A novel coronavirus SARS-CoV-2 mutant vaccine and its application

Abstract

The present disclosure relates to a novel coronavirus SARS-CoV-2 mutant vaccine and its application, the vaccine comprising a fusion protein comprising: (1) interferon or a functional fragment thereof; (2) novel coronavirus SARS-CoV-2 or a functional fragment thereof; and (3) an immunoglobulin Fc region. The mutant vaccine disclosed herein has long-lasting efficacy, is amenable to industrial production, has activity comparable to that of conventional IFN, has higher immunogenicity and neutralizing antibody titers, and can increase defense against mutant strains. It can be used as a new generation of mutant vaccine drugs to combat the spread of the COVID-19 pandemic.

Description

Technical Field

[0001] The present disclosure belongs to the field of biotechnology, and specifically relates to a novel coronavirus SARS-CoV-2 mutant vaccine and its application. Background Art

[0002] The new coronavirus (2019-nCoV, SARS-CoV-2) is a beta coronavirus and the seventh known coronavirus that can infect humans. Infection with this virus can cause patients to experience symptoms such as fever, dry cough, and fatigue; some patients will develop severe pneumonia, and then develop acute respiratory distress syndrome, septic shock, coagulation dysfunction, multiple organ failure, and even death.

[0003] The new coronavirus is composed of four structural proteins (spike protein, envelope protein, membrane protein and nucleocapsid protein) and RNA nucleic acid chains. Among them, the spike protein (S protein) is a glycoprotein located on the surface of the new coronavirus membrane, which mainly acts on cell adhesion and cell membrane fusion. The S protein is composed of two subunits, S1 and S2. The S1 subunit contains the receptor binding domain (RBD), which is responsible for recognizing the host cell receptor ACE2. It is a key factor in the interaction between the virus and the receptor and the virus invading the cell. It is also a key target for vaccine design. The S2 subunit contains the basic elements required for the membrane fusion process and can promote the fusion of the virus and the host cell membrane.

[0004] The novel coronavirus is constantly mutating, and many prevalent SARS-CoV-2 mutants have been linked to a rapid increase in the number of cases, such as the UK mutant B.1.1.7 / 501Y.V1, the South African mutant B.1.351 / 501Y.V2, and the Brazilian mutant P.1 / 501Y.V3. These novel coronavirus mutants, as well as other novel coronavirus variants that may carry the K417N / T, E484K, and N501Y mutations, can reduce the neutralizing capacity of vaccine-induced plasma neutralizing antibodies.

[0005] Most of the COVID-19 vaccines currently on the market or under development utilize wild-type S proteins or RBDs. While these vaccines offer good protection against wild-type or early-stage COVID-19 mutants, they have shown varying degrees of decreased protection against currently prevalent variants (primarily the South African variant B.1.351 / 501Y.V2). Therefore, there is an urgent need to develop new vaccines against the novel coronavirus, particularly mutants. Summary of the Invention

[0006] In order to avoid the limitations of existing vaccines, the present disclosure provides a fusion protein vaccine comprising interferon, a novel coronavirus antigen and an immunoglobulin Fc region. The vaccine can enhance the immunogenicity of mutant novel coronavirus antigens and the titer of neutralizing antibodies through the fused expressed IFN, thereby ensuring the efficient production of neutralizing antibodies and significantly improving the defense capability against mutant strains.

[0007] In one aspect, the present disclosure provides a fusion protein comprising:

[0008] (1) interferon or its functional fragments;

[0009] (2) novel coronavirus SARS-CoV-2 or its functional fragments; and

[0010] (3) Immunoglobulin Fc region.

[0011] In one aspect, the present disclosure provides a nucleic acid encoding the fusion protein of any one of claims 1-6.

[0012] In one aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid.

[0013] In one aspect, the present disclosure provides a host cell expressing the aforementioned fusion protein, comprising the aforementioned nucleic acid and / or comprising the aforementioned vector.

[0014] In one aspect, the present disclosure provides a vaccine for treating and / or preventing novel coronavirus SARS-CoV-2 infection or novel coronavirus disease COVID-19, comprising the aforementioned fusion protein, nucleic acid, vector and / or host cell, and optionally, a pharmaceutically acceptable carrier and / or excipient.

[0015] In one aspect, the present disclosure provides a use of the aforementioned fusion protein, nucleic acid, vector, host cell and / or vaccine in the preparation of a drug or product for preventing and / or treating novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19.

[0016] In one aspect, the present disclosure provides a method for preparing a vaccine for preventing and / or treating novel coronavirus SARS-CoV-2 infection or coronavirus disease COVID-19, the method comprising expressing the aforementioned fusion protein.

[0017] In one aspect, the present disclosure provides a method for preventing and / or treating novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19, comprising administering to a subject an effective amount of the aforementioned fusion protein, nucleic acid, vector, host cell and / or vaccine.

[0018] In one aspect, the present disclosure provides a method for inducing a neutralizing antigen-specific immune response in an individual, comprising administering to the subject an effective amount of the aforementioned fusion protein, nucleic acid, vector, host cell and / or vaccine.

[0019] Different from the known inactivated, adenovirus and mRNA new coronavirus vaccines, the antigen used in the mutant strain vaccine disclosed herein is the antigen part (especially the RBD part) of the mutant new coronavirus strain, and forms a fusion protein with the IFN and Fc domains that enhance the immune activation ability. In order to prolong the half-life, the present disclosure adopts the method of fusion with the Fc fragment to increase the half-life of the mutant strain vaccine; the immunogenicity of the new coronavirus mutant antigen and the titer of neutralizing antibodies are improved by the IFN domain to ensure the efficient production of neutralizing antibodies; the defense ability against mutant strains is increased by the variant RBD; the IgG1 type Fc is used, and the downstream purification method is relatively simple, which is conducive to industrial production. This mutant strain vaccine can induce a stronger immune response in animals and humans. Therefore, the mutant strain fusion protein vaccine disclosed in the present disclosure has a longer half-life, higher immunogenicity, and stronger defense ability compared to the traditional single antigen new coronavirus vaccine. The present disclosure also developed a CHO protein expression and preparation method suitable for transient expression of larger protein molecules, which reduces the difficulty of early preparation and ensures that the product quality meets the scale-up production standards. Combining all the above innovative designs and optimizations, the mutant strain vaccine disclosed herein has long-lasting effect, is conducive to industrial production, has activity comparable to that of ordinary IFN, has higher immunogenicity and neutralizing antibody titers, and can increase defense capabilities against mutant strains. It can be used as a new generation of mutant strain vaccine drugs to resist the spread of the new coronavirus epidemic. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the molecular structure of the parent strain vaccine (V-01) and the mutant strain vaccine (British strain and South African strain).

[0021] Figure 2 This is the expression plasmid map of the parent strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain). Figure 2 a is the map of the parental strain vaccine (V-01); Figure 2 b is the UK strain vaccine map; Figure 2 c is the map of the South African strain vaccine.

[0022] Figure 3The results of enzyme identification of the plasmids expressing the parental vaccine (V-01) and mutant vaccines (UK and South Africa). M1: DL15000 nucleic acid marker; 1. Parental vaccine (V-01) HindIII single digestion; 2. Parental vaccine (V-01) HindIII / PacI double digestion; 3. UK vaccine HindIII single digestion; 4. UK vaccine HindIII / PacI double digestion; 5. South Africa vaccine HindIII single digestion; 6. South Africa vaccine HindIII / PacI double digestion; M2: DL15000 nucleic acid marker.

