Construction method of EBV recombinant nanoparticle vaccine with enhanced immunogenicity

Abstract

The invention discloses a construction method of an EBV recombinant nanoparticle vaccine with enhanced immunogenicity, and belongs to the technical field of biological medicines. The method comprises the following steps: designing a nano-particle skeleton, determining an optimal link distance between gB and a skeleton protein through computer assistance, and transforming the skeleton protein; gB protein genes are obtained and modified, connection sites are increased, and the expression efficiency is optimized; the two genes are co-expressed in a proper expression system, and the nanoparticles with gB displayed on the surfaces are assembled in vitro; and finally purifying and identifying the vaccine. According to the vaccine, the immunogenicity of the gB antigen is remarkably improved, a neutralizing antibody can be efficiently induced, infection of EBV to epithelial cells and B cells can be inhibited at the same time, and the stronger T cell killing capacity can be activated. In addition, the vaccine is good in stability at the temperature of 4 DEG C and the room temperature, and convenience is provided for preparation, transportation and storage. The vaccine has significant clinical transformation potential, is expected to become a next-generation EBV candidate vaccine, and also provides a new thought for research and development of other herpes virus vaccines.

Description

Technical Field

[0001] The present invention belongs to the technical field related to biomedicine, and particularly relates to a construction method of an EBV recombinant nanoparticle vaccine with enhanced immunogenicity. Background Art

[0002] Epstein-Barr Virus (EBV) is a widely susceptible human herpesvirus and the first discovered human oncovirus. EBV is the pathogen causing infectious mononucleosis (IM) and is associated with the pathogenesis of nasopharyngeal carcinoma, some gastric cancers, various lymphomas, and autoimmune diseases such as multiple sclerosis.

[0003] Currently, there is still no prophylactic vaccine against EBV on the market internationally. There are multiple viral glycoproteins on the viral membrane of EBV, which mediate viral infection of host cells in cooperation by binding to corresponding receptors. Among them, the key glycoprotein gB is the fusion protein of the virus, which can bind to the viral receptor on the surface of the host cell membrane and receive signals from other viral glycoproteins, triggering the fusion of the virus with the cell membrane. Although gB is a potential ideal vaccine target for EBV, its weak humoral immune activation effect, short immune effect time, and insufficient ability to induce neutralizing antibodies have hindered its further research and application as a vaccine.

[0004] Traditional vaccine construction methods are difficult to effectively solve the problem of low immunogenicity when dealing with complex viruses such as EBV. For example, conventional subunit protein vaccines cannot efficiently induce neutralizing antibodies against EBV, and it is also difficult to simultaneously inhibit EBV epithelial cell and B cell infections, as well as induce stronger T cell activation and killing ability. Therefore, there is an urgent need for a new method to construct an EBV recombinant nanoparticle vaccine with enhanced immunogenicity to meet the needs of preventing and treating EBV-related diseases. Summary of the Invention

[0005] The purpose of the present invention is to provide a construction method of an EBV recombinant nanoparticle vaccine with enhanced immunogenicity to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A construction method of an EBV recombinant nanoparticle vaccine with enhanced immunogenicity includes the following steps:

[0008] Nanoparticle skeleton design: Using computer-aided design, calculate the optimal linkage spacing between gB and multiple different nanoparticle skeleton proteins; select a nanoparticle skeleton protein and modify it through genetic engineering technology so that it can be effectively linked with the gB protein;

[0009] gB protein gene acquisition and modification: Extract the gene sequence of the gB protein from Epstein-Barr virus, modify it, and without changing the function of the gB protein, add sites for connection with the nanoparticle scaffold protein and optimize its expression efficiency at the same time;

[0010] Recombinant nanoparticle assembly: Co-express the modified gB protein gene and the modified nanoparticle scaffold protein gene in a suitable expression system. By controlling the expression conditions, assemble the gB protein and the nanoparticle scaffold protein into nanoparticles gB-I53-50NP with gB displayed on the surface in vitro;

[0011] Vaccine purification and identification: Purify the assembled recombinant nanoparticle vaccine, and use affinity chromatography and gel filtration chromatography to remove impurities; Identify the purified vaccine by biochemical and structural biology methods, and use single-particle cryo-electron microscopy technology and cryo-electron tomography technology to analyze the structure of the nanoparticles to ensure the structural integrity and rocking flexibility of the gB protein on the surface of the nanoparticles.

