Fusion molecule capable of promoting secretion of membrane immobilized protein along with exosome and application of fusion molecule

Abstract

The invention relates to a fusion molecule capable of promoting secretion of membrane immobilized protein along with exosome, application thereof and the like. The invention specifically provides a design method and application for promoting secretory expression of an antigen in a mode of immobilizing the antigen on an exosome membrane, a core element of the antigen is a highly conserved N-terminal structural domain of beta-galactoside binding lectin Galectin-3, and the antigen can be widely applied to I type, II type, multi-transmembrane type and secretory type antigen proteins. According to the present invention, with the combination of the EPM (Endocytosis prevalence motif), the expression efficiency of the antigen can be effectively improved, the antigen protein can be promoted to enter the exosome secretion pathway, the immune response level can be improved, and the new technology is provided for the field of the preparation of the immune drug capable of efficiently inducing the immune response;

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, in particular the field of immunopharmaceutical technology, and specifically relates to fusion molecules that can promote the secretion of membrane-anchored proteins with exosomes and their applications. Background Art

[0002] The immune system is the body's key defense against pathogen invasion and maintains health. It consists of two major components: innate immunity and adaptive immunity. Adaptive immunity is further divided into humoral immunity and cellular immunity, which work together to defend against pathogens.

[0003] Humoral immunity is mainly mediated by B cells. The process is as follows: when pathogens invade the body, the receptors (BCR) on the surface of B cells recognize and internalize antigens, which are degraded into peptide fragments within the cells and then presented to helper T cells. The activated helper T cells provide co-stimulatory signals for B cells, prompting B cells to proliferate and differentiate, producing a large number of antibodies against the antigen. At the same time, a small number of B cells differentiate into memory B cells, providing long-term immune protection for the body.

[0004] Cellular immunity, primarily mediated by T cells, plays an equally important role. The process is as follows: Antigen-presenting cells (such as dendritic cells) present antigens to helper T cells (Th). Activated Th cells regulate the immune response by secreting cytokines and other molecules. Cytotoxic T cells (Tc) then recognize and bind to infected host or cancer cells, releasing perforins and granzymes to induce apoptosis in target cells, thereby eliminating pathogens or abnormal cells. Cellular immunity also involves other cell types, such as γδ T cells and NKT cells, which have unique functions in immune surveillance and anti-tumor activities.

[0005] Vaccination is an important means of preventing disease. Vaccines can be categorized as preventive or therapeutic. Preventive vaccines are primarily used to prevent infectious diseases, providing lasting immune protection by generating long-term memory B and T cells. Therapeutic vaccines, on the other hand, treat existing conditions, such as cancer and chronic infectious diseases, by activating the immune system to eliminate cancer cells or viruses. Humoral immunity vaccines, such as inactivated vaccines, live attenuated vaccines, and recombinant protein vaccines, are mostly preventive. They stimulate the body to produce targeted antibodies through antigenic molecules contained in the vaccine. In recent years, nucleic acid vaccines have shown great potential. These DNA or mRNA molecules carrying viral antigen genes, upon in vivo expression, produce antigenic proteins that can induce both humoral and cellular immune responses. These vaccines offer advantages such as good safety, ease of large-scale production, and the ability to rapidly design vaccines specifically for novel viruses. Therapeutic vaccines primarily focus on activating cellular immunity. For example, the BNT162b2 vaccine is being used to treat malignant gliomas, and ongoing clinical trials have shown promising results. Additionally, numerous other therapeutic mRNA vaccines are in clinical trials, such as those targeting melanoma, which have demonstrated promising efficacy and safety.

[0006] Exosomes, vesicles produced and secreted from living cells, have a double-layered membrane structure similar to that of cell membranes and play a crucial role in intercellular molecular transfer. Exosomes have garnered widespread attention for their use in immunotherapy, serving as natural antigen-presenting vehicles, delivering antigens to immune cells and stimulating immune responses. For example, dendritic cell (DC)-derived exosomes (DEX) hold promise as a cell-free vaccine containing antigen-presenting and immune cell-activating molecules. They are less susceptible to the immunosuppressive tumor microenvironment. Early studies have demonstrated their feasibility in tumor therapy and have found that DEX participates in NK cell-mediated innate immunity. However, existing approaches to antigen presentation and exosome application remain challenging. For example, the method disclosed in Chinese patents CN115708871A and CN115737796A, which fuses antigens to the extracellular domain of the CD63 protein, suffers from limitations such as limited numbers of EVs secreted extracellularly and potential changes in the conformation of the antigenic protein.

[0007] Galectin-3 is a β-galactoside-binding lectin that plays an important role in various physiological and pathological processes, including immune regulation and inflammatory responses. Galectin-3 is distributed in the nucleus, cytoplasm, and cell surface, and is also secreted into the extracellular matrix, where it performs distinct functions in different subcellular locations. The N-terminal domain of galectin-3, composed of a proline-, glycine-, and tyrosine-rich sequence, exhibits highly conserved and lectin-independent activity. This domain is involved in oligomerization upon ligand binding.

[0008] At present, in the field of immunotherapy, although there are some patents and research reports on improving the efficiency of antigen expression and presentation, the existing technology has many shortcomings. On the one hand, conventional humoral immune nucleic acid vaccines have difficulty in efficiently expressing target proteins such as exogenous membrane proteins and secretory proteins, and the target proteins cannot be fully presented outside the cells, resulting in low levels of antibodies induced by the body and limited immune protection. On the other hand, in the field of therapeutic vaccines, the existing technology also has obvious defects. For example, the delivery system of therapeutic vaccines is not efficient, and lacks regulators, making it difficult to deliver antigens in a targeted manner, thereby affecting the immune activation effect. At the same time, some therapeutic vaccines, such as DNA vaccines, have poor immunogenicity, the risk of DNA integration into chromosomes, and the intensity and persistence of the cellular immune response induced by them are insufficient.

[0009] In summary, there is an urgent need for a technical solution that can improve antigen expression levels and enhance immune response levels. Summary of the Invention

[0010] To address the shortcomings of the existing technology, the present invention fuses the highly conserved N-terminal domain of the β-galactoside-binding lectin Galectin-3 to the intracellular end of the antigen and uses it in combination with the endocytosis prevention motif (EPM) to promote the entry of the antigen into the exosome secretion pathway, which can overcome the shortcomings of the existing technology and provide a more efficient and higher-quality solution for immunotherapy.

[0011] The present invention relates to a method for designing and applying an immune vaccine framework. The core component is the highly conserved N-terminal domain of the β-galactoside-binding lectin Galectin-3. Specifically, the present invention provides a fusion molecular framework that can be widely applied to type I, type II, multi-transmembrane, and secreted antigenic proteins. Combined with an endocytosis prevention motif (EPM), it effectively improves antigen expression efficiency and promotes entry of the antigenic protein into the exosome secretory pathway, thereby enhancing immune response. The key components of this fusion molecular framework include: antigen, transmembrane region (this component is not required if the antigen already contains a transmembrane region), EPM, and the highly conserved N-terminal domain of Galectin-3. This framework facilitates the anchoring of the fusion protein to the exosome membrane and its extracellular secretion with the exosomes, promoting antigen delivery to immune organs or germinal centers, effectively increasing the probability of antigenic protein recognition by the immune system, thereby enhancing immune response. This provides a new technology for the development of immunotherapeutics that effectively induce immune responses.

[0012] One aspect of the present invention provides a fusion protein comprising: an N-terminal conserved domain of β-galactoside-binding lectin protein (Galectin-3); preferably, the N-terminal conserved domain of Galectin-3 comprises a non-lectin region comprising 7 to 14 repeats of a 9-amino acid sequence (YPG-X3-P-[G / S]-A), wherein X represents any amino acid residue and X3 represents a combination of three consecutive amino acid residues. More preferably, the N-terminal conserved domain of Galectin-3 comprises any one of the amino acid sequences of SEQ ID NOs. 3, 4, 5, 15, 17, 19, or 20.

[0013] Furthermore, the fusion protein further comprises an endocytosis prevention motif (EPM); preferably, the EPM comprises the intracellular segment of FcγRII; more preferably, the EPM comprises any one of the amino acid sequences in SEQ ID NO. 2 and 6.

[0014] Furthermore, the fusion protein also contains an antigen; preferably, the antigen is the complete sequence of an immune protein or a fragment sequence that partially contains an immune epitope; more preferably, the immune protein contains a membrane protein or a secretory protein; most preferably, the membrane protein contains a type I transmembrane protein or a type II transmembrane protein or a multi-transmembrane protein. When the antigen is a secretory protein, the fusion protein needs to additionally introduce a transmembrane region sequence; optionally, the transmembrane region is located inside the antigen sequence, or at one end of the antigen sequence.

