag-ST-RBD蛋白与SP-His-tag-SC-HPF蛋白共同孵育，组成一个新的RBD-HPF亚单位多聚体蛋白后作为抗原。或，所述方法构建的SP-His-tag-ST-RBD蛋白与SP-His-tag-ST-HR蛋白以一定的比例混合后再与SP-His-tag-SC-HPF蛋白共同孵育，组成一个新的RBD-HR-HPF双亚单位多聚体蛋白后作为抗原。
上述方案中，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HPF这样一种来源于幽门螺旋杆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，解决RBD单体疫苗引发较弱免疫的缺点。
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HR的原因在于：①RBD是和受体结合的区域；②HR是S蛋白介导的膜融合的结构基础。“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HPF的多聚化，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+HR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。
具体地，上述方案应用在新型冠状病毒疫苗制备时：
首先，新型冠状病毒SARS-CoV-2的RBD的氨基酸序列如SEQ ID NO:1所示；ST标签的氨基酸序列如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ST-RBD。
或SEQ ID NO:2与SEQ ID NO:1以铰链区Linker相连构成一个新的融和蛋白ST-RBD。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，新型冠状病毒SARS-CoV-2的ST-RBD的氨基酸序列如SEQ ID NO:3所示。当在ST-RBD蛋白N端添加信号肽SP，C端添加His标签时，新型冠状病毒SARS-CoV-2的ST-RBD的氨基酸序列如SEQ ID NO:9所示。
另外，新型冠状病毒SARS-CoV-2的HR的氨基酸序列如SEQ ID NO:4所示；ST的氨基酸序列如SEQ ID NO:2所示。
SEQ ID NO:2与SEQ ID NO:4可以直接连接得到融合蛋白ST-HR。
或SEQ ID NO:2与SEQ ID NO:4以铰链区Linker相连构成一个新的融和蛋白ST-HR。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，新型冠状病毒SARS-CoV-2的ST-HR的氨基酸序列如SEQ ID NO:5所示。当在ST-HR蛋白N端添加信号肽SP，C端添加His标签时，新型冠状病毒SARS-CoV-2的ST-HR的氨基酸序列如SEQ ID NO:10所示。
另外，所述HPF的氨基酸序列如SEQ ID NO:6所示，SC的氨基酸序列如SEQ ID NO:7所示。
SEQ ID NO:6与SEQ ID NO:7可以直接连接得到融合蛋白SC-HPF。
或SEQ ID NO:6与SEQ ID NO:7以铰链区Linker相连构成一个新的融和蛋白SC-HPF。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，新型冠状病毒SARS-CoV-2的SC-HPF的氨基酸序列如SEQ ID NO:8所示。当在ST-HR蛋白N端添加信号肽SP，C端添加His标签时，新型冠状病毒SARS-CoV-2的SC-HPF的氨基酸序列如SEQ ID NO:11所示。
然后，SEQ ID NO:9蛋白与SEQ ID NO:11蛋白通过共孵育可通过共价键连接得到新的RBD-HPF亚单位多聚体蛋白。SEQ ID NO:9蛋白与SEQ ID NO:10蛋白以不同比例混合后与SEQ ID NO:11蛋白通过共孵育可通过共价键连接得到新的RBD-HR-HPF双亚单位多聚体蛋白。
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，所述抗原是利用幽门螺旋杆菌铁蛋白自行组装为二十四聚体化的亚单位蛋白RBD-HPF或者二十四聚体化的双亚单位蛋白RBD-HR-HPF(如图1所示)。抗原将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，接受RBD-HPF蛋白以及RBD-HR-HPF蛋白免疫的小鼠均产生中和SARS-CoV-2入侵靶细胞的抗体。本发明的二十四聚体化的RBD-HPF蛋白以及RBD-HR-HPF蛋白可克服RBD单体免疫原性不足的缺点，显著提高受体针对SARS-CoV-2的中和抗体产生。
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(二十四聚体RBD-HPF蛋白)包含本文公开的SP-His-tag-ST-RBD蛋白与SP-His-tag-SC-HPF蛋白通共价键连接，其中所述SP-His-tag-SC-HPF蛋白能够自组装为纳米颗粒，其在表面上展示SP-His-tag-RBD-ST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-HPF亚单位多聚体疫苗具备保护SARS-CoV易感人群的潜力。
