Self-presenting antigen-ligand fusion protein, vaccine containing fusion protein and application of vaccine

Abstract

The invention discloses a self-presenting antigen-ligand fusion protein, a vaccine containing the fusion protein and application of the self-presenting antigen-ligand fusion protein and the vaccine, and relates to the technical field of vaccines, the antigen-ligand fusion protein comprises a first structural unit and a second structural unit, the first structural unit is a virus antigen, and the second structural unit is a CD91 ligand. The antigen-ligand fusion protein provided by the invention can provide a strong and lasting antibody level for an organism.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, in particular to a self-presenting antigen-ligand fusion protein, a vaccine containing the fusion protein and applications thereof. Background Art

[0002] Vaccines are biological products used to prevent infectious diseases. They stimulate the body's immune system to produce specific immune responses, allowing the body to quickly identify and eliminate pathogens when encountering real pathogens, thereby achieving the purpose of preventing diseases. Vaccines have played an important role in the COVID-19 pandemic, effectively reducing the severity and mortality rates of patients. Vaccines generally consist of two parts: antigens and delivery systems. Antigens are substances that can induce the body to produce an immune response and can specifically bind to the immune response product. They can be part of the pathogen or a complete pathogen. The delivery system refers to a substance that safely and effectively delivers the antigens in the vaccine to specific parts of the body so that the immune system can recognize and produce an immune response. It mainly includes lipid nanoparticles (LNP), polymer carriers, and extracellular vesicles.

[0003] The efficient production and maintenance of antibodies are related to the germinal center reaction, in which follicular dendritic cells (FDC) play a key role. Follicular dendritic cells (FDC) are cells located in the follicles of lymph nodes and spleen, but they are not true dendritic cells. They have long processes, can closely contact with B cells and express Fc receptors, but do not mediate endocytosis, so that antigen-antibody complexes form immune complex-coated bodies on their dendrites and are preserved for a long time. They play an important role in activating B cells and are also involved in the immune memory response of B cells.

[0004] gp96 belongs to the heat shock protein 90 (HSP90) family and is a highly conserved and ubiquitous glycoprotein. It acts as a molecular chaperone in cells, helping proteins to fold and assemble correctly. gp96 can bind to a variety of new proteins, damaged proteins, tumor antigens, viral antigens, etc., and initiate specific T cell immune responses by presenting antigen epitopes to MHC class I and class II molecules. In natural immunity, gp96 interacts with Toll-like receptors (TLRs), CD91 and other protein molecules to stimulate antigen-presenting cells (such as dendritic cells DC) to produce various cytokines to activate the immune system.

[0005] For viruses with poor immunogenicity, such as SARS-CoV-2, FluA, HIV, HPV, and HBV, existing vaccines are difficult to induce strong antibody levels, and recipients need to undergo multiple vaccinations to produce higher antibody levels. In addition, the cross-immunity response generated by multiple vaccinations is weak, which is also a pain point in the current research and development of such vaccines. How to effectively utilize potential antigens with weak immunogenicity, increase the titer and persistence of neutralizing antibodies produced by vaccine immunity, and produce effective T cell cross-protection against different mutant strains, etc., urgently need to be solved. Summary of the invention

[0006] The inventors of the present invention have found that the CD91 (LRP1) receptor is expressed on the surface of FDC cells and can specifically bind to the CD91 ligand. Therefore, the present invention provides a self-presenting antigen-ligand fusion protein, a vaccine containing the fusion protein and its application, wherein the antigen-ligand fusion protein can spontaneously specifically bind to the CD91 receptor on the surface of FDC and reside on the surface of FDC, and participate in the immune memory response of B cells together with FDC, thereby providing a strong and lasting antibody level.

[0007] In view of this, the present invention provides an antigen-ligand fusion protein, which comprises a first structural unit and a second structural unit, wherein the first structural unit is a viral antigen, and the second structural unit is a CD91 ligand; the CD91 ligand is gp96 as shown in the amino acid sequence of SEQ ID NO.1, or a mutant thereof with substitution and / or deletion and / or addition of no more than 10 amino acid residues, or HSP70 as shown in the amino acid sequence of SEQ ID NO.2, or a mutant thereof with substitution and / or deletion and / or addition of no more than 10 amino acid residues, or α2 macroglobulin as shown in the amino acid sequence of SEQ ID NO.3, or a mutant thereof with substitution and / or deletion and / or addition of no more than 10 amino acid residues; the C-terminus of the first structural unit is connected to the N-terminus of the second structural unit through a flexible polypeptide sequence, or is directly covalently connected through a peptide bond.

[0008] The CD91 ligand structure segment in the antigen-ligand fusion protein of the present invention can be targeted to specifically bind to the CD91 receptor on the surface of the FDC cell and reside on the FDC surface for a long time. The FDC then presents the antigen-ligand fusion protein to the B cell, and the antigen structure segment of the antigen-ligand fusion protein can be recognized and bound by the BCR of the B cell, so that the B cell begins to proliferate, differentiate and produce specific antibodies. In this process, since the antigen-ligand fusion protein resides on the FDC surface for a long time, the antigen-ligand fusion protein can continuously stimulate the B cell to differentiate into memory B cells, thereby further enhancing the antibody immune response. At the same time, due to the expression of CD91 molecules on the surface of DC cells, the antigen-ligand fusion protein can also cause a strong CD8-specific T cell immune response by targeting DC cross-presentation, thereby providing cross-immune protection. Therefore, the antigen-ligand fusion protein of the present invention can enhance the residence and presentation of antigens on the surface of FDC cells, thereby improving the titer and persistence of neutralizing antibodies produced by vaccine immunity, and at the same time, it can also improve the intensity of antigen-specific CD8+T cell immune response, providing effective T cell cross-protection for different mutant strains. On the other hand, when viral antigens are linked to CD91 ligands, the stability of viral antigens is significantly increased, which is conducive to retaining the antigens on FDCs for a longer time and promoting the differentiation of memory B cells.

