WO2022048415A1 - 新型冠状病毒s蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物 - Google Patents

新型冠状病毒s蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物 Download PDF

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WO2022048415A1
WO2022048415A1 PCT/CN2021/112043 CN2021112043W WO2022048415A1 WO 2022048415 A1 WO2022048415 A1 WO 2022048415A1 CN 2021112043 W CN2021112043 W CN 2021112043W WO 2022048415 A1 WO2022048415 A1 WO 2022048415A1
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novel coronavirus
protein
polyploid
fragment
rbd
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PCT/CN2021/112043
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French (fr)
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童贝
董咸池
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江苏省中国科学院植物研究所
南京大学
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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present application relates to the field of biomedical technology, in particular to a polyploid of a novel coronavirus S protein fragment and its preparation method, detection kit, vaccine and medicine.
  • the new coronavirus is a new strain of coronavirus that has never been found in humans before.
  • the new coronavirus S protein (SARS-CoV-2 spike protein) is a type I transmembrane glycoprotein and the largest structural protein of the virus. It consists of 1255 amino acids. Residue composition that can stimulate the body to produce neutralizing antibodies and mediate immune responses.
  • the S protein is closely related to the process of virus invasion into cells.
  • the S protein is composed of two domains, the part near the N-terminus forms the globular domain, and the part near the C-terminus forms the rod-like domain that penetrates the membrane.
  • S1 spherical part
  • S2 rod-shaped part
  • the S1 part is involved in the recognition and binding of receptors, which contains the receptor binding domain (Receptor Binding Domain, RBD).
  • the S2 part is involved in virus invasion of host cells.
  • the recombinant S protein can be used for antigen detection and vaccine preparation of coronavirus infection. Therefore, how to efficiently utilize the S protein is an important basis for the diagnosis and treatment of the new coronavirus.
  • the present application provides a novel coronavirus S protein fragment polyploid and its preparation method, detection kit, vaccine and medicine to solve the technical defects existing in the prior art.
  • the application provides a novel coronavirus S protein fragment polyploid, characterized in that the novel coronavirus S protein fragment polyploid is composed of at least two novel coronavirus S protein receptor binding region fragments in series.
  • GG or GS sequences are connected in series between the novel coronavirus S protein receptor binding region fragments.
  • GGS, GGGS, GSGS, SGGS, GGGGS, GSGGS, GGSGS, GGSGGS, GGGSGS, GGGGGS or GGGSGGS are connected in series between the novel coronavirus S protein receptor binding region fragments.
  • the C-terminus of the novel coronavirus S protein receptor binding region fragment is connected to the Fc fragment of human immunoglobulin.
  • one or more of the novel coronavirus S protein receptor binding region fragments form a polyploid unit, and the polyploid unit forms two Fc fragments connected by the novel coronavirus S protein receptor binding region fragments.
  • the dimer was used as a polyploid of the novel coronavirus S protein fragment.
  • fragments of the novel coronavirus S protein receptor binding region or the polyploids of the novel coronavirus S protein fragments are connected by a protease or a protein cross-linking agent.
  • the enzymes include transpeptidase and combined protein ligase
  • the protein cross-linking agents include polyethylene glycol, suberic acid disuccinimidyl ester, succinic acid suberic acid glutarate, dimethyl diimide, dimethylimide, dimethylimide, and maleimide hexane.
  • novel coronavirus S protein receptor binding region fragment is selected from (RBD) fragment itself and/or (RBD) fragment mutant.
  • the polyploid of the new coronavirus S protein fragment can be composed of only multiple (RBD) fragment ontology, only multiple (RBD) fragment mutants, or can be composed of one or more (RBD) fragments
  • the fragment ontology is constructed with one or more (RBD) fragment polyploids.
  • the types of (RBD) segment mutants may be the same or different.
  • composition of the triploid of the new coronavirus S protein fragment can be: fragment ontology-segment ontology-segment ontology, segment ontology-segment ontology-segment A mutation, segment ontology-segment A mutation-segment A mutation, segment ontology-segment A mutation-fragment B mutation, fragment body-fragment A mutation-fragment A+B mutation, fragment A mutation-fragment B mutation-fragment C mutation, fragment A+B+C mutation-fragment B+C mutation-fragment C mutation, Fragment A+B+C mutation-fragment A+B+C mutation-fragment A+B+C mutation-fragment A+B+C mutation, etc.
  • Other methods can be deduced in the same way, and will not be repeated here.
  • mutation sites of the (RBD) fragment mutants include: one or more of N501Y, K417N, K417T, E484K, L452R, T478K, L452Q, F490S, E484Q and T478Q.
  • the (RBD) fragment mutant provided by the present application can be obtained by mutation of any one of the above mutation sites, or by simultaneous mutation of any two or more mutation sites.
  • the (RBD) fragment mutants include: B.1.1.7 (Alpha) mutants obtained from N501Y mutation, K417N mutant mutants, and E484K mutant B1.525 (Eta) mutants , N501Y, K417N and E484K mutant B.1.351 (Beta) mutant, N501Y, K417T and E484K mutant P.1 (Gamma) mutant, L452R and T478K mutant B.1.617.2 ( Delta) mutant, L452Q and F490S mutant C.37 (Lambda) mutant, E484Q mutant mutant, L452R and E484Q mutant B.1.617.1 (Kappa) mutant, L452R mutant The B.1.427 (Epsilon) mutant strain was formed, and the B.1.1.222 mutant strain formed by the T478Q mutation.
  • the application also provides a detection kit, which is a kit capable of detecting the presence or absence of the novel coronavirus, and the detection kit includes the polyploid of the S protein fragment of the novel coronavirus as described above.
  • the present application also provides a vaccine, which is a vaccine capable of preventing novel coronavirus infection, and the vaccine includes the novel coronavirus S protein fragment polyploid as described above.
  • the vaccine is an mRNA vaccine, a protein vaccine or an adenovirus vaccine.
  • polyploid of the novel coronavirus S protein fragment in the vaccine activates the aggregated B cell receptors to produce novel coronavirus antibodies.
  • the application also provides a medicine, which is a medicine capable of preventing or treating the novel coronavirus, and the medicine includes the polyploid of the S protein fragment of the novel coronavirus as described above.
  • polyploid of the novel coronavirus S protein fragment in the medicine binds to the ACE2 receptor to inhibit the invasion of the novel coronavirus.
  • the application provides a method for preparing a novel coronavirus S protein fragment polyploid, including:
  • the novel coronavirus S protein fragment polyploid provided by this application is composed of at least two novel coronavirus S protein receptor binding domain (RBD) fragments in series, and has strong binding ability to ACE2, high expression level and good stability, and can be applied It has strong applicability and wide application range in the antibody detection and vaccine preparation of the new coronavirus. Among them, the application of the new coronavirus S protein fragment polyploid in the antibody detection of the new coronavirus can effectively improve the specificity of antibody detection.
  • RBD coronavirus S protein receptor binding domain
  • the new coronavirus S protein fragment polyploid is applied to vaccine preparation, the vaccine can stimulate the body to quickly produce antibodies, so that the body can produce immunity, and the stability of the vaccine Well, the quality is easy to monitor.
  • polyploid of the new coronavirus S protein fragment provided by this application can also be applied to the detection and treatment of various mutant strains of the new coronavirus, with high detection accuracy, good treatment effect, and high application flexibility, and is suitable for large-scale promotion. and use.
  • the method for preparing the polyploid of the S protein fragment of the novel coronavirus which comprises obtaining the nucleotide sequence of the polyploid, inserting the nucleotide sequence into an expression vector, transfecting mammalian cells with the target expression vector, Purifying the culture supernatant can effectively increase the expression of the polyploid of the new coronavirus S protein fragment, and effectively improve the purity of the polyploid of the new coronavirus S protein fragment, which is the most important part of the antibody for the polyploid of the new coronavirus S protein fragment.
  • Fig. 1 is the electrophoresis result figure of the S protein fragment polyploid of the novel coronavirus described in an embodiment of the present application;
  • Fig. 2 is the electrophoresis result diagram of the novel coronavirus S protein fragment with Fc fragment and its polyploid described in an embodiment of the present application;
  • ACE2 coronavirus S protein receptor angiotensin-converting enzyme 2
  • FIG. 4 is a graph of the binding ability of a novel coronavirus S protein fragment without an Fc fragment and its polyploid to ACE2-expressing cells using flow analysis according to an embodiment of the present application;
  • FIG. 5 is a graph showing the binding ability of a novel coronavirus S protein fragment with an Fc fragment and its polyploid to ACE2 expressing cells using flow analysis according to an example of the present application;
  • Fig. 6 is the end-point titer comparison diagram of the novel coronavirus S protein fragment polyploid described in an embodiment of the present application and the commercialized RBD-S protein and RBD monomer protein;
  • Figures 7 to 10 are graphs showing the comparison of the binding ability of the RBD fragment mutant described in an example of the present application to cells expressing the novel coronavirus spike protein receptor ACE2.
  • angiotensin-converting enzyme 2 (ACE2), a carboxypeptidase that efficiently degrades angiotensin II to angiotensin 1-7, has been identified as a functional receptor and Potential receptor for SARA-CoV-2.
  • Immunoglobulin G is the main component of immunoglobulins in serum, accounting for about 75% of the total immunoglobulin content in serum. There are 4 subtypes of IgG, namely IgG1, IgG2, IgG3, and IgG4. IgG is the most important antibody in the body and has the functions of anti-virus, virus neutralization, antibacterial and immune regulation.