[0023] Figure 4 The results of the instantaneous transfection method used in this disclosure are compared with those of the commercially available method. Figure 4 a is the test results of living cell density and cell viability; Figure 4 b is the expression level result.

[0024] Figure 5 The expression levels of the parent strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) expressed in Example 1 of the present disclosure ( Figure 5 a) and the electrophoresis detection results after purification ( Figure 5 b). M1: 180 kDa protein marker.

[0025] Figure 6 The in vitro affinity diagram of the parental strain vaccine (V-01), mutant strain vaccine (UK strain, South Africa strain) and IFNR / ACE2 was determined based on the SPR method. Figure 6 a is the affinity fitting curve between the parent strain (V-01) and ACE2; Figure 6 b is the affinity fitting curve of the UK strain vaccine and ACE2; Figure 6 c is the affinity fitting curve of South African vaccine and ACE2; Figure 6 d is the affinity fitting curve between the parent strain (V-01) and IFNAR2; Figure 6 e is the affinity fitting curve of the UK strain vaccine and IFNAR2; Figure 6 f is the affinity fitting curve between the South African strain vaccine and IFNAR2.

[0026] Figure 7 The graph shows the biological activities of the parental strain vaccine (V-01), mutant strain vaccine (British strain, South African strain) and IFNR based on the indicator cell method.

[0027] Figure 8 To evaluate the in vivo efficacy of the parental strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) in mice.

[0028] Figure 9The neutralization potency of the parental strain vaccine (V-01) and the mutant strain vaccine (British strain, South African strain) against three pseudoviruses was evaluated. Figure 9 a is the neutralization titer of the parental strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) against three pseudoviruses; Figure 9 b is the comparison of the neutralization titers of the parental strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) against pseudovirus. DETAILED DESCRIPTION

[0029] I. Definition

[0030] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0031] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0032] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0033] The term "fusion protein" refers to a natural or synthetic molecule composed of one or more molecules in which two or more peptide- or protein-based (including glycoprotein) molecules with different specificities are fused together, optionally via a chemical or amino acid-based linker molecule. The connection can be achieved by CN fusion or NC fusion (in the 5'→3' direction), preferably CN fusion.

[0034] The term "interferon" (IFN) refers to a class of cytokines produced by the cellular genome in response to viral infection or other interferon-inducing agents. These cytokines possess a variety of biological activities, including antiviral, anti-tumor, and immunomodulatory activities. Interferons can be divided into three main categories based on their biological and physical properties: type I, type II, and type III interferons.

[0035] Type I interferons constitute a family of structurally related genes (IFN-α (α), IFN-β (β), IFN-κ (κ), IFN-δ (δ), IFN-ε (ε), IFN-τ (τ), IFN-ω (ω), and IFN-ζ (ζ)). IFN-δ and IFN-τ do not occur in humans. Human type I interferon (IFN) genes are clustered on human chromosome 9p21, while mouse genes are located in a conserved colinear region on mouse chromosome 4. To date, 14 IFN-α genes and three pseudogenes have been identified in mice. In humans, 13 kinds of IFN-α (or IFNA) genes (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17 and IFNA21) and 1 pseudogene have been identified, wherein two kinds of human IFN-α genes (IFNA1 / IFN-α1 and IFNA13 / IFN-α13) are directed against identical protein coding.All human type I interferons are bound to the cell surface receptors (IFNα receptor, IFNAR) consisting of two transmembrane proteins (IFNAR-1 and IFNAR-2), which cause the formation of JAK-STAT activation, ISGF3 and the gene expression that begins subsequently.Interferon gamma (IFN-γ) is the only known type II interferon, which is mainly involved in inducing antibacterial and antitumor mechanisms by macrophage stimulation. The IFN-γ receptor (IFNGR) is a heterodimeric receptor composed of two ligand-binding IFNGR1 chains associated with two signaling IFNGR2 chains. Type III interferons consist of three subtypes and are also known as IFNλ (IFNλ1 or IL-29, IFNλ2 or IL-28A, and IFNλ3 or IL-28B) and have antiviral, antitumor, and immunomodulatory activities. The IFN-λ receptor is also a heterodimeric complex composed of the sole ligand-binding chain, IFN-λR1 (also designated IL-28Rα), and a side chain, IL-10R2, shared with the receptor for the IL-10-related cytokine.

[0036] The term "coronavirus" belongs to the Coronaviridae family, Coronavirus genus, which can infect mammals and poultry, causing various diseases of the respiratory system, digestive system and central nervous system. Coronaviruses can be divided into four different genera based on genomic and serological differences: α, β, γ and δ. Currently, only α and β coronaviruses infect humans. To date, six human coronaviruses (HCoV) from two genera (α and β) have been identified. The α coronaviruses include NL63 and 229E, and the β coronaviruses include OC43, HKU1, acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and novel coronavirus pneumonia virus (SARS-CoV-2).

[0037] The term "antibody" or "immunoglobulin" has the broadest meaning and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies composed of at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term generally includes hybrid antibodies composed of two or more antibodies or antibody fragments with different binding specificities linked together.

[0038] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from position Cys226, or from the amino acid residue at Pro230, to the carboxyl terminus of the heavy chain. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, the composition of a complete antibody can include an antibody population from which all K447 residues have been removed, an antibody population from which no K447 residues have been removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0039] Sequence "identity" or "identity" has an art-recognized meaning, and published techniques can be used to calculate the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0040] The term "Th cell helper epitope" refers to all epitopes that activate helper T cells, including PADRE.

[0041] PADRE is a short 13-amino acid peptide sequence that binds to different DR molecules in humans and various animals, presenting them on the cell surface and activating CD4+ T helper cells, exerting an immunomodulatory effect. PADRE's ability to induce T cell responses is over 1000 times greater than that of the native epitope, thus possessing some of the characteristics of an immune adjuvant (PMID: 7895164). In vivo, the PADRE peptide can activate helper T cells (Th1) to assist in CTL activation, and can also activate helper T cells (Th2) to assist B cells in secreting specific antibodies, further enhancing the antigenic immune response induced by the recombinant protein.

[0042] The term "treat" refers to any indication of success in treating or ameliorating an injury, disease, disorder, or condition, including any objective or subjective parameter, such as palliation; remission; a decrease in symptoms or making the injury, disorder, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making a degenerative endpoint less debilitating; or improving the patient's physical or mental well-being. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric testing, and / or psychiatric evaluation.

[0043] The term "effective amount" is an amount sufficient to achieve the stated purpose (e.g., to achieve the effect for which it is administered, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce protein function, to alleviate one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to promote the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount."

[0044] II. Fusion Proteins and Vaccines

[0045] In one aspect, the present disclosure provides a fusion protein comprising:

[0046] (1) interferon or its functional fragments;

[0047] (2) novel coronavirus SARS-CoV-2 or its functional fragments; and

[0048] (3) Immunoglobulin Fc region.

[0049] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: interferon or a functional fragment thereof, SARS-CoV-2 or a functional fragment thereof, and an immunoglobulin Fc region.

[0050] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus: SARS-CoV-2 or a functional fragment thereof, interferon or a functional fragment thereof, and an immunoglobulin Fc region.

[0051] In some embodiments, the interferon is selected from type I interferon, type II interferon, and / or type III interferon.

[0052] In some embodiments, the interferon may be of human or murine origin.

[0053] In some embodiments, the type I interferon is selected from IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ.

[0054] In some embodiments, the Type II interferon is interferon gamma.

[0055] In some embodiments, the Type III interferon is selected from IFN-λ1 (IL-29), IFN-λ2 (IL-28a), and IFN-λ (IL-28b).