[0009] In some embodiments, the viral antigen is one of the antigens of HBV, HPV, VZV, EBV, HSV-2, HIV, FluA or SARS-CoV-2 and mutants thereof, and the mutant includes natural point mutations / deletion mutations / increase mutations / truncations at one or more sites, artificial point mutations / deletion mutations / increase mutations / truncations, any combination of natural or artificial mutations, and subtypes produced after mutation. Mutants refer to mutant viral proteins expressed by mutant virus strains encoded by mutant genes derived from mutations of wild-type genes, such as point mutations of different mutant novel coronavirus S proteins that have been found: NTD region 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutations, RBD region K417, E484, N501Y and other mutations, L452R mutation, T478K mutation, D614G, H655Y mutations. These point mutations exist in different combinations in mutated coronaviruses such as the British B.1.1.7 (Alpha) mutant, the South African B.1.617.2 (Beta) mutant, the Brazilian P1 (Gamma) mutant, the Indian B.1.617, B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutant, and the California B.1.429 mutant.

[0010] In some embodiments, the viral antigen is one of the SARS-CoV-2RBD antigen shown in the amino acid sequence of SEQ ID NO.4, the FluA HA1 antigen shown in the amino acid sequence of SEQ ID NO.5, the HIVGAG antigen shown in the amino acid sequence of SEQ ID NO.6, the HPV L1 antigen shown in the amino acid sequence of SEQ ID NO.7, and the HBV HBc antigen shown in the amino acid sequence of SEQ ID NO.8, and their mutants.

[0011] In some embodiments, the C-terminus of the viral antigen is directly covalently linked to the N-terminus of the CD91 ligand through a peptide bond, and has an amino acid sequence as shown in SEQ ID NO.9, or an amino acid sequence as shown in SEQ ID NO.10, or an amino acid sequence as shown in SEQ ID NO.11, or an amino acid sequence as shown in SEQ ID NO.12, or an amino acid sequence as shown in SEQ ID NO.13, or an amino acid sequence as shown in SEQ ID NO.14, or an amino acid sequence as shown in SEQ ID NO.15, or an amino acid sequence as shown in SEQ ID NO.16, or an amino acid sequence as shown in SEQ ID NO.17, or an amino acid sequence as shown in SEQ ID NO.18, or an amino acid sequence as shown in SEQ ID NO.19, or an amino acid sequence as shown in SEQ ID NO.20, or an amino acid sequence as shown in SEQ ID NO.21, or an amino acid sequence as shown in SEQ ID NO.22, or an amino acid sequence as shown in SEQ ID NO.23. The N-terminus of the CD91 ligand is a sequence with a flexible structure, which can be directly covalently linked to the C-terminus of the viral antigen and still maintain the correct spatial structure of the fusion protein.

[0012] In some embodiments, the N-terminus and / or C-terminus of the fusion protein are connected or not connected with a tag that facilitates the expression, detection, tracing, and purification of the target protein. The tag refers to a polypeptide or protein that is fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein, and can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc. In the present invention, unless otherwise clearly stated or clearly limited by the context, the N-terminus of the fusion protein of the present invention may be connected to a tag or not; the C-terminus of the fusion protein may also be connected to a tag or not; and the N-terminus and C-terminus of the fusion protein are independent of each other, and do not need to be connected to tags at the same time or not connected to tags at all.

[0013] The antigen-ligand fusion protein of the present invention can be prepared by conventional methods, such as transfection. The antigen-ligand fusion protein disclosed in the present invention can be prepared by the following steps:

[0014] -Synthesize nucleic acid molecules based on nucleic acid sequences;

[0015] - Connect the target nucleic acid fragment molecule to the expression vector;

[0016] - After recombinant screening of the expression vector, transfection into host cells;

[0017] -Expand and subculture host cells;

[0018] -Separate host cells and target protein, purify the target protein, and obtain antigen-ligand fusion protein.

[0019] Therefore, in another aspect, the present invention provides a nucleic acid molecule encoding the above fusion protein, an expression vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule or the expression vector.

[0020] The present invention also provides the use of the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned host cell in the preparation of a vaccine.

[0021] In some embodiments, the above-mentioned vaccines are used to prevent and / or treat infections caused by the new coronavirus, influenza virus, human immunodeficiency virus, human papillomavirus and hepatitis B virus.

[0022] On the other hand, the present invention provides the use of the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned host cell in the preparation of a product for enhancing antigen retention and presentation.

[0023] On the other hand, the present invention provides the use of the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned host cell in the preparation of a product for improving the body's antibody secretion level and / or antibody maintenance time.

[0024] On the other hand, the present invention provides the use of the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned host cell in the preparation of a product for improving the level of memory B cells in an organism.

[0025] The present invention has the following advantages and effects:

[0026] 1. The antigen-ligand fusion protein provided by the present invention can enhance the retention and presentation of antigens on the surface of FDC cells, thereby improving the titer and persistence of neutralizing antibodies produced by vaccine immunization.

[0027] 2. The antigen-ligand fusion protein provided by the present invention can enhance the intensity of antigen-specific CD8+T cell immune response, provide effective T cell cross-protection against different mutant strains, and has important application value for preventing viral infection and establishing long-term protection and cross-protection functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the description of the present invention will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments and application examples of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a schematic diagram of the connection of the structural units of the antigen-ligand fusion protein.

[0030] Figure 2 The figure is the SDS-PAGE electrophoresis result of antigen-ligand fusion protein; wherein, lane 1 of (a) is SARS-CoV-2RBD-gp96 fusion protein, lane 2 is SARS-CoV-2RBD-HSP70 fusion protein, and lane 3 is SARS-CoV-2RBD-α2 macroglobulin fusion protein; lane 1 of (b) is FluAHA1-gp96 fusion protein, lane 2 is FluA HA1-HSP70 fusion protein, and lane 3 is FluA HA1-α2 macroglobulin fusion protein; lane 1 of (c) is HIVGAG-gp96 fusion protein, lane 2 is HIVGAG-HSP70 fusion protein, and lane 3 is HIVGAG-α2 macroglobulin fusion protein; lane 1 of (d) is HPV L1-gp96 fusion protein, lane 2 is HPV L1-HSP70 fusion protein, and lane 3 is HPV L1-α2 macroglobulin fusion protein; lane 1 of (e) is HBV HBc-gp96 fusion protein, lane 2 is HBV HBc-HSP70 fusion protein, and lane 3 is HBV HBc-α2 macroglobulin fusion protein.