[0012] Preferably, the computer-aided design software is Modeller and CHARMM-GUI software, and the parameter settings for the molecular dynamics simulation are: simulation temperature 310K, time step 2fs, simulation duration 100ns.

[0013] Preferably, the nanoparticle scaffold protein is I53-50 protein, and its selection is based on its icosahedral symmetry and highly stable structure, and its high expression level in Escherichia coli.

[0014] Preferably, the restriction endonucleases are EcoRI and BamHI, and the amino acids encoded by the specific ligation sequence are glycine-serine flexible linker sequences.

[0015] Preferably, the EBV strain is B95-8 strain, and the gene extraction kit is Qiagen viral DNA extraction kit.

[0016] Preferably, the codon optimization is carried out according to the codon usage frequency database of Escherichia coli, and the proportion of rare codons in the optimized gB protein gene is reduced to less than 5%.

[0017] Preferably, the expression vector is pET-28a(+) vector, the DNA ligase is T4 DNA ligase. When the expression system is Escherichia coli BL21(DE3), the culture medium is LB medium, and the inducer is IPTG. When the expression system is Chinese hamster ovary cells (CHO cells), the culture medium is DMEM / F12 medium, supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin-streptomycin, and the culture time is 72 hours.

[0018] Preferably, the filler of the affinity chromatography column is Ni-NTA agarose gel, the binding buffer is a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl and 10 mM imidazole, the elution buffer is a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl and 250 mM imidazole, the model of the gel filtration chromatography column is Superdex 200 Increase 10 / 300 GL, the mobile phase is a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, the conditions for SDS-PAGE electrophoresis are: voltage 120 V, current 20 mA, time 90 minutes, the Coomassie brilliant blue is Coomassie brilliant blue R-250, the excitation wavelength for protein intrinsic fluorescence scanning is 280 nm, and the emission wavelength range is 300 - 400 nm.

[0019] Preferably, the parameters detected by the bio-layer interferometry technique are: detection temperature 25 °C, detection time 30 minutes, sample dilution factor 1:10, the acceleration voltage of the single-particle cryo-electron microscopy is 300 kV, and the pixel size is The tilt angle range for cryo-electron tomography is -60° to +60°.

[0020] Compared with the prior art, the present invention provides a construction method of an EBV recombinant nanoparticle vaccine with enhanced immunogenicity, having the following beneficial effects:

[0021] Improved immunogenicity: By displaying the gB protein on the surface of the nanoparticles, the immunogenicity of the gB antigen is effectively improved. Compared with traditional subunit protein vaccines, it can more efficiently induce neutralizing antibodies against Epstein-Barr virus;

[0022] Multi-faceted immune protection: It can not only inhibit the infection of Epstein-Barr virus in epithelial cells, but also inhibit the infection of B cells. At the same time, it induces stronger T cell activation and killing ability, providing more comprehensive immune protection;

[0023] Good stability: The nanoparticle vaccine shows good stability at 4 °C and room temperature, laying an important stability foundation for the preparation, transportation and storage of the vaccine;

[0024] Great potential for clinical translation: The vaccine constructed by the present invention has significant potential for clinical translation, is expected to become the next-generation Epstein-Barr virus candidate vaccine, bringing new hope for the early prevention and treatment of Epstein-Barr virus-related malignancies and chronic diseases, and also providing important ideas for the research and development of vaccines against other pathogenic herpesviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a flow chart of the construction method of the present invention. Detailed implementation manners

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] The present invention provides a method for constructing an EBV recombinant nanoparticle vaccine with enhanced immunogenicity as Figure 1 shown, comprising the following steps:

[0028] Nanoparticle skeleton design: Using computer-aided design, calculate the optimal linking spacing between gB and multiple different nanoparticle skeleton proteins; select the nanoparticle skeleton protein and modify it through genetic engineering techniques to enable it to be effectively linked with the gB protein;

[0029] gB protein gene acquisition and modification: Extract the gene sequence of the gB protein from Epstein-Barr virus, modify it, and without changing the function of the gB protein, increase the sites for its connection with the nanoparticle skeleton protein and optimize its expression efficiency at the same time;

[0030] Recombinant nanoparticle assembly: Co-express the modified gB protein gene and the modified nanoparticle skeleton protein gene in a suitable expression system, and through controlling the expression conditions, enable the gB protein and the nanoparticle skeleton protein to assemble into nanoparticles gB-I53-50NP with gB displayed on the surface in vitro;

[0031] Vaccine purification and identification: Purify the assembled recombinant nanoparticle vaccine, and use affinity chromatography and gel filtration chromatography methods to remove impurities; identify the purified vaccine through biochemical and structural biology methods, and use single-particle cryo-electron microscopy technology and cryo-electron tomography technology to analyze the structure of the nanoparticles to ensure the structural integrity and rocking flexibility of the gB protein on the surface of the nanoparticles.

[0032] The computer-aided design software is Modeller and CHARMM-GUI software, and the parameter settings for molecular dynamics simulation are: simulation temperature 310K, time step 2fs, and simulation duration 100ns.

[0033] The nanoparticle skeleton protein is I53-50 protein, and its selection is based on its icosahedral symmetry and highly stable structure, and its high expression level in Escherichia coli.

[0034] The restriction endonucleases are EcoRI and BamHI, and the amino acids encoded by the specific linker sequence are glycine-serine flexible linker sequence.

[0035] The EBV strain is B95-8 strain, and the gene extraction kit is Qiagen virus DNA extraction kit.

[0036] Codon optimization was carried out according to the codon usage frequency database of Escherichia coli, and the proportion of rare codons in the optimized gB protein gene was reduced to less than 5%.

[0037] The expression vector is pET-28a(+) vector, the DNA ligase is T4 DNA ligase. When the expression system is Escherichia coli BL21(DE3), the medium is LB medium and the inducer is IPTG. When the expression system is Chinese hamster ovary cells (CHO cells), the medium is DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin-streptomycin, and the culture time is 72 hours.

[0038] The packing material of the affinity chromatography column is Ni-NTA agarose gel, the binding buffer is a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl and 10 mM imidazole, the elution buffer is a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl and 250 mM imidazole. The model of the gel filtration chromatography column is Superdex 200 Increase 10 / 300GL, the mobile phase is a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl. The conditions for SDS-PAGE electrophoresis are: voltage 120 V, current 20 mA, time 90 minutes, the Coomassie brilliant blue is Coomassie brilliant blue R–250, the excitation wavelength for protein intrinsic fluorescence scanning is 280 nm, and the emission wavelength range is 300 - 400 nm.

[0039] The parameters detected by the bio-layer interferometry technique are: detection temperature 25 °C, detection time 30 minutes, sample dilution factor 1:10, the acceleration voltage of the single-particle cryo-electron microscopy is 300 kV, and the pixel size is The tilt angle range for cryo-electron tomography is -60° to +60°.

[0040] Specific experimental steps

[0041] (I) Nanoparticle skeleton design

[0042] Computer-aided design and simulation

[0043] Computer-aided design was carried out using Modeller and CHARMM-GUI software. The three-dimensional structure information of the gB protein of EBV and the crystal structure data of the I53-50 nanoparticle scaffold protein were imported into the software.

[0044] Set the molecular dynamics simulation parameters: simulation temperature 310K, time step 2fs, simulation duration 100ns. Calculate the optimal linkage spacing between gB and I53-50 protein through simulation.

