Methods for enhancing the immunogenicity of protein / peptide antigens by fusion to altered fcregions

Abstract

A method for enhancing the immunogenicity of a protein / peptide antigen, wherein the protein / peptide antigen forms a fusion protein with an engineered antibody Fc fragment, which has an altered amino acid sequence and / or glycosylation form, and has an improved binding ability to Fc receptors and / or complement protein C1q compared with its native form. The SARS-CoV-2 vaccine prepared with the fusion protein as an immunogen has a higher binding ability to Fc receptors, can maintain long-term humoral and cellular immune responses, and the immunized animals can produce higher titers of neutralizing antibodies. The recombinant protein vaccine can be used for preventing SARS-CoV-2 infection-related diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202010394463.6, filed on May 11, 2020, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of immunology, specifically relating to a method for enhancing the immunogenicity of protein / peptide antigens, wherein the protein / peptide antigen forms a fusion protein with a modified antibody Fc fragment. This Fc fragment, due to alterations in its amino acid sequence and / or glycosylation, exhibits enhanced binding affinity to Fc receptors and / or complement protein C1q compared to its native form. SARS-CoV-2 vaccines prepared using the fusion protein of this invention as an immunogen demonstrate high binding affinity to Fc receptors, maintain long-term humoral and cellular immune responses, and produce high titers of neutralizing antibodies in immunized animals. The vaccine can be used to prevent SARS-CoV-2 infection-related diseases. Background Technology

[0004] 1. Fc receptor fusion protein vaccines and FcRs-enhanced Fc fragment fusion protein vaccines

[0005] B cell-mediated humoral immunity is one of the vaccine-mediated protective mechanisms. Studies have shown that the interaction between Fc and Fc receptors (FcRs) and complement receptors (CRs) can mediate better antigen capture and presentation, promote B cell affinity maturation, and generate high-affinity antibodies [1-6]. In addition, after Fc binds to receptors on follicular dendritic cells (fDCs) (mediated by FcRs or CRs), it is displayed on the surface of fDCs. This antigen is extremely important for maintaining the long-term presence of antigens and maintaining the survival of antigen-specific B cells [7].

[0006] Furthermore, cytotoxic T lymphocytes (CTLs) play a crucial role in resisting viral infection and clearing virus-infected cells. Antigen cross-presentation allows exogenous antigens to enter the cell's endogenous processing and presentation mechanisms, thereby displaying exogenous antigen peptides on MHC class I molecules for T cell recognition and initiation of a CTL cell response. Cross-presentation of exogenous antigens is significant for effectively activating CTLs and triggering an antiviral immune response; therefore, enhancing the cross-presentation of subunit vaccines is one of the effective strategies for improving vaccine efficacy. Dendritic cells (DCs) are currently known as the most potent professional antigen-presenting cells and are the primary cells responsible for cross-presentation. Exogenous antigens enter DCs mainly through three pathways: phagocytosis, pinocytosis, and receptor-mediated endocytosis. Currently known endocytosis receptors involved in cross-presentation of exogenous antigens include C-type lectin receptors (CLRs), Fc receptors (FcRs) that recognize immune complex IgG, scavenger receptors that recognize apoptotic cells, and chemokine receptors. These receptors mediate antigen endocytosis, allowing the antigen to enter specific endosomes and bind to MHC class I molecules, thereby activating CD8+. + T cells. Antigen-antibody complexes (immune complexes) can be recognized by FcRs of DC cells, and the resulting cross-linking can internalize antigens and cross-process and present them, specifically activating CTL responses [8-10]. This FcR-mediated antigen cross-presentation has been shown to induce strong CTL responses

[11] .

[0007] The Fc receptors (FcγR) that bind to IgG in the human body mainly include FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32B), and FcγRIII (CD16)

[12] . Among them, FcγRIIB is an inhibitory receptor, mainly expressed on B cells, macrophages, and mast cells

[13] . It can be further divided into FcγRIIB-1 and FcγRIIB-2. FcγRIIB-1 is expressed only on B cells and controls the overactivation of B cells and the recognition of self-antigens during B cell development. It transmits B cell apoptosis signals through the intracellular receptor tyrosine inhibitory motif (ITIM) to achieve the negative selection process of B cells and regulate the development process of B cells. FcγRIIB-2 is expressed on other immune cells except NK and T cells. Through receptor cross-linking, it can effectively induce the phagocytosis of antigen-antibody complexes [14, 15]. The other Fc receptors are all activating receptors. Among them, CD64 is mainly expressed on monocytes, macrophages, and dendritic cells, CD32a is mainly expressed on neutrophils, monocytes, macrophages, and dendritic cells, and CD16a is mainly expressed on NK cells, monocytes, and macrophages

[15] . After recognizing antigen-antibody complexes, the intracellular receptor tyrosine activation motif (ITAM) determines the antigen uptake initiated by antigen-presenting cells (APCs) and the antigen presentation function performed by MHC molecules after uptake

[16] .

[0008] Fc fusion proteins are novel recombinant proteins produced by fusing a bioactive functional protein with an Fc fragment using genetic engineering and other technologies. They not only retain the biological activity of the functional protein molecule but also possess some antibody properties, such as the binding and related biological functions mediated by Fc receptors. Antigen-Fc fusion proteins can serve as antigen delivery vehicles, using Fc fragments to target and bind to antigen-presenting cells, shortening the free time of antigens in plasma, increasing the antigen half-life, and thus enhancing antigen presentation and cross-presentation reactions. Based on this, modifying the Fc fragment to enhance Fc receptor binding yields Fe4-Fc modified molecules, which can increase the binding of Fc to complement protein C1q and Fc receptors CD16, CD32, CD64, etc. [17-21]. This may further enhance Fc and its receptor-mediated antigen capture and presentation, improve B cell maturation and the production of high-affinity antibodies, maintain long-term humoral immunity, and enhance CTL immune responses mediated by antigen cross-presentation, thereby enhancing the immune effect of Fc fusion protein vaccines.

[0009] 2. Existing clinical cases of Fc fusion protein vaccines

[0010] Currently, there are relatively few Fc fusion protein vaccines being developed in clinical practice. In a phase IIb clinical trial, chronic hepatitis B cHBV infected patients were treated with a YIC immunogenic complex (a mixture of yeast-expressed hepatitis B surface antigen HBsAg and HBsAg-immunized human serum neutralizing antibody HBIG in a specific ratio, with the addition of aluminum adjuvant) at a dose of 60 μg / 4 weeks. Compared with the control group that received only aluminum adjuvant, the seroconversion rate of serum hepatitis B virus e antigen (HBeAg) was significantly increased (21.8% vs 9%), the viral titer was significantly decreased, and anti-E antigen antibodies were produced

[22] . At the same time, under in vitro conditions, YIC can increase the maturation (high expression of CD83), antigen recognition and presentation (expression of HLA-II, CD86, CD80, CD40 markers) of DC cells in cHBV infected patients, and secrete more inflammatory factors (IL-12). The patient’s DC-PBMC mixed cells produced more T lymphocyte cytokines (Th1 cells: IL-2, IFNγ) (Th2 cells: IL-5, IL-10) under YIC stimulation than under HBsAg antigen stimulation alone

[23] . However, overstimulation by YIC may induce immune fatigue in the body, thereby reducing the cellular immune response

[24] . Therefore, appropriate immunization protocols are also very important in order to achieve better immune effects.

[0011] Other vaccines based on viral antigen-Fc fusion proteins (RSV, HBV, DENV, TB) have also been tested in animal models, achieving effective activation of the immune system and producing antibodies that reduce viral antigen levels in the serum [25-28].

[0012] The safety risks posed by the activation of the immune system by Fc fusion protein vaccines are also a topic worthy of consideration. In the chronic hepatitis B cHBV infected patients mentioned above, a small number of patients experienced a transient increase in alanine aminotransferase (ALT) after YIC treatment. The proportion of patients with ALT increase was similar to that of the control group that received only aluminum adjuvant. The increase in transaminase levels reflects liver damage to some extent, but subsequent observations showed that ALT could return to normal levels

[24] . In its phase IIa clinical trials, some patients with HBeAg antigen seroconversion were also observed to have elevated ALT

[23] . The clinical adverse events (AEs) data of YIC in Phase IIb showed that the proportion of serious AEs in the YIC 30 μg dose group, YIC 60 μg dose group and aluminum adjuvant control group were similar (3.6% vs 5% vs 5.1%). The most common AEs in the YIC group were injection site-related reactions, including inflammatory reactions such as rash, swelling and itching. Other systemic AEs such as fever, headache and nausea were not significantly different from those in the control group

[22] . Therefore, immune complexes containing Fc have relatively good safety in clinical practice, and no serious adverse reactions related to excessive activation of the immune system occurred. Summary of the Invention

[0013] In one aspect, the present invention provides a method for enhancing the immunogenicity of a protein / peptide antigen, the method comprising fusing the protein / peptide antigen with a modified antibody Fc fragment having an enhanced binding capacity to an Fc receptor and / or complement protein C1q compared to its native form, thereby mimicking the antigen-antibody complex to enhance phagocytosis by DC / B antigen-presenting cells and enhance antigen-immune effects.

[0014] In one embodiment, the protein / peptide antigen in the method is a pathogen-associated protein / peptide antigen or a tumor-associated protein / peptide antigen.

[0015] In one embodiment, the pathogen in the method is selected from:

[0016] Coronavirus, Human Immunodeficiency Virus (HIV-1), Human Herpes Simplex Virus, Cytomegalovirus, Rotavirus, Epstein-Barr Virus, Varicella-Zoster Virus, Hepatitis Virus, Respiratory Syncytial Virus, Parainfluenza Virus, Measles Virus, Mumps Virus, Human Papillomavirus, Flavivir or Influenza Virus, Neisseria spp., Moraxella spp., Bordetella spp., Mycobacterium spp., including Mycobacterium tuberculosis; Escherichia spp., including Enterotoxigenic Escherichia coli; Salmonella spp., Listeria spp., Helicobacter spp., Staphylococcus spp., including Staphylococcus aureus, Staphylococcus epidermidis; Treponema spp., Chlamydia spp., including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium spp., including Plasmodium falciparum; Toxoplasma gondii, Candida albicans;

[0017] The tumors were selected from:

[0018] Diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyosarcoma, hepatocellular carcinoma, prostate cancer, breast cancer, cholangiocarcinoma and gallbladder cancer, bladder cancer, brain tumors including neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcoma.