[0031] Figure 3 This is the result graph of the CD91 expression intensity on the surface of FDC cells in Test Example 1.

[0032] Figure 4 This is a graph showing the retention intensity of the antigen binding to CD91 in Test Example 2.

[0033] Figure 5 Cy5 of lymph node FDC cells 24 hours after the first immunization in test example 3 + Result graph of antigen ratio.

[0034] Figure 6This is a graph showing the antibody titer test results after the third immunization for the first to fourth groups of the antibody level test in Test Example 4.

[0035] Figure 7 This is a graph showing the antibody titer test results after the third immunization for the fifth to eighth groups of the antibody level test in Test Example 4.

[0036] Figure 8 This is a graph showing the results of detecting the proportion of IgG1 memory B cells 6 months after the third immunization in the memory B cell level test in Test Example 4.

[0037] Fig. 9 This is a graph showing the result of the antigen-specific T cell response ratio 7 days after the third immunization in the antigen-specific CD8+ T cell response detection in Test Example 4. DETAILED DESCRIPTION

[0038] In order to make the objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Unless otherwise specified, the experimental reagents and materials used in the present invention can be obtained commercially.

[0040] Example 1 Design of fusion protein

[0041] The antigen-ligand fusion protein consists of two structural units, the first structural unit is the viral antigen, and the second structural unit is the CD91 ligand. In actual construction, the viral antigen can be connected to the CD91 ligand through any flexible structural polypeptide. When the C-terminus of the viral antigen is connected to the N-terminus of the CD91 ligand, it can be directly connected without the flexible structural polypeptide. The representative form of this connection is as follows Figure 1 shown.

[0042] Example 2 Preparation of SARS-CoV-2 RBD-gp96 Fusion Protein

[0043] SARS-CoV-2RBD-gp96 fusion protein was prepared by the following steps:

[0044] S1. Construction of vector and transfection of host cells

[0045] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 24 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-RBD / gp96. The recombinant expression vector pFastBac1-RBD / gp96 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0046] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-RBD / gp96; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0047] S2. Purification of target protein using ion exchange chromatography

[0048] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it on a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the post-column solution and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains SARS-CoV-2RBD-gp96 fusion protein. Aliquot and store at -80°C.

[0049] S3. Electrophoresis verification of fusion protein

[0050] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (a), the molecular weight of the protein in lane 1 is 120 kDa, and the SARS-CoV-2 RBD-gp96 fusion protein was successfully produced.

[0051] Example 3 Preparation of FluAHA1-gp96 fusion protein

[0052] FluA HA1-gp96 fusion protein was prepared by the following steps:

[0053] S1. Construction of vector and transfection of host cells

[0054] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 25 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-HA1 / gp96. The recombinant expression vector pFastBac1-HA1 / gp96 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0055] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-HA1 / gp96; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0056] S2. Purification of target protein using ion exchange chromatography

[0057] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains FluA HA1-gp96 fusion protein. Package and store at -80°C.

[0058] S3. Electrophoresis verification of fusion protein

[0059] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (b), the molecular weight of the protein in lane 1 is 130 kDa, and the FluA HA1-gp96 fusion protein was successfully prepared.

[0060] Example 4 Preparation of HIVGAG-gp96 Fusion Protein

[0061] HIVGAG-gp96 fusion protein is prepared by the following steps:

[0062] S1. Construction of vector and transfection of host cells

[0063] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 26 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-GAG / gp96. The recombinant expression vector pFastBac1-GAG / gp96 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0064] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-GAG / gp96; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0065] S2. Purification of target protein using ion exchange chromatography

[0066] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains HIVGAG-gp96 fusion protein. Package and store at -80°C.

[0067] S3. Electrophoresis verification of fusion protein

[0068] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (c), the molecular weight of the protein in lane 1 is 150 kDa, and HIVGAG-gp96 fusion protein was successfully obtained.

[0069] Example 5 Preparation of HPV L1-gp96 fusion protein

[0070] The HPV L1-gp96 fusion protein is prepared by the following steps:

[0071] S1. Construction of vector and transfection of host cells

[0072] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 27 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-L1 / gp96. The recombinant expression vector pFastBac1-L1 / gp96 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0073] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-L1 / gp96; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0074] S2. Purification of target protein using ion exchange chromatography

[0075] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains HPV L1-gp96 fusion protein. Package and store at -80°C.

[0076] S3. Electrophoresis verification of fusion protein

[0077] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (d), the molecular weight of the protein in lane 1 is 140 kDa, and the HPV L1-gp96 fusion protein was successfully produced.

[0078] Example 6 Preparation of HBV HBc-gp96 fusion protein

[0079] HBV HBc-gp96 fusion protein is prepared by the following steps:

[0080] S1. Construction of vector and transfection of host cells

[0081] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 28 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-HBc / gp96. The recombinant expression vector pFastBac1-HBc / gp96 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0082] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-HBc / gp96; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0083] S2. Purification of target protein using ion exchange chromatography

[0084] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains HBV HBc-gp96 fusion protein. Package and store at -80°C.

[0085] S3. Electrophoresis verification of fusion protein

[0086] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (e), the molecular weight of the protein in lane 1 is 110 kDa, and the HBV HBc-gp96 fusion protein was successfully obtained.

[0087] Example 7 Preparation of SARS-CoV-2 RBD-HSP70 Fusion Protein

[0088] SARS-CoV-2RBD-HSP70 fusion protein was prepared by the following steps:

[0089] S1. Construction of vector and transfection of host cells

[0090] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 29 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-RBD / HSP70. The recombinant expression vector pFastBac1-RBD / HSP70 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0091] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-RBD / HSP70; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0092] S2. Purification of target protein using ion exchange chromatography

[0093] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it on a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the post-column solution and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains SARS-CoV-2RBD-HSP70 fusion protein. Aliquot and store at -80°C.

[0094] S3. Electrophoresis verification of fusion protein

[0095] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (a), the molecular weight of the protein in lane 2 is 100 kDa, and the SARS-CoV-2 RBD-HSP70 fusion protein was successfully produced.