[0015] Furthermore, the N-terminus of the fusion protein further comprises a signal peptide and / or an IgG Fc domain; or the C-terminus of the fusion protein further comprises the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit; or the additionally introduced transmembrane region comprises the transmembrane domain of CD80; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 10, and / or the amino acid sequence of the IgG Fc domain is as shown in SEQ ID NO: 11, and / or the amino acid sequence of STABILON is as shown in SEQ ID NO: 12, and / or the amino acid sequence of the transmembrane domain of CD80 is as shown in SEQ ID NO: 13.

[0016] Furthermore, different elements of the fusion protein may be optionally connected via a linker sequence; preferably, the linker comprises a flexible linker sequence and / or a rigid linker sequence.

[0017] Furthermore, the fusion protein comprises any one of the following four fusion proteins: (1) Fusion protein A: From N-terminus to C-terminus, it contains: signal peptide, secretory antigen, transmembrane region, EPM, and amino acids 1-120 of the N-terminal conserved domain of Galectin-3; (2) Fusion protein B: From N-terminus to C-terminus, it contains: amino acids 1-120 of the N-terminal conserved domain of type I transmembrane antigen, EPM, and Galectin-3; (3) Fusion protein C: From N-terminus to C-terminus, it contains: amino acids 1-120 of the N-terminal conserved domain of Galectin-3, EPM, and type II transmembrane antigen. (4) Fusion protein D: From N-terminus to C-terminus, it contains: multiple transmembrane antigens, EPM, and amino acids 1-120 of the N-terminal conserved domain of Galectin-3.

[0018] Furthermore, the fusion protein comprises any one of the following: (1) Fusion protein 5: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, mouse EPM, and an amino acid fragment at positions 1-120 of the N-terminus of hamster Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 4; (2) Fusion protein 12: comprising, from N-terminus to C-terminus, a signal peptide, a bovine IgG Fc domain, a secretory protein antigen, a transmembrane domain of bovine CD80, a mouse EPM, and an amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein; preferably, the secretory protein antigen is a BVDV 3E2 fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 9, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 10, the amino acid sequence of the bovine IgG Fc domain is shown in SEQ ID NO: 11, the amino acid sequence of the transmembrane domain of bovine CD80 is shown in SEQ ID NO: 13, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 4; (3) Fusion protein 1: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, guinea pig EPM, and an amino acid fragment at positions 1-104 of the N-terminus of a hamster Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the guinea pig EPM is shown in SEQ ID NO: 2, and the amino acid sequence of the amino acid fragment at positions 1-104 of the N-terminus of a hamster Galectin-3 protein is shown in SEQ ID NO: 3; (4) Fusion protein 2: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, guinea pig EPM, and an amino acid fragment at positions 1-120 of the N-terminus of a hamster Galectin-3 protein (SEQ ID NO: 4); preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the guinea pig EPM is shown in SEQ ID NO: 2, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of a hamster Galectin-3 protein is shown in SEQ ID NO: 4; (5) Fusion protein 3: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, guinea pig EPM, and an amino acid fragment at positions 30-104 of the N-terminus of the hamster Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the guinea pig EPM is shown in SEQ ID NO: 2, and the amino acid sequence of the amino acid fragment at positions 30-104 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 5; (6) Fusion protein 20: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, mouse EPM, and an amino acid fragment at positions 1-101 of the N-terminus of chicken Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-101 of the N-terminus of chicken Galectin-3 protein is shown in SEQ ID NO: 19; (7) Fusion protein 21: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, mouse EPM, and a fragment of amino acids 1-109 at the N-terminus of human Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the fragment of amino acids 1-109 at the N-terminus of human Galectin-3 protein is shown in SEQ ID NO: 20; (8) Fusion protein 13: from N-terminus to C-terminus, it comprises: a signal peptide, a bovine IgG Fc domain, a secretory protein antigen, a transmembrane domain of bovine CD80, a mouse EPM, and an amino acid fragment of positions 1-120 at the N-terminus of the bovine Galectin-3 protein; preferably, the secretory protein antigen is a BVDV 3E2 fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 9, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 10, the amino acid sequence of the bovine IgG Fc domain is shown in SEQ ID NO: 11, the amino acid sequence of the transmembrane domain of bovine CD80 is shown in SEQ ID NO: 13, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment of positions 1-120 at the N-terminus of the bovine Galectin-3 protein is shown in SEQ ID NO: 15; (9) Fusion protein 15: comprising, from N-terminus to C-terminus, the following: an amino acid fragment at positions 1 to 120 of the N-terminus of the hamster Galectin-3 protein, mouse EPM, and a type II transmembrane protein antigen; preferably, the type II transmembrane protein antigen is a full-length H1N1 NA protein, the amino acid sequence of which is shown in SEQ ID NO: 16, the amino acid sequence of the amino acid fragment at positions 1 to 120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 4, and the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6; (10) Fusion protein 16: comprising, from N-terminus to C-terminus, the following: an amino acid fragment at positions 1-119 of the N-terminus of porcine Galectin-3 protein, mouse EPM, and a type II transmembrane protein antigen; preferably, the type II transmembrane protein antigen is a full-length H1N1 NA protein, the amino acid sequence of which is shown in SEQ ID NO: 16, the amino acid sequence of the amino acid fragment at positions 1-119 of the N-terminus of porcine Galectin-3 protein is shown in SEQ ID NO: 17, and the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6; (11) Fusion protein 18: From the N-terminus to the C-terminus, it comprises: a multi-transmembrane protein antigen, a mouse EPM, and an amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein; preferably, the multi-transmembrane protein antigen is a full-length CXCR2 protein, the amino acid sequence of which is shown in SEQ ID NO: 17, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 4.

[0019] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises a nucleic acid encoding any one of the fusion proteins of the present invention; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

[0020] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule of the present invention.

[0021] Furthermore, the recombinant gene expression cassette further includes one or more of a promoter, a terminator, and a regulatory sequence.

[0022] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule of the present invention or the recombinant gene expression cassette of the present invention.

[0023] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0024] Furthermore, the prokaryotic vector includes but is not limited to an Escherichia coli vector.

[0025] Furthermore, the E. coli vector includes but is not limited to pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0026] Furthermore, the eukaryotic vector includes but is not limited to yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0027] Furthermore, the yeast expression vector includes but is not limited to pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0028] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule of the present invention, or the recombinant gene expression cassette of the present invention, or the recombinant vector of the present invention.

[0029] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0030] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0031] Furthermore, the yeast cells include but are not limited to Saccharomyces cerevisiae, Pichia pastoris, and Hansenula.

[0032] Furthermore, the prokaryotic cells include but are not limited to Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0033] Furthermore, the Escherichia coli cells include but are not limited to BL21 (DE3), DH5α, TOP10, and Rosetta.

[0034] Another aspect of the present invention provides a pharmaceutical composition, characterized in that it comprises one or more fusion proteins of any one of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0035] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins of any one of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more pharmaceutical compositions of the present invention; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; more preferably, the recombinant vaccine is a nucleic acid vaccine.

[0036] Another aspect of the present invention provides a method for promoting the extracellular secretion of a fusion protein with exosomes, characterized in that the method comprises administering to cells and / or other organisms one or more fusion proteins of any one of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention.

[0037] Another aspect of the present invention provides a method for improving the immunogenicity of an immune antigen, characterized in that the method comprises administering to cells and / or other immune organisms one or more fusion proteins of any one of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention.

[0038] Another aspect of the present invention provides the use of one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more pharmaceutical compositions according to the present invention, and / or one or more recombinant vaccines according to the present invention in any of the following: (1) Preparation of drugs for promoting the secretion of antigens into the extracellular space along with exosomes; (2) Preparation of drugs for improving the immunogenicity of antigens; (3) Preparation of drugs for treatment, vaccination or biological immunity.

[0039] Furthermore, the drug is a drug for preventing and / or treating viral infectious diseases; preferably, the virus includes influenza virus, diarrhea virus, herpes virus, Epstein-Barr virus, hepatitis virus, or HPV virus; more preferably, the influenza virus is avian influenza virus, and the diarrhea virus is viral diarrhea virus; most preferably, the avian influenza virus is avian influenza H9N2 or H1N1, and the viral diarrhea virus is BVDV.