作为本发明可选择的另一种优选实施方案，新型冠状病毒SARS-CoV-2抗原(二十四聚体RBD-HR-HPF蛋白)包含本文公开的His-tag-ST-RBD蛋白与His-tag-ST-HR蛋白以7：3的比例混合后与His-tag-SC-HPF蛋白通共价键连接，其中所述His-tag-SC-HPF蛋白能够自组装为纳米颗粒，其在表面上展示His-tag-ST-RBD蛋白以及His-tag-ST-HR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-HR-HPF亚单位多聚体疫苗具备保护SARS-CoV 易感人群的潜力。
基于上述方案，所获得的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RBD-HPF蛋白以及RBD-HR-HPF蛋白，以及有该抗原所制备获得的冠状病毒疫苗，也均应在本发明保护范围之内。
同时，所述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-HPF蛋白或者RBD-HR-HPF蛋白与SAS佐剂合用制备抗SARS-CoV-2疫苗。
另外本发明还提供一种表达上述抗原(二十四聚体RBD-HPF蛋白或者二十四聚体RBD-HR-HPF蛋白)的重组载体、表达盒、转基因细胞系或重组菌。
最后本发明还提供上述抗原的一种可选择的制备方法，具体是在SEQ ID NO:1与SEQ ID NO:2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:2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:7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:3、SEQ ID NO:5、SEQ ID NO:8所示氨基酸对应的核苷酸序列、或SEQ ID NO:9、SEQ ID NO:10、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，经酶切以及测序正确后，瞬时转染真核表达系统(如293F细胞)进行纳米抗原的表达，表达后收集细胞上清，纯化，His-tag-ST-RBD蛋白(SEQ ID NO:9)单独与His-tag-SC-HPF(SEQ ID NO:11)蛋白共孵育，通过共价作用组成RBD-HPF二十四聚体蛋白，或者将His-tag-ST-RBD蛋白与His-tag-ST-HR(SEQ ID NO:10)蛋白以一定的比例混合后再与His-tag-SC-HPF蛋白共孵育，通过共价作用组成RBD-HR-HPF二十四聚体蛋白。
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、定点敲入、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(His-tag-ST-RBD蛋白、His-tag-ST-HR蛋白、His-tag-SC-HPF蛋白)的捕获，共孵育经共价键获得的RBD-HPF以及RBD-HR-HPF二十四聚体融合蛋白通过使用Superose 6Increase 10/300GL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3-6所示)。
作为可选择的实施方案，超滤洗脱的缓冲液是：pH 7.4的Tris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Tris缓冲液，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Tris缓冲液。
本发明所得纳米疫苗是经纯化的二十四聚体RBD-HPF蛋白或者RBD-HR-HPF二十四聚体蛋白；所述RBD-HPF蛋白单体大小约为65kD。所述RBD-HR-HPF蛋白单体大小为65kD(RBD-HPF单体蛋白)和80kD(HR-HPF单体蛋白)。
最后，编码表达本发明上述抗原的核苷酸序列，以及含有该核苷酸序列、编码表达所述抗原的载体或转基因细胞系，也应在本发明的保护范围之内。
本发明具有以下有益效果：
本发明提供了一种提高抗原免疫原性的方法，制备了免疫原性提高的冠状病毒抗原，具体是将His-tag-ST-RBD蛋白单独与His-tag-SC-HPF蛋白共孵育，通过共价作用组成RBD-HPF二十四聚体蛋白，此RBD-HPF二十四聚体蛋白作为抗原用于制备疫苗；或者将His-tag-ST-RBD蛋白与His-tag-ST-HR蛋白以一定的比例混合后再与His-tag-SC-HPF蛋白共孵育，通过共价作用组成RBD-HR-HPF二十四聚体蛋白，此RBD-HR-HPF二十四聚体蛋白作为抗原用于制备疫苗。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SARS-CoV-2的中和抗体的水平。本发明通过RBD-HPF纳米抗原以及RBD-HR-HPF纳米抗原免疫BALB/c小鼠的实验已证实产生的抗体具有可强力阻挡SARS-CoV-2野生病毒入侵靶细胞的能力(如图7所示)。