[0056] In some embodiments, the interferon is selected from human IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17, and IFN-α21.

[0057] In some further embodiments, the interferon is IFN-α2a; preferably, the amino acid sequence of the IFN-α2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the IFN-α-2a is as shown in SEQ ID NO: 1.

[0058] In one embodiment, the IFN active domain is preferably connected to the N-terminus after the mutant new crown RBD antigen (Gly4Ser) 3 linker sequence. In a preferred embodiment of the present disclosure, a mutated IFN is used, which differs from the wild-type IFN in only one amino acid sequence. According to the position of the mutation site of the mutant IFN in the complete interferon α-2b of GenBank (sequence number: AAP20099.1), the mutation is named recombinant interferon α-2b (Q124R). The purpose of this mutation is to achieve partial binding of human IFNα to heterologous mouse IFN receptors, thereby detecting the activity and function of human IFNα using a mouse model. The mutation has been documented to not affect the affinity of interferon α-2b to human interferon receptors, and is intended to enhance the binding activity to mouse interferon receptors, which is beneficial for in vivo efficacy evaluation in mice. The extracellular secretory sequence of human IFN-α2a is then fused to the C-terminus of the targeted IFN bifunctional molecule Fc fragment, and its amino acid sequence is not changed. The structures of the parental vaccine (V-01) and mutant vaccines (UK and South African strains), made from fusion proteins of the IFN\COVID-19 RBD domain and Fc, are shown in Figure 1. After expression in mammalian cells, the fusion protein self-assembles into a dimer.

[0059] In some embodiments, the SARS-CoV-2 is a mutant SARS-CoV-2.

[0060] In some embodiments, the mutant SARS-CoV-2 is the British mutant strain B.1.1.7 / 501Y.V1 or the South African mutant strain 501Y.V2 (South African strain B.1.351). In particular, the South African strain RBD mutation contains 3 point mutations compared to the wild-type new crown strain, namely K417N / T, E484K, and N501Y. The above three point mutations not only change the conformation of RBD, but also change the antigenicity of RBD. Among them, E484K has been shown to increase the immune escape of the mutant strain, N501Y can significantly enhance the binding force with ACE2, and K417N affects the formation of salt bridges and enhances the escape mechanism of E484K. Based on the above effects, the use of the mutant strain RBD domain as a delivery antigen can make up for the shortcomings of the wild-type new crown vaccine.

[0061] In some embodiments, the functional fragment of SARS-CoV-2 is its receptor binding domain RBD.

[0062] In some embodiments, the RBD comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 2, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the RBD is as shown in SEQ ID NO: 2.

[0063] In some embodiments, the RBD comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 3, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequence of the RBD is as shown in SEQ ID NO: 3.

[0064] In some embodiments, the immunoglobulin Fc region is selected from the constant region amino acid sequences of IgG1, IgG2, IgG3 and / or IgG4.

[0065] In some embodiments, the immunoglobulin Fc region is the Fc region of IgG1; preferably, the IgG1 Fc region comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO 4, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; more preferably, the amino acid sequence of the IgG1 Fc region is as shown in SEQ ID NO 4.

[0066] In one embodiment of the present disclosure, IgG1 Fc is selected, wherein the IgG Fc has a T250Q / M428L mutation (amino acids are numbered according to EU nomenclature), which increases the affinity for human FcRn and can prolong the half-life in vivo.

[0067] In some embodiments, the fusion protein further comprises one or more Th cell helper epitopes and / or linking fragments.

[0068] In some embodiments, the Th cell helper epitope is PADRE or a derivative thereof; the amino acid sequence of PADRE or a derivative thereof is selected from SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10.

[0069] In some embodiments, the connecting segment is a flexible polypeptide sequence; preferably, the amino acid sequence of the flexible peptide is selected from SEQ ID NO 11 and SEQ ID NO 12.

[0070] The linker sequence can be 2-8 repeats of a (Gly4Ser)x or (GlySer)x linker. The specific length depends on the actual size of the fusion protein molecule and needs to take into account the differences in steric hindrance. The linker sequence used in this disclosure is a (Gly4Ser)3 linker, and the sequence information is as follows:

[0071] GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID NO 11)

[0072] GlySer GlySer GlySer (SEQ ID NO 12)

[0073] In some embodiments, the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence selected from SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; preferably, the amino acid sequence of the fusion protein is selected from the amino acid sequences shown in SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15; more preferably, the amino acid sequence of the fusion protein is SEQ ID NO 14 or SEQ ID NO 15; more preferably, the amino acid sequence of the fusion protein is SEQ ID NO 15.

[0074] In one aspect, the present disclosure provides a nucleic acid encoding the aforementioned fusion protein.

[0075] In some embodiments, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 having 80% or greater identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably 98% or greater identity; preferably, the nucleic acid is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18; more preferably, the nucleic acid is SEQ ID NO: 17 or SEQ ID NO: 18; more preferably, the nucleic acid is SEQ ID NO: 18.

[0076] In one aspect, the present disclosure provides a vector of the aforementioned nucleic acid.

[0077] In one aspect, the present disclosure provides a host cell expressing the aforementioned fusion protein, comprising the aforementioned nucleic acid and / or comprising the aforementioned vector.

[0078] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0079] In some embodiments, the prokaryotic cell is a bacterial cell. In some specific embodiments, the prokaryotic cell is an Escherichia coli cell.

[0080] In some embodiments, the eukaryotic cell is selected from yeast cells, insect cells and mammalian cells. In some embodiments, the mammalian cell is selected from CHO, HEK293, SP2 / 0, BHK, C127, etc. In some specific embodiments, the eukaryotic cell is a CHO cell.

[0081] In one aspect, the present disclosure provides a vaccine for treating and / or preventing novel coronavirus SARS-CoV-2 infection or novel coronavirus disease COVID-19, comprising the aforementioned fusion protein, nucleic acid, vector and / or host cell, and optionally, a pharmaceutically acceptable carrier and / or excipient.

[0082] The pharmaceutically acceptable carrier may contain a liquid such as water, saline, glycerol and sorbitol. In addition, these carriers may also contain auxiliary substances such as lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and stabilizers such as albumin.

[0083] In some embodiments, the vaccine is in the form of a recombinant protein subunit vaccine, a recombinant protein mRNA vaccine, or a recombinant protein adenovirus vector vaccine.

[0084] In some embodiments, the vaccine can be prepared into various dosage forms suitable for administration to mammals, including but not limited to injections, capsules, tablets, emulsions, suppositories, and lyophilized powders; preferably injections.

[0085] In one aspect, the present disclosure provides a method for preparing a vaccine for preventing and / or treating novel coronavirus SARS-CoV-2 infection or coronavirus disease COVID-19, the method comprising expressing the aforementioned fusion protein.

[0086] III. Treatment Methods

[0087] In one aspect, the present disclosure provides a use of the aforementioned fusion protein, nucleic acid, vector, host cell and / or vaccine in the preparation of a drug or product for preventing and / or treating novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19.

[0088] In one aspect, the present disclosure provides a method for preventing and / or treating novel coronavirus SARS-CoV-2 infection and / or novel coronavirus disease COVID-19, comprising administering to a subject an effective amount of the aforementioned fusion protein, nucleic acid, vector, host cell and / or vaccine.

[0089] In one aspect, the present disclosure provides a method for inducing a neutralizing antigen-specific immune response in an individual, comprising administering to the subject an effective amount of the aforementioned fusion protein, nucleic acid, vector, host cell and / or vaccine.