[0040] Furthermore, the drug is a drug for preventing and / or treating diseases related to CXCR2 (CXC chemokine receptor 2); preferably, the diseases related to CXCR2 include inflammatory diseases, tumor-related diseases, cardiovascular diseases, metabolic diseases, nervous system diseases, and kidney diseases; more preferably, the inflammatory diseases include chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), and psoriasis; the tumor-related diseases include cancer progression and metastasis, and acute myeloid leukemia (AML); the cardiovascular diseases include atherosclerosis, myocardial infarction, and heart failure; the metabolic diseases include obesity and insulin resistance, and non-alcoholic fatty liver disease (NAFLD); the nervous system diseases include multiple sclerosis (MS) and stroke; and the kidney diseases include acute kidney injury (AKI) and chronic kidney disease (CKD).

[0041] The fusion protein molecular architecture and the nucleic acid architecture encoding the fusion protein molecular architecture, pharmaceutical composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects: 1. The present invention provides a fusion molecular architecture comprising a highly conserved N-terminal domain of Galectin-3, which can be widely applied to type I, type II, multiple transmembrane and secretory antigen proteins. When used in combination with an anti-endocytosis motif, it can effectively improve the expression efficiency of the antigen, promote the entry of the antigen protein into the exosome secretion pathway, and improve the immune response level.

[0042] 2. The key components of the fusion molecule architecture of the present invention include: antigen, transmembrane region (if the antigen itself contains a transmembrane region, this component is not required), EPM, and the highly conserved N-terminal domain of Galectin-3. This architecture enables the fusion protein to be anchored to the exosome membrane and secreted into the extracellular space with the exosomes, promoting antigen delivery to immune organs or germinal centers, effectively increasing the probability of immune recognition of the antigen protein, thereby enhancing the immune response. This provides a new technology for the preparation of immunotherapeutics that effectively induce immune responses.

[0043] 3. Example 1 demonstrates that: ① the present invention can be fused to the intracellular end of a type I transmembrane protein, thereby promoting protein secretion and expression; ② the present invention, when used in combination with an EPM element, can further improve the efficiency of promoting antigen secretion and expression, with significantly better results than the prior art; ③ the present invention, when used in combination with a mouse EPM element, achieves the best results, not only promoting antigen secretion and expression, but also promoting high-abundance expression and accumulation of antigens within cells; ④ the core elements of the present invention are functionally conserved across species, and the N-terminal peptide segments of Galectin-3 from different species exhibit similar effects.

[0044] 4. Example 2 demonstrates that the present invention can be applied to secretory antigen expression, significantly improving antigen expression and secretion efficiency, and the effect is superior to the existing technology.

[0045] 5. Example 3 demonstrates that the present invention can be applied to the expression of type II transmembrane proteins, significantly improves the expression level, and is more effective than conventional technical means.

[0046] 6. Examples 2 and 3 both demonstrate once again that the functions of the element sequences of the present invention are relatively conserved in different species, and element sequences from different species can all exert similar expression-promoting and secretion-promoting effects.

[0047] 7. Example 4 demonstrates that the present invention can be applied to the expression of multiple transmembrane proteins, significantly improves the expression level, and is more effective than conventional technical means.

[0048] 8. Example 5 shows that mRNA vaccine 5, incorporating elements of the present invention, induced significantly higher levels of neutralizing antibodies in guinea pigs than mRNA vaccine 19 (p < 0.05). This demonstrates that mRNA vaccines based on the present invention can induce higher immune titers by improving antigen presentation. Analysis of the reasons for this result suggests that the present invention promotes extracellular secretion of antigens in the form of exosomes, increasing the probability of antigen delivery to immune organs or germinal centers, thereby improving the efficiency of B cell antigen recognition and ultimately inducing a high-level immune response.

[0049] 9. Example 6 shows that the mRNA vaccine 12 based on the present invention induced a higher level of immune response in the guinea pig model, which was significantly better than the mRNA vaccine 9 with conventional secretion design (p < 0.01), once again proving that the mRNA vaccine based on the present invention can not only improve protein expression and secretion effects in in vitro cell experiments (Example 2), but also effectively improve the immune effect of mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a non-limiting schematic diagram of the fusion molecules involved in the present invention.

[0051] Figure 2 Schematic diagram of a non-limiting molecular cassette for expressing the fusion protein of the present invention.

[0052] Figure 3 Schematic diagram of the molecular design framework of the vaccine used in Example 1.

[0053] Figure 4 for Figure 3 Quality control chart corresponding to the vaccine.

[0054] Figure 5 This is the Western blot result of vaccines 1-5.

[0055] Figure 6 These are the Western blot results for vaccines 2, 5, and 7.

[0056] Figure 7 These are the Western blot results for vaccines 5, 20, and 21.

[0057] Figure 8 Schematic diagram of the molecular design framework of the vaccine used in Example 2.

[0058] Figure 9 for Figure 8 Quality control chart corresponding to the vaccine.

[0059] Figure 10 This is the Western blot result of vaccine 8-13.

[0060] Figure 11 Schematic diagram of the molecular design framework of the vaccine used in Example 3.

[0061] Figure 12 for Figure 11 Quality control chart corresponding to the vaccine.

[0062] Figure 13 This is the Western blot result of vaccine 14-16.

[0063] Figure 14 Schematic diagram of the molecular design framework of the vaccine used in Example 4.

[0064] Figure 15 for Figure 14 Quality control chart corresponding to the vaccine.

[0065] Figure 16 This is the Western blot result of vaccine 17-18.

[0066] Figure 17 Schematic diagram of the molecular design architecture of vaccine 19.

[0067] Figures 18A-18B Comparison of the immune effects of vaccine 5 and vaccine 19 in the guinea pig model.

[0068] Figures 19A-19B Comparison of the immune effects of vaccine 9 and vaccine 12 in the guinea pig model. DETAILED DESCRIPTION

[0069] Terms and Definitions The term "Galectin-3" refers to a β-galactoside-binding lectin protein encoded by the LGALS3 gene and widely expressed in various cell types. Galectin-3 has three domains: a highly conserved N-terminal domain, a long N-terminal domain, and a C-terminal carbohydrate recognition domain (CRD). The N-terminal conserved domain of Galectin-3 is a non-lectin region consisting of 7 to 14 repeats of a 9-amino acid sequence (YPG-X3-P-[G / S]-A), where X represents any amino acid residue and X3 represents a combination of three consecutive amino acid residues. Galectin-3 can be derived from any species, preferably from mammals or birds, and more preferably from hamsters, chickens, or humans. Preferably, the truncated sequence of the N-terminal domain of Galectin-3 comprises any of the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 20.

[0070] The term "exosome" refers to membrane vesicles secreted by cells, which are approximately 30-160 nm in size and can be released into the extracellular space by fusion of multivesicular bodies with the cell membrane or by budding from the cell membrane.

[0071] The term "endocytosis prevention motif (EPM)" refers to an endocytosis-preventing motif that can block endocytosis. EPMs can tether target proteins to the cytoskeleton, thereby preventing localization to coated pits and endocytosis. EPMs can be derived from any species, preferably mammalian, more preferably guinea pig, mouse, or human. Preferably, the EPM comprises the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 6.

[0072] The terms "administering" or "vaccinating" refer to administration of a nucleic acid vaccine or vaccine composition of the present invention, preferably via intramuscular or subcutaneous routes, although other routes of administration can also be used, for example, oral, intranasal (e.g., aerosol or other non-injectable administration), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or a combination thereof. Administration into the animal's neck muscle is preferred. Boosting regimens can be used to adjust the dosing regimen to provide optimal immunity.

[0073] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0074] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," "recombinant protein," "fusion protein," and the like.

[0075] The term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, and DNA from viruses such as lentiviruses, retroviruses, vaccinia, adenoviruses, fowlpox, baculoviruses, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0076] The term "mRNA" refers to messenger RNA, which is translated into "messenger ribonucleic acid" in Chinese. It is a type of single-stranded ribonucleic acid that is transcribed from a chain of DNA as a template, carries genetic information and can guide protein synthesis.

[0077] The term "5'-UTR," referring to the "5' untranslated region" or "5'UTR," is a portion of a gene that is transcribed into the primary RNA transcript (pre-mRNA) and located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced by transcription from DNA. Many primary transcripts must undergo RNA processing to form biologically active RNA. Processing to form mature mRNA includes terminal modification, intron removal, capping, and / or splicing of individual rRNA molecules from the pre-RNA. Therefore, the 5'UTR of an mRNA is the portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In a genomic sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The 5' untranslated region (5'UTR) of vertebrate mRNAs can range from tens to hundreds of bases in length.

[0078] The term "3'-UTR," referring to a "3'-untranslated region" or "3'UTR," refers to a region located at the 3' end of a gene, downstream of the stop codon of the protein-coding region, that is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that is typically attached after the transcription process. The 3'-UTR of mammalian mRNA often has a homology region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats and may fold to create a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins known to enhance RNA stability (e.g., RNA-binding proteins).

