EBV vaccine as well as preparation method and application thereof

Abstract

The invention relates to three fusion proteins. The fusion proteins comprise an EBV gp350 polypeptide variant, a gh / gl / gp42 compound and a gB polypeptide variant. According to the present invention, the main target EBV gp350, gh / gl / gp42 and gB polypeptide of the neutralizing antibody is optimized, and the main target EBV gp350, gh / gl / gp42 and gB polypeptide can further comprise the dioxotetrahydropteridine synthase so as to form the nanoparticles for preparing the vaccine, and the vaccine can stimulate the body to generate the neutralizing antibody against the EBV, can prevent the in-vivo infection, and has good neutralizing effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an EBV vaccine, a preparation method and an application thereof. Background Art

[0002] Epstein-Barr virus (EBV) is a member of the genus Lymphotropic virus of the family Herpesviridae, and its genome is DNA. EBV is a disease that is widely present in the population. Most infected people have no obvious symptoms, and a small number of infected adults may present with infectious mononucleosis. The virus is also associated with diseases such as nasopharyngeal carcinoma and lymphoproliferative diseases, and is mainly transmitted through oral contact.

[0003] Epstein-Barr virus (EBV) is the leading cause of infectious mononucleosis and has been associated with a variety of human cancers and, more recently, multiple sclerosis. Despite its impact on human health, there is currently no vaccine or treatment. Four viral glycoproteins (gp), gp350 and the gH / gL / gp42 complex, mediate entry into the major sites of viral replication, B cells and epithelial cells. gH (glycoprotein H) and gL (glycoprotein L) are core proteins in the EBV infection process, which form heterodimers and play a key role in viral infection. gp42 (glycoprotein 42) is another important glycoprotein that interacts with the gH / gL complex and together mediates viral infection of B cells. During EBV infection of B cells, the gH / gL / gp42 complex triggers the fusion of the viral envelope with the cellular membrane by interacting with the cell receptor CD21 (also known as CR2) and cellular MHC class II molecules, thereby allowing EBV entry into B cells. gB is a key element in the EBV viral infection process. It has the highest sequence and structural conservation in the herpesviridae family. gB is initially anchored to the viral envelope in a pre-fusion conformation, and after receiving activation signals transmitted upstream, it undergoes conformational changes and refolds into a post-fusion conformation. This conformation can insert its own hydrophobic residues into the host cell membrane, promote the formation of a fusion pore, and thus drive the fusion of the viral envelope and the host cell membrane.

[0004] Given that there is currently no effective treatment for EBV infection or vaccine to prevent it, and previous vaccine development attempts have focused on a viral envelope glycoprotein, gp350, a vaccine with a higher neutralization efficiency is still needed to improve neutralization efficiency and effectively treat EBV infection. Summary of the invention

[0005] In order to overcome the above defects, the present invention provides a technical solution comprising a fusion protein including an EBV gp350 polypeptide variant, a gh / gl / gp42 complex or a gB polypeptide variant, a preparation method and its application in the preparation of an EBV nanoparticle vaccine. Specifically,

[0006] In a first aspect, the present invention provides a fusion protein, the fusion protein comprising:

[0007] (1), EBV gp350 polypeptide variant, gh / gl / gp42 complex or gB polypeptide variant, and,

[0008] (2) Lumazine synthase (LS).

[0009] In one embodiment, the protein sequence of the polypeptide variant includes a modification, wherein the modification includes an extension of the amino acid sequence.

[0010] Preferably, the extension of the amino acid sequence comprises adding 1-20 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids.

[0011] Preferably, the modification is located at the N-terminus or C-terminus of the polypeptide.

[0012] Preferably, the modification comprises adding positions 451-464 of SEQ ID No: 1, positions 431-435 of SEQ ID No: 5 and / or positions 675-688 of SEQ ID No: 5.

[0013] Preferably, the protein sequence of the EBV gp350 polypeptide includes the amino acid sequence shown at positions 26-450 of SEQ ID No:1.

