Preparation and application of nanoparticle vaccine of EB (Epstein-Barr) virus gp350 protein polypeptide and combination of nanoparticle vaccine

Abstract

The invention discloses a nanoparticle vaccine of EB (Epstein-Barr) virus gp350 protein polypeptide and preparation and application of a combination of the nanoparticle vaccine. The invention belongs to the field of biological medicine, and particularly relates to a nanoparticle vaccine of EB virus gp350 protein polypeptide and preparation and application of a combination of the nanoparticle vaccine. The immunogen complex provided by the invention is formed by assembling polypeptide related to gp350 glycoprotein of EB (Epstein-Barr) virus and carrier protein Ferritin, wherein the assembling molar ratio of the polypeptide to the carrier protein Ferritin is 5: 1. The immunogen complex provided by the invention can be used for preparing a nanoparticle vaccine, compared with an ungranulated vaccine, the total antititer is improved, the neutralizing titer and the immune memory stimulation of the vaccine effect are obviously improved, and the immunogen complex can be used for preventing or treating diseases caused by EB (Epstein-Barr) virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the preparation and application of a nanoparticle vaccine of Epstein-Barr virus (EBV) gp350 protein polypeptide and its combination. Background Art

[0002] Epstein-Barr virus (EBV), also known as Human herpesvirus 4 (HHV-4), is the only virus in the γ-subfamily of Herpesviridae that can cause human infection, and it is also the first discovered tumor virus. EBV infection is closely related to the occurrence of various human malignancies, such as Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma, etc. It has been found that EBV can also be detected in leiomyoma, hepatic sarcoma, thymic carcinoma, and cholangiocarcinoma. Therefore, the development of EBV vaccines is of great significance.

[0003] The research on EBV vaccines mainly focuses on the glycoproteins of EBV, including gp350 / 220, gp85, gp25, etc. EBV gp350 is the protein with the highest content in the outer membrane protein of EBV and is also the main surface antigen of EBV, playing an important role in the invasion of EBV into CD21-positive cells (such as B lymphocytes). EBV mediates its entry into cells through the interaction of gp350 with the surface receptor CD21 of B lymphocytes. Many research results have proved that gp350 can be used as a potential vaccine for preventing EBV.

[0004] Due to the potential carcinogenic characteristics of EBV, inactivated or attenuated vaccines are not feasible, and single antigens isolated from the virus have low immunogenicity. Therefore, how to obtain an EBV vaccine with high immunogenicity has become the current research focus. Summary of the Invention

[0005] The main problem to be solved by the present invention is how to obtain an EBV vaccine with high immunogenicity.

[0006] To solve the above problems, the present invention provides an immunogenic complex.

[0007] The immunogenic complex of the present invention is assembled from a polypeptide related to the gp350 glycoprotein of Epstein-Barr virus and the carrier protein Ferritin.

[0008] The Epstein-Barr virus gp350 glycoprotein is the main surface antigen of EBV. Fusing it with the carrier protein can significantly improve the immunogenicity of the antigen peptide.

[0009] In the above immunogenic complex, the polypeptide is connected with a flexible linker, and a SPY tag sequence and a 6×His sequence are connected to the C-terminus of the polypeptide.

[0010] In the above immunogenic complex, the N-terminus of the carrier protein Ferritin can be linked to the Catcher sequence and the 6×His sequence.

[0011] In the above immunogenic complex, the polypeptide can be any of the following: a1) The amino acid sequence is SEQ ID No:1; a2) A polypeptide having the same function with one or several amino acid residues substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No:1; a3) A polypeptide having more than 75% identity with the amino acid sequence defined in any of a1) or (a2) and having the same function; a4) A polypeptide obtained by linking a tag to the end of the protein defined in any of a1)-(a3). The carrier protein Ferritin is any of the following: b1) A protein with the amino acid sequence SEQ ID No:2; b2) A protein having the same function with one or several amino acid residues substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No:2; b3) A protein having more than 75% identity with the amino acid sequence defined in any of b1) or (b2) and having the same function; b4) The carrier protein Ferritin obtained by linking a tag to the end of the protein defined in any of b1)-(b3).

[0012] In this article, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0013] In this article, the above 90% or more identity can be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0014] The protein tag refers to a polypeptide or protein that is expressed in fusion with the target protein to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a His tag, Flag tag, MBP tag, HA tag, myc tag, GST tag, Fc fragment of immunoglobulin G, and / or SUMO tag, etc.