[0090] In some embodiments, the vaccine is administered by intradermal injection, subcutaneous injection, intramuscular injection, or intravenous injection.

[0091] In some embodiments, the vaccine is administered by injection.

[0092] For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0093] Example

[0094] Example 1: Preparation of fusion protein

[0095] 1.1 Expression plasmid construction

[0096] The mutant vaccine is a fusion protein of the IFN\COVID-19 RBD domain and Fc. It consists of genetically fusing the C-terminus of IFN to RBD-Fc via a flexible (Gly4Ser)3 linker ( Figure 1To compare the activity and function, mutant vaccines (South African strain and British strain) and the parental strain vaccine (V-01) were constructed simultaneously, differing in that the RBD antigen had different point mutations. All genes involved in the present disclosure were synthesized by whole gene synthesis and then ligated into the mammalian cell expression vector pCGS3 (purchased from Sigma-Aldrich) for expression by double enzyme digestion. Figure 2 The maps of the parental strain vaccine (V-01) and mutant strain vaccines (British strain, South African strain) are shown. Figure 3 The electrophoresis results of enzyme digestion identification are shown, and the enzyme digestion identification is correct. The plasmid was extracted using Omega's endotoxin-free plasmid extraction kit (Cat. No.: D6926-03B; purchased from Omega Bio-tek) and stored at -80°C.

[0097] Among them, the amino acid sequence of the parent strain vaccine (V-01) is shown in SEQ ID NO 13, and its encoding nucleic acid sequence is shown in SEQ ID NO 16; the amino acid sequence of the British strain vaccine is shown in SEQ ID NO 14, and its encoding nucleic acid sequence is shown in SEQ ID NO 17; the amino acid sequence of the South African strain vaccine is shown in SEQ ID NO 15, and its encoding nucleic acid sequence is shown in SEQ ID NO 18.

[0098] 1.2 Protein expression

[0099] For the expression of the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South African strain), the present disclosure uses the ExpiCHO expression system to express the protein. The expression scale is amplified by a bioreactor (15L), that is, a large-volume transient transfection scheme is adopted for expression, thereby improving the commercially available ExpiCHO expression system.

[0100] (1) 24 hours before transfection, adjust the density of Expi-CHO cells to 1.5×10 6 -3×10 6 Cells / ml were plated into a 15L bioreactor and cultured overnight. Control parameters were set consistent with those used in later-stage 200L or larger bioreactors to ensure that the quality of the expressed product was as consistent as possible with commercial scale production.

[0101] (2) One hour before transfection, measure the viable cell density and viability. The viable cell density should reach 6×10 6 -10×10 6 cells / ml, and the cell viability should be no less than 95%.

[0102] (3) First, use Opti-MEM to dilute the expression plasmid and transfection reagent separately. The plasmid dilution solution needs to be filtered and sterilized. Then, slowly add the diluted transfection reagent to the plasmid, mix evenly, and incubate at room temperature for 5 minutes. Then add the mixture to the cell culture at a uniform speed. The commercial method is to pour it in manually, according to the operating instructions of the commercial ExpiCHO expression system (purchased from: Lifetechnologies, product number: A29133), and the reaction scale is 1L shake flask production. The transfection mixture of the present invention is prepared and configured in the same proportion as the commercial instructions. After the mixture is prepared, the present invention uses a bioreactor rehydration device to add it at a uniform speed. The bioreactor is controlled at 37°C, 5% CO2, pH7.0, dissolved oxygen 40%, rotation speed 150rpm, and deep air constant flow 20ml / min for culture parameters.

[0103] (4) 24 hours after transfection, the temperature was lowered to 31°C and an appropriate amount of sodium butyrate was added; 5% feed was added on the first, third, and fifth days after transfection, and the sugar concentration was controlled in the range of 3-6 g / L.

[0104] (5) When the cell viability is lower than 80% after transfection or on the 6th day, the supernatant is collected for protein quantification and subsequent purification.

[0105] Following the steps shown above, the parent strain vaccine (V-01) expression vector was used to prepare the transfection mixture, and transient transfection was performed using the commercially available manual direct addition method and the reactor rehydration device disclosed in the present invention. The changing trends of cell viability and viable cell density during the expression culture process were similar, and high cell viability and cell density were always maintained (Figure 4a). Under the bioreactor addition mode adopted in the present invention, the expression level of the parent strain vaccine (V-01) was significantly improved, and the yield was increased by nearly 2 times compared to the traditional commercial model ( Figure 4 b) The transient expression system provided by the bioreactor disclosed herein enables producer cells to maintain consistently high viability and expression yields, significantly improving transient expression yields compared to traditional commercially available transfection processes. The transient expression system employed in this disclosure is suitable for transient expression production of larger molecules, reducing the difficulty of early sample preparation. Precise control of reaction conditions in the bioreactor is essentially consistent with later commercial-scale production controls, ensuring more representative transient product quality.

[0106] The parent strain vaccine (V-01) and mutant strain vaccines (British strain and South African strain) were transiently expressed using the large-volume transient expression system provided by the present disclosure. Using the same expression process, signal peptide and expression vector conditions, the parent strain vaccine had the highest expression level, followed by the British strain vaccine and the South African strain vaccine ( Figure 5a) The difference in expression levels may be caused by different point mutations, especially the South African vaccine has added K417T and E484K mutations, and the expression level has decreased significantly.

[0107] 1.3 Protein purification

[0108] Affinity chromatography capture

[0109] The clarified cell culture fluid was directly loaded onto a Protein A affinity column for capture to obtain the purified fusion protein. The results were analyzed by reducing SDS-PAGE. Figure 5 As shown in b, the purity of the parental strain vaccine (V-01) and the mutant strain vaccine (British strain, South African strain) is greater than 95%.

[0110] Example 2: Affinity determination

[0111] The affinity of the parental vaccine (V-01) and the mutant vaccine (UK strain and South African strain) was tested using a molecular interaction analyzer (SPR method), including the affinity of the RBD domain to the ACE2 receptor and the affinity of the IFNα domain to the IFNAR2 receptor. The details are as follows:

[0112] 2.1 Affinity determination with ACE2

[0113] Research methods: HBS-EP+ (item number BR100826, purchased from GE Healthcare) was used as the experimental buffer. Each cycle included the capture of different vaccines, injection of different concentrations of ACE2 proteins, and regeneration. After the mutant strains (South Africa, the United Kingdom) and the parent strain (V-01) vaccines prepared in Example 1 were diluted to 1 μg / mL, they were injected into 2 channels at a flow rate of 10 μL / min for 40 seconds, so that they were captured on this channel by Protein A, and 1 channel was used as a blank reference channel. Using the High Performance model, ACE2 (200, 100, 50, 20, 12.5, 0nM) was injected into chip 1 and 2 channels at a flow rate of 30 μL / min according to a concentration gradient, with a binding time of 150 seconds and a dissociation time of 300 seconds. 10mM glycine (pH 1.5) was injected at a flow rate of 10 μL / min for 30 seconds to regenerate the chip. The instrument was set to a temperature of 25°C. The data were analyzed using Biacore T200 analysis software (Version: 1.0, General Electric Company). Channel 1 was used as a blank reference channel, and the binding of each sample was analyzed after subtracting the background signal. The analysis model used was 1:1 binding.

[0114] Results and Conclusions: The results are shown in Table 1 and Figure 6a-6c, the parent strain vaccine (V-01) and the mutant strain vaccine (British strain and South African strain) can all bind to ACE2 with affinities of 1.05E-08M, 1.65E-08M and 7.41E-09M, respectively, which are basically consistent with the affinity of ACE2.