[0019] In one embodiment, the protein / peptide antigen in the method is selected from secreted proteins or full-length membrane proteins, or their functional domains, mutant proteins, truncated proteins, or modified proteins composed of one or more antigenic polypeptide epitopes.

[0020] In one embodiment, the Fc fragment in the method is derived from the heavy chain constant region of a human antibody, mouse antibody, rabbit antibody, or other animal antibody.

[0021] In one embodiment, the Fc fragment in the method is derived from an IgG, IgM, or IgA subtype antibody of a human antibody.

[0022] Preferably, the antibody is derived from IgG1, IgG2, IgG3 or IgG4 subtypes;

[0023] More preferably, it is an IgG1-modified Fc fragment with altered amino acid sequence and / or glycosylation form for the purpose of improving its binding function with Fc receptor and C1q complement.

[0024] In one embodiment, the Fc receptor of the method is selected from CD16, CD32a, CD32b or CD64.

[0025] In one embodiment, the protein / peptide antigen in the method is the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein.

[0026] In one embodiment, the modified antibody Fc fragment in the method is an Fc receptor CD32a, CD32b and CD64 binding enhancement fragment / complement C1q binding enhancement fragment;

[0027] Its amino acid sequence is shown in SEQ ID NO: 30.

[0028] In one implementation, the method

[0029] The modified antibody Fc fragment is an enhanced fragment for binding to Fc receptors CD16a, CD32a, CD32b, and CD64 / complement C1q binding.

[0030] Its amino acid sequence is shown in SEQ ID NO: 30, and it is produced using Fucose knockout mammalian cells, preferably, the mammalian cells are HEK-293 cells with the fut8 gene knocked out.

[0031] In one embodiment, the Fc fragment in the method is derived from mouse antibody IgG, IgM, or IgA subtype antibody.

[0032] Preferably, the antibody is derived from IgG1, IgG2a, IgG2b or IgG3 subtypes.

[0033] In one embodiment, the antigen in the method is preferably conjugated to other macromolecules via a linker, preferably, the other macromolecules being polysaccharides, peptides / proteins.

[0034] In another aspect, the present invention provides an immunogenically enhanced protein / peptide antigen, wherein

[0035] The protein / peptide antigen is fused with a modified antibody Fc fragment, which, compared to its natural form, has an enhanced binding capacity to the Fc receptor and / or complement protein C1q. This can mimic the antigen-antibody complex to enhance phagocytosis by DC / B antigen-presenting cells and improve antigen-immune efficacy.

[0036] In one embodiment, the antigen in the protein / peptide antigen is a pathogen-associated protein / peptide antigen or a tumor-associated protein / peptide antigen.

[0037] In one embodiment, the pathogen in the protein / peptide antigen is selected from:

[0038] Coronavirus, Human Immunodeficiency Virus (HIV-1), Human Herpes Simplex Virus, Cytomegalovirus, Rotavirus, Epstein-Barr Virus, Varicella-Zoster Virus, Hepatitis Virus, Respiratory Syncytial Virus, Parainfluenza Virus, Measles Virus, Mumps Virus, Human Papillomavirus, Flavivir or Influenza Virus, Neisseria spp., Moraxella spp., Bordetella spp., Mycobacterium spp., including Mycobacterium tuberculosis; Escherichia spp., including Enterotoxigenic Escherichia coli; Salmonella spp., Listeria spp., Helicobacter spp., Staphylococcus spp., including Staphylococcus aureus, Staphylococcus epidermidis; Treponema spp., Chlamydia spp., including Chlamydia trachomatis, Chlamydia pneumoniae; Plasmodium spp., including Plasmodium falciparum; Toxoplasma gondii, Candida albicans;

[0039] The tumors were selected from:

[0040] Diffuse large B-cell lymphoma, follicular lymphoma, other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdomyosarcoma, hepatocellular carcinoma, prostate cancer, breast cancer, cholangiocarcinoma and gallbladder cancer, bladder cancer, brain tumors including neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcoma.

[0041] In one embodiment, the protein antigen in the protein / peptide antigen is selected from secretory proteins or full-length membrane proteins, or their functional domains, mutant proteins, truncated proteins, or modified proteins composed of one or more of their antigenic polypeptide epitopes.

[0042] In one embodiment, the Fc fragment in the protein / peptide antigen is derived from the heavy chain constant region of a human antibody, mouse antibody, rabbit antibody, or other animal antibody.

[0043] In one embodiment, the Fc fragment in the protein / peptide antigen is derived from an IgG, IgM, or IgA subtype antibody.

[0044] Preferably, the antibody is derived from IgG1, IgG2, IgG3 or IgG4 subtypes;

[0045] More preferably, it is an IgG1-modified Fc fragment with altered amino acid sequence and / or glycosylation form for the purpose of improving binding function with Fc receptor and / or C1q complement.

[0046] In one embodiment, the Fc receptor in the protein / peptide antigen is selected from CD16, CD32a, CD32b, or CD64.

[0047] In one embodiment, the antigen in the protein / peptide antigen is the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein.

[0048] In one embodiment, the protein / peptide antigen wherein

[0049] The modified antibody Fc fragment is an enhanced fragment for binding to Fc receptors CD32a, CD32b, and CD64 / an enhanced fragment for binding to complement C1q;

[0050] Its amino acid sequence is shown in SEQ ID NO: 30.

[0051] In one embodiment, the protein / peptide antigen wherein

[0052] The modified antibody Fc fragment is an enhanced fragment for binding to Fc receptors CD16a, CD32a, CD32b, and CD64 / complement C1q binding.

[0053] Its amino acid sequence is shown in SEQ ID NO: 30, and it is produced using Fucose knockout mammalian cells.

[0054] Preferably, the mammalian cells are HEK-293 cells with the fut8 gene knocked out.

[0055] In one embodiment, the Fc fragment in the protein / peptide antigen is derived from mouse antibody IgG, IgM, or IgA subtype antibody.

[0056] Preferably, the antibody is derived from IgG1, IgG2a, IgG2b or IgG3 subtypes.

[0057] In one embodiment, the antigen in the protein / peptide antigen is preferably conjugated to other macromolecules via a linker, preferably, the other macromolecules are polysaccharides, peptides / proteins.

[0058] In another aspect, the present invention provides a conjugate comprising, preferably by means of a linker, a protein / peptide antigen as described herein and other macromolecules, preferably polysaccharides, peptides / proteins.

[0059] In another aspect, the present invention provides a nucleic acid encoding the protein / peptide antigen described herein, which is mRNA and / or DNA.

[0060] In one embodiment, the nucleic acid sequence is shown in SEQ ID NO:31.

[0061] In another aspect, the present invention provides an expression vector comprising the nucleic acid described herein.

[0062] In another aspect, the present invention provides a host cell comprising the nucleic acid or expression vector described herein.

[0063] In another aspect, the present invention provides a method for producing the protein / peptide antigen described herein, comprising culturing the host cells described herein under conditions suitable for the expression of the aforementioned protein molecules, and recovering the expressed product from the culture medium.

[0064] In another aspect, the present invention provides an immune composition comprising...

[0065] a) The protein / peptide antigen, the conjugate, the nucleic acid, or the expression vector described in this invention; and / or

[0066] b) Adjuvants; and

[0067] c) Pharmaceutically acceptable carriers, excipients, or stabilizers, preferably

[0068] Pharmaceutically acceptable carriers, excipients, or stabilizers in lyophilized or aqueous solutions.

[0069] In one embodiment, the adjuvant in the immune composition may be selected from at least one of aluminum adjuvant, MF59, QS-21, or MPL.

[0070] In another aspect, the present invention provides the protein / peptide antigen, the conjugate, the nucleic acid, the expression vector, or the immune composition of the present invention for use in preventing pathogens, preferably coronaviruses, more preferably diseases / tumors caused by SARS-CoV-2.

[0071] In another aspect, the present invention provides the use of the protein / peptide antigen, the conjugate, the nucleic acid, the expression vector, or the immune composition of the present invention in the preparation of a vaccine against pathogens, preferably coronaviruses, more preferably SARS-CoV-2-induced diseases / tumors.

[0072] In another aspect, the present invention provides an immune combination comprising...

[0073] The protein / peptide antigen, the conjugate, the nucleic acid, the expression vector, or the immune composition described in this invention; and

[0074] One or more other immunogenic agents.

[0075] In another aspect, the present invention provides a kit comprising...

[0076] The protein / peptide antigen, the conjugate, the nucleic acid, the expression vector, or the immune composition described in this invention;

[0077] Preferably,

[0078] It also includes a device for administering the vaccine.

[0079] In another aspect, the present invention provides a method for preventing diseases caused by pathogens, preferably coronaviruses, more preferably SARS-CoV-2, or for preventing tumors, comprising administering to a subject the protein / peptide antigen, conjugate, nucleic acid, expression vector, or immune composition, vaccine combination, or kit described herein.

[0080] In another aspect, the present invention provides a method for immunizing animals, comprising administering to the animal the protein / peptide antigen, the conjugate, the nucleic acid, the expression vector, the immune composition, the vaccine combination, or the kit described herein to generate neutralizing antibodies. Attached Figure Description

[0081] Figure 1 : RBD-mFc binds to mouse Fc receptors.

[0082] Figure 2 : The binding of different RBD-Fc fusion proteins to human Fc receptor and C1q.

[0083] Figure 3 MDM phagocytosis assay of different RBD-Fc fusion proteins.

[0084] Figure 4 Comparison of antibody titers and neutralizing titers in RBD-his and RBD-mFc immune serum (Mean ± SEM, n = 5).