  • This embodiment provides a polyploid of a novel coronavirus S protein fragment, and the novel coronavirus S protein fragment polyploid is composed of at least two novel coronavirus S protein receptor binding region fragments in series.
  • the new coronavirus S protein exists in the form of trimers. There are about 1300 amino acids in each monomer, of which more than 300 amino acids constitute the "receptor binding region fragment” (RBD), that is, the S protein is linked to ACE2. place.
  • RBD receptor binding region fragment
  • the polyploid of the novel coronavirus S protein fragment can be the diploid, triploid, tetraploid, pentaploid, hexaploid, Hepploid, octaploid, 9ploid, decaploid, etc.
  • novel coronavirus S protein receptor binding region fragments are preferably connected in series by GG or GS sequences, more specifically, the novel coronavirus S protein receptor binding region fragments can be connected by GGS, GGGS, Sequences such as GSGS, SGGS, GGGGS, GSGGS, GGSGS, GGSGGS, GGGSGS, GGGGGS or GGGSGGS are concatenated.
  • the GGGS sequence can be connected in series.
  • its nucleotide sequence can be two S proteins.
  • the fragments are formed by concatenation of GGGS sequences, see SEQ ID NO: 1; when the polyploidy of the novel coronavirus S protein fragment is triploid, its nucleotide sequence can be three S protein fragments concatenated by GGGS sequences.
  • the polyploid of the new coronavirus S protein fragment is tetraploid
  • its nucleotide sequence can be four S protein fragments connected by GGGS sequence in series
  • in the new coronavirus S protein fragment polyploid is
  • its nucleotide sequence may be five S protein fragments connected in series by GGGS sequences, and other cases can be deduced by analogy, which will not be repeated.
  • RBD diploid, RBD triploid, RBD tetraploid and RBD pentploid were subjected to protein SDS-PAGE electrophoresis respectively. The results are shown in Figure 1. The four lanes represent RBD diploid and RBD triploid, respectively. , RBD tetraploid and RBD pentaploid SDS-PAGE electrophoresis results, it can be seen from Figure 1 that RBD diploid, RBD triploid, RBD tetraploid and RBD pentaploid can be stably expressed.
  • the polyploid of the novel coronavirus S protein fragment provided in this example is composed of at least two novel coronavirus S protein receptor binding domain (RBD) fragments, with high expression and good stability, and can be applied to novel coronaviruses In the antibody detection and vaccine preparation of coronavirus, it has strong applicability and a wide range of applications.
  • the application of the polyploid of the new coronavirus S protein fragment in the antibody detection of the new coronavirus can effectively improve the specificity and sensitivity of antibody detection. And sensitivity, and then effectively improve the accuracy of antibody detection, the new coronavirus S protein fragment polyploid is used in vaccine preparation, the vaccine can stimulate the body to quickly produce antibodies.
  • This embodiment provides a novel coronavirus S protein fragment polyploid, and the novel coronavirus S protein fragment polyploid is composed of at least two novel coronavirus S protein receptor binding region fragments.
  • the C-terminus of the novel coronavirus S protein receptor binding region fragment is linked to the Fc fragment of human immunoglobulin.
  • one or more of the novel coronavirus S protein receptor binding region fragments form a polyploid unit, and the polyploid unit forms two Fc fragments through which the novel coronavirus S protein receptor binding region fragments are connected.
  • the dimer was used as a polyploid of the novel coronavirus S protein fragment.
  • the polyploid unit can be either an RBD fragment (haploid) or an RBD polyploid.
  • haploid an RBD fragment
  • RBD polyploid an RBD polyploid
  • two new coronavirus diploids can form a new coronavirus tetraploid through the Fc fragment
  • two new coronavirus triploids can form a new coronavirus hexaploid through the Fc fragment
  • two new coronavirus tetraploids can form
  • the Fc fragment can form a new coronavirus octaploid
  • two new coronavirus pentaploids can form a new coronavirus decaploid through the Fc fragment, etc.
  • an Fc fragment is innovatively added to the C-terminus of the RBD fragment and RBD polyploid, so that between the RBD fragment and the RBD fragment, between the RBD fragment and the RBD polyploid, and between the RBD polyploid and the RBD polyploid It can be combined more flexibly to form a dimer with stronger activity and higher binding ability to ACE2, that is, the new novel coronavirus S protein fragment polyploid, which provides more information for the detection and prevention of novel coronavirus. choose.
  • Example 1 and Example 2 provide two polyploids of the novel coronavirus S protein fragment with different structures.
  • n-ploid As an example, it can be achieved by the combination of n RBDs described in Example 1.
  • the fragments are formed by concatenating similar sequences such as GGGS, and can also be obtained by forming dimers from two n/2 ploids with Fc fragments as described in Example 2, wherein the new coronavirus S protein fragment with Fc fragments is obtained.
  • Polyploid has a relatively higher protein expression, a relatively longer half-life in vivo, and a longer duration of drug effect, which is more conducive to later development.
  • This embodiment provides a novel coronavirus S protein fragment polyploid, and the novel coronavirus S protein fragment polyploid is composed of at least two novel coronavirus S protein receptor binding region fragments.
  • novel coronavirus S protein receptor binding region fragments or the novel coronavirus S protein fragment polyploids are connected by protease or protein cross-linking agent.
  • the protease includes, but is not limited to, a transpeptidase (Sortase) and a combined protein ligase (Oldenlandia affinis asparaginyl endopeptidases 1, OaAEP1)
  • the protein cross-linking agent includes but is not limited to polyethylene glycol, suberic acid two Disuccinimidyl Suberate, Glutarate Succinate Suberate, Dimethyl Diimide, Dimethyl Diimide, Dimethyl Phthalate and Hexyl Maleimide alkyl.
  • the RBD fragments can be linked by the above-mentioned enzymes or protein cross-linking agents to obtain RBD polyploids, or the above-mentioned enzymes or protein cross-linking agents can be used as described in Example 1 or 2.
  • the RBD polyploids described above were ligated to obtain a higher aggregated form of the RBD protein.
  • the method is: on the basis of the original RBD triploid sequence, insert GL at the N-terminus , and insert NGL at the C-terminus to form the expression plasmid of GL-RBD-RBD-RBD-NGL.
  • the above expression vector was transfected into mammalian cells, and after culturing for 5-7 days, the culture supernatant was collected, and the GL-RBD-RBD-RBD-NGL protein was purified by the method of affinity purification.
  • the purified protein was added to the combined protein ligase in an appropriate proportion, and the reaction was terminated after incubation for an appropriate time. Take an appropriate amount of protein, analyze the results of enzymatic linkage by SDS-PAGE, and finally separate the linked high-polymer fragments with molecular sieves, that is, to obtain a higher aggregated form of RBD protein formed by multiple RBD triploids.
  • the polyploid of the novel coronavirus S protein fragment provided in this example is composed of at least two novel coronavirus S protein receptor binding region (RBD) fragments, has good stability, and can be applied to the antibody of the novel coronavirus In detection and vaccine preparation, it has strong applicability and a wide range of applications.
  • the application of the polyploid of the new coronavirus S protein fragment to the antibody detection of the new coronavirus can effectively improve the specificity, sensitivity and sensitivity of antibody detection.
  • the polyploid of the new coronavirus S protein fragment is used in vaccine preparation, and the vaccine can stimulate the body to quickly produce antibodies.
  • the effect of the polyploidy of the novel coronavirus S protein fragment provided in Examples 1-3 is obtained based on its own polyploid structure, connecting fragments, etc., and neither is indispensable. If only RBD is made into a polyploid , instead of using the connecting fragments provided in the above examples, the effect will be greatly reduced. For example, if RBD polyploids are formed through disulfide bonds, their binding ability to ACE2 receptors and their ability to generate antibodies and inhibit viruses are far Not as RBD polyploids formed by tandem as provided in Examples 1-3.
  • the polyploid of the novel coronavirus S protein fragment provided in Examples 1-3 is a targeted RBD, and targeting RBD is a safer choice compared to the entire S protein, which can trigger less Production of neutralizing antibodies.
  • the new coronavirus S protein polyploid provided in Examples 1-3 is formed through a specific structure, a specific connecting fragment, and a specific connection method, which can greatly improve its receptor binding ability, improve stability, It is safe, increases the scope of application, and has good economic benefits. It is suitable for testing kits, vaccines and drugs with good effects.
  • this embodiment provides a detection kit, which is a kit capable of detecting the presence or absence of a novel coronavirus, and the detection kit includes any one of Embodiments 1-3 Said novel coronavirus S protein fragment polyploid.
  • the polyploid of the new coronavirus S protein fragment as described above can be used as a coating antigen in the detection kit, and the IgM+IgG joint inspection of the serum of the subject is carried out, that is, the whole cycle (early, middle and late) is used to monitor the new crown infection.
  • IgM and IgG are two different antibodies produced by the human body against pathogenic microorganisms (including viruses).
  • IgM is the first antibody that appears after infection. It peaks after 3-7 days of new coronavirus infection, and then gradually decreases.
  • IgM detection can indicate early infection.
  • IgG is a large amount of antibodies produced when B cells enter the lymph nodes to differentiate into plasma cells in the middle and late stages of infection. IgG detection indicates recovery or infection after infection.
  • polyploid of the new coronavirus S protein fragment described above can also be used in other products with the detection function of the new coronavirus, such as test strips and testing instruments.
  • the application principle is the same as that of the detection kit, and will not be repeated here. .
  • the detection kit provided in this embodiment can detect the presence or absence of the new coronavirus through the polyploidy of the new coronavirus S protein fragment, which can effectively improve the specificity, sensitivity and sensitivity of the detection of the new coronavirus antibody, thereby effectively improving the antibody detection. detection accuracy.
