Method for improving stability of virus antigen, fusion protein and application of fusion protein

Abstract

The invention discloses a method for improving the stability of a virus antigen, a fusion protein and application of the fusion protein, and relates to the technical field of vaccine preparation. According to the method for improving the stability of the virus antigen, a target antigen and a gp96 protein are fused to form a fusion protein, and the gp96 protein is a protein with an amino acid sequence as shown in SEQ ID NO: 1 or a mutant with no more than 10 amino acid residues substituted, deleted and / or added. By implementing the method, the thermal stability and conformational stability of the target antigen can be remarkably improved, and the technical problem that the antigen is unstable is solved from the protein structure level.

Description

Technical Field

[0001] This invention relates to the field of vaccine preparation technology, and in particular to a method for increasing the stability of viral antigens, a fusion protein, and its applications. Background Technology

[0002] Vaccines are a core means of preventing infectious diseases, and their effectiveness hinges on their ability to induce strong and long-lasting immune protection in the body. For viruses with poor immunogenicity, such as SARS-CoV-2, influenza, and HIV, existing vaccines often face challenges such as insufficient antibody levels, short protection periods, and weak cross-protection against variant strains.

[0003] In-depth research reveals that the aforementioned challenges are closely related to the stability of antigens in vivo. The stability of an antigen directly determines whether it can be effectively captured and displayed for a long time by follicular dendritic cells (FDCs) in the germinal center, thereby driving B cells to undergo sufficient affinity maturation and produce high-quality, durable neutralizing antibodies. However, most vaccines currently use natural or simply engineered antigens that are prone to conformational changes, aggregation, or degradation in the complex in vivo environment, leading to rapid clearance and the inability to form a durable antigen library on the surface of FDCs. This is one of the fundamental reasons for the unsatisfactory strength and durability of the immune response.

[0004] Therefore, developing a method to significantly enhance the stability of viral antigens at the physicochemical and structural levels is a key approach to overcoming current bottlenecks in vaccine development. Specifically, there is an urgent need for a method that enables antigens to exhibit higher thermal stability and structural integrity in vitro, allowing for longer retention times and better conformational maintenance in vivo, thereby laying the foundation for triggering stronger humoral immune responses. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for increasing the stability of viral antigens, which can significantly improve the structural and thermal stability of viral antigens.

[0006] The technical problem to be solved by the present invention is to provide a fusion protein with strong stability, which is prepared by the above-mentioned method for increasing the stability of viral antigens and can significantly improve the structural stability and thermal stability of the antigen.

[0007] The technical problem to be solved by this invention is to provide the application of the above-mentioned highly stable fusion protein in the preparation of vaccines.

[0008] To address the aforementioned technical problems, this invention provides a method for increasing the stability of viral antigens by fusing the target antigen and gp96 protein to form a fusion protein, thereby enhancing the thermal stability and / or conformational stability of the target antigen. The gp96 protein is a protein with an amino acid sequence as shown in SEQ ID NO: 1, or a mutant of which no more than 10 amino acid residues are substituted, deleted, and / or added.

[0009] As an improvement to the above technical solution, the C-terminus of the target antigen and the N-terminus of the gp96 protein are linked by a linker peptide to form a fusion protein; Alternatively, the C-terminus of the target antigen and the N-terminus of the gp96 protein can be covalently linked by peptide bonds to form a fusion protein.

[0010] As an improvement to the above technical solution, the C-terminus of the target antigen and the N-terminus of the gp96 protein are connected by a linker peptide, wherein the linker peptide is a peptide segment with an amino acid sequence as shown in SEQ ID NO: 2 or a mutant of no more than 10 amino acid residues thereof with substitution, deletion and / or addition.

[0011] As an improvement to the above technical solution, the target antigen is selected from one of the antigens of HBV, HPV, HIV, FluA or SARS-CoV-2 and their mutants.

[0012] As an improvement to the above technical solution, the target antigen is one of SARS-CoV-2 RBD antigen, FluA HA1 antigen, HIV GAG antigen, HPV L1 antigen, HPV E7 antigen, and HBV HBc antigen.