[0014] Preferably, the protein sequence of the EBV gp350 polypeptide variant comprises:

[0015] (A1), shown at positions 26-464 of SEQ ID No: 1,

[0016] (A2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (A1);

[0017] (A3) A protein having 80% or more identity with any one of (A1) to (A2) and having the same function.

[0018] Preferably, the gh / gl / gp42 complex comprises gh, gl and gp42 polypeptides. Further preferably, the gl polypeptide comprises the amino acid sequence shown at positions 26-140 of SEQ ID No: 3, the gh polypeptide comprises the amino acid sequence shown at positions 187-792 of SEQ ID No: 3, and the gp42 polypeptide comprises the amino acid sequence shown at positions 827-1023 of SEQ ID No: 3.

[0019] Preferably, the gh, gl and gp42 are directly connected and / or indirectly connected, and more preferably, the indirect connection includes connection via a connecting peptide.

[0020] In a specific embodiment, the gh / gl / gp42 complex comprises gl-linker peptide-gh-linker peptide-gp42.

[0021] Preferably, the protein sequence of the gh / gl / gp42 complex includes:

[0022] (B1), shown at positions 26-1023 of SEQ ID No: 3,

[0023] (B2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (B1);

[0024] (B3) A protein having 80% or more identity with any one of (B1) to (B2) and having the same function.

[0025] Preferably, the protein sequence of the gB polypeptide variant comprises:

[0026] (C1), shown at positions 26-688 of SEQ ID No: 5,

[0027] (C2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of (C1);

[0028] (C3) A protein having 80% or more identity with any one of (C1) to (C2) and having the same function.

[0029] Preferably, the protein sequence of the dioxotetrahydropterin synthase comprises:

[0030] (D1), 473-624 of SEQ ID No: 1, 1109-1260 of SEQ ID No: 3, or 736-887 of SEQ ID No: 5,

[0031] (D2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (D1);

[0032] (D3) A protein having 80% or more identity with any one of (D1) to (D2) and having the same function.

[0033] Preferably, the fusion protein further comprises (3), a trimer-forming tag. Further preferably, the tag comprises adiponectin or its collagen-like domain, a T4 fibrin trimer domain or a trimer structure of human collagen. More preferably, the fusion protein comprises a T4 fibrin trimer domain, and the T4 fibrin trimer domain comprises the amino acid sequence shown in SEQ ID No: 7.

[0034] Preferably, the polypeptide variant or complex is connected in tandem with a dioxotetrahydropterin synthase or trimer-forming tag.

[0035] Preferably, the tandem connection may be directly connected and / or indirectly connected, more preferably, the indirect connection comprises connection via a connecting peptide.

[0036] Preferably, the sequence of the connecting peptide comprises:

[0037] (E1), SEQ ID No: 8 (GSGGGGSG), SEQ ID No: 9 (GGGGSGGGGSGGGGS), SEQ ID No: 10 (GGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGSGGSG), SEQ ID No: 11

[0038] (GSGGGSGGGGSGGGGSAGSGGGSGGGGSGGGGSA) or SEQ ID No: 12

[0039] (GSGGGSGGGGGSGGGGSAGSGGGSGGGGSGGGGSAGSGGGSGGGGSGGGGSAGSGGGSGGGGSGGGGSAGSG GGSGGGGSGGGGSA),

[0040] (E2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (E1);

[0041] (E3) A polypeptide having 80% or more identity with any one of (E1) to (E2) and having the same function.

[0042] Preferably, the fusion protein further comprises a signal peptide.

[0043] More preferably, the sequence of the signal peptide comprises:

[0044] (F1), SEQ ID No: 1, SEQ ID No: 1, SEQ ID No: 3, or SEQ ID No: 5,

[0045] (F2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (F1);

[0046] (F3) A polypeptide having 80% or more identity with any one of (F1) to (F2) and having the same function.

[0047] Herein, identity refers to amino acid sequence identity. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of amino acid sequences, the value of identity (%) can then be obtained.

[0048] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0049] Preferably, the fusion protein also includes a tag protein.

[0050] The tag protein refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0051] In a specific embodiment, the fusion protein is composed of signal peptide-EBV gp350 polypeptide variant-connecting peptide-LS from N-terminus to C-terminus.