[0085] Figure 5 Comparison of antibody titers and neutralizing titers in immune serum of RBD and different RBD-Fc fusion proteins (Mean ± SEM, n = 5). Detailed Implementation

[0086] The inventors have made groundbreaking discoveries that by fusing protein / peptide antigens with modified antibody Fc fragments, their immunogenicity is enhanced. These Fc fragments, due to alterations in their amino acid sequence and / or glycosylation, possess increased binding capacity to Fc receptors and / or complement protein C1q.

[0087] The inventors used this discovery to develop a vaccine for the novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2).

[0088] SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, namely angiotensin-converting enzyme 2 (ACE2)

[29] . After the viral trimeric S protein binds to the ACE2 receptor, it is cleaved by the host protease into an S1 polypeptide containing a receptor-binding domain (RBD) and an S2 polypeptide responsible for mediating viral fusion with the cell membrane

[30] . The specific interaction between S1 and ACE2 triggers a conformational change in the S2 subunit, which leads to the fusion of the viral envelope with the cell membrane or lysosomal membrane and release of viral nucleic acid into the cytoplasm

[31] . Data show that COVID-19 patients, especially those with severe symptoms, have significantly reduced lymphocytes and significantly increased plasma pro-inflammatory factors, suggesting that the immune system plays an important role in the disease process [32-34]. Analysis of serum antibodies in 23 COVID-19 patients after the onset of symptoms showed that most patients developed an antibody response against the RBD protein 10 days after the onset of symptoms.

[35] In the early stages of the disease, the proportion of patients with positive RBD protein antibodies was higher than that of patients with positive N protein antibodies, indicating that the body may first produce neutralizing antibodies to inhibit viral invasion of cells via RBD. Analysis of cellular immunity showed that the specific T cells against different antigens in newly discharged patients differed significantly from those in uninfected individuals, with RBD-specific T cells being the most widely distributed. Follow-up patients two weeks after recovery showed a significant decrease in cellular immunity levels. RBD not only induces humoral immunity and the production of neutralizing antibodies but also induces T-cell immune responses; therefore, RBD protein is an effective target for SARS-CoV-2 vaccines.

[0089] One particularly preferred embodiment of the invention is the RBD-Fe4-Fc fusion protein, which maximizes the efficacy advantages of antigen-antibody complex vaccines. Based on the clinical adverse events (AEs) assessment of antigen-antibody complexes, it is inferred that Fc fusion protein vaccines are safe and controllable.

[0090] definition

[0091] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of this invention, the following terms are further defined.

[0092] When used herein and in the appended claims, the singular forms “a,” “an,” “another,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0093] The terms "comprising" or "including" mean that a specific ingredient is included without excluding any other ingredients. Phrases such as "consistently composed of" allow the inclusion of other ingredients or steps that do not impair the novelty or essential characteristics of the invention; that is, they exclude other unlisted ingredients or steps that would impair the novelty or essential characteristics of the invention. The term "composed of" means that a specific ingredient or group of ingredients is included and all other ingredients are excluded.

[0094] The term "RBD receptor-binding domain" specifically refers to the "ACE2 receptor-binding domain of the coronavirus spike protein (SARS-CoV-2 RBD)" in this specification and the appended claims. These terms are used interchangeably. SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin-converting enzyme 2 (ACE2). After the viral trimeric S protein binds to the ACE2 receptor, it is cleaved by host proteases into an S1 polypeptide containing the receptor-binding domain (RBD) and an S2 polypeptide responsible for mediating viral fusion with the cell membrane.

[0095] The term "antigen" refers to a foreign substance that is recognized (specifically bound) by an antibody or T-cell receptor, but which cannot definitively induce an immune response. Foreign substances that induce specific immunity are called "immunogenic antigens" or "immunogens." A "hapten" is an antigen that cannot elicit an immune response on its own (although a combination of several hapten molecules, or a combination of a hapten and a large molecular carrier, can elicit an immune response).

[0096] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” encompass chains of amino acids of any length, with relatively short chains (e.g., shorter than 100 amino acids) often referred to as peptides. This chain can be linear or branched, and may contain modified amino acids and / or intercalated non-amino acids.

[0097] The term "antibody" refers to an immunoglobulin molecule, meaning any form of antibody that expresses desired biological activity. This includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), and even antibody fragments. Typically, a full-length antibody structure preferably comprises four polypeptide chains, typically two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. Besides this typical full-length antibody structure, other derived forms are also possible.

[0098] Based on the amino acid sequence of their heavy chain constant region, complete antibodies can be classified into five classes: IgA, IgD, IgE, IgG, and IgM. IgG and IgA can be further subdivided into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Correspondingly, the heavy chains of these five antibody classes are classified as α, δ, ε, γ, and μ chains, respectively. Based on the amino acid sequence of their light chain constant region, the light chains of antibodies can be classified as κ and λ.

[0099] The term "variable region" refers to the domain in the antibody heavy or light chain that is involved in antibody binding to the antigen.

[0100] The term "constant region" refers to certain amino acid sequences on the light and heavy chains of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit a variety of effector functions, such as antibody-dependent cytotoxicity.

[0101] The term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as an amino acid residue extending from Cys226 or from Pro230 to its C-terminus. The residues in the Fc region are numbered as per the EU index in Kabat. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The Fc region of IgG typically has two constant regions, CH2 and CH3.

[0102] The term “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody. Preferred are human FcRs of natural sequence, and more preferably are receptors (γ receptors) that bind to IgG antibodies, including FcγRI, FcγRII, and FcγRIII subtypes, as well as variants of these receptors. Other FcRs are included in the term “FcR”. The term also includes the neonatal receptor (FcRn), which is responsible for transporting maternal IgG to the fetus (Guyer et al., Journal of Immunology 117: 587 (1976) and Kim et al., Journal of Immunology 24: 249 (1994)).

[0103] The term "neonatal Fc receptor," or simply "FcRn," refers to the receptor that binds to the Fc region of IgG antibodies. Neonatal Fc receptors (FcRn) play a crucial role in the metabolic fate of IgG antibodies in vivo. FcRn functions to rescue IgG from lysosomal degradation pathways, thereby reducing its clearance in serum and prolonging its half-life. Therefore, the in vitro FcRn binding properties / characteristics of IgG indicate its in vivo pharmacokinetic properties in the bloodstream.

[0104] The terms “modified Fc peptide”, “modified Fc region” and “modified Fc” are used interchangeably herein to refer to an Fc peptide or a portion thereof that contains at least one amino acid modification or a modified glycosylation modification thereof.

[0105] The term "Fc fusion protein" refers to a novel recombinant protein produced by fusing a biologically active functional protein with an Fc fragment using techniques such as genetic engineering. It not only retains the biological activity of the functional protein molecule, but also has some antibody properties, such as the binding of FcRs and related biological functions.

[0106] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary from antibody isotype to antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor (such as CD16, CD32, CD64) binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (such as B cell receptors), and B cell activation.

[0107] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fcγ receptors present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigens, which are then killed using, for example, cytotoxins. To assess the ADCC activity of a target antibody, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337 or 6,737,056 (Presta), or the methods described in the embodiments of this application. Useful effector cells for such assays include PBMCs and NK cells.

[0108] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component (C1q) of the complement system to an antibody (of an appropriate subclass) which binds to its corresponding antigen. To assess complement activation, CDC assays can be performed, such as those described in Gazzano-Santoro et al., J. Immunol Methods 202: 163 (1996), methods described in the examples of this application, and methods described in U.S. Patent Nos. 6,194,551 B1 and WO1999 / 51642, which describe peptide variants with altered Fc region amino acid sequences (peptides with variant Fc regions) and peptide variants with enhanced or reduced C1q binding.

[0109] "Humoral immune response" is an antibody-mediated immune response and involves the introduction and generation of antibodies that recognize and bind to antigens in the immunogenic compositions of the present invention with a certain affinity. "Cell-mediated immune response" is an immune response mediated by T cells and / or other leukocytes. "Cell-mediated immune response" is induced by the provision of antigenic epitopes associated with class I or II molecules of the major histocompatibility complex (MHC), CD1, or other atypical MHC-like molecules.

[0110] The term "conjugate" refers to a protein / peptide that is covalently conjugated with other molecules.

[0111] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, such as a microorganism or a component thereof, which can be used to induce an immune response in an individual.

[0112] As used herein, “immunogenicity” means the ability of an antigen (or an epitope of an antigen), such as the receptor-binding region of a coronavirus spike protein or a glycoconjugate or immunogenic composition containing that antigen, to induce a humoral or cellular immune response in a host (e.g., a mammal) or both.

[0113] A “protective” immune response refers to the ability of an immunogenic composition to induce a humoral or cellular immune response, or both, to protect an individual from infection. The protection provided need not be absolute, i.e., it need not completely prevent or eradicate the infection, as long as there is a statistically significant improvement relative to a control population (e.g., infected animals that have not been administered the vaccine or the immunogenic composition). Protection may be limited to mitigating the severity or rapid onset of infection symptoms.

[0114] The terms “immunogenic amount” and “immunogenically effective amount” are used interchangeably herein and refer to the amount of an antigen or immunogenic composition sufficient to elicit an immune response (cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to those skilled in the art).

[0115] The effectiveness of an antigen as an immunogen can be measured by proliferation assay, cell lysis assay, or by measuring B cell activity levels.

[0116] The method of the present invention for improving the immunogenicity of protein / peptide antigens

[0117] This invention is groundbreaking, in which the inventors have discovered that the immunogenicity of a protein / peptide antigen is enhanced by fusing it with a modified antibody Fc fragment, the Fc fragment having an improved binding capacity to the Fc receptor and / or complement protein C1q due to changes in its amino acid sequence and / or glycosylation.

[0118] Prior to this invention, no studies had reported an increase in the immunogenicity of protein / peptide antigens after forming fusion proteins with Fc fragments that have enhanced binding ability to Fc receptors and / or complement protein C1q.