  • this embodiment provides a vaccine, which is a vaccine capable of preventing novel coronavirus infection, and the vaccine includes the above-mentioned novel coronavirus S protein fragment polyploid.
  • the vaccine can be an mRNA vaccine, a protein vaccine or an adenovirus vaccine.
  • the polyploid of the new coronavirus described in any one of Examples 1-3 is used for the vaccine to prevent the new coronavirus, it can express the protein antigen through the transcription system of the host cell after entering the body cell, and induce the host cell to produce cells and body fluids. immune response to achieve the effect of preventing the new coronavirus.
  • BCR B cell receptor
  • the polyploid of the new coronavirus S protein fragment can stimulate the body to rapidly produce antibodies, so that the body can produce immunity, and the vaccine has good stability and easy quality monitoring.
  • this embodiment provides a medicine, which is a medicine capable of preventing or treating the novel coronavirus, and the medicine comprises the novel coronavirus S as described in any one of Embodiments 1-3 Protein fragment polyploidy.
  • the medicines provided in this embodiment may be medicines in various dosage forms such as tablets, capsules, and pills, and may also contain other components that can prevent or treat the novel coronavirus. There is no restriction on this.
  • the laser confocal results showed that the ACE2 receptor protein exhibited a highly aggregated form on the cell membrane surface, so the highly aggregated form of the ACE2 receptor protein was more likely to bind to the highly aggregated form of RBD fragments, namely RBD polyploids, The binding ability of RBD polyploid to ACE2 receptor protein is stronger.
  • ACE2 is the receptor protein of the new coronavirus
  • the strong binding force of the RBD polyploid to the ACE2 receptor protein also means that it can effectively inhibit the binding of the new coronavirus and the ACE2 receptor protein, thereby inhibiting the invasion of the new coronavirus to the human body.
  • the polyploid of the new coronavirus S protein fragment has a strong binding ability to the ACE2 receptor protein, which can effectively inhibit the binding of the new coronavirus and the ACE2 receptor protein to treat the new coronavirus, and the drug is stable Well, the quality is easy to monitor.
  • This embodiment provides a method for preparing a polyploid of a novel coronavirus S protein fragment, including steps S1 to S4.
  • a DNA fragment can be synthesized based on the nucleotide sequence of each polyploid of the S protein fragment of the novel coronavirus, and a histone tag is added to the C-terminus of the DNA fragment, and cloned into an expression vector, Obtain the target expression vector.
  • feed can be added 24 hours after mammalian cells are transfected, and the transfected cells can be cultured on a shaker with serum-free medium at 37°C, 5%-8% CO 2 , and 100-150 rpm for 5 For 7 days, cell numbers and viability were recorded daily until cell viability was below 50%.
  • Serum-free medium is a synthetic medium that can maintain the growth and reproduction of cells in vitro for a long time without adding serum.
  • Serum-free medium consists of basal medium and supplementary components.
  • Gibco-Expi293 expression medium Expi293TM Expression Medium, GibcoTM
  • FreeStyteTM 293 expression medium FreeStyleTM 293Expression Medium, GibcoTM
  • OPM-293CD05 medium etc.
  • the cells are cultured, depending on the specific situation, which is not limited in this application.
  • the use of serum-free medium to culture the cells can effectively avoid the quality change between serum batches, improve the cell culture effect, avoid the toxic effect of serum on cells and serum-derived contamination, and avoid serogroups.
  • the effect of fractionation on the results is beneficial to the differentiation of cells cultured in vitro, and also helps to increase the expression level of protein fragments and facilitate the purification of cell products.
  • the cell culture supernatant can be collected, centrifuged, the first centrifugation supernatant is collected, the first centrifugation supernatant is centrifuged again, the second centrifugation supernatant is collected, and filtered , collect the filtrate; add the filtrate to a balanced nickel column (Ni-NTA), add low-concentration imidazole to remove impurity proteins, add high-concentration imidazole to elute, and collect the eluate to obtain the high-efficiency as described in Example 1.
  • Ni-NTA balanced nickel column
  • the expressed polyploid of the novel coronavirus S protein fragment The expressed polyploid of the novel coronavirus S protein fragment.
  • the rotational speed and time of the centrifugal treatment, the filter membrane selected for the filtration treatment, the concentration value of imidazole, etc. can all be determined according to the actual situation, which is not limited in this application.
  • the rotation speed of the first centrifugation is preferably 2000rpm, and the time is preferably 10min.
  • the rotation speed of the second centrifugation is preferably 15000rpm, and the time is preferably 60min.
  • the concentration is preferably 10 mM, and the concentration of high-concentration imidazole is preferably 250 mM.
  • the cell culture supernatant can be collected, centrifuged, the first centrifugation supernatant is collected, the first centrifugation supernatant is centrifuged again, the second centrifugation supernatant is collected, and filtered , collect the filtrate; add the filtrate to the balanced Protein A column, add phosphate buffer saline (PBS) to remove impurities, add Glycine-HCl solution to elute, and immediately add Tris- The HCl solution is used to neutralize the eluate, and the neutralized eluate is collected to obtain the novel coronavirus S protein fragment polyploid as described in Example 2.
  • PBS phosphate buffer saline
  • the rotation speed and time of the centrifugal treatment, the filter membrane selected for the filtration treatment, the concentration of the Glycine-HCl solution and the Tris-HCl solution, the pH, etc. can all be determined according to the actual situation, which is not limited in this application.
  • the rotation speed of the first centrifugation is preferably 2000rpm, and the time is preferably 10min.
  • the rotation speed of the second centrifugation is preferably 15000rpm, and the time is preferably 60min.
  • the concentration of Tris-HCl is preferably 0.1M, the pH value is preferably 2.8, the concentration of Tris-HCl solution is preferably 1M, and the pH value is preferably 8.5.
  • the method for preparing the polyploid of the S protein fragment of the novel coronavirus provided in this example is obtained by obtaining the nucleotide sequence of the polyploid, inserting the nucleotide sequence into an expression vector, and transfecting mammalian cells with the target expression vector. . Purifying the culture supernatant can effectively increase the expression of the polyploid of the new coronavirus S protein fragment, and effectively improve the purity of the polyploid of the new coronavirus S protein fragment, which is the most important part of the polyploid of the new coronavirus S protein fragment. The application of antibody detection, vaccine preparation, etc. provides assistance.
  • test groups 1-10 are set.
  • test groups 1-5 are RBD fragments without Fc fragment (haploid), diploid, triploid, tetraploid and pentaploid respectively
  • test groups 6-10 are respectively with Fc fragment The RBD fragments (haploid), diploid, triploid, tetraploid and pentaploid.
  • RBD fragments and RBD polyploids were prepared by the following methods:
  • test groups 2-10 were subjected to protein SDS-PAGE electrophoresis, and the results are shown in Figure 1 and Figure 2 .
  • Fig. 1 shows the SDS-PAGE results of the RBD polyploid expression products without Fc fragments in test groups 2-5
  • Fig. 2 shows the RBD fragments and polyploid expression products with Fc fragments in test groups 6-10 SDS-PAGE results.
  • various polyploids of RBD can be stably expressed in mammalian cells.
  • FIG. 4 is a line comparison diagram of the interaction between RBD fragments without Fc fragment, RBD diploid, RBD triploid, RBD tetraploid, and RBD pentaploid and ACE-2 expressing cells in test groups 1-5.
  • FIG. 5 is a line comparison chart of the interaction between RBD fragments with Fc fragments, RBD diploids, RBD triploids, RBD tetraploids, RBD pentaploids and ACE-2 expressing cells in test groups 6-10, in which the horizontal axis represents the log value (M) of the molar concentration of RBD fragments and RBD polyploids, and the vertical axis represents the mean fluorescence intensity (MFI) of Alexa488 in mCherry-positive cells, that is, the number of RBD fragments or RBD polyploids bound to ACE2.
  • MFI mean fluorescence intensity
  • the RBD polyploid provided by this application that is, the new coronavirus S protein fragment polyploid not only has stable expression, but more importantly, its binding ability with the new coronavirus protein receptor ACE2 has further improved. This means that its ability to detect, prevent, and treat new coronaviruses has also been further improved.
  • the RBD-S group, the RBD group, the 3RBD group and the 4RBD-Fc group were set, and 6 female Balb/C mice were selected in each group.
  • mice in RBD-S group were intramuscularly injected with 10ug of RBD-S protein and 100ug of aluminum hydroxide adjuvant
  • mice in RBD group were intramuscularly injected with 10ug of RBD fragments and 100ug of aluminum hydroxide adjuvant.
  • mice were intramuscularly injected with 10ug of RBD triploid and 100ug of aluminum hydroxide adjuvant, and the mice in the 4RBD-Fc group were intramuscularly injected with 10ug of RBD tetraploid with Fc fragment and 100ug of aluminum hydroxide adjuvant. Serum was collected on the 28th day, and the RBD-specific antibody content of each group of mice was detected, that is, the endpoint titer.
  • polyploid of the new coronavirus S protein fragment provided in this application has high expression level, good stability, and has a relatively longer half-life in vivo and a longer duration of drug effect, which is more conducive to later development and application. It has strong applicability and wide application range in antibody detection and vaccine preparation of new coronavirus.
  • This embodiment provides a novel coronavirus S protein fragment polyploid, and the novel coronavirus S protein fragment polyploid is composed of at least two novel coronavirus S protein receptor binding region fragments.
  • the connection between the fragments can be found in Examples 1-3.