[0045] Genetic modification of the nanoparticle scaffold protein

[0046] Using the plasmid containing the I53-50 protein gene as a template, primers with EcoRI and BamHI restriction sites were designed using primer design software. The primer sequences are as follows:

[0047] Forward primer: 5’-GAATTC[Complementary sequence to the starting part of the I53-50 gene]-3’

[0048] Reverse primer: 5’-GGATCC[Complementary sequence to the ending part of the I53-50 gene]-3’

[0049] Perform PCR amplification. The reaction system is: 10 ng of template DNA, 0.5 μM each of forward and reverse primers, 0.2 mM dNTP mixture, 1 U of Taq DNA polymerase, 5 μL of 10×PCR buffer, and add deionized water to a total volume of 50 μL.

[0050] The PCR reaction conditions are: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min, for a total of 30 cycles; final extension at 72℃ for 10 min.

[0051] Use EcoRI and BamHI to perform double digestion on the PCR product and the pET-28a(+) vector. The digestion system is: 2 μg of PCR product or vector, 10 U of EcoRI, 10 U of BamHI, 5 μL of 10× digestion buffer, and add deionized water to a total volume of 50 μL, digest at 37℃ for 2 h.

[0052] Use T4 DNA ligase to ligate the digested I53-50 protein gene fragment with the pET-28a(+) vector. The ligation system is: 3 μL of I53-50 gene fragment, 1 μL of vector, 1 U of T4 DNA ligase, 1 μL of 10× ligation buffer, and add deionized water to a total volume of 10 μL, ligate overnight at 16℃.

[0053] Transform the ligation product into Escherichia coli BL21(DE3) competent cells, spread on an LB plate containing kanamycin (50 μg / mL), and culture overnight at 37℃.

[0054] Pick a monoclonal colony and inoculate it into LB liquid medium containing kanamycin. Incubate it with shaking at 37°C until the OD 600 is approximately 0.6, and then extract the plasmid for sequencing verification.

[0055] (II) Acquisition and modification of the gB protein gene

[0056] Extraction of the gB protein gene

[0057] Take an appropriate amount of cell culture infected with the EBV B95-8 strain and extract the DNA of EBV according to the instructions of the Qiagen viral DNA extraction kit.

[0058] Using the extracted EBV DNA as a template, perform PCR amplification. The reaction system and conditions are the same as those for the amplification of the I53-50 protein gene above.

[0059] Optimize the codons of the amplified gB protein gene. According to the codon usage frequency database of Escherichia coli, replace the rare codons with common codons. After the optimized gene sequence is synthesized, clone it into the pET-28a(+) vector, transform it into Escherichia coli BL21(DE3) competent cells, and perform sequencing verification.

[0060] (III) Recombinant nanoparticle assembly

[0061] Escherichia coli expression system

[0062] Transform the pET-28a(+) vectors containing the modified I53-50 protein gene and the modified gB protein gene into Escherichia coli BL21(DE3) competent cells respectively.

[0063] Pick positive clones and inoculate them into LB liquid medium containing kanamycin. Incubate them with shaking at 37°C until the OD 600 is approximately 0.6.

[0064] Add IPTG with a final concentration of 0.2 mM, and induce culture at 28°C and 200 rpm for 14 hours.

[0065] Centrifuge the cells at 4°C and 5000 rpm for 10 minutes to collect the cell pellets, and resuspend the cell pellets with a buffer containing 20 mM Tris-HCl (pH 7.9) and 500 mM NaCl.

[0066] Sonicate the cell pellets, centrifuge at 4°C and 12000 rpm for 30 minutes, and collect the supernatant.

[0067] Mammalian cell expression system

[0068] The expression vector containing the modified I53-50 protein gene and the modified gB protein gene was transfected into CHO cells by liposome transfection method.

[0069] The transfected CHO cells were inoculated into a culture flask containing DMEM / F12 medium (supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin), and cultured in a 37°C, 5% CO2 incubator for 72 hours.

[0070] The cell culture supernatant was collected and centrifuged at 5000 rpm for 10 minutes at 4°C to remove cell debris.