[0079] The term "polyA," meaning "polyadenylic acid sequence," "poly(A) sequence," or "poly(A) tail," refers to a sequence of adenylate residues typically located at the 3' end of an RNA molecule. The present invention allows for the attachment of such a sequence during RNA transcription by a DNA template based on repeated thymidylate residues in the strand complementary to the coding strand, although such a sequence is not normally encoded in DNA and is instead attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription in the nucleus.

[0080] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells.

[0081] The terms "individual," "patient," or "subject" include animals. Animals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, horses, chickens, and ducks), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0082] The terms "transformation, transfection, and transduction" have the meanings generally understood by those skilled in the art, namely, the process of introducing exogenous DNA or RNA into a host.

[0083] The term "pharmaceutical combination" refers to auxiliary materials widely used in pharmaceutical production. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe, stable, and / or possesses specific functionality, and also to provide a method for effective absorption in a subject. A pharmaceutically acceptable carrier can be an inert filler or an active ingredient that provides a specific function to the pharmaceutical combination (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0084] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0085] The present invention discloses a fusion molecule architecture (including a fusion molecule nucleic acid architecture and a protein architecture) that promotes extracellular secretion of a target protein in the form of exosomes, as well as methods for preparing and applying a recombinant vaccine based on this architecture. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve this goal. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and it is apparent that those skilled in the art will be able to modify, alter, and combine the methods and applications described herein without departing from the disclosure, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0086] The fusion proteins and encoding nucleic acids and their components, as well as their preparation methods and applications, provided herein can all be commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression constructs, vaccine preparation, and immunization, those skilled in the art can implement the methods of the present invention.

[0087] The present invention is further illustrated below with reference to the following examples, wherein, as a preferred embodiment, a nucleic acid vaccine framework is selected for the preparation of a recombinant vaccine.

[0088] Example 1 Application of the fusion molecules of the present invention in type I transmembrane proteins and screening of the best combination Figure 1 The figure is a non-limiting schematic diagram of the fusion molecules involved in the present invention, which explains the mechanism of action of the fusion molecules of the present invention. Figure 2 Schematic diagram of a non-limiting molecular cassette for expressing the fusion protein of the present invention. Figure 2Four exemplary fusion proteins AD are given, wherein fusion protein A comprises a signal peptide, a transmembrane segment, an EPM, and the N-terminal 1-120 amino acids of Galectin3 from N-terminus to C-terminus, fusion protein B comprises a type I transmembrane antigen, an EPM, and the N-terminal 1-120 amino acids of Galectin3 from N-terminus to C-terminus, fusion protein C comprises the N-terminal 1-120 amino acids of Galectin3, an EPM, and a type II transmembrane antigen from N-terminus to C-terminus, and fusion protein D comprises multiple transmembrane antigens, an EPM, and the N-terminal 1-120 amino acids of Galectin3 from N-terminus to C-terminus. This example mainly describes the application of the fusion molecules of the present invention in type I transmembrane proteins and the screening of the best combination.

[0089] The core functional element used in the present invention is the N-terminal domain of Galectin-3 protein. In order to verify that the present invention can be applied to type I transmembrane proteins and to compare and screen the best combination of the present invention, it is necessary to prepare a method that can produce the following: Figure 3 The recombinant nucleic acid vaccine shown. First, Figure 3 This is a schematic diagram of the molecular design architecture of the vaccine used in Example 1, including the structural diagrams of vaccines 1-5, 7, and vaccines 20-21. Figure 3 The desired gene expression cassette is constructed based on the molecular schematic. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription, obtaining a vector plasmid for preparing a recombinant nucleic acid vaccine. For example, a capped mRNA vaccine is produced for subsequent validation experiments. The capped mRNA production process is as follows: Step a: The vector plasmid used to produce the capped mRNA vaccine is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0090] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0091] In order to more rigorously demonstrate the effects of the present invention, the mRNA vaccine antigens in Example 1 are all H9N2 HA protein (SEQ ID NO: 1), and the sequence optimization method remains consistent.

[0092] According to the above method, mRNA vaccines 1-5, 7, and mRNA vaccines 20-21 (schematic diagrams shown in FIG. 20 ) for comparative experiments in this embodiment were prepared. Figure 3 ): mRNA vaccine 1, the core expression elements from N-terminus to C-terminus include: H9N2 HA full-length sequence (SEQ ID NO: 1), guinea pig-derived EPM (SEQ ID NO: 2), and the 1-104 amino acid fragment of the N-terminus of hamster Galectin-3 protein (SEQ ID NO: 3); mRNA vaccine 2, the core expression elements from N-terminus to C-terminus include: H9N2 HA full-length sequence (SEQ ID NO: 1), guinea pig-derived EPM (SEQ ID NO: 2), and the 1-120 amino acid fragment of the N-terminus of hamster Galectin-3 protein (SEQ ID NO: 4); mRNA vaccine 3, the core expression elements from N-terminus to C-terminus include: H9N2 HA full-length sequence (SEQ ID NO: 1), guinea pig-derived EPM (SEQ ID NO: 2), and the N-terminal amino acid fragment of hamster Galectin-3 protein at positions 30-104 (SEQ ID NO: 5); mRNA vaccine 4, the core expression elements from N-terminus to C-terminus include: the full-length sequence of H9N2 HA (SEQ ID NO: 1), the N-terminal amino acid fragment of hamster Galectin-3 protein (SEQ ID NO: 4); mRNA vaccine 5, the core expression elements from N-terminus to C-terminus include: H9N2 HA full-length sequence (SEQ ID NO: 1), mouse-derived EPM (SEQ ID NO: 6), and the 1-120 amino acid fragment of the N-terminus of hamster Galectin-3 protein (SEQ ID NO: 4); mRNA vaccine 7, prior art (CN118620085B), the core expression elements from N-terminus to C-terminus include: H9N2 HA full-length sequence (SEQ ID NO: 1), guinea pig-derived EPM (SEQ ID NO: 2), and chicken ITCH WW domain amino acid fragment (SEQ ID NO: 8).

[0093] mRNA vaccine 20, the core expression elements from N-terminus to C-terminus include: H9N2 HA full-length sequence (SEQ ID NO: 1), mouse-derived EPM (SEQ ID NO: 6), and the N-terminal 1-101 amino acid fragment of chicken Galectin-3 protein (SEQ ID NO: 19); mRNA vaccine 21, the core expression elements from N-terminus to C-terminus include: H9N2 HA full-length sequence (SEQ ID NO: 1), mouse-derived EPM (SEQ ID NO: 6), and the N-terminal 1-109 amino acid fragment of human Galectin-3 protein (SEQ ID NO: 20); The quality control results of the above vaccines are as follows Figure 4 shown.

[0094] The comparative experiment is divided into three groups, the specific groupings are as follows: Group A: includes mRNA vaccine 1, mRNA vaccine 2, mRNA vaccine 3, mRNA vaccine 4, and mRNA vaccine 5. They are tested in parallel to compare whether Galectin-3 N-terminal fragments with different truncation methods can promote protein secretion and expression, and whether the combination with endocytosis prevention motifs (EPMs) from different sources can further improve the secretion and expression effect.

[0095] Group B: Contains mRNA vaccine 2, mRNA vaccine 5, and mRNA vaccine 7, which are tested in parallel to compare whether the effect produced by the optimal combination of the present invention is better than the existing technology.

[0096] Group C: Contains mRNA vaccine 5, mRNA vaccine 20, and mRNA vaccine 21, which were tested in parallel to prove that the core elements involved in the present invention are functionally conserved in different species and that the Galectin-3 N-terminal peptide segments of different species have similar effects.

[0097] For the parallel testing of Groups A, B, and C, equal amounts of the mRNA vaccines were transfected into HEK293 cells using in vitro transfection. Cells and culture supernatants were harvested 48 hours after transfection for protein concentration and extraction. Protein concentration was determined using the BCA assay. Forty micrograms of each sample were loaded equally for SDS-PAGE electrophoresis and Western blot analysis. The estimated molecular weights of mRNA vaccines 1-5, 7, and 20-21 are shown in Table 1.

[0098] Table 1 Estimated molecular weight of mRNA vaccines 1-5, 7, and 20-21

[0099] The test results of group A are as follows Figure 5As shown. The detection of target protein signals in the supernatant protein of mRNA vaccine 4 indicates that the element of the present invention (the amino acid peptide fragment of the N-terminal domain of galectin-3) alone can promote antigen secretion and expression, but the effect is modest. Comparative results from mRNA vaccines 2 and 4 demonstrate that the addition of an EPM element can further enhance antigen secretion and expression. Comparative results from mRNA vaccines 1, 2, and 3 demonstrate that the elements of the present invention of varying truncation lengths, when combined with an EPM element, can all promote antigen secretion and expression. Among them, the peptide fragment containing amino acids 1-120 (mRNA vaccine 2) exhibits the greatest secretion and expression-enhancing effects. Comparative results from mRNA vaccines 2, 4, and 5 demonstrate that both mouse-derived and guinea pig-derived EPM elements can be used in combination with the present invention to further enhance antigen secretion and expression, with the mouse EPM element exhibiting the greatest efficacy.