[0119] The immunogenicity-enhanced protein / peptide antigen of the present invention

[0120] In one embodiment of the invention, the starting protein / peptide antigen is the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein, and its immunogenicity is enhanced by forming a fusion protein with an Fc fragment that has enhanced binding ability to the Fc receptor and / or complement protein C1q. In a particularly preferred embodiment, the Fc fragment is an Fc receptor CD32a, CD32b, and CD64 binding enhancement fragment / complement C1q binding enhancement fragment; its amino acid sequence is shown in SEQ ID NO: 30. In a most preferred embodiment, the modified antibody Fc fragment is an Fc receptor CD16a, CD32a, CD32b, and CD64 binding enhancement fragment / complement C1q binding enhancement fragment; its amino acid sequence is shown in SEQ ID NO: 30, and it is produced using Fucose knockout CHO cells.

[0121] Coronaviruses primarily mediate viral invasion by binding to host cell receptors via their spike protein (S protein), which also determines the virus's tissue or host tropism. The host cell receptor protein for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2). After the viral trimeric spike protein (S protein) binds to the ACE2 receptor, it is cleaved by host proteases into an S1 polypeptide containing a receptor-binding domain (SARS-CoV-2 RBD) and an S2 polypeptide responsible for mediating viral fusion with the cell membrane, thereby allowing the virus to invade the body.

[0122] The immunogenic composition of the present invention

[0123] In one embodiment, the immunogenic composition of the present invention further comprises at least one of an adjuvant, a buffer, a cryoprotectant, a salt, a divalent cation, a nonionic detergent, a free radical oxidation inhibitor, a diluent, or a carrier.

[0124] An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen. The immunogenic compositions of the present invention may or may not contain a vaccine adjuvant. Adjuvants that can be used in the compositions of the present invention include, but are not limited to, at least one of MF59, QS-21, or MPL.

[0125] In one embodiment, the adjuvant in the immunogenic composition of the present invention is an aluminum-based adjuvant. The adjuvant used will depend on the individual being administered the immunogenic composition, the prescribed route of injection, and the number of injections.

[0126] The immunogenic composition may optionally contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include those used in national pharmacopoeias for animals (including humans and non-human mammals). The term carrier can be used to refer to a diluent, adjuvant, excipient, or medium administered with the pharmaceutical composition. Water, saline solutions, and solutions containing dextrose and glycerol may be used as liquid carriers, particularly for injectable solutions.

[0127] The immunogenic compositions of the present invention may also contain one or more additional immunogenic agents.

[0128] Administration of the immunogenic composition of the present invention

[0129] The immunogenic compositions of the present invention for therapeutic or prophylactic treatment can be administered via intramuscular, intraperitoneal, intradermal, or subcutaneous injection; or via mucosal administration to the oral / esophagus, respiratory tract, or genitourinary tract. Intranasal administration of the vaccine is preferred for the treatment of certain diseases, such as pneumonia or otitis media. Although the vaccines of the present invention can be administered in a single dose, their components can also be administered simultaneously or at different times. In addition to a single route of administration, two different routes of administration can be used.

[0130] The optimal amount of a component used in a particular immunogenic composition can be determined through standard studies involving the observation of an appropriate immune response in an individual. Following the initial vaccination, an individual may receive one or more adequately spaced booster immunizations.

[0131] Use of the immunogenic composition of the present invention

[0132] The protein / peptide antigens and immune complexes of this invention can prevent or treat diseases caused by pathogens, especially coronaviruses, and more particularly diseases caused by SARS-CoV-2 virus. The protein / peptide antigens and immune complexes of this invention can also prevent or treat tumor diseases. They can also be used to immunize animals to produce neutralizing antibodies.

[0133] Example

[0134] Example 1: Construction and protein production of SARS-CoV-2 RBD and RBD-Fc fusion protein expression vectors

[0135] 1.1 Construction of SARS-CoV-2 RBD expression vector and protein production

[0136] The SARS-CoV-2-Spike-RBD sequence (SEQ ID NO:5) was obtained by PCR amplification (PCR amplification template was obtained from Beijing Yiqiao Shenzhou Technology Co., Ltd., hereinafter the same), containing the signal peptide sequence (SEQ ID NO:3) and the SARS-CoV-2-Spike-RBD sequence (SEQ ID NO:1), and was inserted into the SARS-CoV-2-Spike-RBD sequence using the in-fusion method. Hind III+ Xba I. The pSE-CoV-2-RBD expression vector (SEQ ID NO:5) was obtained by digesting the pSE vector (source: Shenzhou Cell Engineering Co., Ltd., hereinafter the same) with enzymes (source: Fermentas).

[0137] Amplification primers:

[0138]

[0139] pSE-CoV-2-RBD plasmid was extracted, transfected into HEK-293 cells (source: Invitrogen, the same below), and cultured for expression for 7 days. High-purity SARS-CoV-2 RBD protein was obtained after purification.

[0140] 1.2 Construction of SARS-CoV-2 RBD-mFc expression vector and protein production

[0141] The SARS-CoV-2-Spike-RBD sequence was amplified by PCR and inserted into the genome using an in-fusion method. AfeThe pSE-CoV-2-RBD-mFc expression vector (SEQ ID NO:11) was obtained by digesting the pSE-mFc vector containing the signal peptide (SEQ ID NO:3), linker (SEQ ID NO:7) and mouse IgG1 constant region sequence (SEQ ID NO:9) with FastAP.

[0142] Amplification primers:

[0143]

[0144] pSE-CoV-2-RBD-mFc plasmid was extracted, transfected into HEK-293 cells, and cultured for expression for 7 days. High-purity RBD-mFc protein was obtained by purification using a protein A purification column.

[0145] 1.3 Construction of SARS-CoV-2 RBD-Fc expression vector and protein production

[0146] The SARS-CoV-2-Spike-RBD sequence was amplified by PCR and inserted into the genome using an in-fusion method. Afe pSE-CoV-2-RBD-Fc expression vector (SEQ ID NO:15) was obtained by digesting the pSTEP2-Fc vector (source: Shenzhou Cell Engineering Co., Ltd.) containing the signal peptide (SEQ ID NO:3), linker (SEQ ID NO:7) and human IgG1 constant region sequence (SEQ ID NO:13) with FastAP dephosphater.

[0147] Amplification primers:

[0148]

[0149] pSE-CoV-2-RBD-Fc plasmid was extracted, transfected into HEK-293 cells, and cultured for expression for 7 days. High-purity RBD-Fc protein was obtained by purification using a protein A purification column.

[0150] 1.4 Construction of SARS-CoV-2 RBD-Fc-Ce3 expression vector and protein production

[0151] To enhance the CDC function mediated by the antibody Fc fragment, nucleotide mutations were performed on the constant region of the IgG1 subtype according to the literature [36, 37] to obtain the genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fc-Ce3, SEQ ID NO:17).

[0152] The SARS-CoV-2-Spike-RBD-Ce3-Fc sequence (SEQ ID NO:19) was amplified by PCR and inserted into the genome using an in-fusion method. Hind III+ Xba The pSE-nCoV-2-RBD-Fc-Ce3 expression vector (SEQ ID NO:19) was obtained by digesting the pSE vector with enzyme I.

[0153] Amplification primers:

[0154]

[0155] The pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted, transfected into HEK-293 cells, and cultured for expression for 7 days. High-purity RBD-Fc-Ce3 was obtained by purification using a protein A purification column.

[0156] 1.5 Construction of SARS-CoV-2 RBD-Fc-Fe4 expression vector and protein production

[0157] To further enhance the immune function mediated by the antibody Fc fragment, the pSE-nCoV-2-RBD-Fc-Ce3 plasmid was extracted and transfected with... fut8 HEK-293 cells with gene knockout (source: Shenzhou Cell Engineering Co., Ltd.) were cultured and expressed for 7 days, and high-purity defucosylated RBD-Fc-Fe4 was obtained by purification using a protein A purification column.

[0158] 1.6 Construction of SARS-CoV-2 RBD-Fc-Fd11-IgG4 expression vector and protein production

[0159] To reduce the immune function mediated by the antibody Fc fragment, the constant region of the IgG4 subtype was mutated according to the literature

[38] to obtain the genetically engineered heavy chain IgG4 constant region nucleotide sequence (Fd11-IgG4, SEQ ID NO:21).

[0160] The SARS-CoV-2-Spike-RBD-Fd11-IgG4 sequence (SEQ ID NO:23) was amplified by splicing PCR and inserted into the genome using an in-fusion method. Hind III+ Xba The pSE-CoV-2-RBD-Fc-Fd11-IgG4 expression vector (SEQ ID NO:23) was obtained by digesting the pSE vector with enzyme I.

[0161] Amplification primers:

[0162]

[0163] The pSE-CoV-2-RBD-Fc-Fd11-IgG4 plasmid was extracted, transfected into HEK-293 cells, and cultured for expression for 7 days. The high-purity RBD-Fc-Fd11-IgG4 protein was obtained by purification using a protein A purification column.

[0164] Example 2: SARS-CoV-2 RBD and RBD-Fc fusion protein Fc receptor binding assay

[0165] 2.1 RBD-mFc increases binding to mouse Fc receptors

[0166] Avidin protein at a concentration of 10 μg / mL (source: Thermo Fisher, hereinafter the same) was coated onto 96-well plates, 100 μL per well, and incubated overnight at 2-8℃. The plates were washed the following day, blocked at room temperature for 1 h, and then 100 μL of different biotin-labeled Fc receptor proteins (all from Beijing Yiqiao Shenzhou Technology Co., Ltd.) were added: 5 μg / mL mCD16a-AVI-His(V158)+BirA, 5 μg / mL mCD32b-AVI-His+BirA, or 0.5 μg / mL mCD64-AVI-His+BirA. A protein-free control was used. After incubation at room temperature for 1 h, the plates were washed, and then 100 μL of different concentrations of RBD-mFc protein or RBD protein were added. The protein concentrations detected when bound to mCD16a and mCD32b were 40 μg / mL, 10 μg / mL, 2.5 μg / mL, 0.6250 μg / mL, 0.1563 μg / mL, 0.0391 μg / mL, 0.0098 μg / mL, and 0.0024 μg / mL; the protein concentrations detected when bound to mCD64 were 10 μg / mL, 2.5 μg / mL, 0.6250 μg / mL, 0.1563 μg / mL, 0.0391 μg / mL, 0.0098 μg / mL, and 0.0024 μg / mL. After incubation at room temperature for 1 h, the plate was washed to remove unbound proteins. 100 μL of 0.1 μg / mL horseradish peroxidase-labeled CoV-2-RBD antibody (CoV-2-HB27-Fd11-IgG4, source: Shenzhou Cell Engineering Co., Ltd., as the detection antibody) was added. After another 1 h of incubation, the plate was washed again, and substrate chromogenic buffer was added for color development. After termination, the OD values ​​were read using a microplate reader. 450 , with sample OD 450 - Blank control OD 450 The data was analyzed.