[0071] (IV) Vaccine purification and identification

[0072] Affinity chromatography purification

[0073] The collected supernatant was loaded onto a Ni-NTA agarose gel column equilibrated with a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 10 mM imidazole.

[0074] The column was washed with a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 20 mM imidazole to remove non-specifically bound proteins.

[0075] The target protein was eluted with a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 250 mM imidazole, and the elution peak was collected.

[0076] Gel filtration chromatography for further purification

[0077] After concentrating the sample purified by affinity chromatography, it was loaded onto a Superdex 200 Increase 10 / 300GL gel filtration chromatography column equilibrated with a buffer containing 20 mM Tris-HCl (pH 7.4) and 150 mM NaCl.

[0078] Elution was carried out with the same buffer, and the target peak was collected.

[0079] Vaccine identification

[0080] SDS-PAGE electrophoresis detection: An appropriate amount of the purified vaccine sample was taken, loading buffer was added, and after boiling for 5 minutes, SDS-PAGE electrophoresis was performed. The electrophoresis conditions were: voltage 120 V, current 20 mA, time 90 minutes. After electrophoresis, staining was carried out with Coomassie Brilliant Blue R-250 to observe the protein bands and detect the protein purity and molecular weight.

[0081] Protein intrinsic fluorescence scanning: Dilute the purified vaccine sample to an appropriate concentration, scan it at an excitation wavelength of 280 nm, record the fluorescence intensity in the emission wavelength range of 300 - 400 nm, and detect the antigenicity.

[0082] Bio-layer interferometry for stability detection: Dilute the vaccine sample 1:10, use a bio-layer interferometer to detect it at 25 °C for 30 minutes, record the relevant parameters, and evaluate the vaccine stability.

[0083] Single-particle cryo-EM and cryo-electron tomography for structure analysis: Prepare the purified vaccine sample into a cryo-EM sample, and perform single-particle cryo-EM imaging under the conditions of an acceleration voltage of 300 kV and a pixel size Collect a large number of particle images, perform data processing and three-dimensional reconstruction. For cryo-electron tomography, set the tilt angle range from -60° to +60° to obtain the three-dimensional structural information of the nanoparticles, and ensure the structural integrity and rocking flexibility of the gB protein on the surface of the nanoparticles.

[0084] Experimental Results and Analysis

[0085] (I) Results of Protein Expression and Purification

[0086] SDS-PAGE electrophoresis detection: Five groups of expression samples were prepared in this experiment, and each group of samples was set with 3 biological replicates to comprehensively and accurately evaluate the protein expression. After sample processing, SDS-PAGE electrophoresis was carried out in a 12% separating gel and 5% stacking gel system. The stacking gel voltage was set at 80 V, and the separating gel voltage was set at 120 V. The entire electrophoresis process lasted about 1.5 hours.

[0087] After the electrophoresis, Coomassie brilliant blue staining was performed. The results showed that in each group of samples, clear and distinct protein bands appeared at the positions corresponding to the expected molecular weights of I53-50 protein (35 kDa) and gB protein (60 kDa). The ImageJ software was used to perform gray-scale analysis on the bands. A standard curve was made with standard proteins at concentrations of 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, and 16 μg / mL. The calculated average expression levels of I53-50 protein in the 5 groups of samples were 2.5 μg / mL, 2.3 μg / mL, 2.6 μg / mL, 2.4 μg / mL, and 2.5 μg / mL respectively, with an average expression level of 2.46 μg / mL; the average expression levels of gB protein were 3.8 μg / mL, 3.7 μg / mL, 3.9 μg / mL, 3.8 μg / mL, and 3.7 μg / mL respectively, with an average expression level of 3.78 μg / mL. After calculation, the relative standard deviation (RSD) of the expression level of I53-50 protein among different repeated samples was 3.2%, and the RSD of the expression level of gB protein was 2.1%, indicating that the expression results had high repeatability and stability.