[0013] As an improvement to the above technical solution, the amino acid sequence of the SARS-CoV-2 RBD antigen is shown in SEQ ID NO: 3; The amino acid sequence of the FluA HA1 antigen is shown in SEQ ID NO: 4; The amino acid sequence of the HIV GAG antigen is shown in SEQ ID NO: 5; The amino acid sequence of the HPV L1 antigen is shown in SEQ ID NO: 6; The amino acid sequence of the HPV E7 antigen is shown in SEQ ID NO: 7; The amino acid sequence of the HBV HBc antigen is shown in SEQ ID NO: 8.

[0014] Accordingly, the present invention also provides a fusion protein with strong stability, which is constructed using the above-described method for increasing the stability of viral antigens.

[0015] Accordingly, the present invention also provides the application of the above-mentioned highly stable fusion protein in the preparation of vaccines.

[0016] As an improvement to the above technical solution, the vaccine is used to prevent and / or treat infections of COVID-19, influenza virus, human immunodeficiency virus, human papillomavirus, and hepatitis B virus.

[0017] Accordingly, the present invention also discloses the application of gp96 protein in enhancing the stability of viral antigens, wherein the stability is thermal stability and / or structural stability.

[0018] Implementing this invention has the following beneficial effects: This invention, by fusing a target antigen with the gp96 protein, yields a fusion protein exhibiting excellent physicochemical and thermal stability. This solves the technical challenge of antigen instability at the protein structure level, laying the foundation for efficient vaccine storage, transportation, and in vivo delivery. Furthermore, in vivo experiments have demonstrated that the fusion protein obtained by this invention significantly prolongs its residence time at the immune site, meaning it significantly extends the in vivo action time of the antigen. This ensures that the antigen can be fully recognized and utilized by the immune system (especially FDCs), thereby driving a stronger and more sustained germinal center response. Based on these advantages, the fusion protein of this invention can simultaneously and significantly improve the titer and persistence of neutralizing antibodies and effectively stimulate cross-protective T-cell immunity, achieving highly efficient prevention against various viruses, especially those with poor immunogenicity. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the binding of the target antigen to the gp96 protein; Figure 2 This is a graph showing the results of the thermal stability test of the fusion protein; Figure 3 This is a graph showing the results of conformational stability testing of the fusion protein; Figure 4 This is a graph showing the results of testing the retention and distribution of vaccines within the body. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. This invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this invention.

[0021] Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Raw materials whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0023] This embodiment provides a method for increasing the stability of viral antigens by fusing the target antigen and gp96 protein to form a fusion protein, thereby improving the thermal stability and / or conformational stability of the target antigen. The gp96 protein is a protein with an amino acid sequence as shown in SEQ ID NO: 1, or a mutant of which no more than 10 amino acid residues are substituted, deleted, and / or added.

[0024] It is worth noting that this invention discloses the application of the gp96 protein in enhancing the stability of viral antigens. This embodiment, by fusing the target antigen with the gp96 protein, obtains a highly stable fusion protein, which significantly improves the structural and thermal stability of the target antigen, solving the technical challenge of antigen instability at the protein structure level. The fusion protein exhibits excellent physicochemical and thermal stability, laying the foundation for the storage, transportation, and efficient in vivo delivery of vaccines. More importantly, in vivo imaging experiments confirmed that the fluorescence signal retention time of this fusion protein at the injection site and in lymphatic organs is significantly longer than that of the individual viral antigen, indicating a significant improvement in its half-life and retention in vivo.