[0015] In the above immunogen complex, the assembly molar ratio of the polypeptide to the carrier protein Ferritin is 5:1.

[0016] Furthermore, the assembly method is as follows: the polypeptide and the carrier protein Ferritin are assembled by rotary incubation overnight in a PBS buffer at 4°C according to a molar ratio of 5:1.

[0017] The polypeptide in the immunogen complex described above also falls within the scope of protection claimed in the present invention.

[0018] In a specific embodiment, the polypeptide is a combination of epitope peptides MEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEF, WCHHAEMQNPVYLIPETVPYIKWDNCN, MLGNEIDIECIMEDGEISQY, ILYVFYSGNGPKASGGDYCIQS, PSTSSKLRPRWTFTSPPVTT on the gp350 glycoprotein of Epstein-Barr virus. The 5 epitope peptides are linked by a flexible linker GGGGS.

[0019] Furthermore, the polypeptide is the L350-ST protein. The amino acid sequence of the L350-ST protein is SEQ ID No:1.

[0020] The present invention also provides biological materials related to the polypeptide. The biological materials can be any of the following: c1) A nucleic acid molecule encoding the polypeptide described above; c2) An expression cassette containing the nucleic acid molecule of c1); c3) A recombinant vector containing the nucleic acid molecule of c1), or a recombinant vector containing the expression cassette of c2); c4) A recombinant microorganism containing the nucleic acid molecule of c1), or a recombinant microorganism containing the expression cassette of c2), or a recombinant microorganism containing the recombinant vector of c3).

[0021] Among the above biological materials, the nucleic acid molecule of c1) can be a DNA molecule shown as any of the following: d1) A DNA molecule with a nucleotide sequence shown as SEQ ID No:3; d2) A DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) and encoding the polypeptide described above; d3) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the polypeptide described above.

[0022] The nucleic acid molecules described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0023] The vectors described herein are well known to those skilled in the art and include, but are not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids or viral vectors. Specifically, it can be the vector pET-28a(+).

[0024] In a specific embodiment, the recombinant vector in c3) is pET-28a(+)-L350-ST, and the structure of the recombinant vector pET-28a(+)-L350-ST is described as follows: a DNA fragment with the sequence of SEQ ID No:3 is inserted between the XhoI and HindII restriction enzyme sites of the starting vector pET-28a(+), and other sequences of the vector pET-28a(+) are kept unchanged to obtain the recombinant vector. The pET-28a(+)-L350-ST vector can express the L350-ST protein, and its amino acid sequence is SEQ ID No:1.

[0025] Among the above biological materials, the microorganism can be a bacterium (such as Escherichia coli), yeast, alga or fungus.

[0026] The present invention also provides a method for preparing the above polypeptide, which may include the following steps: introducing a nucleic acid molecule encoding the above polypeptide into a recipient cell to obtain a transgenic cell expressing the polypeptide, and culturing the transgenic cell to obtain the polypeptide.

[0027] The recipient cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0028] The polypeptide protein in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other properties. The above methods are well-known to those skilled in the art. These methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0029] The present invention also provides an Epstein-Barr virus vaccine, which comprises the immunogenic complex described above.

[0030] The present invention also provides the use of the immunogenic complex described above in the preparation of an Epstein-Barr virus inhibitor.

[0031] The present invention also provides the use of the immunogenic complex described above in the preparation of a medicament for treating or preventing diseases caused by Epstein-Barr virus infection.

[0032] The present invention also provides the use of the immunogenic complex described above in the preparation of an Epstein-Barr virus vaccine.

[0033] The present invention also provides the use of the vaccine described above in the preparation of a medicament for treating or preventing diseases caused by Epstein-Barr virus infection.

[0034] In practical applications, the immunogenic complex or immunogenic vaccine of the present invention can be directly administered to patients or animals as a drug, or administered to patients or animals after being mixed with a suitable carrier or excipient, so as to achieve the purpose of preventing Epstein-Barr virus infection. The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, the preferred is water-soluble carrier materials. Using these materials, various dosage forms can be prepared, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle drug delivery systems.

[0035] To formulate the unit dosage form into tablets, various carriers well-known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, laminarin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To formulate the unit dosage form into pills, various carriers well-known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. To formulate the unit dosage form into suppositories, various carriers well-known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. To formulate the unit dosage form into injectable preparations such as solutions, emulsions, freeze-dried powder injections and suspensions, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc. Additionally, to prepare isotonic injection solutions, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injectable preparation. In addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. Furthermore, if needed, coloring agents, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; administration via body cavities, such as via the rectum and vagina; administration via the respiratory tract, such as via the nasal cavity; mucosal administration. The preferred administration route among the above is injection administration.