[0096] Example 8 Preparation of FluA HA1-HSP70 fusion protein

[0097] FluA HA1-HSP70 fusion protein was prepared by the following steps:

[0098] S1. Construction of vector and transfection of host cells

[0099] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 30 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-HA1 / HSP70. The recombinant expression vector pFastBac1-HA1 / HSP70 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0100] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-HA1 / HSP70; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0101] S2. Purification of target protein using ion exchange chromatography

[0102] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the post-column solution and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains FluA HA1-HSP70 fusion protein. Aliquot and store at -80°C.

[0103] S3. Electrophoresis verification of fusion protein

[0104] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (b), the molecular weight of the protein in lane 2 is 110 kDa, and the FluA HA1-HSP70 fusion protein was successfully produced.

[0105] Example 9 Preparation of HIVGAG-HSP70 fusion protein

[0106] HIVGAG-HSP70 fusion protein is prepared by the following steps:

[0107] S1. Construction of vector and transfection of host cells

[0108] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO.31 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-GAG / HSP70. The recombinant expression vector pFastBac1-GAG / HSP70 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0109] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-HSP70 / GAG; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0110] S2. Purification of target protein using ion exchange chromatography

[0111] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the post-column solution and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains FluA HA1-HSP70 fusion protein. Aliquot and store at -80°C.

[0112] S3. Electrophoresis verification of fusion protein

[0113] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (c), the molecular weight of the protein in lane 2 is 130 kDa, and the FluA HA1-HSP70 fusion protein was successfully produced.

[0114] Example 10 Preparation of HPV L1-HSP70 fusion protein

[0115] The HPV L1-HSP70 fusion protein is prepared by the following steps:

[0116] S1. Construction of vector and transfection of host cells

[0117] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 32 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-L1 / HSP70. The recombinant expression vector pFastBac1-L1 / HSP70 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0118] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-L1 / HSP70; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0119] S2. Purification of target protein using ion exchange chromatography

[0120] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the post-column solution and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains HPV L1-HSP70 fusion protein. Package and store at -80°C.

[0121] S3. Electrophoresis verification of fusion protein

[0122] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (d), the molecular weight of the protein in lane 2 is 120 kDa, and the HPV L1-HSP70 fusion protein was successfully produced.

[0123] Example 11 Preparation of HBV HBc-HSP70 fusion protein

[0124] HBV HBc-HSP70 fusion protein is prepared by the following steps:

[0125] S1. Construction of vector and transfection of host cells

[0126] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 33 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-HBc / HSP70. The recombinant expression vector pFastBac1-HBc / HSP70 was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0127] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-HBc / HSP70; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0128] S2. Purification of target protein using ion exchange chromatography

[0129] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains HBV HBc-HSP70 fusion protein. Package and store at -80℃.

[0130] S3. Electrophoresis verification of fusion protein

[0131] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (e), the molecular weight of the protein in lane 2 is 90 kDa, and the HBV HBc-HSP70 fusion protein was successfully produced.

[0132] Example 12 Preparation of SARS-CoV-2 RBD-α2 macroglobulin fusion protein

[0133] SARS-CoV-2 RBD-α2 macroglobulin fusion protein was prepared by the following steps:

[0134] S1. Construction of vector and transfection of host cells

[0135] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 34 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-RBD / α2 macroglobulin. The recombinant expression vector pFastBac1-RBD / α2 macroglobulin was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0136] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-RBD / α2 macroglobulin; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0137] S2. Purification of target protein using ion exchange chromatography

[0138] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it on a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the post-column solution and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains SARS-CoV-2RBD-α2 macroglobulin fusion protein. Aliquot and store at -80°C.

[0139] S3. Electrophoresis verification of fusion protein

[0140] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (a), the molecular weight of the protein in lane 3 is 170 kDa, and the SARS-CoV-2 RBD-α2 macroglobulin fusion protein was successfully prepared.

[0141] Example 13 Preparation of FluAHA1-α2 macroglobulin fusion protein

[0142] FluA HA1-α2 macroglobulin fusion protein was prepared by the following steps:

[0143] S1. Construction of vector and transfection of host cells

[0144] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 35 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-HA1 / α2 macroglobulin. The recombinant expression vector pFastBac1-HA1 / α2 macroglobulin was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0145] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-HA1 / α2 macroglobulin; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0146] S2. Purification of target protein using ion exchange chromatography

[0147] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain a concentrate of about 1mL. The concentrate contains FluA HA1-α2 macroglobulin fusion protein. Package and store at -80°C.

[0148] S3. Electrophoresis verification of fusion protein

[0149] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (b), the molecular weight of the protein in lane 3 was 180 kDa, and the FluA HA1-α2 macroglobulin fusion protein was successfully prepared.

[0150] Example 14 Preparation of HIVGAG-α2 macroglobulin fusion protein

[0151] HIVGAG-α2 macroglobulin fusion protein is prepared by the following steps:

[0152] S1. Construction of vector and transfection of host cells

[0153] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 36 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-GAG / α2 macroglobulin. The recombinant expression vector pFastBac1-GAG / α2 macroglobulin was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0154] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-GAG / α2 macroglobulin; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0155] S2. Purification of target protein using ion exchange chromatography

[0156] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains HIVGAG-α2 macroglobulin fusion protein. Package and store at -80°C.

[0157] S3. Electrophoresis verification of fusion protein

[0158] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (c), the molecular weight of the protein in lane 3 is 200 kDa, and HIVGAG-α2 macroglobulin fusion protein was successfully prepared.

[0159] Example 15 Preparation of HPV L1-α2 macroglobulin fusion protein

[0160] The HPV L1-α2 macroglobulin fusion protein is prepared by the following steps:

[0161] S1. Construction of vector and transfection of host cells

[0162] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 37 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-L1 / α2 macroglobulin. The recombinant expression vector pFastBac1-L1 / α2 macroglobulin was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0163] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-L1 / α2 macroglobulin; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8 Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0164] S2. Purification of target protein using ion exchange chromatography

[0165] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain about 1mL of concentrated solution. The concentrated solution contains HPV L1-α2 macroglobulin fusion protein. Package and store at -80°C.