[0052] In a specific embodiment, the fusion protein comprises signal peptide-EBV gh / gl / gp42 complex-connecting peptide-LS from N-terminus to C-terminus.

[0053] In a specific embodiment, the fusion protein is composed of signal peptide-EBV gB polypeptide variant-connecting peptide-trimer-promoting component-connecting peptide-LS from N-terminus to C-terminus.

[0054] Further preferably, the fusion protein comprises the amino acid sequence shown in SEQ ID No: 1, 3 or 5.

[0055] Preferably, the fusion protein includes dioxotetrahydropterin synthase, which self-assembles into nanoparticles after being expressed in cells, more preferably 60-sided nanoparticles.

[0056] In a second aspect, the present invention provides a mixed protein, which comprises a mixture of any two or three fusion proteins of EBV gp350 fusion protein, gh / gl / gp42 fusion protein and gB fusion protein, and the fusion protein is selected from the above fusion proteins.

[0057] Preferably, the mixed protein comprises:

[0058] (1) gp350 fusion protein and gh / gl / gp42 fusion protein;

[0059] (2) gp350 fusion protein and gB fusion protein;

[0060] (3) gh / gl / gp42 fusion protein and gB fusion protein, or,

[0061] (4) gp350 fusion protein, gh / gl / gp42 fusion protein and gB fusion protein.

[0062] The third aspect of the present invention provides a biomaterial, characterized in that the biomaterial comprises:

[0063] (I) a nucleotide molecule encoding any one of the above-mentioned fusion proteins or the above-mentioned mixed proteins;

[0064] (II) a vector, the vector comprising any nucleotide molecule described in (I);

[0065] (III) A host cell, comprising the nucleotide molecule described in any one of (I) or the vector described in (II).

[0066] Preferably, the nucleic acid molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0067] More preferably, the nucleotide molecule comprises:

[0068] (G1), SEQ ID No: 2, 4 or 6,

[0069] (G2), complementary, degenerate or transcribed sequence of (G1),

[0070] (G3) A DNA molecule or mRNA that has 75% or more identity with the DNA molecule or mRNA defined in (G1) or (G2) and encodes the corresponding protein in the fusion protein.

[0071] Those skilled in the art can easily mutate the nucleotide sequence encoding the above fusion protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the above fusion protein are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above fusion protein and have the same function.

[0072] The identity refers to the sequence similarity with the compared nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence shown in SEQ ID No: 2, 4 or 6 of the present invention. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0073] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0074] Preferably, the vector further includes a regulatory factor, such as a promoter for initiating transcription of the above polypeptide or fusion protein encoding gene sequence, and may also include a terminator for terminating transcription of the above polypeptide or fusion protein encoding gene sequence. Further, the vector may also include an enhancer sequence.

[0075] The vector described herein refers to a vector that can carry exogenous DNA, mRNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.). In one or more embodiments of the present invention, the vector is a pKS001 vector.

[0076] The host cell (also referred to as recipient cell) described herein can be a plant cell or an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like, but are not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), African green monkey kidney cell (Vero cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cell), fish cell (e.g., grass carp, carp, rainbow trout or catfish cell), insect cell (e.g., Sf21 cell or Sf-9 cell), etc., but are not limited thereto.

[0077] Preferably, the host cell may also be a microorganism, and the microorganism described herein may be a bacterium, a fungus, an actinomycete, a protozoa, an algae or a virus. Wherein, the bacterium may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto, for example, the bacterium may be Escherichia coli, Bacillus subtilis or Bacillus pumilus.

[0078] In a fourth aspect, the present invention provides a method for preparing the above-mentioned fusion protein, the preparation method comprising introducing the above-mentioned nucleotide molecule or vector into a host cell and culturing the host cell.

[0079] Preferably, the preparation method comprises screening a monoclonal cell line with high and stable expression level.

[0080] More preferably, the screening method includes conventional screening methods in the art, such as adding screening reagents, comparing the biological activity of clone pools by ELISA, and the like.