[0100] The test results of group B are as follows Figure 6 As shown, the target protein signal detected in the supernatant containing mRNA vaccine 2 and mRNA vaccine 5 of the present invention is significantly higher than that of the prior art (mRNA vaccine 7). In addition, the combination of mouse EPM elements and the present invention (mRNA vaccine 5) can not only promote secretory expression, but also promote high-abundance expression and accumulation of the target protein in cells.

[0101] The test results of group C are as follows Figure 7 As shown in the figure, the target protein can be detected in the supernatants of the three vaccines, proving that the N-terminal domain of Galectin-3 from different species has the function of promoting protein secretion and expression.

[0102] Based on the results of the three parallel tests A, B, and C, the following conclusions can be drawn: ① The present invention can be fused to the intracellular end of type I transmembrane proteins, and can promote protein secretion and expression; ② The present invention is used in combination with EPM elements to further improve the efficiency of promoting antigen secretion and expression, and the effect is significantly better than the existing technology; ③ The present invention is used in combination with mouse EPM elements, and the effect is best, which can not only promote antigen secretion and expression, but also promote high-abundance expression and accumulation of antigens in cells; ④ The core elements involved in the present invention are functionally conserved in different species, and the Galectin-3 N-terminal peptide segments of different species have similar effects.

[0103] Example 2 Application of the fusion molecules of the present invention in secretory proteins and screening of the best combination In order to verify that the present invention can be applied to secretory proteins and that the effect is better than the existing technology, it is necessary to prepare a method that can produce secretory proteins. Figure 8 The recombinant nucleic acid vaccine shown includes vaccines 8-13. First, according to Figure 8The desired gene expression cassette is constructed based on the molecular schematic. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription, obtaining a vector plasmid for preparing a recombinant nucleic acid vaccine. For example, a capped mRNA vaccine is produced for subsequent validation experiments. The capped mRNA production process is as follows: Step a: The vector plasmid used to produce the capped mRNA vaccine is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0104] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0105] In order to more rigorously demonstrate the effects of the present invention, the mRNA vaccine antigens in this embodiment are all fusions of the three E2 proteins of BVDV (SEQ ID NO: 9), all adopt the 3E2 fusion protein design disclosed in patent CN116284272B, and the sequence optimization method remains consistent.

[0106] According to the above method, mRNA vaccine 8-13 ( Figure 8 ): mRNA vaccine 8, the core expression elements from N-terminus to C-terminus include: signal peptide (SEQ ID NO: 10), bovine IgG Fc domain (SEQ ID NO: 11), BVDV 3E2 fusion protein (SEQ ID NO: 9); mRNA vaccine 9, the core expression elements from N-terminus to C-terminus include: signal peptide (SEQ ID NO: 10), bovine IgG Fc domain (SEQ ID NO: 11), BVDV 3E2 fusion protein (SEQ ID NO: 9), STABILON peptide (SEQ ID NO: 12); mRNA vaccine 10, the core expression elements from N-terminus to C-terminus include: signal peptide (SEQ ID NO: 10), bovine IgG Fc domain (SEQ ID NO: 11), BVDV 3E2 fusion protein (SEQ ID NO: 9), bovine CD80 transmembrane domain (SEQ ID NO: 13), mouse-derived EPM (SEQ ID NO: 6), EABR amino acid fragment (SEQ ID NO: 7); mRNA vaccine 11, the core expression elements from N-terminus to C-terminus include: signal peptide (SEQ ID NO: 10), bovine IgG Fc domain (SEQ ID NO: 11), BVDV 3E2 fusion protein (SEQ ID NO: 9), bovine CD80 transmembrane domain (SEQ ID NO: 13), guinea pig-derived EPM (SEQ ID NO: 2), and bovine ITCH WW domain amino acid fragment (SEQ ID NO: 14); mRNA vaccine 12, the core expression elements from N-terminus to C-terminus include: signal peptide (SEQ ID NO: 10), bovine IgG Fc domain (SEQ ID NO: 11), BVDV 3E2 fusion protein (SEQ ID NO: 9), bovine CD80 transmembrane domain (SEQ ID NO: 13), mouse EPM (SEQ ID NO: 6), and the 1-120 amino acid fragment of the N-terminus of hamster Galectin-3 protein (SEQ ID NO: 4); mRNA vaccine 13, the core expression elements from N-terminus to C-terminus include: signal peptide (SEQ ID NO: 10), bovine IgG Fc domain (SEQID NO: 11), BVDV 3E2 fusion protein (SEQ ID NO: 9), bovine CD80 transmembrane domain (SEQID NO: 13), mouse-derived EPM (SEQ ID NO: 6), and the 1-124 amino acid fragment of the N-terminus of bovine Galectin-3 protein (SEQ ID NO: 15).

[0107] The quality control results of the above vaccines are as follows Figure 9 shown.

[0108] mRNA vaccines 8-12 were tested in parallel to compare and verify whether the present invention can improve the expression and secretion efficiency of secretory proteins; mRNA vaccine 13 was tested alone to prove that the core elements involved in the present invention are functionally conserved in different species, and the Galectin-3 N-terminal peptide segments of different species have similar effects.

[0109] Equal amounts of the mRNA vaccine were transfected into HEK293 cells in vitro. Cells and culture supernatants were collected 48 hours after transfection for protein concentration and extraction. Protein concentration was determined using the BCA assay. 40 μg of each sample was loaded equally for SDS-PAGE electrophoresis and Western blot analysis. The estimated molecular weight of mRNA vaccine 8-13 is shown in Table 2.

[0110] Table 2 Estimated molecular weight of mRNA vaccine 8-13 name Estimated molecular weight (kDa) mRNA vaccines8 88.6 mRNA vaccines9 90.2 mRNA vaccines10 108.6 mRNA vaccines11 124.3 mRNA vaccines12 111.5 mRNA vaccine 13 113.5 Test results such as Figure 10 As shown. No target protein signal was detected in the supernatant of mRNA vaccine 8 and mRNA vaccine 9, which means that only using the signal peptide or using it in combination with the STABILON expression-promoting element cannot make the 3E2 protein significantly expressed and secreted, and cannot meet the application requirements. mRNA vaccine 10, mRNA vaccine 11, and mRNA vaccine 12 that incorporates the elements of the present invention can all detect high abundance of target proteins in the supernatant, and the target protein signal of mRNA vaccine 12 is the strongest, proving that the present invention can be applied to secretory antigen expression, significantly improving the expression and secretion efficiency of the antigen, and the effect is better than the prior art. mRNA vaccine 13 also detected a large amount of target protein expression in the supernatant, once again proving that the function of the element sequence of the present invention is relatively conservative in different species, and the element sequences from different species can all play a similar role in promoting expression and secretion.

[0111] Example 3 Application of the fusion molecules of the present invention in type II transmembrane proteins and screening of the best combination In order to verify that the present invention can be applied to type II transmembrane proteins and that the effect is better than conventional technology, it is necessary to prepare a method that can produce Figure 11 The recombinant nucleic acid vaccine shown includes vaccines 14-16. First, according to Figure 11 The desired gene expression cassette is constructed based on the molecular schematic. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription, obtaining a vector plasmid for preparing a recombinant nucleic acid vaccine. For example, a capped mRNA vaccine is produced for subsequent validation experiments. The capped mRNA production process is as follows: Step a: The vector plasmid used to produce the capped mRNA vaccine is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0112] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0113] In order to more rigorously demonstrate the effects of the present invention, the mRNA vaccine antigens in this embodiment are all H1N1 NA protein (SEQ ID NO: 16), and the sequence optimization method remains consistent.

[0114] According to the above method, mRNA vaccines 14-16 ( Figure 11 ): mRNA vaccine 14, the expression core element comprises expression of full-length H1N1 NA protein (SEQ ID NO: 16); mRNA vaccine 15, the core expression elements from N-terminus to C-terminus include: the 1-120 amino acid fragment of the N-terminus of hamster Galectin-3 protein (SEQ ID NO: 4), mouse-derived EPM (SEQ ID NO: 6), and the full-length H1N1 NA protein (SEQ ID NO: 16); mRNA vaccine 16, the core expression elements from N-terminus to C-terminus include: the N-terminal amino acid fragment 1-119 of the porcine Galectin-3 protein (SEQ ID NO: 17), the mouse EPM (SEQ ID NO: 6), and the full-length H1N1 NA protein (SEQ ID NO: 16); The quality control results of the above vaccines are as follows Figure 12 shown.