[0036] The antigen described in the present invention is Epstein - Barr virus antigen.

[0037] By granulating the immunogen, the present invention improves the immunogenicity of the corresponding immunogen, significantly stimulating the humoral and cellular immune responses. After immunizing mice with the nanoparticle vaccine prepared by the present invention, the mouse serum can efficiently neutralize EBV-infected B cells in vitro; compared with the non-granulated vaccine, the total antibody titer, the neutralization titer against the virus, and the vaccine efficacy in stimulating immune memory are significantly improved, and the chemical stability, particle size, and binding ability to neutralizing antibodies are all higher than those of the polypeptide vaccine, which is beneficial to increasing its residence time on the B cell antigen receptor and stimulating the production of antibodies; at the same time, the self-assembled nanoparticles can induce a high animal immune antibody titer and can be used to prevent EBV infection and treat diseases caused by EBV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of the connection of the fusion protein of the present invention.

[0039] Figure 2 It is a PAGE diagram of the recombinant nanoparticle protein vaccine.

[0040] Figure 3 It is a transmission electron microscopy negative staining result diagram of the recombinant nanoparticle protein.

[0041] Figure 4 It is the particle size and distribution of the recombinant nanoparticle protein.

[0042] Figure 5 It is the immunogenicity of the recombinant nanoparticle protein in BALB / C mice. Among them, (a) is the immunization procedure of the recombinant nanoparticle protein in BALB / C mice; (b) is the serum antibody titers between different vaccine antigens against the gp350D123 protein and the control; (c) is the serum antibody titers between different vaccine antigens against the gp350D123 protein and the control at the 10th week; (d) is the serum antibody titers between different vaccine antigens against the L350 protein and the control; (e) is the serum antibody titers between different vaccine antigens against the L350 protein and the control at the 10th week.

[0043] Figure 6 It is that the serum of BALB / C mice immunized with the recombinant nanoparticle protein blocks EBV infection at the cellular level and induces a high neutralizing antibody titer at the same time. Among them, (a) is to detect the infection of the purified EBV-GFP reporter virus using AKATA cells. After about 48 hours of infection, the infected cells are observed through the fluorescence of green fluorescent protein (GFP); (b) is to detect the infection of EBV-GFP in AKATA cells; (c) is the infection ratio of cells containing green fluorescent EBV; (d) is the half-maximal inhibitory concentration of the virus.

[0044] Figure 7To establish potent B lymphocyte immune memory in immunized BALB / C mice. Among them, (a) Flow cytometry analysis of the proportion of specific memory B cells of APC-labeled gp350D123 protein. (b) Proportion of specific memory cells against gp350D123 protein; (c) Proportion of specific memory cells against L350 protein; (d) Flow cytometry analysis of the proportion of specific memory B cells of APC-labeled L350 protein. Detailed implementation mode

[0045] The present invention will be further described in detail below in conjunction with the specific implementation mode. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0046] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0047] Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged.

[0048] pET-28a(+) in the following embodiments was purchased from Shanghai Yisheng Biological Co., Ltd., with the product number 11905ES03.

[0049] Akata and Raji lymphocytes in the following embodiments were purchased from Wuhan Punosai Biological Co., Ltd., with the product numbers CL-0763 and CL-0189.

[0050] The following embodiments used SPSS11.5 statistical software to process the data. The experimental results were expressed as mean ± standard deviation, and were tested by One-way ANOVA. P < 0.05 (*) indicates significant difference, P < 0.01 (**) indicates extremely significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0051] Example 1 Preparation of Ferritin-gp350 immunogenic complex 1. Select the epitope peptides MEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEF, WCHHAEMQNPVYLIPETVPYIKWDNCN, MLGNEIDIECIMEDGEISQY, ILYVFYSGNGPKASGGDYCIQS, and PSTSSKLRPRWTFTSPPVTT on the gp350 glycoprotein of Epstein-Barr virus. The 5 epitope peptides were concatenated in the order of their distribution on the gp350 glycoprotein from the N-terminus to the C-terminus. The peptide segments were connected by the flexible linker GGGGS, and a SPY tag sequence and a 6×His sequence were cloned at the C-terminus of the concatenated polypeptide sequence. The cloned sequence was ligated to the pET-28a(+) vector to obtain the recombinant vector pET-28a(+)-L350-ST.