[0167] The results are as follows Figure 1As shown, the binding EC of RBD-mFc with mCD16a and mCD32b 50 The concentrations were 0.19 μg / mL and 0.27 μg / mL, respectively, while RBD protein did not bind to either mCD16a or mCD32b. RBD-mFc and RBD did not bind to mCD64 protein.

[0168] 2.2 RBD-Fc-Fe4 increases the binding of human Fc receptors and C1q.

[0169] To detect binding to the Fc receptor, 10 μg / mL of Avidin protein was coated onto each well of a 96-well plate (100 μL per well) and incubated overnight at 2-8°C. The next day, after washing the plate and blocking at room temperature for 1 h, 100 μL of different biotin-labeled Fc receptor proteins (all from Beijing Yiqiao Shenzhou Technology Co., Ltd.) were added: 5 μg / mL CD16a-AVI-His(V158)+BirA, 5 μg / mL CD32a-AVI-His(R131)+BirA, 5 μg / mL CD32b-AVI-His(R131)+BirA, or 0.5 μg / mL CD64-AVI-His(V158)+BirA. A protein-free control was used. After incubation at room temperature for 1 h, the plate was washed, and 100 μL of different RBD-Fc fusion proteins or RBD proteins were added. The protein concentrations detected when binding to CD16a were 40 μg / mL, 10 μg / mL, 2.5 μg / mL, 0.625 μg / mL, 0.156 μg / mL, 0.039 μg / mL, 0.01 μg / mL, and 0.002 μg / mL; the protein concentrations detected when binding to CD32a and CD32b were 40 μg / mL, 10 μg / mL, 2.5 μg / mL, 0.625 μg / mL, and 0.156 μg / mL; and the protein concentrations detected when binding to CD64 were 10 μg / mL, 2.5 μg / mL, 0.625 μg / mL, 0.156 μg / mL, 0.039 μg / mL, 0.01 μg / mL, and 0.002 μg / mL. After incubation at room temperature for 1 h, the plate was washed to remove unbound proteins. 100 μL of 0.1 μg / mL horseradish peroxidase-labeled CoV-2-RBD antibody (CoV-2-HB27-Fd11-IgG4) without Fc receptor binding function was added. After incubation for 1 h, the plate was washed again, and substrate chromogenic solution was added for color development. After termination, the OD values ​​were read using a microplate reader. 450 , with sample OD 450 - Blank control OD 450 The data was analyzed.

[0170] To detect C1q binding, different concentrations of RBD protein or different RBD-Fc fusion proteins were coated onto 96-well plates at 100 μL / well and incubated overnight at 4°C. The protein concentrations were 200 μg / mL, 40 μg / mL, 8.0 μg / mL, 1.6 μg / mL, 0.32 μg / mL, and 0.064 μg / mL. The plates were washed the following day, blocked at room temperature for 1 h, and then 5 μg / mL of C1q complement protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added at 100 μg / well, followed by incubation for 1 h. After washing to remove unbound protein, 0.5 μg / mL anti-C1q / HRP (source: Abcam) was added for incubation, followed by repeated washing. Finally, substrate chromogenic buffer was added for color development, and OD was measured after termination. 450 .

[0171] The results are as follows Figure 2 As shown, among different RBD-Fc fusion proteins, RBD-Fc-Fe4 exhibits the strongest binding affinity to CD16, CD32a, and CD32b, superior to RBD-Fc and RBD-Fc-Ce3. RBD-Fc-Fd11-IgG4 and RBD proteins show almost no binding affinity to CD16, CD32a, and CD32b. RBD-Fc-Fe4, RBD-Fc-Ce3, and RBD-Fc show high and similar binding affinity to CD64, while RBD-Fc-Fd11-IgG4 protein binds weakly to CD64, suggesting non-specific binding of RBD proteins at high concentrations. RBD-Fc-Fe4 and RBD-Fc-Ce3 bind slightly more strongly to C1q than RBD-Fc, while RBD and RBD-Fc-Fd11-IgG4 do not bind to C1q.

[0172] Example 3: Phagocytosis assay of SARS-CoV-2 RBD and RBD-Fc fusion protein cells

[0173] Monocyte-derived macrophages (MDMs) were obtained by obtaining monocytes from PBMCs isolated from fresh human peripheral blood through cell adhesion, followed by 7 days of induction culture. Phagocytosis was observed under a confocal microscope after co-incubation with FITC-labeled antigen proteins.

[0174] 3×10 4MDM cells (obtained by induction at Shenzhou Cell Engineering Co., Ltd.) were seeded in 50 μL / well and cultured overnight in 1640 + 10% FBS + 1×PS medium (obtained by Shenzhou Cell Engineering Co., Ltd.). Then, 20 μg / mL FITC-labeled different antigen proteins were added to 50 μL / well. The 96-well plates were incubated at 37°C and 5% CO2 in the dark for 2.5 h. After incubation, the cleansing agent was discarded, and unbound antigen proteins were washed away with PBS. 4% paraformaldehyde (obtained by Sigma-Aldrich) was added to 50 μL / well. The plates were fixed at 4°C for 15 min, the fixative was discarded, and the plates were washed once with PBS. 50 μL of PBS was added to each well, and fluorescence images were taken using a confocal microscope with the FITC channel. The fluorescence intensity was analyzed using Image Pro Plus software.

[0175] The results are as follows Figure 3 As shown, RBD-Fc-Fe4-FITC can mediate stronger phagocytosis.

[0176] Example 4: Immunization in mice and evaluation of immunization effects

[0177] 4.1 Mouse Immunization

[0178] Preparation of aluminum-adjuvanted antigen: RBD-his (source: Beijing Sinocare Technology Co., Ltd.) and RBD-mFc antigen were diluted with PBS to 0.06 mg / mL, and aluminum adjuvant (source: Sinocare Cell Engineering Co., Ltd., hereinafter the same) was diluted with PBS to 1 mg / mL. The diluted antigen and aluminum adjuvant were mixed in equal volumes to obtain an antigen containing aluminum adjuvant at a concentration of 0.03 mg / mL.

[0179] Preparation of RBD, RBD-Fc, RBD-Fc-Ce3, and RBD-Fc-Fe4 antigens containing MF59 (source: Shenzhou Cell Engineering Co., Ltd., hereinafter the same) and aluminum adjuvant mixed adjuvant: Take 1.5 mL of aluminum adjuvant, add 1.5 mL of MF59 adjuvant, and then add 0.18 mL of antigen protein with a concentration of 0.53 mg / mL to obtain antigens with a protein concentration of 0.03 mg / mL and mixed adjuvant.

[0180] The mice were 4-6 week old Balb / c mice (source: China National Institutes for Food and Drug Control). They were intraperitoneally injected with 0.1 mL of aluminum adjuvant antigen or antigen containing mixed adjuvants, which contained 3 μg of antigen. They were boosted on day 14 and day 28, respectively.

[0181] 4.2 Determination of antibody titer and neutralization titer in mouse serum

[0182] 5 μg / mL RBD-mFc protein was coated onto 96-well plates at 100 μL / well. CD155(D1)-mFc (source: Shenzhou Cell Engineering Co., Ltd.) was used as an irrelevant control with the same label. After incubation at room temperature for 2 h, the plates were washed and blocked with 2% BSA (bovine serum albumin), and incubated at room temperature for 1 h. Mouse aluminum adjuvant immunogenic serum was diluted 8000× using 0.1% BSA TBST buffer as the sample diluent, and mixed adjuvant immunogenic serum was diluted 1000×. Mouse serum with irrelevant immune targets was used as a negative control. Simultaneously, 100 μL / well each of the test serum, negative control serum, and 80 ng / mL rabbit anti-mouse IgG F(ab)2 / HRP (source: Jackson Immuno Research) was added. After incubation for 2 h, the plates were washed 5 times, and substrate chromogenic solution was added for color development. After termination, the OD was read by a microplate reader. 450 The antibody titer in mouse serum was measured.

[0183] Mouse aluminum adjuvant immunogen serum was diluted 500×, and mixed with adjuvant immunogen serum diluted 10×. An equal volume of this mixture was then added to SARS-CoV-2 pseudovirus (source: China National Institutes for Food and Drug Control). After incubation at 37℃ for 1 h, 293FT-ACE2 cells (source: Shenzhou Cell Engineering Co., Ltd.) were added. The group with pseudovirus but no antibody was used as a positive control, and the group without pseudovirus and antibody was used as a negative control. After infection, cells were cultured at 37℃ and 5% CO2 for 20–28 h. Fluorescence intensity (RLU) values ​​were detected using a microplate chemiluminescence analyzer, and the neutralization inhibition rate was calculated. Neutralization inhibition rate % = (Positive control RLUs – Sample RLUs) / (Positive control RLUs – Negative control RLUs) × 100%.

[0184] The results showed that 7 days after the third immunization of mice with the aluminum adjuvant antigen, the immune serum titer and neutralizing titer of RBD-mFc protein were both superior to those of RBD-his, indicating that RBD-mFc can activate a stronger humoral immune response. Figure 4 ).