  • novel coronavirus S protein receptor binding region fragment is selected from (RBD) fragment itself and/or (RBD) fragment mutant.
  • the polyploid of the new coronavirus S protein fragment can be composed of only multiple (RBD) fragment ontology, only multiple (RBD) fragment mutants, or can be composed of one or more (RBD) fragments
  • the fragment ontology is constructed with one or more (RBD) fragment polyploids.
  • the types of (RBD) segment mutants may be the same or different.
  • composition of the triploid of the new coronavirus S protein fragment can be: fragment ontology-segment ontology-segment ontology, segment ontology-segment ontology-segment A mutation, segment ontology-segment A mutation-segment A mutation, segment ontology-segment A mutation-fragment B mutation, fragment body-fragment A mutation-fragment A+B mutation, fragment A mutation-fragment B mutation-fragment C mutation, fragment A+B+C mutation-fragment B+C mutation-fragment C mutation, Fragment A+B+C mutation-fragment A+B+C mutation-fragment A+B+C mutation-fragment A+B+C mutation, etc.
  • Other methods can be deduced in the same way, and will not be repeated here.
  • mutation sites of the (RBD) fragment mutants include: one or more of N501Y, K417N, K417T, E484K, L452R, T478K, L452Q, F490S, E484Q and T478Q.
  • the (RBD) fragment mutant provided by the present application can be obtained by mutation of any one of the above mutation sites, or by simultaneous mutation of any two or more mutation sites.
  • the (RBD) fragment mutants include: B.1.1.7 (Alpha) mutants obtained from N501Y mutation, K417N mutant mutants, and E484K mutant B1.525 (Eta) mutants , N501Y, K417N and E484K mutant B.1.351 (Beta) mutant, N501Y, K417T and E484K mutant P.1 (Gamma) mutant, L452R and T478K mutant B.1.617.2 ( Delta) mutant, L452Q and F490S mutant C.37 (Lambda) mutant, E484Q mutant mutant, L452R and E484Q mutant B.1.617.1 (Kappa) mutant, L452R mutant The B.1.427 (Epsilon) mutant strain was formed, and the B.1.1.222 mutant strain formed by the T478Q mutation.
  • the RBD fragments (WT) were labeled with Alexa488, and stably bound to the cells expressing the new coronavirus spike protein receptor ACE2, which could be detected by FACs.
  • Different concentrations of N501Y mutated RBD fragments, K417N mutated RBD fragments, E484K mutated RBD fragments, N501Y-K417N-E484K mutated RBD fragments (triple) and other RBD mutant fragments and Alexa488-labeled RBD fragments (WT) and ACE2 were used The co-incubation of expressing cells was performed using FACs to detect the affinity of each RBD mutant fragment and the ACE2 receptor. The results are shown in Figure 7.
  • the results show that the RBD fragment mutant can be expressed stably like the RBD body, and has the ability to bind to the new coronavirus protein receptor ACE2.
  • Figures 9a-9c are line graphs showing the receptor binding ability of RBD fragment, N501Y mutated RBD fragment, and N501Y-K417N-E484K mutated RBD fragment, respectively
  • Figure 9d is RBD fragment, N501Y mutated RBD fragment, N501Y-K417N-E484K A graph comparing the receptor binding ability of mutant RBD fragments.
  • Kd represents the dissociation constant (dissociation constant), which reflects the affinity to the target, and the smaller the value, the stronger the affinity
  • Kon represents the association rate constant (association rate constant), which represents the speed of intermolecular binding
  • Koff represents the solution
  • the dissociation rate constant represents the speed of intermolecular dissociation.
  • the RBD fragment of N501Y mutation and the RBD fragment of N501Y-K417N-E484K mutation have strong receptor binding ability.
  • Figures 10a-10b are line graphs showing the receptor binding ability of the K417N mutated RBD fragment and the E484K mutated RBD fragment, respectively, and Figure 10c is a comparison chart of the receptor binding ability of the K417N mutated RBD fragment and the E484K mutated RBD fragment.