[0081] In a fifth aspect, the present invention provides an application of any of the above fusion proteins, mixed proteins or biomaterials, wherein the application includes any of the following:

[0082] (1) Use in the preparation of products for preventing and / or treating diseases caused by EBV virus infection;

[0083] (2) Application in the preparation of products for inducing immune response to EBV virus antigens;

[0084] (3) Use in the prevention and / or treatment of diseases caused by EBV infection;

[0085] (4) Application in inducing immune response to EBV virus antigens.

[0086] The product described herein may be a reagent, a drug, or a vaccine.

[0087] Preferably, the preparation further comprises screening, for example, using the above mutant protein or fusion protein as a target to screen candidate preventive or therapeutic drugs or diagnostic agents.

[0088] The products described in (1) and (2) may be vaccines or antibodies against EBV virus, wherein the antibodies include full-length antibodies or antigen-binding fragments (such as Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific antibodies or minimal recognition units (MRU), etc., but not limited thereto).

[0089] Furthermore, the EBV antibody may be a neutralizing antibody that specifically binds to EBV. The neutralizing antibody may be a high-titer neutralizing antibody against multiple epidemic strains of EBV.

[0090] In the above application, the diseases caused by EBV virus infection include but are not limited to infectious mononucleosis (IM), oral leukoplakia, X chromosome-related lymphoproliferative syndrome (XLP), virus-related erythrophagocytosis, Burkitts lymphoma, Hodgkin's disease (HD), nasopharyngeal carcinoma (NPC), NK / T cell lymphoma, leiomyosarcoma, large cell anaplastic lymphoma, malignant histiocytosis, rheumatoid arthritis, Kawasaki disease, glomerulonephritis, nephrotic syndrome, viral myocarditis, pericarditis, acute idiopathic thrombocytopenic purpura, multiple sclerosis, viral encephalitis, Guillain-Barré syndrome, aplastic anemia, respiratory system infection, digestive system infection, cardiovascular system infection, and / or nervous system infection.

[0091] Preferably, the respiratory system infection may include respiratory tract infection and / or lung infection.

[0092] Preferably, the digestive system infection may include intestinal diseases, anorexia, nausea, vomiting, abdominal pain and / or diarrhea.

[0093] More preferably, the respiratory tract infection may include severe acute respiratory syndrome, hypoxemic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngitis, tracheitis and / or bronchitis.

[0094] More preferably, the lung infection may include pneumonia and / or lung injury.

[0095] Preferably, the product for preventing and / or treating diseases caused by EBV virus infection comprises a vaccine.

[0096] In a sixth aspect, the present invention provides a drug, which comprises any one of the above-mentioned fusion proteins, mixed proteins or biomaterials.

[0097] Preferably, the drug is a vaccine, and more preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

[0098] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response to the antigen inoculated with it. The adjuvant described herein may be known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factor IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0099] It is well known to those skilled in the art that in order to enhance the immunogenicity of antigenic proteins, in addition to adding compounds with immunopotentiating effects as adjuvants, the gene combination can be adjusted to allow them to be expressed in a granular structure; or they can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0100] The vaccine delivery system can be a type of substance that can carry antigenic substances to the body's immune system, store and exert its antigenic effect therein for a long time. The vaccine delivery system described herein can be an aluminum salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system or a nano adjuvant vaccine delivery system.

[0101] Furthermore, the medicine or vaccine also includes one or more pharmaceutically acceptable carriers.

[0102] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0103] The vaccine for preventing infection of the present invention can be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection.

[0104] In an eighth aspect, the present invention also provides a method for producing an immune response, which may include administering any of the above vaccines to a subject.

[0105] In the above method, after the vaccine is administered to a subject, an immune response against EBV virus can be induced in the subject. The immune response can be a cellular immune response, a humoral immune response, or a cellular immune response and a humoral immune response.

[0106] The cellular immune response may include a B cell immune response and a T cell immune response.

[0107] The subject described herein can be a human or a non-human animal.

[0108] Furthermore, the non-human animal may be a non-human mammal.

[0109] The non-human mammal may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but is not limited thereto.

[0110] The subjects described herein include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).

[0111] The administration described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral and nasal spraying, or aerosol inhalation.