[0115] Table 1 Affinity results of parental strain vaccine (V-01), mutant strain vaccine (UK strain, South Africa strain) and ACE2

[0116]

[0117]

[0118] 2.2 Affinity determination for IFNAR2

[0119] Research methods: HBS-EP+ was used as the experimental buffer, and each cycle included capture vaccine, injection of different concentrations of IFNAR2 protein, and regeneration. After the mutant strains (South Africa, UK) and parent strain (V-01) vaccines prepared in Example 1 were diluted to 4 μg / mL, they were injected into channel 4 at a flow rate of 10 μL / min for 40 seconds, so that they were captured on this channel by Protein A, and channel 3 was used as a blank reference channel. Using the High Performance model, IFNAR2 (100, 50, 20, 12.5, 6.25, 3.125, 0nM) was injected into chip channels 3 and 4 at a flow rate of 30 μL / min according to a concentration gradient, with an association time of 100 seconds and a dissociation time of 150 seconds. 10mM glycine (pH 1.5) was injected at a flow rate of 30 μL / min for 30 seconds to regenerate the chip. The instrument was set to a temperature of 25°C. The data were analyzed using Biacore T200 analysis software (Version: 1.0, General Electric Company). Channel 3 was used as a blank reference channel, and the binding of each sample was analyzed after subtracting the background signal. The analysis model used was 1:1 binding.

[0120] Results and Conclusions: The results are shown in Table 2 and Figure 6 d-6f, both the parental strain vaccine (V-01) and the mutant strain vaccine (British strain and South African strain) can bind to the IFNAR2 protein with affinities of 1.03E-07M, 8.64E-08M and 2.95E-07M, respectively, which are basically consistent with the affinity of IFNAR2.

[0121] Table 2 Affinity results of parental strain (V-01) vaccine, mutant strain vaccine (British strain, South African strain) and IFNAR2

[0122] sample ka(1 / Ms) kd(1 / s) KD(M) Parent strain vaccine (V-01) 1.61E+05 1.65E-02 1.03E-07 British strain vaccine 2.26E+05 1.96E-02 8.64E-08 South African vaccine 6.14E+04 1.81E-02 2.95E-07

[0123] Example 3: Biological activity of parental strain vaccine (V-01) and mutant strain vaccine (UK strain, South Africa strain)

[0124] After the IFNα-2b in the parental strain vaccine (V-01) and the mutant strain vaccine (UK strain, South Africa strain) structures bind to the endogenous receptors IFNAR2 and IFNAR1 on the cell membrane, the interferon-stimulated response element can be activated through signal transduction, and the expression of luciferase is initiated. The expression level is positively correlated with the biological activity of interferon. After adding cell lysate and luciferase substrate, the luminescence intensity is measured to determine its biological activity. Therefore, the cell activity of the IFNα domain in the mutant strain (South Africa, UK) vaccine and the parent strain vaccine (V-01) structure prepared in Example 1 was detected using interferon recombinant cells (reporter gene method). Specifically as follows:

[0125] Research methods: The parent strain vaccine (V-01) and the mutant strain vaccine (British strain, South African strain) were diluted to 12 μg / mL (2×, final concentration of 6 μg / mL) using assay culture medium (DMEM containing 1% GlutaMax, 10% FBS), added to the second column of the dilution plate as the starting concentration, and diluted to the 11th concentration gradient according to the gradient of about 3.5 times in columns 3-7 and about 6 times in columns 8-12. The concentrations were 12, 3.43, 0.98, 0.28, 0.080, 0.023, 0.0065, 0.0011, 0.00018, 0.000030, and 0.0000050 μg / mL, respectively. Collect HEK-Lucia TM Null recombinant cells (catalog number: hkl-null, purchased from: InvivoGen) were cultured and the cell density was adjusted to 8×10 5 Cells / mL, 50 μL / well was added to a 96-well leukocyte plate, i.e. 4×10 cells 4 / well, and then add samples of each dilution gradient at 50 μL / well, with 3 replicates for each well. Set up a negative control (NC) without drug addition and a blank control (Blank) of the culture medium. Culture in a 37°C, 5% CO2 incubator for 18h~24h. After the reaction is completed, remove the culture plate and equilibrate to room temperature, add Bio-Glo luciferase reagent at 100 μL / well, and shake at 200~500 rpm at room temperature in the dark for 10 min~30 min. The chemiluminescence unit RLU value is detected by an enzyme reader, and the four-parameter fitting analysis is performed using Softmax software. And calculate the EC 50 .

[0126] Results and Conclusions: The results are shown in Table 3 and Figure 7 The IFNα domains of the parental vaccine (V-01) and the mutant vaccine (British strain and South African strain) can activate the IFNα signaling pathway and have the expected cell biological activity.50 They were 15.1ng / mL, 12.4ng / mL and 12.8ng / mL respectively.

[0127] Table 3 Biological activities of the IFNα domain of the parental vaccine (V-01) and mutant vaccines (British strain, South African strain)

[0128] sample <![CDATA[EC 50 (ng / mL)]]> Parent strain vaccine (V-01) 15.1 British strain vaccine 12.4 South African vaccine 12.8

[0129] Example 4: In vivo efficacy evaluation in mice - titer

[0130] The in vivo efficacy evaluation of vaccines in animals is crucial, as it directly reflects the immunogenicity of the vaccine and is directly related to the protective effect it produces. Therefore, the in vivo efficacy of the parental strain vaccine (V-01) and mutant strain vaccines (UK strain and South African strain) were evaluated using C57BL / 6 mice.

[0131] Research method: The parent strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) prepared in Example 1 were used to immunize 6-8 week old C57BL / 6 mice. The vaccine concentration was 10 μg / ml. Each mouse was injected with 0.1 mL into the thigh muscle each time. There were 10 mice in each group. Each mouse was immunized twice (once on the 0th day and once on the 14th day). Blood was collected from the eye sockets 28 days after the initial immunization (i.e., 14 days after the second immunization). The blood was allowed to stand at room temperature for coagulation, and then centrifuged at 4000 rpm, 2-8°C for 10 minutes to obtain the supernatant.

[0132] The geometric mean titer (GMT) of anti-RBD antibodies was determined using an enzyme-linked immunosorbent assay (ELISA): The RBD protein corresponding to each vaccine (including wild-type RBD, South African mutant RBD, and UK mutant RBD) was diluted to 1 μg / mL in PBS, and 100 μL / well was added for overnight coating. After washing twice with PBST, the plates were blocked with 1% skim milk powder or BSA. The serum was diluted 1000-fold in PBST and then serially diluted 2-fold using a 96-well plate, for a total of 12 steps. The diluted serum samples were added to the RBD protein-coated and blocked ELISA plate at 100 μL / well, and incubated at 200 rpm for 2 hours. After washing four times with PBST, an HRP-conjugated goat anti-mouse IgG secondary antibody (approximately 1:20,000) was added, and the plates were incubated at 200 rpm for 1 hour. After washing four times with PBST, 100 μL / well of TMB colorimetric solution was added for 10 minutes. After stopping with 0.2 M H2SO4, the absorbance at 450 nm and 620 nm was read. The titer of the immune serum sample was the maximum dilution factor at which the signal value was greater than the cutoff value, and the geometric mean titer of the binding titer was calculated.

[0133] Results and Conclusions: The results are shown in Table 4 and Figure 8Both the parental strain vaccine (V-01) and the mutant strain vaccine (British strain, South African strain) can induce strong immunogenicity, and the average titers of the parental strain vaccine (V-01) and the mutant strain vaccine (British strain, South African strain) are comparable.