[0052] The structure of the pET-28a(+)-L350-ST vector is described as follows: A DNA fragment with the sequence of SEQ ID No:3 was inserted between the XhoI and HindIII restriction enzyme sites of the starting vector pET-28a(+), and the other sequences of the vector pET-28a(+) were kept unchanged to obtain the recombinant vector. The pET-28a(+)-L350-ST vector can express the L350-ST protein, and its amino acid sequence is SEQ ID No:1.

[0053] The successfully constructed recombinant plasmid pET-28a(+)-L350-ST was transformed into BL21 (Shenzhen Kangti Biotechnology Co., Ltd., product number KTSM104L) to obtain the recombinant Escherichia coli BL21 / pET-28a(+)-L350-ST. The recombinant Escherichia coli BL21 / pET-28a(+)-L350-ST was subjected to prokaryotic protein expression and purification ( Figure 1 ), and the fusion peptide L350-ST was obtained. The amino acid sequence of the L350-ST is SEQ ID No:1.

[0054] 2. Clone a Catcher sequence and a 6×His sequence at the N-terminus of the Ferritin ferritin sequence ( Figure 1 ). The Catcher protein can specifically recognize the SPY tag and stably bind under mild conditions, thereby anchoring the L350-ST recombinant protein on the surface of the Ferritin nanoparticles. The cloned sequence was ligated to the pET-28a(+) vector to obtain the recombinant vector pET-28a(+)-SC-Ferritin.

[0055] The structure of the pET-28a(+)-SC-Ferritin vector is described as follows: A DNA fragment with the sequence of SEQ ID No:4 is inserted between the XhoI and HindIII restriction enzyme sites of the starting vector pET-28a(+), and other sequences of the vector pET-28a(+) are kept unchanged to obtain the recombinant vector. The pET-28a(+)-SC-Ferritin vector can express the SC-Ferritin protein, and its amino acid sequence is SEQ ID No:2.

[0056] The successfully constructed recombinant plasmid pET-28a(+)-SC-Ferritin was transformed into BL21 (Shenzhen Kangti Biotechnology, product number KTSM104L) to obtain the recombinant Escherichia coli BL21 / pET-28a(+)-SC-Ferritin. The recombinant Escherichia coli BL21 / pET-28a(+)-SC-Ferritin was subjected to prokaryotic protein expression and purification ( Figure 1 ). The fusion peptide SC-Ferritin was obtained, and the amino acid sequence of the SC-Ferritin is SEQ ID No:2.

[0057] 3. The L350-ST obtained in step 1 and the SC-Ferritin obtained in step 2 were incubated by rotation overnight at 4°C in PBS buffer at a molar ratio of 5:1, and then the fully assembled L350-Ferritin nanoparticles were purified using a size exclusion chromatography column Superose6 Increase 10 / 300GL gel filtration column, and then the purified product was concentrated using a 100KD ultrafiltration tube ( Figure 2 ). The L350-ST protein was efficiently assembled onto the SC-Ferritin nanoparticles to obtain the L350-Ferritin nanoparticle vaccine.

[0058] The particle size of the purified L350-Ferritin nanoparticles was analyzed using a particle size analyzer DLS. The results are as Figure 4 shown: Both Ferritin and L350-Ferritin nanoparticles have only one particle size peak, and the average particle sizes of the two are 32nm and 79nm respectively; the particle size of the L350-Ferritin nanoparticles conjugated with the protein has changed significantly.

[0059] The shape of the nanoparticles was characterized by transmission electron microscopy TEM as follows ( Figure 3), compared with the Ferritin group, the L350-Ferritin nanoparticles had a larger particle size, which was consistent with the results measured by the particle size analyzer. Moreover, compared with the smooth surface of the Ferritin nanoparticles, the surface of the L350-Ferritin nanoparticle vaccine was relatively rough, indicating that the L350-ST protein was successfully assembled on the surface of the SC-Ferritin nanoparticles. Example 2. L350-Ferritin Nanoparticle Vaccine Stimulates Mice to Produce High Levels of Specific Antibodies and Neutralizing Antibodies The experimental mice were SPF-grade Balb / c mice (Zhuhai Bestoon Biotechnology Co., Ltd., cat. no. BALB / c). Six-week-old Balb / c mice (weighing about 20 g) were selected and housed together for at least 1 week before the experiment.