[0112] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0113] The fusion protein of the present invention optimizes EBV gp350 and gB polypeptide sequences on the basis of the prior art, constructs gh / gl / gp42 complex, and increases the selection of fusion expression of EBV gp350 polypeptide variant, gh / gl / gp42 complex, gB polypeptide variant and LS, can be assembled into 60-sided nanoparticles, improves the stability of antigen, and increases the effect of inducing the body to produce antibodies. The selection of signal peptide is optimized at the same time. The fusion protein of the present application has better neutralization effect than the prior art, and the vaccine of the present invention can be used for the effective prevention or treatment of EBV virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 : SDS-PAGE result diagram of fusion protein purification, wherein lane 2 is a marker, lane 3 is the first step ECA purification of gh / gl / gp42-LS, lane 4 is the reduced gh / gl / gp42-LS molecular sieve purification, lane 5 is the non-reduced gh / gl / gp42-LS molecular sieve purification, lane 6 is the reduced gp350, lane 7 is the reduced gB, and lane 8 is a marker.

[0115] Figure 2 : GH / GL / Gp42 antibody ELISA titer after mixed immunization of mice.

[0116] Figure 3 : gp350 antibody ELISA titer after mixed immunization of mice.

[0117] Figure 4 : ELISA titer of gB antibody after mixed immunization of mice.

[0118] Figure 5 : Neutralization experiment of CNE2-EBV virus by serum induced by vaccine. DETAILED DESCRIPTION

[0119] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0120] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials and reagents used in the examples are listed below, and the others can be obtained from commercial sources unless otherwise specified.

[0121] Example 1 Preparation of fusion protein

[0122] The present invention uses gp350, gh / gl / gp42 complex or gB protein and lumazine synthase (LS) to express and assemble into 60-sided nanoparticles. After adding the signal peptide sequence to this amino acid sequence, Nanjing GenScript Biotechnology Co., Ltd. is entrusted to optimize the synthesis. Specifically,

[0123] 1. Sequence Description

[0124] 1. gp350 fusion protein:

[0125] MLRGPGPGLLLAVLCLGTAVRCTEAEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENA TNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTE MLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAPE STTTSPTSNTTGFA GSGGGGSGQIYEGKLTAEEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGSGGITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR (SEQ ID No: 1)

[0126] Among them, positions 1-25 are signal peptides, positions 26-464 are modified gp350, positions 451-464 are newly added amino acids, positions 465-472 are connecting peptides, and positions 473-624 are dioxotetrahydropterin synthases.

[0127] 2. Nucleotide sequence encoding the amino acids of the above fusion protein:

[0128]

[0129] 3. Amino acid sequence of gh / gl / gp42 complex fusion protein

[0130]

[0131] Among them, positions 1-25 are signal peptides, positions 26-140 are gl, positions 141-186 are connecting peptides, positions 187-792 are gH, positions 793-826 are connecting peptides, positions 827-1023 are gp42, positions 1024-1108 are connecting peptides, and positions 1109-1260 are dioxotetrahydropterin synthases.

[0132] 4. Nucleotide sequence of gh / gl / gp42 complex fusion protein

[0133]

[0134] 5. Amino acid sequence of the gB fusion protein

[0135] MLRGPGPGLLLAVLCLGTAVRCTEAQTPEQPAPPATTVQPTATRQQTSFPFRVCELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKIVTNILIYNGHRADSVTNRHEEKFSVDSYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNKETFHERADSFHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQHWQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKTMHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSPPAPSAARGSTPAAVLGGSGGDAGNATTPVPPTAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETMCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIAGLRKDLDNAVSNGSGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFL GGGGSGGGGSGGGGS QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGSGGITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR(SEQ ID No: 5)

[0136] Among them, positions 1-25 are signal peptides, positions 26-688 are gB polypeptide variants (wherein positions 431-435 are newly added amino acids, and positions 675-688 are newly added amino acids), positions 689-694 are connecting peptides, positions 695-720 are T4 fibrin trimer domains, positions 721-735 are connecting peptides, and positions 736-887 are dioxotetrahydropterin synthases.