[0115] mRNA vaccines 14 and 15 were tested in parallel to compare and verify whether the present invention can improve the expression of type II transmembrane proteins; mRNA vaccines 15 and 16 were tested in parallel to prove that the core elements involved in the present invention are functionally conserved in different species, and that the Galectin-3 N-terminal peptide segments of different species have similar effects.

[0116] Equal amounts of the mRNA vaccine were transfected into HEK293 cells in vitro. Cells and culture supernatants were collected 48 hours after transfection for protein concentration and extraction. Protein concentration was determined using the BCA assay. 40 μg of each sample was loaded equally for SDS-PAGE electrophoresis and Western blot analysis. The estimated molecular weight of mRNA vaccine 14-16 is shown in Table 3.

[0117] Table 3 Estimated molecular weight of mRNA vaccine 14-16 name Estimated molecular weight (kDa) mRNA vaccines14 54.8 mRNA vaccines15 70.2 mRNA vaccines16 71.7 Test results such as Figure 13 As shown. Since the NA protein is a type II transmembrane protein and is not secreted during conventional expression, mRNA vaccine 14 only detects the expression level in the cleaved protein. Comparing the target protein signals of mRNA vaccine 14 and mRNA vaccine 15, it was found that the supernatant of mRNA vaccine 15, which incorporates the elements of the present invention, was able to detect significant expression of the target protein, while the conventional mRNA vaccine 14, which expresses the full-length NA alone, failed to detect protein expression. This proves that the present invention can be applied to the expression of type II transmembrane proteins, significantly improves expression levels, and is more effective than conventional technical means.

[0118] Comparing the target protein signals of mRNA vaccine 15 and mRNA vaccine 16, significant expression of the target protein was detected in the supernatant, which once again proves that the function of the element sequence of the present invention is relatively conserved in different species, and the element sequences from different species can exert similar expression-promoting and secretion-promoting effects.

[0119] Example 4 Application of the fusion molecules of the present invention in multi-transmembrane proteins and screening of the best combination In order to verify that the present invention can be applied to multiple transmembrane proteins and that the effect is better than conventional technology, it is necessary to prepare a method that can produce Figure 14 The recombinant nucleic acid vaccine shown includes vaccines 17-18. First, according to Figure 14 The desired gene expression cassette is constructed based on the molecular schematic. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription, obtaining a vector plasmid for preparing a recombinant nucleic acid vaccine. For example, a capped mRNA vaccine is produced for subsequent validation experiments. The capped mRNA production process is as follows: Step a: The vector plasmid used to produce the capped mRNA vaccine is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0120] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0121] In order to more rigorously demonstrate the effects of the present invention, the mRNA vaccine antigens in this embodiment are all seven-transmembrane protein CXCR2 (SEQ ID NO: 18), and the sequence optimization method remains consistent.

[0122] According to the above method, mRNA vaccines 17 and 18 ( Figure 14 ): mRNA vaccine 17, the expression core element comprises expression of full-length CXCR2 (SEQ ID NO: 18); mRNA vaccine 18, the core expression elements from N-terminus to C-terminus include: full-length CXCR2 (SEQ ID NO: 17), mouse-derived EPM (SEQ ID NO: 6), and the 1-120 amino acid fragment of the N-terminus of hamster Galectin-3 protein (SEQ ID NO: 4); The quality control results of the above vaccines are as follows Figure 15 shown.

[0123] mRNA vaccines 17 and 18 were tested in parallel to compare and verify whether the present invention can improve the expression of multiple transmembrane proteins.

[0124] Equal amounts of the mRNA vaccine were transfected into HEK293 cells in vitro. Cells and culture supernatants were collected 48 hours after transfection for protein concentration and extraction. Protein concentration was determined using the BCA assay. 40 μg of each sample was loaded equally for SDS-PAGE electrophoresis and Western blot analysis. The estimated molecular weight of mRNA vaccine 17-18 is shown in Table 4.

[0125] Table 4 Estimated molecular weight of mRNA vaccine 17-18 name Estimated molecular weight (kDa) mRNA vaccines17 45.6 mRNA vaccines18 62.4 Test results such as Figure 16 As shown. Since the CXCR2 protein is a multi-transmembrane protein and is not secreted when expressed in its full length, mRNA vaccine 17 only detects the expression level in the cleaved protein. Comparing the target protein signals of mRNA vaccine 17 and mRNA vaccine 18, it was found that the target protein was significantly expressed in the supernatant of mRNA vaccine 18 incorporating the elements of the present invention, while the conventional mRNA vaccine 17 expressing the full length of CXCR2 alone could not detect protein expression, demonstrating that the present invention can be applied to the expression of multi-transmembrane proteins, significantly improves the expression level, and is more effective than conventional technical means.

[0126] Example 5: Improving the immune effect of guinea pigs based on the H9N2 recombinant nucleic acid vaccine of the present invention To demonstrate the effectiveness of the present invention in animals, this example constructed an mRNA vaccine 19 expressing the full-length H9N2 HA protein (SEQ ID NO: 1). Figure 17 A schematic diagram of the molecular design architecture of vaccine 19 is provided. This vaccine was used in parallel with mRNA vaccine 5 in Example 1 for the mouse immunization experiment in this example. The two vaccines share the same antigen sequence, differing only in their architecture.

[0127] Ten SPF guinea pigs were used in this experiment. After acclimation for 3-7 days, they were randomly divided into two groups based on body weight. Each group consisted of five mice, for a total of two groups. The details are shown in Table 5. Dosages in this table and below refer to the amount of active ingredient.

[0128] Table 5 Grouping and treatment of animals in the immunization experiment

[0129] The guinea pigs in the above groups were immunized once and twice according to the immunization process in Table 1. Blood was collected on day 0 (before the first immunization) and day 28 (Day 28) to measure the hemagglutination inhibition titer (HI titers). Figure 18A shown.

[0130] The results of hemagglutination inhibition titer test are shown in Table 6 and Figure 18B As shown in the experimental results, the mRNA vaccine 5 incorporating the elements of the present invention induced significantly higher levels of neutralizing antibodies in guinea pigs than the mRNA vaccine 19 (p < 0.05). This demonstrates that the mRNA vaccine based on the present invention can induce higher levels of immune titers by improving antigen presentation.

[0131] Table 6 Results of H9 subtype avian influenza HI antibody detection in immune guinea pig serum samples

[0132] Note: The kaolin treatment resulted in a 4-fold dilution of the serum.

[0133] Analysis of the reasons for the above results shows that the present invention can promote the secretion of antigens outside cells in the form of exosomes, increasing the probability of antigen delivery to immune organs or germinal centers, thereby improving the efficiency of B cells in recognizing antigens and ultimately inducing a high level of immune response.

[0134] Example 6: Improving the immune effect of guinea pigs based on the BVDV recombinant nucleic acid vaccine of the present invention To demonstrate the effectiveness of vaccines based on the present invention in different animal models and to compare the present invention with conventional secretory protein designs, this example continued to use the immune antigens from Example 2 and conducted comparative experiments using mRNA vaccines 9 and 12. This example conducted immunization experiments in guinea pigs. The two vaccines had the same antigen sequence and both promoted protein secretion. Vaccine 9 was a conventional secretory protein design, while vaccine 12 was based on the present invention.

[0135] Ten SPF guinea pigs were used in this experiment. After acclimation for 3-7 days, they were randomly divided into two groups based on body weight. Each group consisted of five mice, for a total of two groups. The details are shown in Table 7. Dosages in this table and below refer to the amount of active ingredient.

[0136] Table 7 Grouping and treatment of animals in the immunization experiment

[0137] The guinea pigs in each group were vaccinated once and twice according to the immunization schedule in Table 3. Blood was collected on day 0 (before the first vaccination) and day 28 (Day 28), and serum was isolated. The samples were inactivated at 56°C for 30 minutes, and the neutralizing antibody titers against BVDV-1a, NADL strain (GenBank: M31182.1) were measured (fixed virus-dilution sample method) to evaluate the immune effects of different vaccine designs on guinea pigs. The immunization and sampling procedures are as follows: Figure 19A shown.

[0138] The test results are shown in Table 8 and Figure 19B As shown in the experimental results, it can be seen that the mRNA vaccine 12 based on the present invention induced a higher level of immune response in the guinea pig model, which was significantly better than the mRNA vaccine 9 with conventional secretion design (p < 0.01), once again proving that the mRNA vaccine based on the present invention can not only improve the expression and secretion effect of proteins in in vitro cell experiments (Example 2), but also effectively improve the immune effect of mRNA vaccines.