[0060] Thirty mice were divided into 5 groups: the PBS group, the Ferritin group, the gp350D123 group, the L350 group, and the L350-Ferritin group, with 6 mice in each group. Each mouse was intramuscularly injected with an equal volume of PBS buffer (concentration 0.01 M, pH 7.4), Ferritin (injection dose 15 μg), gp350D123 (injection dose 15 μg), L350 (injection dose 15 μg), and L350-Ferritin (injection dose 15 μg). The PBS group was the control group with PBS solution. Ferritin was obtained by expressing and purifying the pET-28a(+) plasmid in Example 2 and dissolved in PBS buffer. The gp350D123 protein was obtained by expressing and purifying the pET-28a(+) plasmid and dissolved in PBS buffer. The L350 protein was obtained by expressing and purifying the pET-28a(+) plasmid and dissolved in PBS buffer. Taking the first immunization as week 0, an equal amount of booster immunization was given at weeks 2 and 4 after the first immunization.

[0061] Mouse sera were collected at weeks 0, 2, 4, 6, 8, 10, 12, 14, and 16, respectively, to detect the protein-specific antibodies and neutralizing antibodies in the sera ( Figure 5 in (a)).

[0062] The results were as Figure 5 shown in (b)-(d). Serum samples of immunized mice were collected at weeks 0, 2, 4, 6, 8, 10, 12, 14, and 16 for serological detection. The IgG titers against the gp350D123 protein and the L350 protein in these serum samples were detected by ELISA. After two rounds of booster immunization, the antibody titers against the L350 protein and the gp350D123 protein in all immunized groups reached the peak at week 10.

[0063] In addition, L350-ferritin nanoparticles and L350 protein can induce the production of high-titer antibodies against the gp350D123 protein ( Figure 5 in (b)), indicating that these epitopes in the fusion recombinant L350 protein can be fully exposed in vivo. The shown gp350D123 protein can also induce the production of high-titer antibodies against the L350 protein ( Figure 5 in (c)), indicating that these epitopes in the gp350 protein can indeed be recognized in vivo.

[0064] Figure 5 The antibody titers against the gp350D123 protein and the L350 protein at the 10th week in (d) and (e) show that the antigen-specific antibodies induced by L350-ferritin nanoparticles are higher than those induced by the L350 protein. These data indicate that the L350-ferritin nanoparticle vaccine can successfully induce strong antibody recognition of the EBV-encoded gp350 protein.

[0065] The neutralization ability of the immune mouse serum samples was evaluated, and the results are as Figure 6 shown: (a) shows that these cells showed strong GFP fluorescence 48 hours after infection, confirming the ability of the EBV-GFP virus to infect AKATA cells; through flow cytometry analysis of GFP-positive cells, the results are as Figure 6 in (b) to determine that the infection rate of the EBV-GFP reporter virus solution is approximately 17.5%; subsequently, the serum samples of the immune mice at the 10th week were serially diluted to detect their blocking efficiency against the infection of AKATA cells by the EBV-GFP virus, and the results are as Figure 6 shown in (c). Compared with other immune mice, the serum samples of the mice vaccinated with the L350-ferritin nanoparticle vaccine showed stronger neutralization efficiency, while the neutralization efficiency of the serum samples of the mice vaccinated with the L350 protein vaccine was lower than that of the mice vaccinated with the gp350D123 protein vaccine. At the 10th week, the half-maximal inhibitory dose (ID50) values of the serum samples of the mice immunized with the L350 protein, gp350D123 protein, and L350-ferritin protein nanoparticles vaccine were 4.53, 10.03, and 16.98, respectively; calculating the ID50 values of these serum samples at the 8th, 10th, and 12th weeks, the results are as Figure 6 shown in (d), and the results show that the L350-ferritin protein nanoparticle vaccine was significantly stronger than other groups at the 10th and 12th weeks. These data indicate that the L350-ferritin nanoparticle vaccine can induce the production of high-titer neutralizing antibodies in immune mice.