  • the polyploid composed of the RBD mutant provided by the application that is, the polyploid of the new coronavirus S protein fragment not only has stable expression, but also has strong receptor binding ability, and can be flexibly applied to the new coronavirus and various novel coronaviruses. Detection and treatment of coronavirus mutants (such as Alpha mutants, Eta mutants, Beta mutants, Gamma, Delta mutants, Lambda mutants, Kappa mutants, Epsilon mutants, etc.).
  • coronavirus mutants such as Alpha mutants, Eta mutants, Beta mutants, Gamma, Delta mutants, Lambda mutants, Kappa mutants, Epsilon mutants, etc.

Abstract

提供一种新型冠状病毒S蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物，其中，新型冠状病毒S蛋白片段多倍体由至少两个新型冠状病毒S蛋白受体结合区域片段串联构成。提供的新型冠状病毒S蛋白片段多倍体，与ACE2结合能力强，稳定性好，可以应用于新型冠状病毒的抗体检测和疫苗制备中，适用性强，适用范围广，其中，将新型冠状病毒S蛋白片段多倍体应用于新型冠状病毒的抗体检测中，可以有效提高抗体检测的特异性、灵敏度和敏感性，进而有效提高抗体检测的准确率。将新型冠状病毒S蛋白片段多倍体应用于疫苗制备中，疫苗可以刺激机体快速产生抗体，以使机体产生免疫作用。

Description

新型冠状病毒S蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物 技术领域
本申请涉及生物医学技术领域，特别涉及一种新型冠状病毒S蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物。
背景技术
新型冠状病毒是以前从未在人体中发现的冠状病毒新毒株，新型冠状病毒S蛋白(SARS-CoV-2spike蛋白)是Ⅰ型跨膜糖蛋白，也是病毒最大的结构蛋白，由1255个氨基酸残基组成，能够刺激机体产生中和抗体和介导免疫反应。
现有研究证明，S蛋白与病毒侵入细胞的过程密切相关。S蛋白由两个结构域组成，靠近N端的部分形成球形结构域，靠近C端的部分形成穿膜的棒状结构域。S蛋白前体在宿主细胞质中合成后会被切成S1(球形部分)和S2(棒状部分)，其中，S1部分参与受体的识别和结合，其包含受体结合区域(Receptor Binding Domain，RBD)，S2部分参与病毒入侵宿主细胞。
由于S蛋白的RBD区域可以与受体结合，重组S蛋白可以被用于冠状病毒感染的抗原检测和疫苗制备，因此，如何高效利用S蛋白是诊治新型冠状病毒的一个重要基础。
但是，将S蛋白的编码序列克隆入一些表达载体后，其表达量很低，难以有效表达，此外，新型冠状病毒来势汹汹，至今未找到高效、高准确率的新型冠状病毒抗体检测方法和预防方法，因此如何利用新型冠状病毒S蛋白准确高效地检测、预防新型冠状病毒成为了亟待解决的问题。
发明内容
有鉴于此，本申请提供了一种新型冠状病毒S蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物，以解决现有技术中存在的技术缺陷。
本申请提供一种新型冠状病毒S蛋白片段多倍体，其特征在于，所述新型冠状病毒S蛋白片段多倍体由至少两个新型冠状病毒S蛋白受体结合区域片段串联构成。
进一步地，所述新型冠状病毒S蛋白受体结合区域片段之间通过GG或GS序列串联。
进一步地，所述新型冠状病毒S蛋白受体结合区域片段之间通过GGS、GGGS、GSGS、SGGS、GGGGS、GSGGS、GGSGS、GGSGGS、GGGSGS、GGGGGS或GGGSGGS序列串联。
进一步地，所述新型冠状病毒S蛋白受体结合区域片段的C端与人免疫球蛋白的Fc片段连接。
进一步地，一个或多个所述新型冠状病毒S蛋白受体结合区域片段构成多倍体单元，所述多倍体单元通过所述新型冠状病毒S蛋白受体结合区域片段连接的Fc片段形成二聚体，并将所述二聚体作为新型冠状病毒S蛋白片段多倍体。
进一步地，所述新型冠状病毒S蛋白受体结合区域片段之间或所述新型冠状病毒S蛋白片段多倍体之间通过蛋白酶或蛋白交联剂相连。
进一步地，所述酶包括转肽酶和联用蛋白连接酶，所述蛋白交联剂包括聚乙二醇、辛二酸二琥珀酰亚胺酯、琥珀酸辛二酸戊二酸盐、二甲基二亚胺酯、二甲基亚胺酯、二甲基苯二酸亚胺酯和马来酰亚胺己烷。
进一步地，所述新型冠状病毒S蛋白受体结合区域片段选自(RBD)片段本体和/或(RBD)片段突变体。
需要说明的是，新冠病毒S蛋白片段多倍体既可以仅由多个(RBD)片段本体构成，也可以仅由多个(RBD)片段突变体构成，或者可以由一个或多个(RBD)片段本体与一个或多个(RBD)片段多倍体一同构成。
在新冠病毒S蛋白片段多倍体中包含多个(RBD)片段突变体的情况下，(RBD)片段突变体的种类既可以相同，也可以不同。
比如，新冠病毒S蛋白片段三倍体的构成方式可以为：片段本体-片段本体-片段本体、片段本体-片段本体-片段A突变、片段本体-片段A突变-片段A突变、片段本体-片段A突变-片段B突变、片段本体-片段A突变-片段A+B突变、片段A突变-片段B突变-片段C突变、片段A+B+C突变-片段B+C突变-片段C突变、片段A+B+C突变-片段A+B+C突变-片段A+B+C突变等。其他方式均可以此类推，不再赘述。
进一步地，所述(RBD)片段突变体的突变位点包括：N501Y、K417N、K417T、E484K、L452R、T478K、L452Q、F490S、E484Q、T478Q中的一种或几种。
换而言之，本申请提供的(RBD)片段突变体，可以由以上任意一种突变位点的突变得到，也可以由任意两种或多种突变位点同时突变得到。
优选地，所述(RBD)片段突变体包括：N501Y突变而成的B.1.1.7(Alpha)突变株，K417N突变而成的突变株，E484K突变而成的B1.525(Eta)突变株，N501Y、K417N及E484K突变而成的B.1.351(Beta)突变株，N501Y、K417T及E484K突变而成的P.1(Gamma)突变株，L452R及T478K突变而成的B.1.617.2(Delta)突变株，L452Q及F490S突变而成的C.37(Lambda)突 变株，E484Q突变而成的突变株，L452R及E484Q突变而成的B.1.617.1(Kappa)突变株，L452R突变而成的B.1.427(Epsilon)突变株，T478Q突变而成的B.1.1.222突变株。
本申请还提供一种检测试剂盒，所述检测试剂盒为能够检测新型冠状病毒存在与否的试剂盒，所述检测试剂盒包括如上所述的新型冠状病毒S蛋白片段多倍体。
本申请还提供一种疫苗，所述疫苗为能够预防新型冠状病毒传染的疫苗，所述疫苗包括如上所述的新型冠状病毒S蛋白片段多倍体。
进一步地，所述疫苗为mRNA疫苗、蛋白疫苗或腺病毒疫苗。
进一步地，所述疫苗中的新型冠状病毒S蛋白片段多倍体激活聚集的B细胞受体，产生新型冠状病毒抗体。
本申请还提供一种药物，所述药物为能够预防或治疗新型冠状病毒的药物，所述药物包括如上所述的新型冠状病毒S蛋白片段多倍体。
进一步地，所述药物中的新型冠状病毒S蛋白片段多倍体与ACE2受体结合，抑制新型冠状病毒的侵袭。
本申请提供一种新型冠状病毒S蛋白片段多倍体的制备方法，包括：
S1、获取新型冠状病毒S蛋白片段多倍体的核苷酸序列；
S2、将所述新型冠状病毒S蛋白片段多倍体的核苷酸序列插入至真核表达载体中，获得目标表达载体；
S3、采用所述目标表达载体转染哺乳动物细胞，在预设培养条件下采用无血清培养基对转染后的细胞摇床培养5-7天，直至细胞存活率降至50％；
S4、收集哺乳动物细胞培养的上清液，使用亲和层析的方法对上清液进行分离纯化处理，获得如上所述的新型冠状病毒S蛋白片段多倍体。
本申请提供的新型冠状病毒S蛋白片段多倍体，由至少两个新型冠状病毒S蛋白受体结合区域(RBD)片段串联构成，与ACE2结合能力强，表达量高，稳定性好，可以应用于新型冠状病毒的抗体检测和疫苗制备中，适用性强，适用范围广，其中，将新型冠状病毒S蛋白片段多倍体应用于新型冠状病毒的抗体检测中，可以有效提高抗体检测的特异性、灵敏度和敏感性，进而有效提高抗体检测的准确率，将新型冠状病毒S蛋白片段多倍体应用于疫苗制备中，疫苗可以刺激机体快速产生抗体，以使机体产生免疫作用，并且疫苗稳定性好，质量易于监控。
此外，本申请提供的新型冠状病毒S蛋白片段多倍体还可以应用于新冠病毒多种变异毒株的检测与治疗，检测准确率高，治疗效果好，应用灵活度高，适于大规模推广和使用。
本申请提供的新型冠状病毒S蛋白片段多倍体的制备方法，其通过获取多倍体的核苷酸序列、将核苷酸序列插入至表达载体中、采用目标表达载体转染哺乳动物细胞、对培养上清液进行纯化，可以有效提高新型冠状病毒S蛋白片段多倍体的表达量，有效提高新型冠状病毒S蛋白片段多倍体的纯度，为新型冠状病毒S蛋白片段多倍体在抗体检测、疫苗制备等方面的应用提供助力。
附图说明
图1是本申请一实施例所述的新型冠状病毒S蛋白片段多倍体的电泳结果图；
图2是本申请一实施例所述的带有Fc片段的新型冠状病毒S蛋白片段及其多倍体的电泳结果图；
图3是本申请一实施例所述的新型冠状病毒S蛋白受体血管紧张素转换酶2(ACE2)稳定表达细胞株的激光共聚焦图；
图4是本申请一实施例所述的采用流式分析的不带有Fc片段的新型冠状病毒S蛋白片段及其多倍体与ACE2表达细胞的结合能力图；
图5是本申请一实施例所述的采用流式分析的带有Fc片段的新型冠状病毒S蛋白片段及其多倍体与ACE2表达细胞的结合能力图；