[0139] Table 8 Serum antibody test results (BVDV-1a)

[0140] In summary, the present invention provides a fusion molecular architecture with extremely high commercial value and broad application prospects, and has the following excellent characteristics: ① The present invention can be fused to the intracellular end of various types of proteins, and can promote protein expression and secretion; ② The present invention is used in combination with EPM elements to further improve the efficiency of promoting antigen secretion and expression, and the effect is significantly better than the existing technology; ③ The present invention is used in combination with mouse EPM elements, and the effect is best, which can not only promote antigen secretion and expression, but also promote high-abundance expression and accumulation of antigens in cells; ④ The core elements involved in the present invention are functionally conserved in different species, and the Galectin-3 N-terminal peptide segments of different species have similar effects; ⑤ The present invention can be applied to the production and research and development of animal immune drugs, significantly improve the immunogenicity of antigens, induce vaccinated individuals to produce high levels of immune responses, and the effect is better than the current existing technology.

[0141] The amino acid sequences of the proteins involved in all the above examples are shown in Table 9.

[0142] Table 9 Amino acid sequences of proteins involved in the present invention Sequence number sequence SEQ ID NO: 1 METVSLITILLVATVSNADKICIGYQSTNSTETVDTLTENNVPVTHVKELLHTEHNGMLCATSLGHPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIW NVSYSGTSKACSDSFYRSMRWLTQKNNAYPIQDAQYTNNQEKNILFMWGINHPPTDTTQTNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRIDYYWSVLKPGQTLRIRSDGNLIAPWYGHILSGESHGRILKTD LKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGITSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINN KIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI SEQ ID NO:2 GNPEHREMGETLPEDPGEYSVVFGGSMMSCPGLPDGLEPARTDLS SEQ ID NO:3 MADGFSLNDALAGSGNPNPQGWPGAWGNQPGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGPTAPGAYPGPAPGAYPGQPGASGAYPSAPGAYPAAGPYGA SEQ ID NO:4 MADGFSLNDALAGSGNPNPQGWPGAWGNQPGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGPTAPGAYPGPAPGAYPGQPGASGAYPSAPGAYPAAGPYGAPTGALTVPYKLPLAGG SEQ ID NO:5 PGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGPTAPGAYPGPAPGAYPGQPGASGAYPSAPGAYPAAGPYGA SEQ ID NO:6 ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY SEQ ID NO:7 FNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP SEQ ID NO:8 GPLPPGWEQRVDQHGRVYYVDHVEKRTTWDRPEPLPPSWERRVDNMGRIYYVDHFTRTTTWQRPTLESVRNYEQWQLQRSQLQGAMQQFNQRFIYGNQDFSSTQNKEFDPLGPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRSQGQLNEKPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRT SEQ ID NO:9 HLDCKPEFSYAIAKSDRIGPLGAEGLTTTWKDYSHEMKLEDTMVIAWCKDGKFVYLQRCTRETRYLAILHSRALPTSVVFKKLFDGRRQEDTVEMDDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCMLANRDTLDTAVVRTYRRSKPFPYRQGCGGSGGGGSGGLPDCKPDFSYAIAKNDEIGPLGATGLTTQWYEYSDGMRLQDTEVVVWCKDGEFKYLIRCEREARYLAILHTRALPTSVVFEKILNGKEQEDVVEMDDNFEFGLCPCDAKPLVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTLANKDTLATTVVRTYKRHRPFPYRQGCGGSGGGGSGGFPECKEGFQYAISKDKKMGLLGPESLTTTWHLPTKKIVDSMVQVWCEGKDLKILRTCTKEERYLVAVHERALSTSAEFLQISDGTTGPEVIDMPDDFEFGLCPCDSKPVIKGKFNTSLLNGPAFQMVCPQGWTGTIECILANQDTLDTTVIRTYRRTTPFQRRKWC SEQ ID NO:10 MNPLWTLLFVLSAPRGVLS SEQ ID NO:11 DPRCKTTCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPAPIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGK SEQ ID NO:12 KDGKKDKKEEDKK SEQ ID NO:13 LTWTIIIPVSAFGISVIIAVILTCLTCRNAAIRRQRRENEVEMQSCSQSP SEQ ID NO:14 APLPPGWEQRVDQHGRVYYVDHIEKRTTWDRPEPLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLESVRNYEQWQLQRSQLQGAMQQFNQRFIYGNQDLFATSQNKEFDPLGPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRNQGQLNEKPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRT SEQ ID NO:15 MADGFSLNDALSGSGKPNPQGWPGSWGNQPAGAGGYPGAAYPGAYPGQAPPGPYPGQGPPGAYPGQGPPGAYPGQGPPGAYPGPTAPAYPGPTAPSAYPGPGAYPPPAQPSAPGAYPAAGPYGI SEQ ID NO:16 MSPNQKIITIGSICMTIGIASLILQIGNIISIWISHSIQTENQNQSEICNQNVITYENNTWVNQTYVNVSNTNFVAEQTVASVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRKNILRTQESECACVNGSCFTVMTDGPSNGQASYKIFKIEKGKIVKSVELNAPNYHYEECSCYPESSEIICVCRDNWHGSNRPWVSFNQNLEYQIGYICSGIFGDNPRPNDKTGSCGPVFLNGANGVKGFSFKYGNGVWIGRTKSTNSRMGFEMIWDPDGWTRTDDKFSVKQDIIGITDWSGYSGSFVQHPELTGLDCMRPCFWVELIRGRPKENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK SEQ ID NO:17 MADGFSLNDALSGSGKPNPQGWPGAWGNQPAGPGGYPGASYPGTYPGQGPPGAYPGQAPPGAYPGQAPPGAYPGPTAPGYPGPAAPGAYPGQPGGPGAYPPPAQPSAPGAYPATGPYGA SEQ ID NO:18 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL SEQ ID NO:19 MSDGFSLSDALPAHNPGAPPPQGWNRPPGPGAFPAYPGYPGAYPGAPGPYPGAPGPHHGPPGPYPGGPPGPYPGGPPGPYPGGPPGPYPGGPTAPYSEAPA SEQ ID NO:20 MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGA The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the embodiments of the present disclosure. For those skilled in the art, other variations or changes in different forms can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that Comprising: the N-terminal conserved domain of β-galactoside-binding lectin protein (Galectin-3); preferably, the N-terminal conserved domain of Galectin-3 comprises a non-lectin region, and the non-lectin region comprises 7 to 14 repeats of a 9-amino acid sequence (YPG-X3-P-[G / S]-A), wherein X represents any amino acid residue, and X3 represents a combination of 3 consecutive arbitrary amino acid residues; more preferably, the N-terminal conserved domain of Galectin-3 comprises any one of the amino acid sequences of SEQ ID NO. 3, 4, 5, 15, 17, 19 or 20.

2. The fusion protein according to claim 1, characterized in that The fusion protein further comprises an endocytosis prevention motif (EPM); preferably, the EPM comprises the intracellular segment of FcγRII; more preferably, the EPM comprises any one of the amino acid sequences of SEQ ID NO. 2 and 6.

3. The fusion protein according to claim 1 or 2, characterized in that The fusion protein also contains an antigen; preferably, the antigen is the complete sequence of an immune protein or a fragment sequence that partially contains an immune epitope; more preferably, the immune protein contains a membrane protein or a secretory protein; most preferably, the membrane protein contains a type I transmembrane protein or a type II transmembrane protein or a multi-transmembrane protein. When the antigen is a secretory protein, the fusion protein needs to additionally introduce a transmembrane region sequence; optionally, the transmembrane region is located inside the antigen sequence, or at one end of the antigen sequence.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The N-terminus of the fusion protein further comprises a signal peptide and / or an IgG Fc domain; or the C-terminus of the fusion protein further comprises the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit; or the additionally introduced transmembrane region comprises the transmembrane domain of CD80; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 10, and / or the amino acid sequence of the IgG Fc domain is as shown in SEQ ID NO: 11, and / or the amino acid sequence of STABILON is as shown in SEQ ID NO: 12, and / or the amino acid sequence of the transmembrane domain of CD80 is as shown in SEQ ID NO:

13.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The different elements of the fusion protein may optionally be connected via a linker sequence; preferably, the linker comprises a flexible linker sequence and / or a rigid linker sequence.