[0166] S3. Electrophoresis verification of fusion protein

[0167] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (d), the molecular weight of the protein in lane 3 is 190 kDa, and the HPV L1-α2 macroglobulin fusion protein was successfully prepared.

[0168] Example 16 Preparation of HBV HBc-α2 macroglobulin fusion protein

[0169] HBV HBc-α2 macroglobulin fusion protein is prepared by the following steps:

[0170] S1. Construction of vector and transfection of host cells

[0171] S1.1. A nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO. 38 was synthesized using artificial bases, and the nucleic acid molecule was connected to the insect cell expression vector pFastBac1 to construct a recombinant expression vector pFastBac1-HBc / α2 macroglobulin. The recombinant expression vector pFastBac1-HBc / α2 macroglobulin was transformed into DH10Bac competent cells, and recombinant bacmid DNA was obtained through recombination screening.

[0172] S1.2, transfect the recombinant bacmid DNA into Sf9 cells (1×10 6 Each Sf9 cell was transfected with 4 μg of recombinant plasmid pFastBac1-HBc / α2 macroglobulin; during the transfection process, the transfection reagent was Cellfectin II reagent), incubated at 27°C for 72 h, and centrifuged. The supernatant was the P1 virus. 8Sf9 cells) were cultured at 27°C for 8-10 hours to obtain cultured cells; then, P1 virus (at a dose of 0.05-0.1 MOI) was added to the cultured cells, incubated at 27°C for 72 hours, and centrifuged at 4000 rpm for 5 minutes. The supernatant was the P2 virus. 8 P2 virus (at a dose of 0.05-0.1 MOI) was added to 300 mL of Sf9 cell suspension 3 (containing 4.5×10 8 P3 virus (at a dose of 5 MOI) was added to each Sf9 cell) and cultured at 27° C. and 100-120 rpm for 72 h to obtain a suspension.

[0173] S2. Purification of target protein using ion exchange chromatography

[0174] Take the suspension, centrifuge at 7000rpm for 20min, and take the supernatant. After filtering the supernatant through a 0.22mm filter membrane, load it onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate of 1mL / min), then rinse with 5mL of pH7.5, 200mM PBS buffer (flow rate of 1mL / min); then rinse with 10mL of pH7.5, 300mM PBS buffer (flow rate of 1mL / min); finally rinse with 3mL of pH7.5, 600mM PBS buffer (flow rate of 1mL / min), collect the solution after the column and use an ultrafiltration tube with a molecular weight cutoff of 50KD for ultrafiltration concentration to obtain a concentrate of about 1mL. The concentrate contains HBV HBc-α2 macroglobulin fusion protein. Package and store at -80°C.

[0175] S3. Electrophoresis verification of fusion protein

[0176] The concentrated solution was subjected to SDS-PAGE electrophoresis analysis. The experimental results were as follows: Figure 2 As shown in (e), the molecular weight of the protein in lane 3 is 160 kDa, and the HBV HBc-α2 macroglobulin fusion protein was successfully obtained.

[0177] Test Example 1 Verification of CD91 receptor expression on the surface of FDC cells

[0178] 1.1 Reagents and Materials

[0179] Bovine serum albumin (BSA, B265994), phosphate buffered saline (PBS, P903593), anti-mouse CD45 APC-Cy7 (Biolegend, 157203), anti-mouse podoplanin PE-Cy7 (Biolegend, 127411), anti-mouse CD31 APC (Biolegend, 102509), anti-mouse CD21 / 35 FITC (Biolegend, 123407), anti-mouse CD16 / 32 PerCP-Cy5.5 (Biolegend, 101323), Purified anti-mouse CD91 (Biolegend, 111902), Goat Anti-Rabbit IgG H&L (PE) preadsorbed (abcam, ab72465).

[0180] 1.2 Instruments

[0181] Centrifuge (TD5A-WS), flow cytometer (BD Fortessa).

[0182] 1.3 Experimental methods

[0183] Isolate spleen cells from 6-week-old female BALB / c (H-2d) mice and prepare single-cell suspension. Distribute cells into 1.5mL EP tubes, 500,000 cells per tube, and add PBS containing 10% BSA to block for at least 10 minutes. Add 1mL of PBS to the cells, centrifuge at 1500rmp for 5 minutes, and discard the supernatant. Resuspend the cells with 100μL of PBS containing 10% BSA, add 0.5μL of CD45, CD31, podoplanin, CD16 / 32, CD21 / 35 and CD91 surface staining antibodies respectively, and stain for 30 minutes in the dark. Add 1mL of PBS to the cells, centrifuge at 1500rmp for 5 minutes, and discard the supernatant. Resuspend the cells with 200μL of PBS and directly detect on the flow cytometer.

[0184] 1.4 Experimental Results

[0185] The results are as follows Figure 3 As shown, the fluorescence intensity of CD91 protein detected on the surface of FDC cells was significantly higher than that of the isotype control group, that is, the expression intensity of CD91 on the surface of FDC cells was significantly higher than that of the control group, indicating that the CD91 receptor was highly expressed on the surface of FDC cells.

[0186] Test Example 2 Fusion protein retention intensity test

[0187] 2.1 Reagents and Materials

[0188] PBST buffer (biosharp, BL314B), Anti-SARS-CoV-2 (COVID-19) Spike RBD antibody produced in rabbit (Sigma-Aldrich, SAB3501120), HA1 Antibody (SinoBiological, 40016-T46-100), GAG Antibody (Abmart, PH18493S), Anti-Cathepsin L1Antibody (Sigma-Aldrich, ZRB1636-25UL), anti-HBc antibody (FineTest, FNab09775), HRP Conjugated Goat anti-Rabbit IgG Antibody secondary antibody (HUABIO, HA1001), TMB, sulfuric acid.

[0189] 2.2 Instruments

[0190] ELISA reader, 96-well plate washer, and clean bench.

[0191] 2.3 Samples to be tested

[0192] Control group (antigen): SARS-CoV-2RBD, FluA HA1, HIVGAG, HPV L1, HBV HBc.