[0185] Seven days after the first immunization of mice with antigens containing aluminum adjuvant and MF59 mixed adjuvant, the serum titers and neutralizing titers of different RBD-Fc fusion proteins were superior to those of RBD proteins. Among them, RBD-Fc-Ce3 and RBD-Fc-Fe4 had higher serum neutralizing titers, indicating that RBD-Fc fusion proteins can activate a stronger humoral immune response. Figure 5 ).

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[0225] The main sequence involved in this invention

[0226] SEQUENCE LISTING <110> Shenzhou Cell Engineering Co., Ltd. <120> Methods to enhance the immunogenicity of protein / peptide antigens by forming fusion proteins with modified Fc fragments. <130> PCT69298SXB <150> CN202010394463.6 <151> 2020-05-11 <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 672 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 1 agcagggtcc aaccaacaga gagcattgtg aggtttccaa acatcaccaa cctgtgtcca 60 tttggagagg tgttcaatgc caccaggttt gcctctgtct atgcctggaa caggaagagg 120 attagcaact gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 180 aagtgttatg gagtgagccc aaccaaactg aatgacctgt gtttcaccaa tgtctatgct 240 gactcctttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 300 attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 360 agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 420 agagcaacc tgaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 480 acaccatgta atggagtgga gggctcaac tgttacttc cactccaatc ctatggcttc 540 caccaacca atggagtggg ctaccacca tacaggtgg tggtgctgtc ctttgaactg 600 ctccatgccc ctgccacagt gtgtggacca aagagagca ccaacctggt gagaacaag 660 tgtgtgaact tc 672 <210> 2 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 2 Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 1 5 10 15 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 20 25 30 Val Tyr Only Trp Asn Arg Lys Arg Ile Ser Asn Cys Only Only Asp Tyr 35 40 45 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 50 55 60 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 65 70 75 80 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 85 90 95 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 100 105 110 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 115 120 125 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 130 135 140 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 145 150 155 160 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 165 170 175 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 180 185 190 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 195 200 205 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 3 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 3 atggatgcca tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtgtttgtc 60 tctccc 66 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 4 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro 20 <210> 5 <211> 738 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 5 atggatgcca tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtgtttgtc 60 tctcccagca gggtccaacc aacagagagc attgtgaggt ttccaaacat caccaacctg 120 tgtccatttg gagaggtgtt caatgccacc aggtttgcct ctgtctatgc ctggaacagg 180 aagaggatta gcaactgtgt ggctgactac tctgtgctct acaactctgc ctccttcagc 240 accttcaagt gttatggagt gagcccaacc aaactgaatg acctgtgttt caccaatgtc 300 tatgctgact cctttgtgat taggggagat gaggtgagac agattgcccc tggacaaaca 360 ggcaagattg ctgactacaa ctacaaactg cctgatgact tcacaggctg tgtgattgcc 420 tggaacagca acaacctgga cagcaaggtg ggaggcaact acaactacct ctacagactg 480 ttcaggaaga gcaacctgaa accatttgag agggacatca gcacagagat ttaccaggct 540 ggcagcacac catgtaatgg agtggagggc ttcaactgtt actttccact ccaatcctat 600 ggcttccaac caaccaatgg agtgggctac caaccataca gggtggtggt gctgtccttt 660 gaactgctcc atgcccctgc cacagtgtgt ggaccaaaga agagcaccaa cctggtgaag 720 aacaagtgtg tgaacttc 738 <210> 6 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 6 Met Asp Ala Met Lys Arg Gly Leu Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln With Pro Gly Gln Thr Gly Lys With Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 225 230 235 240 Asn Lys Cys Val Asn Phe 245 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 7 gctgatgacg atgacaag 18 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 8 Ala Asp Asp Asp Asp Lys 1 5 <210> 9 <211> 687 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 9 gctgtgccca gggattctgg tgtaagcct tgcatatgta cagtcccaga agtatcatct 60 gtcttcatct tccccaaa gcccaggat gtgctcacca ttactctgac tcctaggtc 120 acgtgtgttg tggtagacat cagcaggat gatcccgagg tccagttcag ctggtttgta 180 gatgatgtgg aggtgcacac agctcagacg caacccggg aggagcagtt cacacact 240 ttccgctcag tcagtgaact tcccatcatg caccaggact ggctcaatgg tcagtg 300 aaatgcaggg tcacagtgc agctttccct gccccatcg agaaaccat ctccaaaacc 360 aaaggcagac cgaaggctcc acaggtgtac accattccac ctcccagga gcagatggcc 420 aaggataaag tcagtctgac ctgcatgata acagacttct tccctgaaga cattactgtg 480 gagtggcagt ggaatgggca gccagcggag aactacaga acaccagcc catcatggac 540 acagatggct cttacttcgt ctacagcaag ctcaatgtgc agaagagcaa ctgggaggca 600 ggaaatactt tcacctgctc tgtgttacat gagggcctgc acaaccacca tactgagaag 660 agcctctccc actctcctgg taaatga 687 <210> 10 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 10 Ala Val Pro Arg Asp Ser Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 1 5 10 15 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 20 25 30 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 35 40 45 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 50 55 60 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 115 120 125 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 130 135 140 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 145 150 155 160 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 165 170 175 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 180 185 190 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 195 200 205 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 210 215 220 Ser Pro Gly Lys 225 <210> 11 <211> 1443 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 11 atggatgcca tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc aggtttgtc 60 tctcccagca gggtccaacc aacagagagc attgtgaggt ttccaaacat caccaacctg 120 tgtccattg gagaggtgtt caatgccacc agttttgcct ctgtctatgc ctggaacagg 180 aagaggatta gcaactgtgt ggctgactac tctgtgctct acaactctgc ctccttcagc 240 accttcaagt gttatggagt gagcccaacc aaactgaatg acctgtgttt caccaatgtc 300 tatgctgact cctttgtgat taggggagat gaggtgagac agattgcccc tggacaaaca 360 ggcaagattg ctgactacaa ctacaaactg cctgatgact tcacaggctg tgtgattgcc 420 tggaacagca acaacctgga cagcaaggtg ggaggcaact acaactacct ctacagactg 480 ttcaggaaga gcaacctgaa accatttgag agggacatca gcacagagat ttaccaggct 540 ggcagcacac catgtaatgg agtggagggc ttcaactgtt actttccact ccaatcctat 600 ggcttccaac caaccaatgg agtgggctac caaccataca gggtggtggt gctgtccttt 660 gaactgctcc atgcccctgc cacagtgtgt ggaccaaaga agagcaccaa cctggtgaag 720 aacaagtgtg tgaacttcgc tgatgacgat gacaaggctg tgcccaggga ttctggttgt 780 aagccttgca tatgtacagt cccagagta tcatctgtct tcatcttccc cccaaagccc 840 areatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc 900 areatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct 960 cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc 1020 atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct 1080 ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag 1140 gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc 1200 atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca 1260 gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac 1320 1380 ttacatgagg gcctgcacaa ccaccatact gagagagcc tctcccactc tcctgtaaa 1440 made 1443 <210> 12 <211> 480 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 12 Met Asp Ala Met Lys Arg Gly Leu Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 225 230 235 240 Asn Lys Cys Val Asn Phe Ala Asp Asp Asp Asp Lys Ala Val Pro Arg 245 250 255 Asp Ser Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser 260 265 270 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu 275 280 285 Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro 290 295 300 Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala 305 310 315 320 Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val 325 330 335 Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe 340 345 350 Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr 355 360 365 Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile 370 375 380 Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys 385 390 395 400 Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp 405 410 415 Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp 420 425 430 Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Ser Lys Ser 435 440 445 Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly 450 455 460 Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 465,470,475,480 <210> 13 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 13 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120 acccctgagg tcacgtgcgt ggtggtggac gtgagccacg aagaccccga ggtcaagttc 180 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540 cccgtgctgg actccgacgg ctccttctttc ctctacagca agctcaccgt ggacaagagc 600 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660 tacacgcaga agagcctctc cctgtctccg ggtaaatga 699 <210> 14 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 14 Glu Pro Lys Ser Ser 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 Thr 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 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Dear Leu Dear Leu Dear Pro Gly Lys 225 230 <210> 15 <211> 1455 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 15 atggatgcca tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc aggtttgtc 60 tctcccagca gggtccaacc aacagagagc attgtgaggt ttccaaacat caccaacctg 120 tgtccattg gagaggtgtt caatgccacc agttttgcct ctgtctatgc ctggaacagg 180 aagaggatta gcaactgtgt ggctgactac tctgtgctct acaactctgc ctccttcagc 240 accttcaagt gttatggagt gagcccaacc aaactgaatg acctgtgttt caccaatgtc 300 tatgctgact cctttgtgat taggggagat gaggtgagac agattgcccc tggacaaaca 360 ggcaagattg ctgactacaa ctacaaactg cctgatgact tcacaggctg tgtgattgcc 420 tggaacagca acaacctgga cagcaaggtg ggaggcaact acaactacct ctacagactg 480 ttcaggaaga gcaacctgaa accatttgag agggacatca gcacagagat ttaccaggct 540 ggcagcacac catgtaatgg agtggagggc ttcaactgtt actttccact ccaatcctat 600 ggcttccaac caaccaatgg agtgggctac caaccataca gggtggtggt gctgtccttt 660 gaactgctcc atgcccctgc cacagtgtgt ggaccaaaga agagcaccaa cctggtgaag 720 aacaagtgtg tgaacttcgc tgatgacgat gacaaggagc ccaaatcttc tgacaaaact 780 cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 840 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 900 gtggacgtga gccacgaaga ccccgaggtc aagttcaact ggtacgtgga cggcgtggag 960 gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 1020 agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 1080 tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 1140 cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 1200 agcctgacct gcctggtcaa aggctcttat cccagcgaca tcgccgtgga gtgggagagc 1260 aatgggcagc cggagacaa ctacagacc acgcctcccg tgctggactc cgacggctcc 1320 ttctcctct acagcaagct caccgtggac agagcaggt ggcagcaggg gaacgctctc 1380 tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagg cctctccctg 1440 tctccgggta atga 1455 <210> 16 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 16 Met Asp Ala Met Lys Arg Gly Leu Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 225 230 235 240 Asn Lys Cys Val Asn Phe Ala Asp Asp Asp Asp Lys Glu Pro Lys Ser 245 250 255 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Pro Gly Lys <210> 17 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 17 gagcccaaat cttctgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120 acccctgagg tcacgtgcgt ggtggtggac gtgagccacg aagaccccga ggtcaagttc 180 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540 cccgtgctgg actccgacgg ctccttctttc ctctacagca agctcaccgt ggacaagagc 600 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atggcgctct gcacaaccac 660 tacacgcaga agagcctctc cctgtctccg ggtaaataa 699 <210> 18 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 18 Glu Pro Lys Ser Ser 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 Thr 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 Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Dear Leu Dear Leu Dear Pro Gly Lys 225 230 <210> 19 <211> 1455 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 19 atggatgcca tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc aggtttgtc 60 tctcccagca gggtccaacc aacagagagc attgtgaggt ttccaaacat caccaacctg 120 tgtccattg gagaggtgtt caatgccacc agttttgcct ctgtctatgc ctggaacagg 180 aagaggatta gcaactgtgt ggctgactac tctgtgctct acaactctgc ctccttcagc 240 accttcaagt gttatggagt gagcccaacc aaactgaatg acctgtgttt caccaatgtc 300 tatgctgact cctttgtgat taggggagat gaggtgagac agattgcccc tggacaaaca 360 ggcaagattg ctgactacaa ctacaaactg cctgatgact tcacaggctg tgtgattgcc 420 tggaacagca acaacctgga cagcaaggtg ggaggcaact acaactacct ctacagactg 480 ttcaggaaga gcaacctgaa accatttgag agggacatca gcacagagat ttaccaggct 540 ggcagcacac catgtaatgg agtggagggc ttcaactgtt actttccact ccaatcctat 600 ggcttccaac caaccaatgg agtgggctac caaccataca gggtggtggt gctgtccttt 660 gaactgctcc atgcccctgc cacagtgtgt ggaccaaaga agagcaccaa cctggtgaag 720 aacaagtgtg tgaacttcgc tgatgacgat gacaaggagc ccaaatcttc tgacaaaact 780 cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 840 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 900 gtggacgtga gccacgaaga ccccgaggtc aagttcaact ggtacgtgga cggcgtggag 960 gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 1020 agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 1080 tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 1140 cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 1200 agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 1260 aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1320 ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1380 tcatgctccg tgatgcatgg cgctctgcac aaccactaca cgcagaagag cctctccctg 1440 tctccgggta aataa 1455 <210> 20 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 20 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln With Pro Gly Gln Thr Gly Lys With Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 225 230 235 240 Asn Lys Cys Val Asn Phe Ala Asp Asp Asp Asp Lys Glu Pro Lys Ser 245 250 255 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Pro Gly Lys <210> 21 <211> 691 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 21 gagtccaaat atggtccccc atgcccaccc tgcccagcac ctgaggccgc cgggggacca 60 tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 120 gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 180 gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 240 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 300 tacaagtgca aggtccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 360 gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 420 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 