[0134] Table 4 In vivo efficacy of parental strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) in mice

[0135] sample Wild-type RBD UK mutant RBD South African mutant RBD Parent strain vaccine (V-01) 406375 322540 237024 British strain vaccine 406375 512000 203187 South African vaccine 376252 298631 348362

[0136] Example 5: Pseudovirus Neutralization Titer-Protection Assessment

[0137] After the vaccine is immunized in animals, specific antibodies are produced. However, whether the antibodies can protect the body from being attacked by the virus requires the evaluation of the protective power of the serum after immunization for the virus attack. Due to the danger of the new coronavirus, it is very difficult to evaluate the attack with a real virus. Therefore, the pseudovirus protective power of the parental strain vaccine (V-01) and mutant strain vaccine (British strain, South African strain) prepared in Example 1 was evaluated using a recombinant new coronavirus S protein, a pseudovirus packaged with VSV G as the backbone, and a pseudovirus carrying a luciferase reporter gene.

[0138] Research methods: Serum samples were inactivated in a 56°C water bath for 30 minutes in advance. Anti-RBD mouse neutralizing antibodies were taken and first diluted to 25 μg / mL with detection culture medium (10% FBS DMEM) as a positive quality control (PC). Take the serum sample to be tested, dilute it to a first-well concentration of 5% (ie 1:20) in a 96-well all-leukocyte plate, and then dilute it with the positive quality control product at a ratio of 1:3, for a total of 8 dilution gradients, and the serum sample for each dilution gradient is 100 μL / well. Melt the pseudovirus at 4°C in advance, and dilute the pseudovirus corresponding to each vaccine (including wild-type pseudovirus, South African mutant pseudovirus, and British mutant pseudovirus) to 20,000 TCID with detection culture medium. 50 / mL, add 50μL / well of pseudovirus dilution solution to the serum dilution plate, the pseudovirus amount is 1000TCID 50 / well, the initial serum dilution is 1:30. Simultaneously set up a pseudovirus control (VC, without serum) and a cell control (CC, without serum and pseudovirus), and then place the whole white blood cell plate in a 37°C, 5% CO2 incubator for 1-2 hours. Collect HEK293T-ACE2 cells, resuspend and count in the assay medium, and adjust the cell concentration to 2.5×10 5 Cells were added at 100 μL / well, i.e. 2.5×10 cells per well. 4Incubate the all-leukocyte plate at 37°C in a 5% CO2 incubator for 20–28 hours. Before testing, aspirate and discard 150 μL / well of supernatant. Then, add 100 μL / well of luciferase assay reagent. Use a multichannel pipette to pipette the liquid in the reaction well 6–8 times. Incubate at room temperature in the dark for 5 minutes. Read the chemiluminescence units (RLUs) using a microplate reader. Calculate the inhibition rate using the following formula: Inhibition rate (%) = [1 – (RLU value of sample group – mean CC value of cell control) / (mean VC value of pseudovirus control – mean CC value of cell control)] × 100%.

[0139] The inhibition rate data results were imported into the software, with the horizontal axis representing the logarithm of the dilution ratio (Log Titer) and the vertical axis representing the percentage of inhibition (% Inhibition). Four-parameter fitting analysis was performed, and the software automatically calculated pNT 50 The value is rounded to the nearest integer and is the pseudovirus neutralization titer.

[0140] Results and Conclusions: The results are shown in Table 5 and Figure 9 The serum after immunization with the parental strain vaccine (V-01) can neutralize the wild-type pseudovirus, the British mutant pseudovirus and the South African mutant pseudovirus. 50 They are 1977, 1131 and 418 respectively. Compared with the wild-type pseudovirus, their neutralizing effects on the British mutant pseudovirus and the South African mutant pseudovirus decreased by 1.7 times and 4.7 times respectively. The neutralizing effect on the South African mutant pseudovirus was greatly reduced, and the difference was significant.

[0141] The serum after immunization with the British strain vaccine can also neutralize the wild-type pseudovirus, the British mutant pseudovirus and the South African mutant pseudovirus. 50 The neutralization efficacy of the UK strain vaccine against the wild-type and UK mutant pseudoviruses was better than that against the South African mutant pseudovirus. Compared with the parental strain vaccine (V-01), the UK strain vaccine was designed to significantly increase its protection against the UK mutation by approximately 1.8 times, but its protection against the South African mutation was slightly lower.

[0142] The serum after vaccination with the South African strain vaccine can neutralize the wild-type pseudovirus, the British mutant pseudovirus and the South African mutant pseudovirus. 50 The neutralization efficacy of the South African mutant pseudovirus was significantly higher than that of the parental vaccine (V-01), increasing by approximately 4.1 times, and the difference was extremely significant. Therefore, the design of the South African vaccine significantly improved protection against the South African mutation.

[0143] Table 5 Neutralization titers of pseudoviruses of parental strain vaccine (V-01) and mutant strain vaccine (UK strain, South Africa strain)