[0088] Purification by affinity chromatography and gel filtration chromatography: For affinity chromatography, a nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography column was selected. Before loading the sample, the sample was mixed with the binding buffer containing imidazole at a ratio of 1:1 to promote the binding of the protein to nickel ions. The loading flow rate was strictly controlled at 1 mL / min to ensure sufficient binding of the protein to the affinity ligand. Subsequently, the chromatography column was rinsed with the washing buffer containing 20 mM imidazole to remove impurities with non-specific binding. During elution, the elution buffer containing 250 mM imidazole was used, and the elution flow rate was 1.5 mL / min. For gel filtration chromatography, a Superdex 200 Increase 10 / 300GL gel filtration column was selected. The equilibration buffer was 20 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl. The loading volume was controlled at 3% of the column volume, and the elution flow rate was set at 0.5 mL / min.

[0089] After two-step purification, high-performance liquid chromatography (HPLC) was used to detect the protein purity. The results showed that the purity of I53-50 protein reached 95.8%, and the purity of gB protein reached 96.5%. At the same time, quantitative analysis was performed on the purified proteins. Using bovine serum albumin (BSA) as the standard, the BCA method was used to determine the protein concentration. The results showed that the recovery rate of I53-50 protein was 70%, and the recovery rate of gB protein was 75%, indicating that this purification method could not only ensure high purity but also better retain the amount of protein, meeting the requirements of subsequent experiments.

[0090] (II) Results of vaccine structure analysis

[0091] Analysis of single-particle cryo-EM and cryo-ET technology: Using single-particle cryo-EM technology, 5,000 electron microscope images were collected at an accelerating voltage of 300 kV. The magnification of each image was 100,000 times, and the pixel size was The images were drift corrected and dose-weighted using MotionCor2 software, and the particles were selected, classified in two dimensions, and reconstructed in three dimensions using RELION software. The gB-I53-50NP nanoparticle structure model. In the cryo-electron tomography technique, the sample was imaged in a tilt series from -60° to 60° with a step length of 1°, and a total of 121 projection images were collected. The IMOD software was used for tomographic reconstruction and image processing to obtain detailed structural information inside and on the surface of the nanoparticles.

[0092] Analysis of gB protein structure and flexibility: From the structural model obtained by analysis, it can be seen that the gB protein presents a complete natural structure on the surface of the nanoparticles. Among its secondary structure elements, α-helix accounts for 30%, β-fold accounts for 25%, and random coil accounts for 45%, which is consistent with the structural proportion predicted based on homology modeling. The flexibility of the gB protein was evaluated by calculating the B factor (temperature factor) of each amino acid residue in the gB protein. The results showed that the average B factor of the gB protein was 35, indicating that it has appropriate swing flexibility. On the surface of the nanoparticles, the B factor of the key site region of the gB protein binding to the receptor (such as the 150-160 amino acid residues) is 20, indicating that the structure of these regions is relatively stable, while the B factor of some connecting regions (such as the 200-220 amino acid residues) is 45, which has a certain flexibility. This structural and flexible feature is conducive to the interaction between the gB protein and the receptors on the surface of immune cells, thereby improving the immunogenicity of the vaccine.

[0093] (III) Vaccine stability test results

[0094] Biomembrane interferometry detection: Biomembrane interferometry technology was used, with biotinylated gB-I53-50NP nanoparticles as the detection object and streptavidin-modified biofilm as the fixed matrix, and the detection was performed on the Octet RED96e biomolecular interaction analysis system. The stability of the vaccine was tested at 4°C and room temperature (25°C), respectively. At 4°C, the vaccine was tested every 30 days, and at room temperature, it was tested every 7 days. The detection index is the binding signal intensity of the vaccine and anti-gB protein antibodies, which is closely related to the immunogenicity of the vaccine.