图6是本申请一实施例所述的新型冠状病毒S蛋白片段多倍体与商品化的RBD-S蛋白及RBD单体蛋白的终点效价对比图；
图7-图10是本申请一实施例所述的RBD片段突变体与新型冠状病毒刺突蛋白受体ACE2表达细胞的结合能力对比图。
具体实施方式
下面结合附图对本申请的具体实施方式进行描述。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的试剂、材料和操作步骤均为相应领域内广泛使用的试剂、材料和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，血管紧张素转换酶2(ACE2)是一种可将血管紧张素II有效降解为血管紧张素1-7的羧肽酶，其已被确定为SARS-CoV的功能受体和SARA-CoV-2的潜在受体。
免疫球蛋白G(IgG)是血清中免疫球蛋白的主成分，约占血清中免疫球蛋白总含量的75％。IgG有4个亚型，即IgG1、IgG2、IgG3、IgG4。IgG是体内最主要的抗体，具有抗病毒、中和病毒、抗菌及免疫调节的功能。
实施例1
本实施例提供一种新型冠状病毒S蛋白片段多倍体，该新型冠状病毒S蛋白片段多倍体由至少两个新型冠状病毒S蛋白受体结合区域片段串联构成。
新型冠状病毒S蛋白以三聚体形态存在，每一个单体中约有1300多个氨基酸，其中300多个氨基酸构成了“受体结合区域片段”(RBD)，即S蛋白与ACE2相联结的地方。
在本实施例中，新型冠状病毒S蛋白片段多倍体可以为新型冠状病毒S蛋白受体结合区域片段即RBD的二倍体、三倍体、四倍体、五倍体、六倍体、七倍体、八倍体、九倍体、十倍体等。
具体地，所述新型冠状病毒S蛋白受体结合区域片段之间优选通过GG或GS序列串联，更为具体地，所述新型冠状病毒S蛋白受体结合区域片段之间可以通过GGS、GGGS、GSGS、SGGS、GGGGS、GSGGS、GGSGS、GGSGGS、GGGSGS、GGGGGS或GGGSGGS等序列串联。
以新型冠状病毒S蛋白受体结合区域片段之间可以通过GGGS序列串联为例，在新型冠状病毒S蛋白片段多倍体为二倍体的情况下，其核苷酸序列可以为两个S蛋白片段通过GGGS序列串联而成，参见SEQ ID NO：1；在新型冠状病毒S蛋白片段多倍体为三倍体的情况下，其核苷酸序列可以为三个S蛋白片段通过GGGS序列串联而成；在新型冠状病毒S蛋白片段多倍体为四倍体的情况下，其核苷酸序列可以为四个S蛋白片段通过GGGS序列串联而成，在新型冠状病毒S蛋白片段多倍体为五倍体的情况下，其核苷酸序列可以为五个S蛋白片段通过GGGS序列串联而成，其他情况可以此类推，不再赘述。
分别对RBD二倍体、RBD三倍体、RBD四倍体和RBD五倍体进行蛋白质SDS-PAGE电泳处理，结果如图1所示，四个泳道分别表示RBD二倍体、RBD三倍体、RBD四倍体和RBD五倍体的SDS-PAGE电泳结果，从图1中可以看出，RBD二倍体、RBD三倍体、RBD四倍体和RBD五倍体均能够稳定表达。
由此可见，本实施例提供的新型冠状病毒S蛋白片段多倍体，由至少两个新型冠状病毒S蛋白受体结合区域(RBD)片段构成，表达量高，稳定性好，可以应用于新型冠状病毒的抗体检测和疫苗制备中，适用性强，适用范围广，其中，将新型冠状病毒S蛋白片段多倍体应用于新型冠状病毒的抗体检测中，可以有效提高抗体检测的特异性、灵敏度和敏感性，进而有效提高抗体检测的准确率，将新型冠状病毒S蛋白片段多倍体应用于疫苗制备中，疫苗可以刺激机体快速产生抗体。
实施例2
本实施例提供一种新型冠状病毒S蛋白片段多倍体，该新型冠状病毒S蛋白片段多倍体由至少两个新型冠状病毒S蛋白受体结合区域片段构成。
在本实施例中，新型冠状病毒S蛋白受体结合区域片段的C端与人免疫球蛋白的Fc片段连接。
其中，一个或多个所述新型冠状病毒S蛋白受体结合区域片段构成多倍体单元，所述多倍体单元通过其中所述新型冠状病毒S蛋白受体结合区域片段连接的Fc片段形成二聚体，并将所述二聚体作为新型冠状病毒S蛋白片段多倍体。
具体地，该多倍体单元既可以为RBD片段(单倍体)，也可以为RBD多倍体。比如，两个新型冠状病毒二倍体通过Fc片段能够形成新型冠状病毒四倍体；两个新型冠状病毒三倍体通过Fc片段能够形成新型冠状病毒六倍体；两个新型冠状病毒四倍体通过Fc片段能够形成新型冠状病毒八倍体；两个新型冠状病毒五倍体通过Fc片段能够形成新型冠状病毒十倍体等。
在本实施例中，创新的为RBD片段、RBD多倍体的C端添加Fc片段，使RBD片段与RBD片段之间、RBD片段与RBD多倍体之间、RBD多倍体与RBD多倍体之间能够更加灵活的组合，以形成活性更强、与ACE2结合能力更高的二聚体，即新的新型冠状病毒S蛋白片段多倍体，为新型冠状病毒的检测与预防提供更多选择。
需要说明的是，实施例1与实施例2提供了两种结构不同的新型冠状病毒S蛋白片段多倍体，以n倍体为例，其既可以通过实施例1所述的由n个RBD片段通过GGGS等类似序列串联起来形成，也可以通过实施例2所述的由2个带有Fc片段的n/2倍体形成二聚体得到，其中带有Fc片段的新型冠状病毒S蛋白片段多倍体，其蛋白表达量相对更高，且在体内的半衰期相对更长，药效持续时间更久，更加有利于后期的开发。
实施例3
本实施例提供一种新型冠状病毒S蛋白片段多倍体，该新型冠状病毒S蛋白片段多倍体由至少两个新型冠状病毒S蛋白受体结合区域片段构成。
在本实施例中，新型冠状病毒S蛋白受体结合区域片段之间或新型冠状病毒S蛋白片段多倍体之间通过蛋白酶或蛋白交联剂相连。
其中，所述蛋白酶包括但不限于转肽酶(Sortase)和联用蛋白连接酶(Oldenlandia affinis asparaginyl endopeptidases 1，OaAEP1)，所述蛋白交联剂包括但不限于聚乙二醇、辛二酸二琥珀酰亚胺酯(Disuccinimidyl Suberate)、琥珀酸辛二酸戊二酸盐、二甲基二亚胺酯、二甲基亚胺酯、二甲基苯二酸亚胺酯和马来酰亚胺己烷。
需要说明的是，在本实施例中，既可以通过上述酶或蛋白交联剂将RBD片段进行连接获得RBD多倍体，也可以通过上述酶或蛋白交联剂将如实施例1或2所述的RBD多倍体进行连接获得更高聚集形式的RBD蛋白。
以通过使用蛋白连接酶(OaAEP1)将RBD三倍体片段连接成为多个RBD三倍体片段的串联为例，其方法为：在原有的RBD三倍体序列的基础上，在N端插入GL，在C端插入NGL，构成GL-RBD-RBD-RBD-NGL的表达质粒。将上述表达载体转染哺乳动物细胞，培养5-7天后，收集培养上清，采用亲和纯化的方法纯化GL-RBD-RBD-RBD-NGL蛋白。将纯化后的蛋白以适当比例加入联用蛋白连接酶，孵育适当时间后，终止反应。取适量蛋白，SDS-PAGE分析酶链接的结果，最后用分子筛分离链接好的高聚片段，即得由多个RBD三倍体形成的更高聚集形式的RBD蛋白。
由此可见，本实施例提供的新型冠状病毒S蛋白片段多倍体，由至少两个新型冠状病毒S蛋白受体结合区域(RBD)片段构成，稳定性好，可以应用于新型冠状病毒的抗体检测和疫苗制备中，适用性强，适用范围广，其中，将新型冠状病毒S蛋白片段多倍体应用于新型冠状病毒的抗体检测中，可以有效提高抗体检测的特异性、灵敏度和敏感性，进而有效提高抗体检测的准确率，将新型冠状病毒S蛋白片段多倍体应用于疫苗制备中，疫苗可以刺激机体快速产生抗体。
对于上述实施例1-3提供的新型冠状病毒S蛋白多倍体，有以下三点需要说明：
第一，并不是所有蛋白的多倍体均能够具有强于其单倍体的效果，在实施例1-3中，由于与RBD片段及其多倍体结合的ACE2受体蛋白以及B细胞受体在细胞膜表面均是呈现高度聚集形式，因此高度聚集形式的ACE2受体蛋白、B细胞受体才会更易与高度聚集形式的RBD片段即RBD多倍体结合，这是通过一系列的机理研究及试验得到的。相反，如果受体不是以聚集形式存在的，相对应的蛋白单倍体和多倍体对于受体的结合能力几乎没有差异。并且，蛋白截取片段长度及其所加连接片段存在差异，也会对多倍体的结合能力产生影响，进而导致多倍体与受体结合能力不如单倍体。所以，不论是单倍体还是多倍体，其受体结合能力、使用效果等均是由其自身的特性而决定的，而并不是所有多倍体的效果均强于单倍体。
第二，实施例1-3提供的新型冠状病毒S蛋白片段多倍体的效果是基于其自身多倍体结构、连接片段等共同得到的，缺一不可，若仅仅将RBD制成多倍体，而不采用上述实施例提供的连接片段连接，则效果会大打折扣，比如若通过二硫键形成RBD多倍体，其与ACE2受体的结合能力以及其产生抗体、抑制病毒的能力远远不如实施例1-3提供的通过串联形成的RBD多倍体。
第三，实施例1-3提供的新型冠状病毒S蛋白片段多倍体是一种靶向RBD，靶向RBD相对于整个S蛋白而言是一种更安全的选择，能触发较少的非中和抗体的产生。
综上所述，实施例1-3提供的新型冠状病毒S蛋白多倍体，通过特定的结构、特定的连接片段、特定的连接方式形成，能够大幅提高其受体结合能力，提高稳定性、安全性，增大适用范围，经济效益好，适用于检测试剂盒、疫苗及药物中，效果佳。
实施例4
在实施例1-3的基础上，本实施例提供一种检测试剂盒，该检测试剂盒为能够检测新型冠状病毒存在与否的试剂盒，该检测试剂盒包括实施例1-3任意一项所述的新型冠状病毒S蛋白片段多倍体。
如上所述的新型冠状病毒S蛋白片段多倍体可以作为包被抗原用于检测试剂盒中，对受试者血清进行IgM+IgG联检，即全周期(早、中晚)监控新冠感染。
其中，IgM和IgG是人体对病原微生物(含病毒)感染产生的两种不同抗体。IgM是感染后最先出现的抗体，新型冠状病毒感染3-7天后达到峰值，后续逐步减少，IgM检测可提示早期感染。IgG是感染中后期时，B细胞进入淋巴结分化成浆细胞，大量产生的抗体，IgG检测提示感染后康复或感染中。
此外，如上所述的新型冠状病毒S蛋白片段多倍体还可以用于检测试纸、检测仪器等其他具有新型冠状病毒检测功能的产品中，其应用原理与检测试剂盒相同，在此不再赘述。