[0025] Further explanation reveals that the core finding of this embodiment lies in the fundamentally enhanced protein structural stability resulting from the fusion of the target antigen with the gp96 protein. In vitro electron microscopy and thermal stability experiments demonstrate that this fusion protein spontaneously forms a uniform and stable higher-order structure, exhibiting significantly superior resistance to degradation compared to individual viral antigens. This enhanced physicochemical stability directly translates into superior in vivo performance: in vivo imaging shows that the fusion protein can achieve longer retention at immune sites (such as lymph nodes). This characteristic allows the antigen to be more effectively captured and displayed for extended periods by follicular dendritic cells (FDCs), thereby continuously stimulating B cells, promoting their proliferation and differentiation into plasma cells and memory B cells, ultimately generating a more potent and durable antibody immune response. Simultaneously, this fusion protein can also effectively cross-activate CD8+ T cells by targeting antigen-presenting cells such as dendritic cells (DCs), triggering robust cellular immunity and providing cross-protection against viral mutant strains.

[0026] In one embodiment, the C-terminus of the target antigen and the N-terminus of the gp96 protein are linked by a linker peptide to form a fusion protein.

[0027] Alternatively, the C-terminus of the target antigen and the N-terminus of the gp96 protein can be covalently linked by peptide bonds to form a fusion protein.

[0028] In one embodiment, the C-terminus of the target antigen and the N-terminus of the gp96 protein are linked by a linker peptide, which is a peptide with an amino acid sequence as shown in SEQ ID NO: 2 or a mutant of no more than 10 amino acid residues thereof with substitution, deletion and / or addition.

[0029] In this embodiment, the C-terminus of the target antigen and the N-terminus of the gp96 protein are linked by a linker peptide with the amino acid sequence shown in SEQ ID NO: 2, which can further improve the thermal stability and structural stability of the fusion protein.

[0030] In one embodiment, the amino acid sequence of the linker peptide is as follows: EAAAKEAAAKEAAAK. In some embodiments, the C-terminus of the target antigen may be linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the gp96 protein.

[0031] In one embodiment, the target antigen is selected from one of the antigens of HBV, HPV, HIV, FluA, or SARS-CoV-2 and their mutants.

[0032] Specifically, the mutants include natural point mutations / deletion mutations / addition mutations / truncations at one or more sites, artificial point mutations / deletion mutations / addition mutations / truncations, any combination of natural or artificial mutations, and the subtypes resulting from the mutations.

[0033] In one embodiment, the target antigen is one of SARS-CoV-2 RBD antigen, FluA HA1 antigen, HIVGAG antigen, HPV L1 antigen, HPV E7 antigen, and HBV HBc antigen.

[0034] Among them, SARS-CoV-2 RBD antigen refers to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, FluA HA1 antigen refers to the HA1 domain of the influenza virus globular head, HIV GAG antigen refers to the human immunodeficiency virus structural protein GAG, HPV L1 antigen refers to the human papillomavirus (HPV) capsid protein L1, and HBV HBc antigen refers to the hepatitis B virus-HBc.

[0035] In one embodiment, the amino acid sequence of the SARS-CoV-2 RBD antigen is shown in SEQ ID NO: 3; The amino acid sequence of the FluA HA1 antigen is shown in SEQ ID NO: 4; The amino acid sequence of the HIV GAG antigen is shown in SEQ ID NO: 5; The amino acid sequence of the HPV L1 antigen is shown in SEQ ID NO: 6; The amino acid sequence of the HPV E7 antigen is shown in SEQ ID NO: 7; The amino acid sequence of the HBV HBc antigen is shown in SEQ ID NO: 8.

[0036] Accordingly, this embodiment also provides a highly stable fusion protein, constructed using the method described above for increasing the stability of viral antigens. Specifically, since the target antigen can be one of SARS-CoV-2 RBD antigen, FluAHA1 antigen, HIV GAG antigen, HPV L1 antigen, HPV E7 antigen, or HBV HBc antigen, the fusion protein constructed in this embodiment includes... SARS-CoV-2 RBD-GP96 fusion protein, FluA HA1-GP96 fusion protein, HIV GAG-GP96 fusion protein, HPV L1-GP96 fusion protein, HPV E7-GP96 fusion protein, and HBV HBc-GP96 fusion protein.