[0193] Sample group (antigen-ligand fusion protein): SARS-CoV-2RBD-gp96 fusion protein, FluA HA1-gp96 fusion protein, HIVGAG-gp96 fusion protein, HPV L1-gp96 fusion protein, HBV HBc-gp96 fusion protein, SARS-CoV-2RBD-HPS70 fusion protein, FluAHA1-HPS70 fusion protein, HIVGAG-HPS70 fusion protein, HPV L1-HPS70 fusion protein, HBV HBc-HPS70 fusion protein, SARS-CoV-2RBD-α2 macroglobulin fusion protein, FluA HA1-α2 macroglobulin fusion protein, HIVGAG-α2 macroglobulin fusion protein, HPV L1-α2 macroglobulin fusion protein, HBV HBc-α2 macroglobulin fusion protein.

[0194] 2.4 Experimental methods

[0195] Take a 96-well plate, coat each well with 10μg CD91 protein, incubate overnight at 4℃, and then wash 3 times with PBS-T, each time for 5min. Add 5μg antigen or antigen-ligand fusion protein to each well, add 100μL to each well, add 3 wells in total, and detect the binding strength. Incubate at 37℃ for 2h, then wash 3 times with PBS-T, each time for 5min. Add the corresponding antigen antibody (such as RBD group, add RBD antibody, HA1 group, add HA1 antibody), add 100μL to each well, incubate at 37℃ for 2h, then wash 3 times with PBS-T, each time for 5min. Add HRP-labeled IgG antibody and incubate at 37℃ for 1h. Wash 3 times with PBS-T, each time for 5min. Add 100μL TMB colorimetric solution to each well, place at 37℃ for 15min, add 50μl 2M sulfuric acid to terminate the reaction, and detect on the microplate reader at a wavelength of 450nm.

[0196] 2.5 Experimental Results

[0197] The results of the retention intensity of viral antigen binding to CD91 are shown in Figure 4 The experimental results showed that compared with the control group, the binding strength of the fusion protein in the sample group to CD91 was significantly improved (**** indicates P<0.0001, with statistically significant difference), proving that the antigen-ligand fusion protein of the present invention can bind to the CD91 receptor on the surface of FDC, achieving the purpose of long-term retention of the antigen-ligand fusion protein on the surface of FDC cells.

[0198] Test Example 3: Detection of in vivo residence time of antigen-ligand fusion protein

[0199] 3.1 Reagents and Materials

[0200] R-Cy5, bovine serum albumin (BSA, B265994), phosphate buffered saline (PBS, P903593), anti-mouse CD45 APC-Cy7 (Biolegend, 157203), anti-mouse podoplanin PE-Cy7 (Biolegend, 127411), anti-mouse CD31 APC (Biolegend, 102509), anti-mouse CD21 / 35FITC (Biolegend, 123407), anti-mouse CD16 / 32PerCP-Cy5.5 (Biolegend, 101323).

[0201] 3.2 Instruments

[0202] Centrifuge (TD5A-WS), flow cytometer (BD Fortessa).

[0203] 3.3 Grouping

[0204] Female BALB / c (H-2d) mice aged 6 weeks were randomly divided into single antigen group, antigen-gp96 fusion protein treatment group, antigen-HSP70 fusion protein treatment group, antigen-α2 macroglobulin treatment group, single antigen mixed with CD91 antibody treatment group, antigen-gp96 fusion protein mixed with CD91 antibody treatment group, antigen-HSP70 fusion protein mixed with CD91 antibody treatment group, antigen-α2 macroglobulin mixed with CD91 antibody treatment group. The above antigens are divided into five types: SARS-CoV-2RBD, FluA HA1, HIVGAG, HPV L1 and HBV HBc, with 10 mice in each group.

[0205] 3.4 Experimental methods

[0206] For antigen and antigen fusion protein, use R-Cy5 kit was used to label Cy5 fluorescence. Each group of mice was immunized three times, on day 0, day 14 and day 28, respectively, and the immunization method was subcutaneous immunization.

[0207] Group 1 (antigen): 10 μg of antigen (diluted to 200 μL with PBS) was administered to each mouse for each immunization.

[0208] The second group (antigen-gp96 fusion protein): each mouse was immunized with 40 μg of antigen-gp96 fusion protein (diluted to 200 μL with PBS).

[0209] The third group (antigen-HSP70 fusion protein): each mouse was immunized with 40 μg of antigen-HSP70 fusion protein (diluted to 200 μL with PBS).

[0210] The fourth group (antigen-α2 macroglobulin): each mouse was immunized with 40 μg of antigen-α2 macroglobulin (diluted to 200 μL with PBS).

[0211] Group 5 (antigen mixed with CD91 antibody): Each mouse was immunized with 10 μg of antigen mixed with 5 μg of CD91 antibody (diluted to 200 μL with PBS).

[0212] Group VI (antigen-gp96 fusion protein mixed with CD91 antibody): each mouse was immunized with 40 μg antigen-gp96 fusion protein mixed with 5 μg CD91 antibody (diluted to 200 μL with PBS).

[0213] Group 7 (antigen-HSP70 fusion protein mixed with CD91 antibody): each mouse was immunized with 40 μg antigen-HSP70 fusion protein mixed with 5 μg CD91 antibody (diluted to 200 μL with PBS).

[0214] Group 8 (antigen-α2 macroglobulin mixed with CD91 antibody): each mouse was immunized with 40 μg antigen-α2 macroglobulin mixed with 5 μg CD91 antibody (diluted to 200 μL with PBS).

[0215] 24 hours after the first immunization, the inguinal lymph nodes of mice were taken to prepare single cell suspensions, and flow cytometry was used to detect the proportion of Cy5+ antigen-specific FDC cells, that is, the degree to which different antigens were retained on the surface of FDC cells. When staining cells, 1 million cells were taken from each sample, washed with 1 mL PBS, centrifuged, and 0.5 μL CD45, CD31, podoplanin, CD16 / 32, CD21 / 35 and Cy5+ antigen staining antibodies were added to each sample. After staining in the dark for 30 minutes, the cells were washed with 1 mL PBS, centrifuged, and resuspended in 200 μL PBS for flow cytometry analysis.