[0144] sample Wild-type pseudovirus British mutant pseudovirus South African mutant pseudovirus Parent strain vaccine (V-01) 1977 1131 418 British strain vaccine 846 1535 217 South African vaccine 279 496 1136 SEQUENCE LISTING <110> Zhuhai Livzon Monoclonal Antibody Biotechnology Co., Ltd. <120> A novel coronavirus SARS-CoV-2 mutant vaccine and its application <130> MTI21145 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 165 <212> PRT <213> Artificial Sequence <400> 1 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210> 2 <211> 223 <212> PRT <213> Artificial Sequence <400> 2 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 3 <211> 223 <212> PRT <213> Artificial Sequence <400> 3 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 4 <211> 232 <212> PRT <213> Artificial Sequence <400> 4 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <400> 5 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <400> 6 Ala Trp Phe Val Ala Ala Asn Thr Leu His Ala Ala Ala 1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <400> 7 Ala Lys Tyr Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <400> 8 Ala Lys Phe Val Ala Ala Tyr Thr Leu Lys Ala Ala Ala 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <400> 9 Ala Phe Phe Val Ala Ala Asn Thr Leu Lys Ala Ala Ala 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <400> 10 Ala Lys Tyr Val Ala Ala Tyr Thr Leu Lys Ala Ala Ala 1 5 10 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <400> 11 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <400> 12 Gly Ser Gly Ser Gly Ser 1 5 <210> 13 <211> 654 <212> PRT <213> Artificial Sequence <400> 13 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 165 170 175 Gly Gly Gly Ser Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala 180 185 190 Ala Gly Ser Gly Ser Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val 195 200 205 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 210 215 220 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 225 230 235 240 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 245 250 255 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 260 265 270 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 275 280 285 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 290 295 300 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 305 310 315 320 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 325 330 335 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 340 345 350 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 355 360 365 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 370 375 380 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 385 390 395 400 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 405 410 415 Asn Lys Cys Val Asn Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr 420 425 430 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 435 440 445 Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro 450 455 460 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 465 470 475 480 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 485 490 495 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 500 505 510 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 515 520 525 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 530 535 540 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 545 550 555 560 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 565 570 575 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 580 585 590 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 595 600 605 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 610 615 620 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His 625 630 635 640 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 645 650 <210> 14 <211> 654 <212> PRT <213> Artificial Sequence <400> 14 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 165 170 175 Gly Gly Gly Ser Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala 180 185 190 Ala Gly Ser Gly Ser Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val 195 200 205 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 210 215 220 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 225 230 235 240 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 245 250 255 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 260 265 270 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 275 280 285 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 290 295 300 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 305 310 315 320 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 325 330 335 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 340 345 350 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 355 360 365 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val 370 375 380 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 385 390 395 400 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 405 410 415 Asn Lys Cys Val Asn Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr 420 425 430 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 435 440 445 Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro 450 455 460 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 465 470 475 480 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 485 490 495 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 500 505 510 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 515 520 525 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 530 535 540 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 545 550 555 560 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 565 570 575 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 580 585 590 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 595 600 605 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 610 615 620 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His 625 630 635 640 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 645 650 <210> 15 <211> 654 <212> PRT <213> Artificial Sequence <400> 15 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Arg Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 165 170 175 Gly Gly Gly Ser Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala 180 185 190 Ala Gly Ser Gly Ser Gly Ser Arg Val Gln Pro Thr Glu Ser Ile Val 195 200 205 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 210 215 220 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 225 230 235 240 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 245 250 255 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 260 265 270 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 275 280 285 Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr 290 295 300 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 305 310 315 320 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 325 330 335 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 340 345 350 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn 355 360 365 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val 370 375 380 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 385 390 395 400 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 405 410 415 Asn Lys Cys Val Asn Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr 420 425 430 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 435 440 445 Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro 450 455 460 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 465 470 475 480 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 485 490 495 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 500 505 510 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 515 520 525 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 530 535 540 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 545 550 555 560 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 565 570 575 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 580 585 590 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 595 600 605 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 610 615 620 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His 625 630 635 640 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 645 650 <210> 16 <211> 1962 <212> DNA <213> Artificial Sequence <400> 16 tgtgacctgc ctcagaccca tagcctgggc tctaggcgga cactgatgct gctggcccag 60 atgagacgca tctccctgtt tagctgcctg aaggacagac acgatttcgg ctttccacag 120 gaggagttcg gcaatcagtt tcagaaggct gagaccatcc ccgtgctgca tgagatgatc 180 cagcagatct tcaacctgtt ttctacaaag gattccagcg ccgcttggga cgagaccctg ctggataagt tctatacaga gctgtaccag cagctgaatg acctggaggc ctgcgtgatc cagggagtgg gagtgaccga gacaccactg atgaaggagg attctatcct ggctgtgagg aagtatttca ggcggatcac cctgtatctg aaggagaaga agtactcccc ttgtgcctgg 420 gaggtggtga gagctgat catgcgctct ttttccctga gcacaaacct gcaggagtct ctgcggtcca aggaggagg aggaggatcc ggcggaggag gcagcggagg aggaggatct 540 gccaagtttg tggctgcttg gaccctgaag gctgctgctg gatctggatc cggaagcaga 660. gtgcagccaa ccgagagcat cgtgcgcttc cctaacatca caaatctgtg cccattcggc gaggtgttta atgctacccg ctttgcctcc gtgtacgctt ggaatagaaa gcgcatcagc 720 aactgcgtgg ccgactattc tgtgctgtac aactctgctt ccttcagcac ctttaagtgc 780 840. tatggcgtga gccccaccaa gctgaatgac ctgtgcttca caaacgtgta cgccgactct tttgtgatca ggggcgatga ggtgcggcag atcgctcctg gacagaccgg caagatcgct gactacaatt ataagctgcc agacgatttc acaggctgcg tgatcgcttg gaactccaac 960 aatctggata gcaaagtggg cggcaactac aattatctgt acaggctgtt ccggaagtcc 1020 aatctgaagc cttttgagag agacatctct accgagatct accaggctgg ctccacacca 1080 tgcaatggcg tggagggctt caactgttat tttcccctgc agtcctacgg cttccagcct 1140 accaacggcg tgggctatca gccataccgc gtggtggtgc tgagctttga gctgctgcac 1200 gctccagcta ccgtgtgcgg acccaagaag tctacaaacc tggtgaagaa taagtgcgtg 1260 aacttcgagc ctaagtcctg tgacaagacc catacatgcc caccttgtcc agctccagag 1320 ctgctgggag gaccaagcgt gttcctgttt ccacccaagc ctaaggatca gctgatgatc 1380 tctaggaccc ccgaggtgac atgcgtggtg gtggacgtgt cccacgagga tcctgaggtg 1440 aagtttaatt ggtacgtgga cggcgtggag gtgcataacg ctaagaccaa gccaagggag 1500 gagcagtata actctaccta ccgggtggtg tccgtgctga cagtgctgca ccaggattgg 1560 ctgaatggca aggagtacaa gtgcaaggtg agcaacaagg ctctgcctgc cccaatcgag 1620 aagaccatct ctaaggccaa gggccagccc agagagcctc aggtgtatac actgcctcca 1680 tcccgcgacg agctgaccaa gaatcaggtg agcctgacat gtctggtgaa gggcttctac 1740 ccaagcgata tcgctgtgga gtgggagtct aacggccagc ccgagaacaa ttataagacc 1800 acaccccctg tgctggacag cgatggctct ttctttctgt actccaagct gaccgtggat 1860 aagagccggt ggcagcaggg caacgtgttc tcctgctccg tgctgcatga ggccctgcac 1920 aaccattaca cacagaagag cctgtctctg tcccccggca ag 1962 <210> 17 <211> 1962 <212> DNA <213> Artificial Sequence <400> 17 tgtgacctgc cacagacaca ctccctgggc tccagacgga ccctgatgct gctggcccag 60 atgcggagaa tctccctctt tagctgtctg aaggacagac acgacttcgg attccctcaa 120 gaggaattcg gcaaccagtt ccagaaggcc gaaaccatcc ccgtcctgca tgagatgatc 180 cagcagatct tcaacctgtt cagcaccaag gactcctctg ccgcctggga cgaaaccctg 240 ctggacaagt tttatacaga actgtaccag cagctgaacg acctggaagc ctgcgtgatc 300 caaggcgtcg gcgtgaccga gacccctctg atgaaggaag attccatcct ggcagtgcgg 360 aagtacttca gacggatcac cctgtacctg aaagaaaaga agtactcccc atgcgcctgg 420 gaagtggtga gagccgagat catgagatcc ttctctctga gtaccaatct gcaagagtca 480 ctgagaagca aagagggcgg aggaggctct ggcggcggag gctccggtgg gggcggctct 540 gctaagttcg tggctgcttg gaccctgaag gccgctgctg gctctggctc cggctccaga 600 gtgcagccca ccgagtcaat cgtgcggttt cctaacatca ccaacctgtg ccctttcggc 660 gaggtgttca acgctaccag attcgcctcc gtgtacgctt ggaacagaaa gcggatttct 720 aactgcgtgg ccgattactc cgtcctgtac aattccgctt ctttttccac cttcaagtgc 780 tacggcgtgt ctcccaccaa gctgaacgat ctgtgcttca ccaacgtgta cgccgattcc 840 ttcgtgatca gaggcgacga ggtcagacag atcgcccctg gacagacagg caagatcgcc 900 gactacaact acaagctgcc cgacgacttc accggctgcg tcatcgcctg gaactccaac 960 aacctggatt ctaaagtggg cggaaattac aactacctgt atcggctgtt ccggaagagc 1020 aacctcaagc ccttcgagag ggacatctcc acagagatct accaggccgg ctctacccct 1080 tgcaacggcg tggaaggctt caattgctac ttccctctgc agtcctacgg ctttcagcct 1140 acctatggcg tgggctacca gccttaccgg gtggtggtgc tgagcttcga gctcctgcac 1200 gctcccgcca ccgtctgcgg cccaaagaaa tctaccaacc tggtgaagaa caagtgcgtg 1260 aacttcgagc ctaagtcctg tgataagacc cacacctgtc ctccttgtcc tgctccagag 1320 ctgctgggcg gacctagcgt gttcctgttt cctccaaagc ccaaggacca gctgatgatc 1380 tccagaaccc ccgaggtgac ctgtgtggtc gtggacgtgt ctcatgagga ccctgaagtg 1440 aagttcaact ggtacgtgga tggcgtggaa gtgcacaacg ccaagaccaa gcctagagaa 1500 gagcagtaca acagcaccta cagggtggtg tccgttttaa ctgtgctgca ccaggactgg 1560 ctgaacggca aagagtacaa atgcaaggta agcaacaagg ctctgcctgc tcctatcgag 1620 aaaaccatct ctaaagccaa gggccagcct cgcgagcctc aagtgtacac cctgccacct 1680 tcccgggacg agctgacaaa gaatcaggtg tccctgacct gcctggtcaa gggcttctac 1740 ccttccgata tcgccgtgga gtgggagtct aatggccagc ccgagaacaa ctacaagaca 1800 acccctcctg tgctggactc ggacggctct ttcttcctgt actccaagct gaccgtggac 1860 aagtctagat ggcagcaggg caacgtgttc tcctgctccg tgctgcacga ggccctgcac 1920 aaccactaca cccagaaatc cctttctctg tctcctggca ag 1962 <210> 18 <211> 1962 <212> DNA <213> Artificial Sequence <400> 18 tgcgacctgc ctcagaccca ctccctgggc tctaggagga ccctcatgct gctggctcag 60 atgagacgga tctctctgtt ctcttgtttg aaggaccggc acgacttcgg ctttcctcag 120 gaagagttcg gcaatcagtt ccagaaggcc gaaaccatcc cagtgctgca cgagatgatc 180 cagcagatct tcaacctgtt ttccaccaag gattcctccg cagcttggga cgagacactg 240 ctggacaagt tctacaccga gctgtaccag cagctgaacg atctggaagc ctgcgtgatc 300 cagggcgtgg gagtgaccga gacacctctg atgaaagagg actctatcct ggccgtgcgg 360 aagtactttc ggagaatcac cctgtacctg aaagaaaaga agtatagccc ctgtgcctgg 420 gaagtggtga gagccgagat catgcggtcc ttcagcctgt ccaccaacct gcaagagagc 480 ctgagatcca aagagggcgg aggcggctct ggcggtggcg gctccggcgg cggagggtcc 540 gccaagttcg tggctgcctg gaccctgaag gccgctgctg gctctggatc tggctccccgc 600 gtgcagccta ccgaaagcat cgtgagattc cctaacatca ccaacctgtg cccctttggc gaggtgttca atgccaccag attcgcctct gtgtacgctt ggaccgga gcggatcagc 720 aattgtgtcg ctgactattc cgtcctgtc aactctgcct ccttttctc cttcaagtgc 780 840. tacggcgtct cgcctaccaa gctgaacgac ctgtgcttca ccaacgtgta cgctgattcc ttcgtgatca gaggcgacga agtccggcag atcgccccag gccaaaccgg aaacattgcc 960. acaaactccc tgacgacttc accggctgcg ttatcgcctg gaactccaac aacctggact ccaaggtggg cggcaactac aactacctgt accggctgtt cagaaagtct aatctgaagc ctttcgagag agacatctcc accgagatct accaggccgg ctccaccccg 1080 tgcaacggcg tgaagggctt caactgctat ttccccctgc agtcctacgg ctttcagccc 1140 acctacggcg tgggctacca accttacaga gtcgtcgtgc tgagcttcga actgcttcac 1200 gcccctgcta ccgtgtgcgg cccaaagaaa tctacaaacc tggtcaagaa caagtgcgtg 1260 aacttcgagc ccaagtcctg tgacaaaact cacacctgtc ctccttgccc tgcccccgaa 1320 ctgctgggag gcccctccgt gtttctgttc ccacctaagc ctaaggacca gctgatgatc 1380 tcccgcacac ccgaggtgac ctgtgtggtg gtcgacgtgt ctcatgagga tcctgaggtg 1440 aagttcaact ggtacgtgga tggcgtggaa gtgcacaacg ccaagacgaa gcccagagag 1500 gaacagtaca actccaccta cagagtggtg tccgtgctga cagttctgca ccaggactgg 1560 ctgaatggca aagagtacaa gtgcaaggtg tccaacaagg ctctgcccgc tcctatcgag 1620 aagacaatca gcaaggccaa aggccagcct agagagcctc aagtgtacac cctgcctcct 1680 tctagagatg agctgactaa gaaccaggtg tctctgacct gcctggtgaa gggcttctac 1740 ccttctgata tcgctgtgga atgggagtct aatggccagc ctgagaacaa ctacaagacc 1800 acccctccag tgctcgattc cgacggatct ttcttcctgt attccaagct gaccgtggac 1860 aagtccagat ggcagcaggg caacgtgttc tcctgctccg tgctgcacga ggccctgcat 1920 aaccactaca cccagaaatc tctgtccctg tctcctggca at 1962