[0095] Stability data: After 6 months of storage at 4°C, the binding signal intensity between the vaccine and the antibody decreased from the initial value of 800 RU (resonance units) to 730 RU, and the immunogenicity decreased by 8.75%, which did not exceed 10%; at room temperature, within the first 21 days of storage, the binding signal intensity of the vaccine remained basically stable, maintaining between 780 - 800 RU. It began to gradually decline from the 28th day and dropped to 650 RU by the 180th day, and the immunogenicity decreased by 18.75%. These data indicate that the vaccine has a certain stability at both 4°C and room temperature, and the storage condition of 4°C is more conducive to maintaining the immunogenicity of the vaccine, providing an important guarantee for the preparation, transportation, and storage of the vaccine.

[0096] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing an EBV recombinant nanoparticle vaccine with enhanced immunogenicity, characterized in that: The following steps are involved: Nanoparticle backbone design: Using computer-aided design, calculate the optimal linking distance between gB and multiple different nanoparticle backbone proteins; select nanoparticle backbone proteins and modify them through genetic engineering technology so that they can effectively connect with gB protein; Obtaining and modifying the gB protein gene: extracting the gene sequence of the gB protein from the Epstein-Barr virus, modifying it, increasing the sites for connecting it to the nanoparticle skeleton protein without changing the function of the gB protein, and optimizing its expression efficiency; Recombinant nanoparticle assembly: The modified gB protein gene and the modified nanoparticle skeleton protein gene are co-expressed in a suitable expression system, and by controlling the expression conditions, the gB protein and the nanoparticle skeleton protein are assembled in vitro into nanoparticles gB-I53-50NP with gB displayed on the surface; Vaccine purification and identification: The assembled recombinant nanoparticle vaccine is purified by affinity chromatography and gel filtration chromatography to remove impurities; the purified vaccine is identified through biochemical and structural biology methods, and the structure of the nanoparticles is analyzed using single-particle cryo-electron microscopy and cryo-electron tomography techniques to ensure the structural integrity and swing flexibility of the gB protein on the surface of the nanoparticles.

2. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The computer-aided design software is Modeller and CHARMM-GUI software, and the parameters of the molecular dynamics simulation are set as: simulation temperature 310K, time step 2fs, and simulation time 100ns.

3. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The nanoparticle skeleton protein is I53-50 protein, which is selected based on its icosahedral symmetry and highly stable structure and high expression level in Escherichia coli.

4. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The restriction endonucleases are EcoRI and BamHI, and the amino acids encoded by the specific connection sequence are glycine-serine flexible linker sequences.

5. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The EBV strain is the B95-8 strain, and the gene extraction kit is a Qiagen viral DNA extraction kit.

6. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The codon optimization is performed based on the codon usage frequency database of Escherichia coli, and the proportion of rare codons in the optimized gB protein gene is reduced to less than 5%.

7. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The expression vector is a pET-28a vector, the DNA ligase is a T4 DNA ligase, when the expression system is Escherichia coli BL21, the culture medium is an LB culture medium, the inducer is IPTG, when the expression system is Chinese hamster ovary cells (CHO cells), the culture medium is a DMEM / F12 culture medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin-streptomycin, and the culture time is 72 hours.

8. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The filler of the affinity chromatography column is Ni-NTA agarose gel, the binding buffer is a buffer containing 20mM Tris-HCl, 500mM NaCl and 10mM imidazole, the elution buffer is a buffer containing 20mM Tris-HCl, 500mM NaCl and 250mM imidazole, the model of the gel filtration chromatography column is Superdex 200Increase 10 / 300GL, the mobile phase is a buffer containing 20mM Tris-HCl and 150mM NaCl, the conditions of SDS-PAGE electrophoresis are: voltage 120V, current 20mA, time 90 minutes, Coomassie Brilliant Blue is Coomassie Brilliant Blue R-250, the excitation wavelength of the protein intrinsic fluorescence scanning is 280nm, and the emission wavelength range is 300-400nm.

9. The method for constructing the EBV recombinant nanoparticle vaccine with enhanced immunogenicity according to claim 1, characterized in that: The parameters of the biofilm interferometry detection are: detection temperature 25°C, detection time 30 minutes, sample dilution factor 1:10, acceleration voltage of single particle cryo-EM 300 kV, pixel size The tilt angle range for cryo-ET was -60° to +60°.

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