480 gtggagtggg aaagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 540 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 600 gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 660 aagagcctct ccctgtctct gggtaaataa a 691 <210> 22 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 22 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 23 <211> 1429 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 23 atggatgcca tgaagagg gctctgctgt gtgctgctgc tgtgtggagc agtgtttgtc 60 tctcccagca gggtccaacc aacagagagc attgtgaggt ttccaaacat caccaacctg 120 tgtccatttg gagaggtgtt caatgccacc aggtttgcct ctgtctatgc ctggaacagg 180 aagagatta gcaactgtgt ggctgactac tctgtgctct acaactctgc ctccttcagc 240 accttcaagt gttatggagt gagcccaacc aaactgaatg acctgtgttt caccaatgtc 300 tatgctgact cctttgtgat taggggagat gaggtgagac agattgcccc tggacaaaca 360 ggcaagattg ctgactacaa ctacaaactg cctgatgact tcacaggctg tgtgattgcc 420 480 540 ggcagcacac catgtaatgg agtggagggc ttcaactgtt actttccact ccaatcctat 600 ggcttccaac caaccaatgg agtgggctac caaccataca gggtggtggt gctgtccttt 660 gaactgctcc atgcccctgc cacagtgtgt ggaccaaaga agagcaccaa cctggtgaag 720 aacaagtgtg tgaacttcga gtccaaatat ggtcccccat gcccaccctg cccagcacct 780 gaggccgccg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 840 atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 900 gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 960 gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1140. gagaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc ccatcccagg aggagatgac caagaccag gtcagcctga cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtggga agcaatgggc agccggaga caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320 gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact cacacacaga gagcctctcc ctgtctctgg gtaaataa <210> 24 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 24 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 225 230 235 240 Asn Lys Cys Val Asn Phe Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 245 250 255 Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 275 280 285 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 370 375 380 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 420 425 430 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 465 470 475 <210> 25 <211> 1377 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 25 agcagggtcc aaccaacaga gagcattgtg aggtttccaa acatcaccaa cctgtgtcca 60 tttggagagg tgttcaatgc caccaggtttt gcctctgtct atgcctggaa caggaagagg 120 attagcaact gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 180 aagtgttatg gagtgagccc aaccaaactg aatgacctgt gtttcaccaa tgtctatgct 240 gactccttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 300 attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 360 agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 420 aagagcaacc tgaaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 480 acaccatgta atggagtgga gggcttcaac tgttactttc cactccaatc ctatggcttc 540 caaccaacca atggagtggg ctaccaacca tacagggtgg tggtgctgtc ctttgaactg 600 ctccatgccc ctgccacagt gtgtggacca aagaagagca ccaacctggt gaagaacaag 660 tgtgtgaact tcgctgatga cgatgacaag gctgtgccca gggattctgg ttgtaagcct 720 tgcatatgta cagtcccaga agtatcatct gtcttcatct tccccccaaa gcccaaggat 780 gtgctcacca ttactctgac tcctaaggtc acgtgtgttg tggtagacat cagcaaggat 840 gatcccgagg tccagttcag ctggtttgta gatgatgtgg aggtgcacac agctcagacg 900 caaccccggg aggagcagtt caacagcact ttccgctcag tcagtgaact tcccatcatg 960 caccaggact ggctcaatgg caaggagttc aaatgcaggg tcaacagtgc agctttccct 1020 gcccccatcg agaaaaccat ctccaaaacc aaaggcagac cgaaggctcc acaggtgtac 1080 accattccac ctcccaagga gcagatggcc aaggataaag tcagtctgac ctgcatgata 1140 acagacttct tccctgaaga cattactgtg gagtggcagt ggaatgggca gccagcggag 1200 aactacaaga acaccagcc catcatggac acgatggct cttactcgt ctacagcaag 1260 ctcaatgtgc agagagcaa ctgggaggca ggaatactt tcacctgctc tgtgttacat 1320 gaggggcctgc acaccacca tactgagaag agcctctccc actctcctgg taaatga 1377 <210> 26 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 26 Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 1 5 10 15 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 20 25 30 Val Tyr Only Trp Asn Arg Lys Arg Ile Ser Asn Cys Only Only Asp Tyr 35 40 45 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 50 55 60 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 65 70 75 80 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 85 90 95 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 100 105 110 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 115 120 125 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 130 135 140 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 145 150 155 160 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 165 170 175 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 180 185 190 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 195 200 205 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 Ala Asp Asp Asp Asp Lys Ala Val Pro Arg Asp Ser Gly Cys Lys Pro 225 230 235 240 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 245 250 255 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 260 265 270 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 275 280 285 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 290 295 300 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 305 310 315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 325 330 335 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 340 345 350 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 355 360 365 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 370 375 380 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 385 390 395 400 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe 405 410 415 There Will Be Tyr Lys On Asn And Gln Lys On Asn On Glu Ala Gly Asn 420 425 430 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 435 440 445 Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450,455 <210> 27 <211> 1389 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 27 agcagggtcc aaccaacaga gagcattgtg aggttccaa acatcaccaa cctgtgtcca 60 tttggagagg tgttcaatgc caccaggttt gcctctgtct atgcctgaa caggaaggg 120 attack gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 180 aagtgttatg gagtgagccc aaccaaactg aatgacctgt gttcaccaa tgtctatgct 240 gactccttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 300 attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 360 agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 420 aagagcaacc tgaaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 480 acaccatgta atggagtgga gggcttcaac tgttactttc cactccaatc ctatggcttc 540 caaccaacca atggagtggg ctaccaacca tacagggtgg tggtgctgtc ctttgaactg 600 ctccatgccc ctgccacagt gtgtggacca aagaagagca ccaacctggt gaagaacaag 660 tgtgtgaact tcgctgatga cgatgacaag gagcccaaat cttctgacaa aactcacaca 720 tgcccaccgt gcccagcacc tgaacctcctg gggggaccgt cagtcttcct cttcccccca 780 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 840 gtgagccacg aagaccccga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 900 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 960 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1020 aaagccctcc cagcccccat cgagaaacc atctccaag cagcggca gccccgagaa 1080 ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccagaacca ggtcagcctg 1140 acctgcctgg tcaaggctt ctatcccagc gatacgccg tggagtggga gagcaatggg 1200 cagccggaga acaactaca gaccacgcct cccgtgctgg actcgacgg ctccttctc 1260 ctctacagca agctcaccgt ggacaagagc aggtggcagc agggaacgt cttctcatgc 1320 tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagccctc cctgtctccg 1380 ggTAATGA 1389 <210> 28 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 28 Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 1 5 10 15 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 20 25 30 Val Tyr Only Trp Asn Arg Lys Arg Ile Ser Asn Cys Only Only Asp Tyr 35 40 45 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 50 55 60 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 65 70 75 80 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 85 90 95 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 100 105 110 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 115 120 125 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 130 135 140 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 145 150 155 160 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 165 170 175 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 180 185 190 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 195 200 205 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 Ala Asp Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 29 <211> 1389 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 29 agcagggtcc aaccaacaga gagcattgtg aggtttccaa acatcaccaa cctgtgtcca 60 tttggagagg tgttcaatgc caccaggttt gcctctgtct atgcctggaa caggaagagg 120 attagcaact gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 180 aagtgttatg gagtgagccc aaccaaactg aatgacctgt gtttcaccaa tgtctatgct 240 gactccttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 300 attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 360 agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 420 aagagcaacc tgaaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 480 acaccatgta atggagtgga gggcttcaac tgttactttc cactccaatc ctatggcttc 540 caaccaacca atggagtggg ctaccaacca tacagggtgg tggtgctgtc ctttgaactg 600 ctccatgccc ctgccacagt gtgtggacca aagaagagca ccaacctggt gaagaacaag 660 tgtgtgaact tcgctgatga cgatgacaag gagcccaaat cttctgacaa aactcacaca 720 tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 780 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 840 gtgagccacg aagaccccga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 900 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 960 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1020 aaagccctcc cagcccccat cgagaaaac atctccaaag ccaaagggca gccccgagaa 1080 ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 1140 acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 1200 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1260 ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1320 tccgtgatgc atggcgctct gcacaaccac tacacgcaga agagcctctc cctgtctcg 1380 ggtaaataa 1389 <210> 30 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 30 Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 1 5 10 15 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 20 25 30 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 35 40 45 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 50 55 60 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 65 70 75 80 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 85 90 95 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 100 105 110 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 115 120 125 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 130 135 140 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 145 150 155 160 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 165 170 175 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 180 185 190 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 195 200 205 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 Ala Asp Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 31 <211> 1363 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 31 agcagggtcc aaccaacaga gagcattgtg aggtttccaa acatcaccaa cctgtgtcca 60 tttggagagg tgttcaatgc caccaggtttt gcctctgtct atgcctggaa caggaagagg 120 attagcaact gtgtggctga ctactctgtg ctctacaact ctgcctcctt cagcaccttc 180 aagtgttatg gagtgagccc aaccaaactg aatgacctgt gtttcaccaa tgtctatgct 240 gactccttg tgattagggg agatgaggtg agacagattg cccctggaca aacaggcaag 300 attgctgact acaactacaa actgcctgat gacttcacag gctgtgtgat tgcctggaac 360 agcaacaacc tggacagcaa ggtgggaggc aactacaact acctctacag actgttcagg 420 aagagcaacc tgaaaccatt tgagagggac atcagcacag agatttacca ggctggcagc 480 acaccatgta atggagtgga gggcttcaac tgttactttc cactccaatc ctatggcttc 540 caaccaacca atggagtggg ctaccaacca tacagggtgg tggtgctgtc ctttgaactg 600 ctccatgccc ctgccacagt gtgtggacca aagaagagca ccaacctggt gaagaacaag 660 tgtgtgaact tcgagtccaa atatggtccc ccatgcccac cctgcccagc acctgaggcc 720 gccggggac catcagtctt cctgttcccc ccaaaaccca aggacactct catgatctcc 780 cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc aggaagaccc cgaggtccag 840 ttcaactggt acgtggatgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagttcaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aacggcaagg agtacaagtg caaggtctcc aaaaggcc tccgtcctc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc 1080 caggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1140 agcgacatcg ccgtggagtg ggaaagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaggctaac cgtggacaag 1260 agcaggtggc aggaggggaa tgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ctgggtaaat aaa 1363 <210> 32 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 32 Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 1 5 10 15 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 20 25 30 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 35 40 45 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 50 55 60 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 65 70 75 80 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 85 90 95 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 100 105 110 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 115 120 125 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 130 135 140 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 145 150 155 160 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 165 170 175 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 180 185 190 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 195 200 205 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 325 330 335 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Leu Gly Lys 450 <210> 33 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 33 gtcaccgtcc tgacacgaag cttggtacc 29 <210> 34 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 34 tatagaatag ggccctctag atttagaagt tcacacactt gttcttcacc 50 <210> 35 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 35 tttgtctctc ccagcagggt ggtgccatct ggagatgt 38 <210> 36 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 36 gtcatcgtca tcagcgaagt tcacacactg gttcttaa 38 <210> 37 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 37 gtcaccgtcc tgacacgaag cttggtacc 29 <210> 38 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 38 gggagaggct cttctgcgtg tagtggttgt gcagagcgcc atgcatcacg gagcat 56 <210> 39 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 39 tatagaatag ggccctctag atttatttac ccggagacag ggagaggctc ttctgcgtgt 60 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 40 gagtccaaat atggtccccc at 22 <210> 41 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 41 atgggggacc atatttggac tcgaagttca cacacttgtt cttcac 46 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 42 actatagaat agggccctct aga 23