本实施例提供的检测试剂盒，其通过新型冠状病毒S蛋白片段多倍体检测新型冠状病毒的存在与否，可以有效提高新型冠状病毒抗体检测的特异性、灵敏度和敏感性，进而有效提高抗体检测的准确率。
实施例5
在实施例1-3的基础上，本实施例提供一种疫苗，该疫苗为能够预防新型冠状病毒传染的疫苗，该疫苗包括如上所述的新型冠状病毒S蛋白片段多倍体。
具体地，所述疫苗可以为mRNA疫苗、蛋白疫苗或腺病毒疫苗。将实施例1-3任意一项所述的新冠病毒多倍体用于预防新型冠状病毒的疫苗时，其进入机体细胞后可以通过宿主细胞的转录系统表达蛋白抗原，诱导宿主细胞产生细胞和体液免疫应答，以达到预防新型冠状病毒的效果。
具体地，由于B细胞受体(BCR)在B细胞表面呈现聚集形式，故相较于单个RBD蛋白，多聚形式的RBD蛋白即RBD多倍体更容易激活BCR，从而更易产生抗新冠病毒的抗体。
本实施例提供的疫苗，其中的新型冠状病毒S蛋白片段多倍体可以刺激机体快速产生抗体，以使机体产生免疫作用，并且疫苗稳定性好，质量易于监控。
实施例6
在实施例1-3的基础上，本实施例提供一种药物，该药物为能够预防或治疗新型冠状病毒的药物，该药物包括如实施例1-3任意一项所述的新型冠状病毒S蛋白片段多倍体。
本实施例提供的药物可以为片剂、胶囊剂、丸剂等各种剂型的药物，其还可以含有其他能够起到预防或治疗新型冠状病毒作用的组分，可视具体情况而定，本申请对此不做限制。
具体地，如图3所示，激光共聚焦结果显示ACE2受体蛋白在细胞膜表面呈现高度聚集形式，因此高度聚集形式的ACE2受体蛋白更易与高度聚集形式的RBD片段即RBD多倍体结合，RBD多倍体与ACE2受体蛋白的结合能力更强。
由于ACE2是新冠病毒的受体蛋白，故RBD多倍体与ACE2受体蛋白的结合力强也意味着其可以有效抑制新冠病毒与ACE2受体蛋白的结合，进而抑制新冠病毒对人体的侵袭。
本实施例提供的药物，其中的新型冠状病毒S蛋白片段多倍体与ACE2受体蛋白结合能力强，可以有效抑制新冠病毒与ACE2受体蛋白的结合，以治疗新型冠状病毒，并且药物稳定性好，质量易于监控。
实施例7
本实施例提供一种新型冠状病毒S蛋白片段多倍体的制备方法，包括步骤S1至步骤S4。
S1、获取新型冠状病毒S蛋白片段多倍体的核苷酸序列。
S2、将所述新型冠状病毒S蛋白片段多倍体的核苷酸序列插入至真核表达载体中，获得目标表达载体。
在实际应用中，可以基于每个所述新型冠状病毒S蛋白片段多倍体的核苷酸序列合成其DNA片段，并在所述DNA片段的C端添加组蛋白标签，克隆至表达载体中，获得目标表达载体。
S3、采用所述目标表达载体转染哺乳动物细胞，在预设培养条件下采用无血清培养基对转染后的哺乳动物细胞摇床培养5-7天，直至细胞存活率降至50％。
具体地，可以在哺乳动物细胞转染24h后加入补料，在37℃、5％-8％CO 2、100-150rpm的条件下采用无血清培养基对转染后的细胞进行摇床培养5-7天，每天记录细胞数目和存活率直至细胞存活率低于50％。
无血清培养基是不需要添加血清就可以维持细胞在体外较长时间生长繁殖的合成培养基。无血清培养基包括基础培养基及添加组分两大部分。
在实际应用中，可以采用Gibco-Expi293表达培养基(Expi293 TM Expression Medium，Gibco TM)、FreeStyte TM 293表达培养基(FreeStyle TM 293Expression Medium，Gibco TM)、OPM-293CD05培养基等对转染后的细胞进行培养，可视具体情况而定，本申请对此不做限制。
在本实施例中，采用无血清培养基对细胞即宿主细胞进行培养可以有效避免血清批次间的质量变动，提高细胞培养效果，避免血清对细胞的毒性作用和血清源性污染，避免血清组分对结果的影响，有利于体外培养细胞的分化，还有助于提高蛋白片段的表达水平并使细胞产品易于纯化。
S4、收集细胞培养的上清液，使用亲和层析的方法对上清液进行分离纯化处理，获得高效表达的新型冠状病毒S蛋白片段多倍体。
在实际应用中，可以收集细胞培养的上清液，离心，收集第一次离心上清液，将所述第一次离心上清液再次进行离心，收集第二次离心上清液，并过滤，收集滤液；将所述滤液加入平衡的镍柱(Ni-NTA)中，加入低浓度咪唑去除杂蛋白，加入高浓度咪唑洗脱，收集洗脱液，即得如实施例1所述的高效表达的新型冠状病毒S蛋白片段多倍体。
其中，离心处理的转速和时间、过滤处理选用的滤膜、咪唑的浓度值等均可以根据实际情况确定，本申请对此不做限制。第一次离心的转速优选为2000rpm，时间优选为10min，第二次离心的转速优选为15000rpm，时间优选为60min，在过滤处理的过程中，优选采用0.22μm的滤膜过滤，低浓度咪唑的浓度优选为10mM，高浓度咪唑的浓度优选为250mM。
在实际应用中，可以收集细胞培养的上清液，离心，收集第一次离心上清液，将所述第一次离心上清液再次进行离心，收集第二次离心上清液，并过滤，收集滤液；将所述滤液加入平衡的Protein A柱中，加入磷酸缓冲盐溶液(phosphate buffer saline，PBS)去除杂蛋白，加入Glycine-HCl溶液洗脱，并在洗脱液中立即加入Tris-HCl溶液以中和洗脱液，收集中和后的洗脱液，即得如实施例2所述的新型冠状病毒S蛋白片段多倍体。
其中，离心处理的转速和时间、过滤处理选用的滤膜、Glycine-HCl溶液及Tris-HCl溶液的浓度、酸碱度等均可以根据实际情况确定，本申请对此不做限制。第一次离心的转速优选为2000rpm，时间优选为10min，第二次离心的转速优选为15000rpm，时间优选为60min，在过滤处理的过程中，优选采用0.22μm的滤膜过滤，Glycine-HCl溶液的浓度优选为0.1M，pH值优选为2.8，Tris-HCl溶液的浓度优选为1M，pH值优选为8.5。
本实施例提供的新型冠状病毒S蛋白片段多倍体的制备方法，其通过获取多倍体的核苷酸序列、将核苷酸序列插入至表达载体中、采用目标表达载体转染哺乳动物细胞、对培养上清液进行纯化，可以有效提高新型冠状病毒S蛋白片段多倍体的表达量，有效提高新型冠状病毒S蛋白片段多倍体的纯度，为新型冠状病毒S蛋白片段多倍体在抗体检测、疫苗制备等方面的应用提供助力。
实施例8
本实施例设置试验组1-10。其中，试验组1-5分别为不带有Fc片段的RBD片段(单倍体)、二倍体、三倍体、四倍体和五倍体，试验组6-10分别为带有Fc片段的RBD片段(单倍体)、二倍体、三倍体、四倍体和五倍体。
在本试验例中，通过以下方法制备RBD片段及RBD多倍体：
(1)分别合成上述试验组1-10的DNA片段并在C端加上组蛋白或者Fc标签，克隆到真核表达载体中；
(2)悬浮培养细胞，于对数生长期时将上述4种质粒分别转染细胞，转染24h后加入补料，每天记录细胞数目和存活率直至细胞存活率低于50％；
(3)收集细胞培养上清液，于2000rpm离心10分钟，以去除Expi293细胞，收集离心上清液，于15000rpm离心60分钟，进一步Expi293去除细胞碎片。收集第二次离心的上清液，并用0.22μm的滤膜过滤。收集滤液，按照实施例7所述采用亲和层析的方法，即得到试验组1-10的蛋白片段。
对试验组2-10的蛋白片段进行蛋白质SDS-PAGE电泳处理，结果如图1和图2所示。
图1是试验组2-5的不带有Fc片段的RBD多倍体表达产物的SDS-PAGE结果图，图2是试验组6-10的带有Fc片段的RBD片段及多倍体表达产物的SDS-PAGE结果图。从图中可以看出，RBD的各种多倍体均能够在哺乳动物细胞中稳定表达。
我们构建了带有mCherry荧光标签的新型冠状病毒刺突蛋白受体ACE2高表达的稳转细胞株，如图3所示，从该激光共聚焦图中可以看出，构建的ACE2稳定表达细胞株中，ACE2受体主要以高度聚集的形式稳定的表达于细胞膜上。
采用Alexa488标记试验组1-10的RBD片段，采用FACs检测试验组1-10的RBD蛋白片段与新型冠状病毒刺突蛋白受体ACE2表达细胞的结合能力，结果如图4和图5所示。图4是试验组1-5不带有Fc片段的RBD片段、RBD二倍体、RBD三倍体、RBD四倍体、RBD五倍体与ACE-2表达细胞的相互作用折线对比图，图5是试验组6-10带有Fc片段的RBD片段、RBD二倍体、RBD三倍体、RBD四倍体、RBD五倍体与ACE-2表达细胞的相互作用折线对比图，其中横轴表示RBD片段及RBD多倍体摩尔浓度的log值(M)，纵轴表示mCherry阳性细胞中Alexa488的平均荧光强度(MFI)，即表示与ACE2结合的RBD片段或RBD多倍体的数量。结果显示，单个RBD片段的IC50值和Kd值最高，RBD五倍体的IC50值和Kd值最低。可以看出，RBD蛋白与新型冠状病毒蛋白受体ACE2结合能力与RBD片段的数量呈正比。
可见，相对于普通RBD片段而言，本申请提供的RBD多倍体即新型冠状病毒S蛋白片段多倍体不仅表达稳定，更重要的是其与新型冠状病毒蛋白受体ACE2结合能力有了进一步 地提升，这意味着其对于新型冠状病毒的检测能力、预防能力、治疗能力同样得到了进一步地提升。
实施例9
本实施例设置RBD-S组、RBD组、3RBD组和4RBD-Fc组，每组均选择6只雌性Balb/C小鼠。在第0天和第14天对RBD-S组小鼠肌肉注射10ugRBD-S蛋白和100ug氢氧化铝佐剂，对RBD组小鼠肌肉注射10ugRBD片段和100ug氢氧化铝佐剂，对3RBD组小鼠肌肉注射10ugRBD三倍体和100ug氢氧化铝佐剂，对4RBD-Fc组小鼠肌肉注射10ug带有Fc片段的RBD四倍体和100ug氢氧化铝佐剂。第28天收集血清，检测各组小鼠RBD特异性的抗体含量，即终点效价(endpoint titer)。
结果如图6所示，可以看出4RBD-Fc组，血清中RBD抗体浓度显著高于商品化的RBD-S组、RBD组。
这足以说明，本申请提供的新型冠状病毒S蛋白片段多倍体，表达量高，稳定性好，且在体内的半衰期相对更长，药效持续时间更久，更加有利于后期的开发，应用于新型冠状病毒的抗体检测和疫苗制备中，适用性强，适用范围广。