[0137] 6. Nucleotide sequence of gB fusion protein

[0138]

[0139] 2. Construction Method

[0140] 1. Expression of fusion protein:

[0141] The nucleotide sequences of the target gene gp350 and gh / gl / gp42 complex or gB fusion protein were synthesized into the expression vector pks001 (synthesized by Nanjing GenScript), and the plasmid was extracted in large quantities and transiently transfected into 293 cells, and the cell supernatant was purified. The expression materials used are shown in Table 1:

[0142] Table 1: Protein expression materials

[0143] Material Suppliers Part Number 293 cells —— —— 293 medium QuaCell A21501 OptiMEM gibco 31985-070 293 transfection reagent Polymer MF388-02

[0144] 1.1 Add fresh solution one day before transfection, the cell concentration is about 1.2×10 6 cell / mL.

[0145] 1.2 Count the cells on the second day and use trypan blue to check the cell viability. The cell viability should be >90%. Dilute the cells to 1.2×10 6 cell / mL.

[0146] 1.3 Prepare the complex. Prepare the 293 transfection reagent-DNA complex:

[0147] a. Dilute 300 μg of plasmid DNA with serum-free medium (such as OPTI-MEM I) to a final volume of 5 mL;

[0148] b. Dilute 700 μL of "suspension-sensitive" transfection reagent with serum-free culture medium (such as OPTI-MEM I) to a final volume of 5 mL; mix gently and incubate at room temperature for 5 minutes.

[0149] c. After incubation for 5 minutes, add the diluted plasmid DNA to the diluted "suspension-sensitive" transfection reagent to make the total volume 2 mL and mix gently.

[0150] d. Incubate at room temperature for 20-30 minutes to allow DNA-"suspension sensitive" transfection reagent complex to form. The solution may become turbid at this time, but it will not affect transfection.

[0151] 1.4 After incubation is complete, add 10 mL of DNA-"suspension sensitive" transfection reagent complex to 28 mL of growth medium containing 293 suspension cells, so that the final cell concentration is about 1.2×10 6 For negative control, replace with 10 mL serum-free medium (such as OPTI-MEM I).

[0152] 1.5 Incubate at 37°C, 5% CO2 in a shaking incubator at 125 rpm until transgene expression analysis is performed. No need to remove the complex or replace the medium. After 7 days of culture, harvest the culture fluid for protein purification.

[0153] 2. Indirect ELISA method was used to detect the protein activity of the target protein.

[0154] The materials used in the test are shown in Table 2:

[0155] Table 2: Materials for indirect ELISA

[0156] Material supplier Part Number Blocking solution (5% skim milk powder) Erie 647FG1905 96-well enzyme plate Costar 42592 10x coating solution solarbio C1055 Lotion TBST Bring Your Own Substrate colorimetric solution (TMB) solarbio PR1200 Stop Solution solarbio C1058 Secondary antibody (goat anti-mouse IgG) GenScript A00612

[0157] 2.1 Reagent preparation

[0158] 2.1.1 Preparation of ELISA coating solution (1X) Take ELISA coating solution (10X) and dilute it to 1X with sterile distilled water.

[0159] 2.1.2 PBS preparation: Take out PBS powder and dissolve each bag with 2L of sterile distilled water.

[0160] 2.1.3 Washing solution: PBST (PBS containing 0.05% Tween-20) Measure 1L of filtered PBS into a blue-capped bottle, add 500μTween-20, mix thoroughly, and store at 2-8℃ for later use. According to the actual situation, prepare the required volume of PBST according to this method.

[0161] Note: PBST should be prepared fresh and used on the same day.

[0162] 2.1.4 Blocking solution and sample diluent (PBS containing 5% skim milk powder. Weigh 5% skim milk powder of the volume of PBS (milk powder mass / g = PBS volume / mL x 5%), equilibrate the PBS taken out from 2-8°C to room temperature, measure the required volume of PBS solution into the centrifuge tube that has been added with milk powder, and then fully dissolve it for use. Note: The blocking solution and sample diluent should be prepared on the spot and used on the same day.