Claims

1. A fusion protein comprising, from N-terminus to C-terminus: (1) Interferon IFN-α2a or its functional fragments; (2) the receptor binding domain (RBD) of the novel coronavirus SARS-CoV-2; and (3) Fc region of immunoglobulin IgG1; The amino acid sequence of the fusion protein is selected from the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO:

15.

2. A nucleic acid encoding the fusion protein of claim 1.

3. The nucleic acid according to claim 2, wherein The nucleic acid is selected from the group consisting of the nucleic acids shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:

18.

4. A vector comprising the nucleic acid according to claim 2 or 3.

5. A host cell expressing the fusion protein of claim 1, comprising the nucleic acid of claim 2 or 3 and / or comprising the vector of claim 4.

6. The host cell according to claim 5, wherein The host cell is a prokaryotic cell or a eukaryotic cell.

7. The host cell according to claim 6, wherein The prokaryotic cell is a bacterial cell.

8. The host cell according to claim 6, wherein The prokaryotic cells are Escherichia coli cells.

9. The host cell according to claim 6, wherein The eukaryotic cell is selected from the group consisting of yeast cells, insect cells and mammalian cells.

10. The host cell according to claim 9, wherein The mammalian cells are selected from CHO, HEK293, SP2 / 0, BHK, and C127.

11. The host cell according to claim 10, wherein The eukaryotic cells are CHO cells.

12. A vaccine for treating and / or preventing novel coronavirus SARS-CoV-2 infection or novel coronavirus disease COVID-19, comprising the fusion protein of claim 1, the nucleic acid of claim 2 or 3, the vector of claim 4 and / or the host cell of any one of claims 5 to 11, and optionally, a pharmaceutically acceptable carrier and / or excipient.

13. The vaccine according to claim 12, wherein the vaccine is in the form of a recombinant protein subunit vaccine, a recombinant protein mRNA vaccine or a recombinant protein adenovirus vector vaccine.

14. A method for preparing a vaccine for preventing and / or treating novel coronavirus SARS-CoV-2 infection or coronavirus disease COVID-19, the method comprising expressing the fusion protein of claim 1.

15. Use of the fusion protein of claim 1, the nucleic acid of claim 2 or 3, the vector of claim 4, the host cell of any one of claims 5 to 11, and / or the vaccine of claim 12 or 13 in the preparation of a medicament or product for preventing and / or treating infection with the novel coronavirus SARS-CoV-2 and / or the novel coronavirus disease COVID-19.

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