实施例10
本实施例提供一种新型冠状病毒S蛋白片段多倍体，该新型冠状病毒S蛋白片段多倍体由至少两个新型冠状病毒S蛋白受体结合区域片段构成。各片段之间的连接方式可参见实施例1-3。
进一步地，所述新型冠状病毒S蛋白受体结合区域片段选自(RBD)片段本体和/或(RBD)片段突变体。
需要说明的是，新冠病毒S蛋白片段多倍体既可以仅由多个(RBD)片段本体构成，也可以仅由多个(RBD)片段突变体构成，或者可以由一个或多个(RBD)片段本体与一个或多个(RBD)片段多倍体一同构成。
在新冠病毒S蛋白片段多倍体中包含多个(RBD)片段突变体的情况下，(RBD)片段突变体的种类既可以相同，也可以不同。
比如，新冠病毒S蛋白片段三倍体的构成方式可以为：片段本体-片段本体-片段本体、片段本体-片段本体-片段A突变、片段本体-片段A突变-片段A突变、片段本体-片段A突变-片段B突变、片段本体-片段A突变-片段A+B突变、片段A突变-片段B突变-片段C突变、片段A+B+C突变-片段B+C突变-片段C突变、片段A+B+C突变-片段A+B+C突变-片段A+B+C突变等。其他方式均可以此类推，不再赘述。
进一步地，所述(RBD)片段突变体的突变位点包括：N501Y、K417N、K417T、E484K、L452R、T478K、L452Q、F490S、E484Q、T478Q中的一种或几种。
换而言之，本申请提供的(RBD)片段突变体，可以由以上任意一种突变位点的突变得到，也可以由任意两种或多种突变位点同时突变得到。
优选地，所述(RBD)片段突变体包括：N501Y突变而成的B.1.1.7(Alpha)突变株，K417N突变而成的突变株，E484K突变而成的B1.525(Eta)突变株，N501Y、K417N及E484K突变而成的B.1.351(Beta)突变株，N501Y、K417T及E484K突变而成的P.1(Gamma)突变株，L452R及T478K突变而成的B.1.617.2(Delta)突变株，L452Q及F490S突变而成的C.37(Lambda)突变株，E484Q突变而成的突变株，L452R及E484Q突变而成的B.1.617.1(Kappa)突变株，L452R突变而成的B.1.427(Epsilon)突变株，T478Q突变而成的B.1.1.222突变株。
采用Alexa488分别标记RBD片段(WT),与新型冠状病毒刺突蛋白受体ACE2表达细胞稳定结合，可以用FACs检测。采用不同浓度的N501Y突变的RBD片段、K417N突变的RBD片段、E484K突变的RBD片段、N501Y-K417N-E484K突变的RBD片段(triple)等RBD突变体片段与Alexa488标记的RBD片段(WT)及ACE2表达细胞的共孵育，采用FACs检测各个RBD突变体片段和ACE2受体的亲和力，结果如图7所示。同上述方法，采用FACs检测L452R-E484Q突变的RBD片段、L452Q-F490S突变的RBD片段、L452R突变的RBD片段、L452R-T478K突变的RBD片段、T478K突变的RBD片段、N501Y-K417T-E484K突变的RBD片段、F490S突变的RBD片段等RBD片段与新型冠状病毒刺突蛋白受体ACE2表达细胞的结合能力，结果如图8所示。
结果证明RBD片段突变体能够如RBD本体一样能够稳定表达，并具备与新型冠状病毒蛋白受体ACE2结合的能力。
分别检测RBD片段、N501Y突变的RBD片段、N501Y-K417N-E484K突变的RBD片段与新型冠状病毒蛋白受体ACE2的结合能力，结果如图9所示。
图9a-图9c分别为RBD片段、N501Y突变的RBD片段、N501Y-K417N-E484K突变的RBD片段受体结合能力的折线图，图9d为RBD片段、N501Y突变的RBD片段、N501Y-K417N-E484K突变的RBD片段受体结合能力的对比表图。其中，Kd表示解离常数(dissociation constant)，反映的是对靶标的亲和力大小，值越小亲和力越强；Kon表示结合速率常数(association rate constant)，代表分子间结合时的快慢；Koff表示解离速率常数(dissociation rate constant)，代表分子间解离时的快慢。
可以看出，N501Y突变的RBD片段、N501Y-K417N-E484K突变的RBD片段均具备较强的受体结合能力。
分别检测K417N突变的RBD片段、E484K突变的RBD片段与新型冠状病毒蛋白受体ACE2的结合能力，结果如图10所示。
图10a-图10b分别为K417N突变的RBD片段、E484K突变的RBD片段受体结合能力的折线图，图10c为K417N突变的RBD片段、E484K突变的RBD片段受体结合能力的对比表图。
可以看出，K417N突变的RBD片段、E484K突变的RBD片段均具备较强的受体结合能力。
因此，本申请提供的由RBD突变体构成的多倍体即新型冠状病毒S蛋白片段多倍体不仅表达稳定，而且具有较强的受体结合能力，可以灵活应用于新型冠状病毒以及各种新型冠状病毒突变株(比如Alpha突变株，Eta突变株，Beta突变株，Gamma，Delta突变株，Lambda突变株，Kappa突变株，Epsilon突变株等)的检测与治疗。
将其应用于新型冠状病毒、新型冠状病毒突变株的抗体检测中，可以有效提高抗体检测的特异性、灵敏度和敏感性，进而有效提高抗体检测的准确率；将其应用于新型冠状病毒疫苗、新型冠状病毒突变株疫苗的制备中，可以刺激机体快速产生抗体，以使机体产生免疫作用，并且疫苗稳定性好，质量易于监控。
在本文中，“相等”、“相同”等并非严格的数学和/或几何学意义上的限制，还包含本领域技术人员可以理解的且制造或使用等允许的误差。
除非另有说明，本文中的数值范围不仅包括其两个端点内的整个范围，也包括含于其中的若干子范围。
上面结合附图对本申请优选的具体实施方式和实施例作了详细说明，但是本申请并不限于上述实施方式和实施例，在本领域技术人员所具备的知识范围内，还可以在不脱离本申请构思的前提下做出各种变化。

Claims (14)

  1. 一种新型冠状病毒S蛋白片段多倍体，其特征在于，所述新型冠状病毒S蛋白片段多倍体由至少两个新型冠状病毒S蛋白受体结合区域片段串联构成。
  2. 根据权利要求1所述的新型冠状病毒S蛋白片段多倍体，其特征在于，所述新型冠状病毒S蛋白受体结合区域片段之间通过GG或GS序列串联。
  3. 根据权利要求2所述的新型冠状病毒S蛋白片段多倍体，其特征在于，所述新型冠状病毒S蛋白受体结合区域片段之间通过GGS、GGGS、GSGS、SGGS、GGGGS、GSGGS、GGSGS、GGSGGS、GGGSGS、GGGGGS或GGGSGGS序列串联。
  4. 根据权利要求1所述的新型冠状病毒S蛋白片段多倍体，其特征在于，所述新型冠状病毒S蛋白受体结合区域片段的C端与人免疫球蛋白的Fc片段连接；
    一个或多个所述新型冠状病毒S蛋白受体结合区域片段构成多倍体单元，所述多倍体单元通过所述新型冠状病毒S蛋白受体结合区域片段连接的Fc片段形成二聚体，并将所述二聚体作为新型冠状病毒S蛋白片段多倍体。
  5. 根据权利要求1所述的新型冠状病毒S蛋白片段多倍体，其特征在于，所述新型冠状病毒S蛋白受体结合区域片段之间或所述新型冠状病毒S蛋白片段多倍体之间通过蛋白酶或蛋白交联剂相连。
  6. 根据权利要求5所述的新型冠状病毒S蛋白片段多倍体，其特征在于，所述蛋白酶包括转肽酶和联用蛋白连接酶，所述蛋白交联剂包括聚乙二醇、辛二酸二琥珀酰亚胺酯、琥珀酸辛二酸戊二酸盐、二甲基二亚胺酯、二甲基亚胺酯、二甲基苯二酸亚胺酯和马来酰亚胺己烷。
  7. 如权利要求1所述的新型冠状病毒S蛋白多倍体，其特征在于，所述新型冠状病毒S蛋白受体结合区域片段选自片段本体和/或片段突变体。
  8. 如权利要求7所述的新型冠状病毒S蛋白多倍体，其特征在于，所述片段突变体的突变位点包括：N501Y、K417N、K417T、E484K、L452R、T478K、L452Q、F490S、E484Q、T478Q中的一种或几种。
  9. 一种检测试剂盒，其特征在于，所述检测试剂盒为能够检测新型冠状病毒存在与否的试剂盒，所述检测试剂盒包括权利要求1-8任意一项所述的新型冠状病毒S蛋白片段多倍体。
  10. 一种疫苗，其特征在于，所述疫苗为能够预防新型冠状病毒传染的疫苗，所述疫苗包括权利要求1-8任意一项所述的新型冠状病毒S蛋白片段多倍体。
  11. 根据权利要求10所述的疫苗，其特征在于，所述疫苗为mRNA疫苗、蛋白疫苗或腺病毒疫苗；
    所述疫苗中的新型冠状病毒S蛋白片段多倍体激活聚集的B细胞受体，产生新型冠状病毒抗体。
  12. 一种药物，其特征在于，所述药物为能够预防或治疗新型冠状病毒的药物，所述药物包括权利要求1-8任意一项所述的新型冠状病毒S蛋白片段多倍体。
  13. 根据权利要求12所述的药物，其特征在于，所述药物中的新型冠状病毒S蛋白片段多倍体与ACE2受体结合，抑制新型冠状病毒的侵袭。
  14. 一种新型冠状病毒S蛋白片段多倍体的制备方法，其特征在于，包括：
    S1、获取新型冠状病毒S蛋白片段多倍体的核苷酸序列；
    S2、将所述新型冠状病毒S蛋白片段多倍体的核苷酸序列插入至真核表达载体中，获得目标表达载体；
    S3、采用所述目标表达载体转染哺乳动物细胞，在预设培养条件下采用无血清培养基对转染后的细胞摇床培养5-7天，直至细胞存活率降至50％；
    S4、收集哺乳动物细胞培养的上清液，使用亲和层析的方法对上清液进行分离纯化处理，获得权利要求1-8任意一项所述的新型冠状病毒S蛋白片段多倍体。
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