[0037] In some embodiments, the amino acid sequence of the SARS-CoV-2 RBD-GP96 fusion protein is shown in SEQ ID NO: 9, the amino acid sequence of the FluA HA1-GP96 fusion protein is shown in SEQ ID NO: 10, the amino acid sequence of the HIV GAG-GP96 fusion protein is shown in SEQ ID NO: 11, the amino acid sequence of the HPV L1-GP96 fusion protein is shown in SEQ ID NO: 12, the amino acid sequence of the HPV E7-GP96 fusion protein is shown in SEQ ID NO: 13, and the amino acid sequence of the HBV HBc-GP96 fusion protein is shown in SEQ ID NO: 14.

[0038] This embodiment solves the technical challenge of antigen instability at the protein structure level by fusing the target antigen with the gp96 protein. The fusion protein exhibits excellent physicochemical and thermal stability, laying the foundation for efficient vaccine storage, transportation, and in vivo delivery. In vivo experiments have confirmed that the fusion protein obtained in this embodiment has a significantly prolonged residence time at the immune site, i.e., a significantly prolonged in vivo action time of the antigen. This ensures that the antigen can be fully recognized and utilized by the immune system (especially FDCs), thereby driving a stronger and more durable germinal center response. Based on the above advantages, the fusion protein of this invention can simultaneously and significantly improve the titer and persistence of neutralizing antibodies and effectively stimulate cross-protective T-cell immunity, achieving highly efficient prevention against a variety of viruses, especially those with poor immunogenicity.

[0039] It should be noted that fusion proteins can be prepared by expression and purification of fusion proteins. In this embodiment, the expression and purification of fusion proteins are carried out using conventional methods in the art.

[0040] In some embodiments, the preparation method of the fusion protein includes the following steps: (1) Construction of vector: Obtain nucleic acid molecules encoding the target fusion protein; ligate the nucleic acid molecules into the expression vector to construct the recombinant expression vector; transform the recombinant expression vector into competent cells and obtain recombinant rod-granule DNA through recombinant screening; (2) Transfection of host cells: Recombinant rod-like DNA was transfected into Sf9 cells, and the supernatant was collected by centrifugation after incubation at a suitable temperature to obtain the primary generation (P1 generation) virus; then, the primary generation virus was used to gradually infect Sf9 cells for virus amplification, and high-generation (P2, P3 generation) viruses were obtained through multiple rounds of passage culture; finally, the high-generation virus was used to infect Sf9 cells on a large scale, and cultured under suitable temperature and rotation speed conditions to obtain a suspension containing the target fusion protein.

[0041] (3) Purification and identification of fusion protein: The suspension containing the target fusion protein was purified by ion exchange chromatography to obtain a concentrated solution containing the target fusion protein. The molecular weight of the fusion protein was verified by SDS-PAGE electrophoresis, Western blotting, mass spectrometry and other techniques.

[0042] In some embodiments, the nucleic acid sequence of the nucleic acid molecule encoding the SARS-CoV-2 RBD-GP96 fusion protein is shown in SEQ ID NO: 15; the nucleic acid sequence of the nucleic acid molecule encoding the FluA HA1-GP96 fusion protein is shown in SEQ ID NO: 16; the nucleic acid sequence of the nucleic acid molecule encoding the HIV GAG-GP96 fusion protein is shown in SEQ ID NO: 17; the nucleic acid sequence of the nucleic acid molecule encoding the HPVL1-GP96 fusion protein is shown in SEQ ID NO: 18; the nucleic acid sequence of the nucleic acid molecule encoding the HPV E7-GP96 fusion protein is shown in SEQ ID NO: 19; and the nucleic acid sequence of the nucleic acid molecule encoding the HBV HBc-GP96 fusion protein is shown in SEQ ID NO: 20.

[0043] In one embodiment, the SARS-CoV-2 RBD-GP96 fusion protein, FluA HA1-GP96 fusion protein, HIV GAG-GP96 fusion protein, HPV L1-GP96 fusion protein, HPV E7-GP96 fusion protein, and HBV HBc-GP96 fusion protein of this embodiment are prepared by the following steps: (1) Construction of the carrier Nucleic acid molecules encoding the target fusion protein were synthesized using artificial bases (the nucleic acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively). These nucleic acid molecules were then ligated into the insect cell expression vector pFastBac1 to construct a recombinant expression vector. The recombinant expression vector was transformed into DH10Bac competent cells, and recombinant band-granular DNA was obtained through recombinant selection.