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Ferritin抗原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。
另外，我们通过比对SARS-CoV-2Wuhan-Hu-1、SARS-CoV-2USA-IA-6399、SARS-CoV-2SYSU-IHV三种毒株发现，三种病毒株的RBD、HR序列高度保守(100％)；通过对来源于2003年的SARS-CoV病毒和SARS-CoV-2SYSU-IHV病毒的序列比对发现，两种病毒株的75.9％的RBD序列，92.6％的HR1，100％的HR2序列保守。因此，本发明以RBD序列和HR序列为基础的方案，有望研发出针对多种冠状病毒有效的疫苗。
附图说明
图1为电镜复染图(左：RBD-HPF，右：RBD-HR-HPF)。
图2为结构示意图(上：SP-His-tag-ST-RBD，中：SP-His-tag-ST-HR，下：SP-His-tag-SC-HPF)。
图3为纯化后RBD-HPF二十四聚体蛋白考马斯亮蓝染色条带。
图4为纯化后RBD-HR-HPF二十四聚体蛋白考马斯亮蓝染色条带。
图5为纯化后RBD-HPF二十四聚体蛋白纯化分子筛图。
图6为纯化后RBD-HR-HPF二十四聚体蛋白纯化分子筛图。
图7为小鼠免疫RBD-HPF纳米疫苗以及RBD-HR-HPF纳米疫苗产生阻挡SARS-CoV-2野生病毒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(双亚单位多聚体蛋白RBD-HPF以及双亚单位多聚体蛋白RBD-HR-HPF)
RBD-HPF亚单位多聚体蛋白以及RBD-HR-HPF双亚单位多聚体蛋白自行组装成纳米颗粒的电镜复染图如图1。
具体地，RBD-HPF亚单位多聚体蛋白的构建制备方法如下：
1、表达His-tag-ST-RBD蛋白的制备
将SP-His-tag-ST-RBD对应的核苷酸序列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)，构建表达载体。
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。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，将表达His-tag-ST-RBD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Tris(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(Tris-HCl，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。
2、表达His-tag-ST-HR蛋白的制备
将SP-His-tag-ST-HR对应的核苷酸序列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)，构建表达载体。
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。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，将表达His-tag-ST-HR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Tris(pH 7.4)缓冲液和低浓度咪唑缓冲液(Tris-HCl，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(Tris-HCl，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。
3、表达His-tag-SC-HPF蛋白的制备
将SP-His-tag-SC-HPF对应的核苷酸序列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)，构建表达载体。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。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，将表达His-tag-SC-HPF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Tris(pH 7.4)缓冲液和低浓度咪唑缓冲液(Tris-HCl，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(Tris-HCl，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。