[0163] 2.1.5 Dilute the goat anti-mouse secondary antibody. Equilibrate 1 mL of the secondary antibody to room temperature, then divide it into aliquots and store it at -20℃±5℃. Take it out before each use and dilute it at 1:4000. The diluent here is PBS. Note: The diluted secondary antibody should be used on the same day.

[0164] 2.2 Coating: Dilute the cell supernatant 10 times, add 90 μl coating solution to 10 μl, coat the ELISA plate, 100 μl / well, set up positive and negative controls, and place at 4°C overnight.

[0165] 2.3 Blocking: After taking out the coated plate from 2-8℃, wash the plate three times, with the volume of each wash solution being 300μl / well. If there is any wash solution remaining in the wells after washing, pat it dry on absorbent paper; then add the pre-prepared blocking solution to the coated wells, 300μl / well, cover with sealing film, and incubate at 37℃ for 60-90min.

[0166] 2.4 Washing: Wash the blocked coated plate three times, with the washing solution volume of 300μl / well each time. If there is any washing solution left in the well after washing, pat it dry on absorbent paper.

[0167] 2.5 Primary antibody dilution: Dilute the primary antibody to an appropriate concentration with PBS in a centrifuge tube. Add the diluted primary antibody to the sample wells, 100 μl / well; cover with a sealing film and incubate at 37°C for 60 minutes.

[0168] 2.6 Add secondary antibody: discard the sample, wash the plate 3 times, each time with a washing volume of 300μl / well. If there is any washing solution left in the well after washing, pat it dry on absorbent paper; add diluted secondary antibody, 100μl / well, cover with sealing film, and incubate at 37℃ for 60min.

[0169] 2.7 Color development: Wash the 96-well plate three times, with a washing volume of 300 μl / well each time. If there is any washing solution left in the wells after washing, pat it dry on absorbent paper, add single-component TMB color developing solution (taken out from 2-8°C in advance and equilibrated to room temperature), 100 μl / well, and develop color at 25°C in the dark for 15 min.

[0170] 2.8 Termination: Add stop solution immediately after color development to terminate the reaction, 50 μl / well, and shake gently to mix.

[0171] 2.9 Detection: Place the ELISA plate into the ELISA reader and measure the absorbance at wavelengths of 450nm and 650nm.

[0172] 3. Antigen Purification

[0173] The materials used for purification are shown in Table 3.

[0174] Table 3: Antigen purification materials

[0175]

[0176]

[0177] Nanoparticles using ECA fillers:

[0178] 3.1 Centrifugation: Centrifuge the cell suspension at 8000 rpm for 20 minutes and collect the supernatant.

[0179] 3.2 PBS pre-washing: After washing the lectin resin (ECA) with PBS, add it to the cell supernatant (add 1 ml to 500 ml supernatant), rotate at room temperature for 2 hours or at 4°C overnight.

[0180] 3.3 Pour the mixed solution onto the column.

[0181] 3.4 Wash with 10 ml PBS.

[0182] 3.5 Add 10 ml of elution buffer D-lactose (0.2 M) and rotate at room temperature for 10-15 minutes.

[0183] 3.6 Collect the eluate and store on ice.

[0184] 3.7 Repeat elution twice more (one elution gives >90% yield, 5 ml can be added for the second elution).

[0185] 3.8 Combine all eluates.

[0186] The results are as follows Figure 1 As shown, SDS-PAGE was used for verification, and the target band size was gh / gl / gp42, the theoretical value size was 137KD, the theoretical value size of gB was 95KD, and the theoretical value size of gp350 was 66KD. Since the proteins are all glycoproteins, the protein bands are larger than the theoretical values.

[0187] Example 2 Protective effect of vaccine containing the above fusion protein on mice

[0188] 1. Mouse immunization

[0189] The specific method is: select 6-8 week old Balb / c mice and randomly divide them into groups, 5 mice in each group, inject various vaccine compositions intramuscularly, immunize at 0 and 3 weeks, and collect blood at 3 and 5 weeks. ELISA method is used to detect the titer of gB, gh / gl / gp42, and gp350 antibodies (i.e., total IgG) in serum. The results of the second immunization are as follows Figure 2-4 As shown in Table 4, Table 5 and Table 6.