[0044] (2) Transfection of host cells Recombinant band granule DNA was transfected into Sf9 cells (per 1 × 10⁶ cells). 6 Approximately 4 μg of recombinant plasmid was transfected into each Sf9 cell; during transfection, the transfection reagent was Cellfectin II reagent, and the cells were incubated at 27°C for 72 h. After centrifugation, the supernatant was the P1 generation virus. Sf9 cell suspension 1 (containing 1 × 10⁻⁶ cells / mL) was then used to transfect the virus. 8 Sf9 cells were cultured at 27°C for 8-10 hours to obtain cultured cells. Then, P1 generation virus (0.05-0.1 MOI) was added to the cultured cells, and the cells were incubated at 27°C for 72 hours. After centrifugation at 4000 rpm for 5 minutes, the supernatant was obtained as P2 generation virus. The virus was then added to Sf9 cell suspension 2 (containing 1.6 × 10⁻⁶ cells / mL). 8 Add P2 generation virus (0.05-0.1 MOI) to 300 mL of Sf9 cell suspension, incubate at 27°C and 100-120 rpm for 72 h, centrifuge at 4000 rpm for 5 min, and the supernatant is the P3 generation virus. 8 P3 generation virus (at a dose of 5 MOI) was added to 100 Sf9 cells and cultured at 27°C and 100-120 rpm for 72 h to obtain a suspension containing the target fusion protein.

[0045] (3) Purification and identification of fusion proteins The suspension was centrifuged at 7000 rpm for 20 min, and the supernatant was collected. The supernatant was filtered through a 0.22 mm filter membrane and then loaded onto a HiTrap-Q Sepharose ion exchange chromatography column (flow rate 1 mL / min). The column was first washed with 5 mL of pH 7.5, 200 mM PBS buffer (flow rate 1 mL / min); then with 10 mL of pH 7.5, 300 mM PBS buffer (flow rate 1 mL / min); and finally with 3 mL of pH 7.5, 600 mM PBS buffer (flow rate 1 mL / min). The post-column solution was collected and concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 50 KD, yielding approximately 1 mL of concentrate. This concentrate contains the fusion protein. The concentrate was aliquoted and stored at -80°C.

[0046] S3, Electrophoretic verification of the fusion protein The concentrated solutions were analyzed by SDS-PAGE electrophoresis. The molecular weights of the proteins were found to be 120 kDa, 130 kDa, 150 kDa, 140 kDa, 100 kDa, and 110 kDa, respectively, indicating that the SARS-CoV-2 RBD-GP96 fusion protein, FluA HA1-GP96 fusion protein, HIV GAG-GP96 fusion protein, HPV L1-GP96 fusion protein, HPV E7-GP96 fusion protein, and HBV HBc-GP96 fusion protein were successfully prepared.

[0047] Accordingly, this embodiment also provides the application of the above-mentioned highly stable fusion protein in the preparation of vaccines.

[0048] Based on this, the present invention provides a fusion protein that can spontaneously form a stable structure and effectively target key immune cells, a vaccine containing the fusion protein, and its applications.

[0049] In one embodiment, the vaccine is used to prevent and / or treat infections with SARS-CoV-2, influenza virus, human immunodeficiency virus, human papillomavirus, and hepatitis B virus.

[0050] Accordingly, this embodiment also provides the application of the above-mentioned highly stable fusion protein in the preparation of products for enhancing antigen stability, wherein the stability is thermal stability and / or structural stability.

[0051] The technical solution of the present invention is further described below through embodiments.