Claims

1. A method for enhancing the immunogenicity of a protein / peptide antigen, the method comprising fusing the protein / peptide antigen with a modified antibody Fc fragment, the Fc fragment having an enhanced binding capacity to Fc receptors and / or complement protein C1q compared to its native form, thereby mimicking the antigen-antibody complex to enhance phagocytosis by DC / B antigen-presenting cells and enhance antigen-immune effects. The amino acid sequence of the modified antibody Fc fragment is shown in SEQ ID NO: 18; The protein / peptide antigen mentioned therein is the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein; in The amino acid sequence of the fusion of the protein / peptide antigen and the modified antibody Fc fragment is shown in SEQ ID NO:

30.

2. A method for enhancing the immunogenicity of a protein / peptide antigen, the method comprising fusing the protein / peptide antigen with a modified antibody Fc fragment, the Fc fragment having an enhanced binding capacity to Fc receptors and / or complement protein C1q compared to its native form, which can mimic antigen-antibody complexes to enhance phagocytosis by DC / B antigen-presenting cells and enhance antigen-immune effects. The amino acid sequence of the modified antibody Fc fragment is shown in SEQ ID NO: 18; The protein / peptide antigen mentioned therein is the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein; in The amino acid sequence of the protein / peptide antigen fused with the modified antibody Fc fragment is shown in SEQ ID NO: 30, and is produced using Fucose knockout mammalian cells.

3. The method of claim 2, wherein the mammalian cell is a fut8 gene knockout HEK-293 cell.

4. The method according to any one of claims 1-3, wherein the antigen is conjugated to other macromolecules via a linker.

5. The method of claim 4, wherein the other macromolecule is a polysaccharide, peptide / protein.

6. An immunogenic protein / peptide antigen fusion protein, wherein... The protein / peptide antigen is fused with a modified antibody Fc fragment, which, compared to its natural form, has an enhanced binding capacity to the Fc receptor and / or complement protein C1q. This can mimic the antigen-antibody complex to enhance phagocytosis by DC / B antigen-presenting cells and improve antigen-immune effects. The amino acid sequence of the modified antibody Fc fragment is shown in SEQ ID NO:18; The antigen mentioned therein is the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein; in The amino acid sequence of the fusion of the protein / peptide antigen and the modified antibody Fc fragment is shown in SEQ ID NO:

30.

7. An immunogenic protein / peptide antigen fusion protein, wherein... The protein / peptide antigen is fused with a modified antibody Fc fragment, which, compared to its natural form, has an enhanced binding capacity to the Fc receptor and / or complement protein C1q. This can mimic the antigen-antibody complex to enhance phagocytosis by DC / B antigen-presenting cells and improve antigen-immune effects. The amino acid sequence of the modified antibody Fc fragment is shown in SEQ ID NO:18; The antigen mentioned therein is the ACE2 receptor-binding domain (RBD) of the coronavirus spike protein; in The amino acid sequence of the protein / peptide antigen fused with the modified antibody Fc fragment is shown in SEQ ID NO: 30, and is produced using Fucose knockout mammalian cells.

8. The protein / peptide antigen fusion protein of claim 7, wherein the mammalian cell is a fut8 gene knockout HEK-293 cell.

9. A conjugate comprising, via a connector, the protein / peptide antigen fusion protein of any one of claims 6-8 and other macromolecules.

10. The conjugate of claim 9, wherein the other macromolecule is a polysaccharide, peptide / protein.

11. A nucleic acid encoding the protein / peptide antigen fusion protein of any one of claims 6-8, wherein the protein is mRNA and / or DNA.

12. The nucleic acid of claim 11, the sequence of which is shown in SEQ ID NO:

29.

13. An expression vector comprising the nucleic acid of claim 11 or 12.

14. A host cell comprising the nucleic acid of claim 11 or 12 or the expression vector of claim 13.

15. A method for producing a protein / peptide antigen fusion protein according to any one of claims 6-8, comprising culturing the host cells of claim 14 under conditions suitable for the expression of the aforementioned fusion protein, and recovering the expressed product from the culture medium.

16. An immune composition comprising the protein / peptide antigen fusion protein of any one of claims 6-8, the conjugate of any one of claims 9-10, the nucleic acid of any one of claims 11-12, or the expression vector of claim 13; and Pharmaceutically acceptable excipients.

17. The immunomodulatory composition of claim 16, wherein the pharmaceutically acceptable excipient is a lyophilized formulation or a pharmaceutically acceptable excipient in aqueous solution form.

18. The immune composition of claim 16, wherein the immune composition further comprises an adjuvant.

19. The immune composition of claim 18, wherein the adjuvant is selected from at least one of aluminum adjuvant, MF59, QS-21, or MPL.

20. Use of the protein / peptide antigen of any one of claims 6-8, the conjugate of any one of claims 9-10, the nucleic acid of any one of claims 11-12, the expression vector of claim 13, or the immune composition of any one of claims 16-19 in the preparation of a vaccine to prevent diseases caused by pathogens.

21. The use as described in claim 20, specifically in the preparation of a vaccine to prevent disease caused by a coronavirus.

22. The use as described in claim 21, specifically in the preparation of a vaccine to prevent disease caused by SARS-CoV-2.

23. A kit comprising the protein / peptide antigen fusion protein of any one of claims 6-8, the conjugate of any one of claims 9-10, the nucleic acid of any one of claims 11-12, the expression vector of claim 13, or the immune composition of any one of claims 16-19.

24. The kit of claim 23, further comprising means for administering the vaccine.

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