6. The fusion protein according to any one of claims 1 to 5, characterized in that Contains any one of the following 4 fusion proteins: Fusion protein A: From N-terminus to C-terminus, it contains: signal peptide, secretory antigen, transmembrane region, EPM, and amino acids 1-120 of the N-terminal conserved domain of Galectin-3; Fusion protein B: From N-terminus to C-terminus, it contains: amino acids 1-120 of the N-terminal conserved domain of type I transmembrane antigen, EPM, and Galectin-3; Fusion protein C: From N-terminus to C-terminus, it contains: amino acids 1-120 of the N-terminal conserved domain of Galectin-3, EPM, and type II transmembrane antigen Fusion protein D: From N-terminus to C-terminus, it contains: multiple transmembrane antigens, EPM, and amino acids 1-120 of the N-terminal conserved domain of Galectin-3.

7. The fusion protein according to any one of claims 1 to 6, characterized in that Contains any of the following fusion proteins: Fusion protein 5: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, mouse EPM, and an amino acid fragment at positions 1-120 of the N-terminus of a hamster Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 4; Fusion protein 12: comprising, from N-terminus to C-terminus, a signal peptide, a bovine IgG Fc domain, a secretory protein antigen, a bovine CD80 transmembrane domain, a mouse EPM, and an amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein; preferably, the secretory protein antigen is a BVDV 3E2 fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 9, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 10, the amino acid sequence of the bovine IgG Fc domain is shown in SEQ ID NO: 11, the amino acid sequence of the bovine CD80 transmembrane domain is shown in SEQ ID NO: 13, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 4; Fusion protein 1: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, guinea pig EPM, and an amino acid fragment at positions 1-104 of the N-terminus of a hamster Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the guinea pig EPM is shown in SEQ ID NO: 2, and the amino acid sequence of the amino acid fragment at positions 1-104 of the N-terminus of a hamster Galectin-3 protein is shown in SEQ ID NO: 3; Fusion protein 2: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, guinea pig EPM, and an amino acid fragment at positions 1-120 of the N-terminus of a hamster Galectin-3 protein (SEQ ID NO: 4); preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the guinea pig EPM is shown in SEQ ID NO: 2, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of a hamster Galectin-3 protein is shown in SEQ ID NO: 4; Fusion protein 3: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, a guinea pig EPM, and an amino acid fragment at positions 30-104 of the N-terminus of a hamster Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the guinea pig EPM is shown in SEQ ID NO: 2, and the amino acid sequence of the amino acid fragment at positions 30-104 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 5; Fusion protein 20: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, mouse EPM, and an amino acid fragment at positions 1-101 of the N-terminus of a chicken Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-101 of the N-terminus of the chicken Galectin-3 protein is shown in SEQ ID NO: 19; Fusion protein 21: comprising, from N-terminus to C-terminus, a type I transmembrane protein antigen, mouse EPM, and an amino acid fragment at positions 1-109 of the N-terminus of a human Galectin-3 protein; preferably, the type I transmembrane protein antigen is the full-length sequence of H9N2 HA, the amino acid sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-109 of the N-terminus of the human Galectin-3 protein is shown in SEQ ID NO: 20; Fusion protein 13: comprising, from N-terminus to C-terminus, a signal peptide, a bovine IgG Fc domain, a secretory protein antigen, a bovine CD80 transmembrane domain, a mouse EPM, and an amino acid fragment 1-120 at the N-terminus of the bovine Galectin-3 protein; preferably, the secretory protein antigen is a BVDV 3E2 fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 9, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 10, the amino acid sequence of the bovine IgG Fc domain is shown in SEQ ID NO: 11, the amino acid sequence of the bovine CD80 transmembrane domain is shown in SEQ ID NO: 13, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment 1-120 at the N-terminus of the bovine Galectin-3 protein is shown in SEQ ID NO: 15; Fusion protein 15: comprising, from N-terminus to C-terminus, the following: an amino acid fragment at positions 1-120 of the N-terminus of a hamster Galectin-3 protein, a mouse EPM, and a type II transmembrane protein antigen; preferably, the type II transmembrane protein antigen is a full-length H1N1 NA protein, the amino acid sequence of which is shown in SEQ ID NO: 16, the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO: 4, and the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6; Fusion protein 16: comprising, from N-terminus to C-terminus, the following: an amino acid fragment at positions 1-119 of the N-terminus of porcine Galectin-3 protein, mouse EPM, and a type II transmembrane protein antigen; preferably, the type II transmembrane protein antigen is a full-length H1N1 NA protein, the amino acid sequence of which is shown in SEQ ID NO: 16, the amino acid sequence of the amino acid fragment at positions 1-119 of the N-terminus of porcine Galectin-3 protein is shown in SEQ ID NO: 17, and the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6; Fusion protein 18: From N-terminus to C-terminus, it comprises: a multi-transmembrane protein antigen, a mouse EPM, and an amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein; preferably, the multi-transmembrane protein antigen is a full-length CXCR2 protein, the amino acid sequence of which is shown in SEQ ID NO: 17, the amino acid sequence of the mouse EPM is shown in SEQ ID NO: 6, and the amino acid sequence of the amino acid fragment at positions 1-120 of the N-terminus of the hamster Galectin-3 protein is shown in SEQ ID NO:

4.

8. A recombinant nucleic acid molecule, characterized in that Comprising a nucleic acid encoding the fusion protein according to any one of claims 1 to 7; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

9. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 8.

10. A recombinant vector, characterized in that Comprising the recombinant nucleic acid molecule according to claim 8 or the recombinant gene expression cassette according to claim 9.

11. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule according to claim 8, or the recombinant gene expression cassette according to claim 9, or the recombinant vector according to claim 10.

12. A pharmaceutical composition, characterized in that Comprising one or more fusion proteins according to any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules according to claim 8, and / or one or more recombinant gene expression cassettes according to claim 9, and / or one or more recombinant vectors according to claim 10, and / or one or more recombinant host cells according to claim 11; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

13. A recombinant vaccine, characterized in that Comprising one or more fusion proteins of any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules according to claim 8, and / or one or more recombinant gene expression cassettes according to claim 9, and / or one or more recombinant vectors according to claim 10, and / or one or more recombinant host cells according to claim 11, and / or one or more pharmaceutical compositions according to claim 12; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; more preferably, the recombinant vaccine is a nucleic acid vaccine.

14. A method for promoting the secretion of fusion proteins into cells along with exosomes, characterized in that: The method comprises administering to cells and / or other organisms one or more fusion proteins of any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules of claim 8, and / or one or more recombinant gene expression cassettes of claim 9, and / or one or more recombinant vectors of claim 10, and / or one or more recombinant host cells of claim 11, and / or one or more pharmaceutical compositions of claim 12, and / or one or more recombinant vaccines of claim 13.

15. A method for improving the immunogenicity of an immune antigen, characterized in that: The method comprises administering to cells and / or other immune organisms one or more fusion proteins of any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules of claim 8, and / or one or more recombinant gene expression cassettes of claim 9, and / or one or more recombinant vectors of claim 10, and / or one or more recombinant host cells of claim 11, and / or one or more pharmaceutical compositions of claim 12, and / or one or more recombinant vaccines of claim 13.

16. Use of one or more fusion proteins according to any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules according to claim 8, and / or one or more recombinant gene expression cassettes according to claim 9, and / or one or more recombinant vectors according to claim 10, and / or one or more recombinant host cells according to claim 11, and / or one or more pharmaceutical compositions according to claim 12, and / or one or more recombinant vaccines according to claim 13 in any of the following: (1) Preparation of drugs for promoting the secretion of antigens into the extracellular space along with exosomes; (2) Preparation of drugs for improving the immunogenicity of antigens; (3) Preparation of drugs for treatment, vaccination or biological immunity.

17. The use according to claim 16, characterized in that The drug is a drug for preventing and / or treating viral infectious diseases; preferably, the virus includes influenza virus, diarrhea virus, herpes virus, Epstein-Barr virus, hepatitis virus, or HPV virus; more preferably, the influenza virus is avian influenza virus, and the diarrhea virus is viral diarrhea virus; most preferably, the avian influenza virus is avian influenza H9N2 or H1N1, and the viral diarrhea virus is BVDV.

18. The use according to claim 16, characterized in that The drug is a drug for preventing and / or treating diseases related to CXCR2 (CXC chemokine receptor 2); preferably, the diseases related to CXCR2 include inflammatory diseases, tumor-related diseases, cardiovascular diseases, metabolic diseases, nervous system diseases, and kidney diseases; more preferably, the inflammatory diseases include chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), and psoriasis; the tumor-related diseases include cancer progression and metastasis, and acute myeloid leukemia (AML); the cardiovascular diseases include atherosclerosis, myocardial infarction, and heart failure; the metabolic diseases include obesity and insulin resistance, and non-alcoholic fatty liver disease (NAFLD); the nervous system diseases include multiple sclerosis (MS) and stroke; and the kidney diseases include acute kidney injury (AKI) and chronic kidney disease (CKD).

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