[0066] Example 3. L350-Ferritin Nanoparticle Vaccine Stimulates Potent B Lymphocyte Immunological Memory in Mice The experimental mice were SPF - level Balb / c mice. Thirty mice were divided into the following 5 groups: PBS group, Ferritin group, gp350D123 group, L350 group, and L350 - Ferritin group, with 6 mice in each group. Each mouse was intramuscularly injected with an equal volume of PBS buffer (concentration 0.01M, pH 7.4), Ferritin (injection dose 15 μg), gp350D123 (injection dose 15 μg), L350 (injection dose 15 μg), and L350 - Ferritin (injection dose 15 μg). The PBS group used the PBS solution as the control group. Ferritin was obtained by expression and purification of the pET - 28a(+) plasmid in Example 2 and dissolved in the PBS buffer. The gp350D123 protein was obtained by expression and purification of the pET - 28a(+) plasmid and dissolved in the PBS buffer. The L350 protein was obtained by expression and purification of the pET - 28a(+) plasmid and dissolved in the PBS buffer. Taking the first immunization as week 0, an equal - amount booster immunization was given at weeks 2 and 4 after the first immunization.

[0067] At week 16, the spleens of the mice were collected for research. The gp350D123 protein and L350 protein were labeled with the APC fluorescent dye (Wuhan Elabscience Biotechnology Co., Ltd., product number E - LK - E006A) and co - incubated with splenocytes. Memory B cells (MBCs) in splenocytes were classified and labeled by anti - B220 and anti - CD38 antibodies to analyze the immune memory of B lymphocytes after vaccination.

[0068] The results are as Figure 7 shown in (a) - (d) below. Compared with the L350 protein monomer, the L350 - ferritin nanoparticle vaccine induced more gp350D123 - protein - specific memory B cells (gp350D123+ / B220+ / CD38+) and L350 - protein - specific memory B cells (APC+ / B220+ / CD38+). However, in the mice inoculated with the gp350D123 protein, the number of gp350D123 - protein - specific memory B cells (gp350D123+ / B220+ / CD38+) was higher than that in other groups, which may be related to multiple epitopes on the surface of the gp350 protein. In addition, the memory B cells induced by the L350 - ferritin nanoparticle vaccine could effectively recognize the gp350D123 protein and the L350 protein, which was consistent with the result that the L350 - ferritin nanoparticle vaccine induced high - titer gp350D123 - protein - specific and L350 - protein - specific antibodies. In summary, the L350 - ferritin nanoparticle vaccine can provide a long - lasting protective humoral immune response.

[0069] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any variations, uses, or improvements of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Immunogenic complex, characterized in that: The immunogen complex is assembled from a polypeptide related to the gp350 glycoprotein of Epstein-Barr virus and the carrier protein Ferritin.

2. The immunogenic complex according to claim 1, wherein: The polypeptides are linked by a flexible linker, and a SPY tag sequence and a 6×His sequence are linked to the C-terminus of the polypeptides; a Catcher sequence and a 6×His sequence are linked to the N-terminus of the carrier protein Ferritin.

3. The immunogenic complex according to claim 1, characterized in that: The molar ratio of assembly of the polypeptides and the carrier protein Ferritin is 5:

1.

4. The immunogen complex according to claims 1-3, wherein: The polypeptide is any one of the following: a1) The amino acid sequence is SEQ ID No:1; a2) A polypeptide having the same function after substitution and / or deletion and / or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID No:1; a3) A polypeptide having more than 75% identity with the amino acid sequence defined in any one of a1) or (a2) and having the same function; a4) A polypeptide obtained by linking a tag to the end of the protein defined in any one of a1)-(a3). The carrier protein Ferritin is any one of the following: b1) A protein with the amino acid sequence of SEQ ID No:2; b2) A protein having the same function after substitution and / or deletion and / or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID No:2; b3) A protein having more than 75% identity with the amino acid sequence defined in any one of b1) or (b2) and having the same function; b4) A protein obtained by linking a tag to the end of the protein defined in any one of b1)-(b3).

5. The polypeptide in the immunogen complex according to claims 1-3.

6. An Epstein-Barr virus vaccine, characterized in that: The active ingredient of the vaccine comprises the immunogen complex according to any one of claims 1-4.

7. Use of the immunogen complex according to any one of claims 1-4 in the preparation of an Epstein-Barr virus inhibitor.

8. Use of the immunogen complex according to any one of claims 1-4 in the preparation of a drug for treating or preventing diseases caused by Epstein-Barr virus infection.

9. Use of the immunogen complex according to any one of claims 1-4 in the preparation of an Epstein-Barr virus vaccine.

10. Use of the vaccine according to claim 6 in the preparation of a drug for treating or preventing diseases caused by Epstein-Barr virus infection.

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