[0216] 3.5 Experimental Results

[0217] The experimental results are as follows Figure 5 As shown, the results indicate that the antigen-ligand fusion protein group can significantly increase the proportion of antigens retained on the FDC cell surface by binding to CD91 on the FDC cell surface compared to the simple antigen group, providing an antigenic basis for the subsequent continuous stimulation of B cells to produce antibodies.

[0218] Test Example 4: Antigen-ligand fusion protein immunological function test

[0219] 4.1 Reagents and Materials

[0220] Aluminum hydroxide adjuvant, PBST buffer (biosharp, BL314B), HRP conjugated rabbit anti-mouse IgG Antibody secondary antibody (Aladdin, Ab176437), TMB, sulfuric acid, bovine serum albumin (BSA, B265994), phosphate buffered saline (PBS, P903593), anti-mouse CD19 BV605 (Biolegend, 115539), anti-mouse B220 BV421 (Biolegend, 103239), anti-mouse IgD PE-Cy7 (Biolegend, 405719), anti-mouse Fas PE (Biolegend, 152607), anti-mouse CD38 AF700 (Biolegend, 102741), anti-mouse CD3 BV421 (Biolegend, 100227), anti-mouse CD8FITC (Biolegend, 100803), anti-mouse IFN-γBV711 (Biolegend, 505836).

[0221] 4.2 Instruments

[0222] Centrifuge (TD5A-WS), flow cytometer (BD Fortessa), microplate reader, cell culture incubator.

[0223] 4.3 Grouping and modeling

[0224] Female BALB / c (H-2d) mice aged 6 weeks were randomly divided into antigen-aluminum hydroxide adjuvant group, antigen-gp96 fusion protein treatment group, antigen-HSP70 fusion protein treatment group, antigen-α2 macroglobulin treatment group, antigen alone mixed with CD91 antibody treatment group, antigen-gp96 fusion protein mixed with CD91 antibody treatment group, antigen-HSP70 fusion protein mixed with CD91 antibody treatment group, antigen-α2 macroglobulin mixed with CD91 antibody treatment group. The above antigens are divided into five types: SARS-CoV-2RBD, FluAHA1, HIVGAG, HPV L1 and HBV HBc. There are 10 mice in each group, and each group is immunized three times, on the 0th day, the 14th day and the 28th day, respectively, and the immunization method is subcutaneous immunization.

[0225] Group 1 (antigen-aluminum hydroxide adjuvant): 10 μg of antigen (diluted to 200 μL with PBS) was administered to each mouse for each immunization.

[0226] The second group (antigen-gp96 fusion protein): each mouse was immunized with 40 μg of antigen-gp96 fusion protein (diluted to 200 μL with PBS).

[0227] The third group (antigen-HSP70 fusion protein): each mouse was immunized with 40 μg of antigen-HSP70 fusion protein (diluted to 200 μL with PBS).

[0228] The fourth group (antigen-α2 macroglobulin): each mouse was immunized with 40 μg of antigen-α2 macroglobulin (diluted to 200 μL with PBS).

[0229] Group 5 (antigen mixed with CD91 antibody): Each mouse was immunized with 10 μg of antigen mixed with 5 μg of CD91 antibody (diluted to 200 μL with PBS).

[0230] Group VI (antigen-gp96 fusion protein mixed with CD91 antibody): each mouse was immunized with 40 μg antigen-gp96 fusion protein mixed with 5 μg CD91 antibody (diluted to 200 μL with PBS).

[0231] Group 7 (antigen-HSP70 fusion protein mixed with CD91 antibody): each mouse was immunized with 40 μg antigen-HSP70 fusion protein mixed with 5 μg CD91 antibody (diluted to 200 μL with PBS).

[0232] Group 8 (antigen-α2 macroglobulin mixed with CD91 antibody): each mouse was immunized with 40 μg antigen-α2 macroglobulin mixed with 5 μg CD91 antibody (diluted to 200 μL with PBS).

[0233] 4.4 Experimental methods

[0234] 4.4.1 Antibody level detection

[0235] 7 days after the third immunization, mouse serum was collected and antibody levels were detected by ELISA. The operation was as follows: 10 μg of the corresponding antigen was coated on each well, incubated at 4°C overnight, and then washed 3 times with PBS-T, each time for 5 minutes. The serum was diluted 10,000 times, 100 μL was added to each well, and 3 wells were added to detect IgG. Incubated at 37°C for 2 hours, and then washed 3 times with PBS-T, each time for 5 minutes. HRP-labeled IgG antibody was added and incubated at 37°C for 1 hour. Then washed 3 times with PBS-T, each time for 5 minutes. 100 μL of TMB colorimetric solution was added to each well, and after standing at 37°C for 15 minutes, 50 μL of 2M sulfuric acid was added to terminate the reaction, and the results were detected on an enzyme reader at a wavelength of 450 nm.

[0236] 4.4.2 Detection of memory B cell levels

[0237] The spleen of mice 6 months after the third immunization was taken to prepare a single cell suspension, and the level of IgG1-specific memory B cells was detected by flow cytometry. The cells were distributed into 1.5mL EP tubes, 500,000 cells per tube, and PBS containing 10% BSA was added to block for at least 10 minutes. 1mL of PBS was added to the cells, centrifuged at 1500rmp for 5min, and the supernatant was discarded. The cells were resuspended in 100μL of PBS containing 10% BSA, and 5μL of CD19, B220, IgD, Fas, CD38 and IgG1 surface staining antibodies were added respectively, and the cells were stained in the dark for 30min. 1mL of PBS was added to the cells, centrifuged at 1500rmp for 5min, and the supernatant was discarded. The cells were resuspended in 200μL of PBS and directly detected on the flow cytometer.