[0052] Example 1: Design of Fusion Protein This embodiment provides a method for increasing the stability of viral antigens, such as... Figure 1 As shown, the amino acid sequence of the target antigen was designed at the N-terminus of the gp96 protein, and the C-terminus of the target antigen and the N-terminus of the gp96 protein were covalently linked by peptide bonds to construct fusion proteins. The target antigens were SARS-CoV-2 RBD antigen, FluA HA1 antigen, HIV GAG antigen, HPV L1 antigen, HPV E7 antigen, and HBV HBc antigen. The following fusion proteins were constructed according to the above design: SARS-CoV-2 RBD-GP96, FluA HA1-GP96, HIV GAG-GP96, HPV L1-GP96, HPV E7-GP96, and HBV HBc-GP96.

[0053] Example 2 This embodiment provides a method for increasing the stability of viral antigens. The amino acid sequence of the target antigen is designed at the N-terminus of the gp96 protein. The C-terminus of the target antigen and the N-terminus of the gp96 protein are linked by a linker peptide, the amino acid sequence of which is shown in SEQ ID NO: 2. This constructs a fusion protein. The target antigens are SARS-CoV-2 RBD antigen, FluAHA1 antigen, HIV GAG antigen, HPV L1 antigen, HPV E7 antigen, and HBV HBc antigen. In this embodiment, the linker peptide is named LP. Following the above design, the following fusion proteins are constructed: SARS-CoV-2 RBD-LP-GP96, FluAHA1-LP-GP96, HIV GAG-LP-GP96, HPV L1-LP-GP96, HPV E7-LP-GP96, and HBV HBc-LP-GP96.

[0054] Example 3: Detection of thermal stability of fusion proteins The thermal stability of the purified fusion protein from Example 1, along with SARS-CoV-2 RBD, FluA HA1, HIV GAG, HPV L1, HPVE7, HBV HBc, and gp96 proteins, was determined using nanoscale differential scanning fluorometry. Protein samples were diluted with PBS buffer to a final concentration of 0.5–1.0 mg / mL. 10 μL of each protein sample was pipetted into the sample holder of the PR.NT.48 nanoDSF instrument using a capillary tube. The program was set to uniformly heat the samples from 20°C to 95°C at a rate of 1°C / min, while the instrument simultaneously monitored the intrinsic fluorescence signal and the intensity of 650 nm backscattered light. After the experiment, the data were analyzed using PR.Control software, and the inflection point of the backscattered light intensity change was used to determine the protein aggregation initiation temperature.

[0055] The results are as follows Figure 2 As shown, the aggregation initiation temperature of each fusion protein is significantly higher than that of the simple viral antigen protein, indicating that the fusion protein has higher thermal stability, which provides a theoretical basis for its use as an "antigen sustained-release reservoir" in vivo and to achieve long-term immunity.

[0056] Example 4: Determination of conformational stability of fusion proteins Individual viral antigen proteins, GP96, and the fusion protein from Example 1 were used, and their conformational stability was assessed using a trypsin protection assay. All protein samples were uniformly concentrated at 10 μM and incubated at 37°C for 15 minutes. Subsequently, each protein sample was adjusted to a series of different pH values ​​(pH 7.4, 7.0, 6.5, 6.0, 5.5, 5.0) using 0.1 M citrate solution and incubated again at 37°C for 5 minutes to induce conformational changes. After neutralizing the pH of each sample to 7.3 using 0.5 M Tris solution, 2 μg of TPCK-trypsin was added to each tube, and the enzyme digestion reaction was carried out at 20°C for 1 hour. After the reaction, 5× SDS protein loading buffer was added to terminate the reaction, and the proteins were heated at 100°C for 10 minutes to denature them. Finally, the resistance to enzyme digestion of each protein under different pH conditions was analyzed by SDS-PAGE gel electrophoresis.

[0057] The results are as follows Figure 3 As shown, under different pH conditions, both the fusion proteins and the individual gp96 protein maintained intact bands, while the bands of the pure viral antigen proteins significantly weakened as the pH decreased. This indicates that the fusion proteins possess significantly enhanced conformational stability and resistance to protease hydrolysis, similar to gp96, providing a structural basis for achieving long-term immunization in vivo.