4、RBD-HPF纳米抗原(RBD-HPF亚单位多聚体蛋白)的制备
检测His-tag-ST-RBD蛋白与His-tag-SC-HPF蛋白的浓度，将制备的His-tag-ST-RBD蛋白与His-tag-SC-HPF蛋白以1：1的摩尔数室温共孵育12小时，使用Superose 6 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-HPF蛋白，即RBD-HPF亚单位多聚体蛋白(如图3、5所示)，分子筛层析的缓冲液是：Tris-HCl，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。
5、RBD-HR-HPF纳米抗原(RBD-HR-HPF双亚单位多聚体蛋白)的构建
检测His-tag-ST-RBD蛋白、His-tag-ST-HR蛋白、His-tag-SC-HPF蛋白的浓度，将制备的His-tag-ST-RBD蛋白与His-tag-ST-HR蛋白以7：3的摩尔比混匀后，再与等量的His-tag-SC-HPF蛋白室温共孵育12小时，使用Superose 6 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-HR-HPF蛋白，即RBD-HR-HPF双亚单位多聚体蛋白(如图4、6所示)，分子筛层析的缓冲液是：Tris-HCl，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-HPF亚单位多聚体蛋白与RBD-HR-HPF亚单位多聚体蛋白按照表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。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-HPF蛋白 10μg SAS 4
RBD-HR-HPF蛋白 10μg SAS 4
RBD蛋白 10μg SAS 4
实施例3 野生病毒中和试验
1、试验材料
新型冠状病毒SARS-CoV-2野生病毒来源：广州市第八人民医院提供的新冠患者的粪便样本中分离。
野生病毒中和试验在中山大学生物安全三级实验室(P3实验室)完成。
2、实验方法
第0天：
将Vero E6细胞以每孔2×10 4个细胞的密度接种到96孔板中。
第1天：(等待细胞生长密度达到100％。)
(1)稀释血清：血清56℃灭活30min(此灭活步骤一般在取得血清后立即操作)，根据实验需求以5倍或10倍稀释血清，此处选取2个稀释梯度即100倍和1000倍。例如要得到终浓度为10倍、100倍和100倍稀释的血清的体系，按如下表2所示稀释：
表2
Figure PCTCN2020108259-appb-000001
(2)稀释病毒：每毫升DMEM培养液中6000-8000FFU，此处根据本实验室经验，按每孔加0.25ul的病毒原液。根据所需孔数，配制足量稀释好的病毒液。
(3)细胞孵育：将96孔板中的细胞培养基去除，然后每孔加入60ul病毒/血清混合物，要做复孔(因此以上过程每个血清要保证有够两份的量，此处180ul足够，注意180ul体系含0.75ul病毒原液)，并在37℃条件下孵育1小时。
(4)细胞培养：去除病毒上清，然后用排枪每孔加入125ul加热至37℃的含1.6％CMC的DMEM培养基，注意液体粘稠需要等待吸入枪头。将96孔板放置在37℃(5％CO 2)下，孵育24小时。
第2天：
(1)固定：去除上清，每孔加入200ul 4％多聚甲醛，4℃固定过夜后传入P2继续实验。除去固定剂，每孔用200ul PBS洗涤3次。
(2)破膜/封闭：每孔加入100ul含有0.2％Triton X-100和1％BSA的PBS，在室温条件下反应30分钟后，每孔用200ul PBS洗涤3次。
(3)孵育一抗：稀释一抗(Anti-SARS-N；40143-T62-100)用含有1％BSA的PBS，1:1000稀释。每孔加50ul稀释后的一抗，在37℃下孵育1小时。弃去一抗，每孔用200ul PBS/T(0.1％Tween)洗涤3次。
(4)孵育二抗：稀释二抗(Goat anti-rabbit IgG HRP；SSA004-1)，用含有1％BSA的PBS溶液，1:2000稀释。每孔加50ul稀释后的二抗，在37℃下孵育1小时。弃去二抗，每孔用200ul PBS/T洗涤3次。
(5)显色：每孔加入50ul TrueBlue(KPL)，避光室温摇动孵育5分钟。
(6)干板：去除显色液，每孔用200ul ddH 2O洗涤，每次3分钟，合计洗2次。并拍干，用ELISPOT计数。
3、结果分析
结果如图7。将RBD-HPF纳米抗原和RBD-HR-HPF免疫BALB/c小鼠后31天血清即检测到对SARS-CoV-2野生病毒的中和活性，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_Ferritin与SAS佐剂合用，经两次免疫后31天即可激发小鼠体液免疫，小于平行对照的二十四聚体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 (19)