[0238] 4.4.3 Antigen-specific CD8+ T cell response detection

[0239] The spleen of mice 7 days after the third immunization was taken to prepare single cell suspension, and the level of antigen-specific CD8+ T cell response was detected by flow cytometry. The cells were added to a 48-well plate, 1 million cells were added to each well, and 5 μg of the corresponding simple antigen was added to each well for stimulation for 24 hours. The cells were incubated at 37°C and 5% CO 2 The cells were cultured in an incubator and stained with flow cytometry after 24 hours. Each sample well was washed with 1 mL of PBS and centrifuged. 0.5 μL of CD3 and CD8 staining antibodies were added to each sample. After staining in the dark for 30 minutes, the cells were washed with 1 mL of PBS and centrifuged. 500 μL of 4% paraformaldehyde was added for light-proof fixation for 30 minutes. The cells were washed with 1 mL of membrane permeabilization solution and centrifuged. 0.5 μL of IFN-γ flow cytometry antibody was added for light-proof staining for 30 minutes. The cells were washed with 1 mL of PBS and centrifuged. The cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0240] 4.5 Experimental Results

[0241] Antibody level test results Figure 6-7 As shown, the results of memory B cell level detection are as follows Figure 8 The results of antigen-specific CD8+ T cell response detection are shown in Fig. 9 shown.

[0242] The results of antibody level detection showed that after three immunizations of mice, the fusion protein group could produce a strong antibody response compared with the antigen-aluminum hydroxide adjuvant group, and the expression of IgG was significantly increased. This shows that the fusion protein group can induce a stronger antibody response. After adding CD91 antibody for mixed immunization, the immune response produced was not significantly different from that of the antigen-aluminum hydroxide adjuvant group, indicating that the difference was mainly mediated by CD91, and it was the antibody response produced by the fusion protein based on its CD91 ligand to retain more antigens on the FDC cells in the germinal center.

[0243] The results of memory B cell level detection showed that the IgG1 memory B cell level in the antigen-CD91 ligand fusion protein group was significantly increased compared with the antigen-aluminum hydroxide adjuvant immunization group, indicating that the antigen-CD91 ligand fusion protein group can further expose the antigen through CD91 on the surface of FDC cells and increase the proportion of memory B cells.

[0244] The results of antigen-specific CD8+T cell response detection showed that the antigen-CD91 ligand fusion protein group can significantly enhance the intensity of antigen-specific CD8+T cell immune response through its binding with CD91, which is significantly higher than the simple antigen immunization group.

[0245] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0246] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or terminal device including the elements.

[0247] The above is a detailed introduction to an anti-aging hexapeptide and its composition and use provided by the present invention. Specific examples are used herein to illustrate the principle and implementation mode of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation mode and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. An antigen-ligand fusion protein, characterized in that: It comprises a first structural unit and a second structural unit, wherein the first structural unit is a viral antigen, and the second structural unit is a CD91 ligand; The CD91 ligand is gp96 as shown in the amino acid sequence of SEQ ID NO.1 or a mutant thereof with substitution and / or deletion and / or addition of no more than 10 amino acid residues, or HSP70 as shown in the amino acid sequence of SEQ ID NO.2 or a mutant thereof with substitution and / or deletion and / or addition of no more than 10 amino acid residues, or α2 macroglobulin as shown in the amino acid sequence of SEQ ID NO.3 or a mutant thereof with substitution and / or deletion and / or addition of no more than 10 amino acid residues; The C-terminus of the first structural unit and the N-terminus of the second structural unit are connected via a flexible polypeptide sequence, or are directly covalently connected via a peptide bond.

2. The fusion protein according to claim 1, characterized in that The viral antigen is one of the antigens of HBV, HPV, VZV, EBV, HSV-2, HIV, FluA or SARS-CoV-2 and their mutants, and the mutants include natural point mutations / deletion mutations / increase mutations / truncations at one or more sites, artificial point mutations / deletion mutations / increase mutations / truncations, any combination of natural or artificial mutations, and subtypes produced after mutation.

3. The fusion protein according to claim 2, characterized in that The viral antigen is one of the SARS-CoV-2RBD antigen shown in the amino acid sequence of SEQ ID NO.4, the FluAHA1 antigen shown in the amino acid sequence of SEQ ID NO.5, the HIVGAG antigen shown in the amino acid sequence of SEQ ID NO.6, the HPV L1 antigen shown in the amino acid sequence of SEQ ID NO.7, and the HBV HBc antigen shown in the amino acid sequence of SEQ ID NO.8, and their mutants.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The C-terminus of the viral antigen is directly covalently linked to the N-terminus of the CD91 ligand through a peptide bond, and has an amino acid sequence as shown in SEQ ID NO.9, or an amino acid sequence as shown in SEQ ID NO.10, or an amino acid sequence as shown in SEQ ID NO.11, or an amino acid sequence as shown in SEQ ID NO.12, or an amino acid sequence as shown in SEQ ID NO.13, or an amino acid sequence as shown in SEQ ID NO.14, or an amino acid sequence as shown in SEQ ID NO.15, or an amino acid sequence as shown in SEQ ID NO.16, or an amino acid sequence as shown in SEQ ID NO.17, or an amino acid sequence as shown in SEQ ID NO.18, or an amino acid sequence as shown in SEQ ID NO.19, or an amino acid sequence as shown in SEQ ID NO.20, or an amino acid sequence as shown in SEQ ID NO.21, or an amino acid sequence as shown in SEQ ID NO.22, or an amino acid sequence as shown in SEQ ID NO.

23.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The N-terminus and / or C-terminus of the fusion protein may or may not be connected to a tag that facilitates the expression, detection, tracing, and purification of the target protein.

6. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 5, an expression vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule or the expression vector.

7. Use of the fusion protein according to any one of claims 1 to 5, or the nucleic acid molecule, expression vector or host cell according to claim 6 in preparing a vaccine.

8. The use according to claim 7, characterized in that: The vaccine is used to prevent and / or treat infection by the new coronavirus, influenza virus, human immunodeficiency virus, human papillomavirus and hepatitis B virus.

9. Use of the fusion protein according to any one of claims 1 to 5, or the nucleic acid molecule, expression vector or host cell according to claim 6 in preparing a product for enhancing antigen retention and presentation.

10. Use of the fusion protein according to any one of claims 1 to 5, or the nucleic acid molecule, expression vector or host cell according to claim 6 in the preparation of a product for improving the level of antibody secretion and / or antibody maintenance time and / or T cell level, or in the preparation of a product for improving the level of memory B cells.

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