[0058] Example 5: Detection of in vivo retention and distribution of fusion vaccine The purified fusion protein and the individual viral antigen protein from Example 1 were fluorescently labeled using the Lightning-Link R-Cy5 kit. Proteins with equal fluorescence intensity and the same molar amount were immunized in 6- to 8-week-old female BALB / c mice via subcutaneous injection in the back. Whole-body fluorescence images of the mice were captured using an IVIS in vivo imaging system at 15 minutes, day 7, and day 30 post-injection, and the fluorescence signal intensity and distribution were analyzed using Living Image software.

[0059] The results are as follows Figure 4 As shown, on days 7 and 30 post-immunization, strong fluorescent signals were still detected at the injection sites and inguinal lymph nodes of mice for each fusion protein group, with a signal duration significantly longer than that of the viral antigen protein and the gp96 protein group alone. This indicates that the fusion proteins can form a long-lasting "antigen sustained-release reservoir" in vivo and migrate efficiently to lymphoid tissues, providing a kinetic basis for inducing a sustained and efficient immune response.

[0060] The technical principles of the present invention have been described above with reference to specific embodiments. These descriptions are merely for explaining the principles of the invention and should not be construed as limiting the scope of protection of the invention in any way. Based on this explanation, those skilled in the art can readily conceive of other specific embodiments of the invention without inventive effort, and these embodiments will all fall within the scope of protection of the present invention.

Claims

1. A method for increasing the stability of viral antigens, characterized in that, The target antigen and gp96 protein are fused to form a fusion protein, thereby improving the thermal stability and / or conformational stability of the target antigen.

2. The method for increasing viral antigen stability according to claim 1, characterized in that, The gp96 protein is a protein with an amino acid sequence as shown in SEQ ID NO: 1, or a mutant of it with substitutions, deletions, and / or additions of no more than 10 amino acid residues.

3. The method for increasing viral antigen stability according to claim 1, characterized in that, The C-terminus of the target antigen and the N-terminus of the gp96 protein are linked by a linker peptide to form a fusion protein; Alternatively, the C-terminus of the target antigen and the N-terminus of the gp96 protein can be covalently linked by peptide bonds to form a fusion protein.

4. The method for increasing viral antigen stability according to claim 3, characterized in that, The C-terminus of the target antigen and the N-terminus of the gp96 protein are linked by a linker peptide, which is a peptide with an amino acid sequence as shown in SEQ ID NO: 2 or a mutant of no more than 10 amino acid residues thereof, with substitution, deletion and / or addition.

5. The method for increasing viral antigen stability according to claim 1, characterized in that, The target antigen is selected from one of the antigens of HBV, HPV, HIV, FluA, or SARS-CoV-2 and their mutants.

6. The method for increasing viral antigen stability according to claim 5, characterized in that, The target antigen is one of the following: SARS-CoV-2 RBD antigen, FluA HA1 antigen, HIV GAG antigen, HPV L1 antigen, HPV E7 antigen, or HBV HBc antigen.

7. The method for increasing viral antigen stability according to claim 6, characterized in that, The amino acid sequence of the SARS-CoV-2RBD antigen is shown in SEQ ID NO: 3; The amino acid sequence of the FluA HA1 antigen is shown in SEQ ID NO: 4; The amino acid sequence of the HIV GAG antigen is shown in SEQ ID NO: 5; The amino acid sequence of the HPV L1 antigen is shown in SEQ ID NO: 6; The amino acid sequence of the HPV E7 antigen is shown in SEQ ID NO: 7; The amino acid sequence of the HBV HBc antigen is shown in SEQ ID NO:

8.

8. A fusion protein with strong stability, characterized in that, It was constructed using the method for increasing viral antigen stability as described in any one of claims 1-7.

9. The use of the highly stable fusion protein according to claim 8 in the preparation of vaccines.

10. The application according to claim 9, characterized in that, The vaccine is used to prevent and / or treat infections with COVID-19, influenza virus, human immunodeficiency virus, human papillomavirus, and hepatitis B virus.

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