  1. 一种提高抗原免疫原性的方法，其特征在于，所述方法是将新型冠状病毒的受体结合域(Receptor binding domain,RBD)进行表达，所得蛋白与幽门螺旋杆菌多聚物蛋白(Helicobacter pylori_Ferritin,HPF)通过共价结合的方式组成一个新的RBD-HPF亚单位多聚体蛋白后作为抗原。
  2. 根据权利要求1所述方法，其特征在于，新型冠状病毒SARS-CoV-2的RBD的氨基酸序列如SEQ ID NO:1所示，RBD先与ST标签连接后进行蛋白表达，ST的氨基酸序列如SEQ ID NO:2所示；SEQ ID NO:2与SEQ ID NO:1直接连接，或两者以铰链区Linker相连构成一个新的融和蛋白ST-RBD。
  3. 根据权利要求2所述方法，其特征在于，当所述Linker为GSG时所得融和蛋白ST-RBD的氨基酸序列如SEQ ID NO:3所示。
  4. 根据权利要求1-3任一所述方法，其特征在于，分别将新型冠状病毒的受体结合域(Receptor binding domain,RBD)与七肽重复区(Heptad Repeat,HR)进行表达，所得蛋白再共同与幽门螺旋杆菌多聚物蛋白(Helicobacter pylori_Ferritin,HPF)通过共价结合的方式组成一个新的RBD-HR-HPF双亚单位多聚体蛋白后作为抗原。
  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HR的氨基酸序列如SEQ ID NO:4所示，HR先与ST标签连接后进行蛋白表达，ST的氨基酸序列如SEQ ID NO:2所示；SEQ ID NO:2与SEQ ID NO:4直接连接，或两者以铰链区Linker相连构成一个新的融和蛋白ST-HR。
  6. 根据权利要求5所述方法，其特征在于，当所述Linker为GSG时所得融和蛋白ST-HR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，幽门螺旋杆菌多聚物蛋白HPF的氨基酸序列如SEQ ID NO:6所示，HPF先与SC标签连接后进行蛋白表达，SC的氨基酸序列如SEQ ID NO:7所示；SEQ ID NO:7与SEQ ID NO:6直接连接，或两者以铰链区Linker相连构成一个新的融和蛋白SC-HPF。
  8. 根据权利要求7所述方法，其特征在于，当所述Linker为GSG时所得融和蛋白SC-HPF的氨基酸序列如SEQ ID NO:8所示。
  9. 根据权利要求1-8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His后，利用真核表达系统表达出抗原。
  10. 根据权利要求9所述方法，其特征在于，所述信号肽为分泌型信号肽(Signal peptide,SP)；所述纯化标签为His标签(His-tag)；
    SP、His-tag、RBD与ST融合的氨基酸序列如SEQ ID NO:9所示；
    SP、His-tag、HR与ST融合的氨基酸序列如SEQ ID NO:10所示；
    SP、His-tag、HPF与SC融合的氨基酸序列如SEQ ID NO:11所示。
  11. 一种His-tag-ST-RBD蛋白，其特征在于，根据权利要求9或10所述方法表达得到，氨基酸序列如SEQ ID NO:9所示。
  12. 一种His-tag-ST-HR蛋白，其特征在于，根据权利要求9或10所述方法表达得到，氨基酸序列如SEQ ID NO:10所示。
  13. 一种His-tag-SC-HPF蛋白，其特征在于，根据权利要求9或10所述方法表达得到，氨基酸序列如SEQ ID NO:11所示。
  14. 一种免疫原性提高的冠状病毒抗原，其特征在于，将权利要求11所述His-tag-ST-RBD蛋白与权利要求13所述His-tag-SC-HPF蛋白室温孵育，两者通过ST-SC的共价结合作用构建得到的新的RBD-HPF亚单位多聚体蛋白。
  15. 一种免疫原性提高的冠状病毒抗原，其特征在于，将权利要求11所述His-tag-ST-RBD蛋白、权利要求12所述His-tag-ST-HR蛋白与权利要求13所述His-tag-SC-HPF蛋白共同孵育，三者通过ST-SC的共价结合作用构建得到的新的RBD-HR-HPF双亚单位多聚体蛋白。
  16. 权利要求14或15所述冠状病毒抗原在制备抗冠状病毒药物方面的应用。
  17. 一种表达权利要求14或15所述抗原的重组载体、表达盒、转基因细胞系或重组菌。
  18. 一种冠状病毒疫苗，其特征在于，以权利要求14或15所述冠状病毒抗原为抗原制备而成。
  19. 编码表达权利要求14或15所述抗原的核苷酸序列，或包含该序列的载体或转基因细胞系。
PCT/CN2020/108259 2020-08-04 2020-08-10 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白多聚体纳米疫苗 WO2022027702A1 (zh)

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