[0190] Table 4 Anti-gB antibody titers produced after mice were immunized with vaccines containing gB or its mixed fusion protein

[0191] Mouse B gh / gl / gp42+gB gp350+gB gh / gl / gp42+gp350+gB Rat 1 17510 21913 28161 27163 Rat 2 21919 29429 22986 25715 Rat 3 27803 24609 23029 38167 Rat 4 21896 35105 30832 37965 Rat 5 26567 33562 24626 38543

[0192] As can be seen from the above results, the vaccine group comprising a single gB fusion protein can effectively induce the body to produce antibodies. And after the gB fusion protein is combined with other fusion proteins as different vaccine groups for immunization, the antibody for gB antigen can also be significantly improved, indicating that there is no inhibitory effect between each fusion protein. And, relative to the vaccine group of independent gB fusion protein, the vaccine group of various fusion protein combinations, its gB antibody titer also has a significant increase, indicating that there is synergy between different antigens inducing the effect of gB antibody.

[0193] Table 5 Anti-gh / gl / gp42 antibody titers produced after mice were immunized with vaccines containing gh / gl / gp42 or its mixed fusion protein

[0194] Mouse gh / gl / gp42 gh / gl / gp42+gp350 gh / gl / gp42+gB gh / gl / gp42+gp350+gB Rat 1 15605 18873 21593 18579 Rat 2 22250 12624 23107 15798 Rat 3 11383 14345 14114 16533 Rat 4 20875 20777 10309 34788 Rat 5 24213 23152 25462 26842

[0195] The above results show that the vaccine group including a single gh / gl / gp42 fusion protein can effectively induce the body to produce antibodies. After immunization with the gh / gl / gp42 fusion protein and other fusion proteins as different vaccine groups, the antibodies against the gh / gl / gp42 antigen can also be significantly improved, indicating that there is no inhibitory effect between the fusion proteins. Moreover, compared with the vaccine group with a single gh / gl / gp42 fusion protein, the vaccine groups with various fusion protein combinations also have a significant increase in the titer of gh / gl / gp42 antibodies, indicating that different antigens have a synergistic effect in inducing gh / gl / gp42 antibodies.

[0196] Table 6 Anti-gp350 antibody titers produced after mice were immunized with vaccines containing gp350 or its mixed fusion protein

[0197]

[0198]

[0199] The above results show that the vaccine group including a single gp350 fusion protein can effectively induce the body to produce antibodies. After immunization with the gp350 fusion protein combined with other fusion proteins as different vaccine groups, the antibodies against the gp350 antigen can also be significantly improved, indicating that there is no inhibitory effect between the fusion proteins. Moreover, compared with the vaccine group with a single gp350 fusion protein, the vaccine groups with various fusion protein combinations also have a significant increase in the gp350 antibody titer, indicating that different antigens have a synergistic effect in inducing gp350 antibodies.

[0200] 2. Neutralization experiment

[0201] The materials used in the neutralization experiment are shown in Table 7.

[0202] Table 7 Materials used in the experiments

[0203] Material Suppliers Part Number Raji cells Wuhan Prosai Life Science Co., Ltd. CL-0189 1640 medium gibco 11875-093 Fetal bovine serum gibco 10091-148

[0204] (1) Raji cells were cultured using RPMI1640 + 5% FBS medium.

[0205] (2) CNE2-EBV virus was removed and the appropriate amount of virus was used, starting with a 1:10 dilution of serum in 60 uL of virus in a 96-well plate based on a 40% infection ratio of Raji cells. The serum was then diluted 10-fold in subsequent wells and placed at 37°C for 2 hours to neutralize the virus.

[0206] (3) Raji cells were cultured at 2×10 4 The amount of cells / 40uL was added to the neutralization plate and infected for 3 hours.

[0207] (4) The neutralization plate was then centrifuged at 800 rpm and 28°C for 10 minutes.

[0208] (5) Discard 80uL of supernatant, making sure it does not touch the bottom of the plate, and add 200uL of fresh RPMI1640 + 5% FBS for culture for 24-48 hours.

[0209] (6) Detect the GFP positive ratio of Raji cells using flow cytometry. Perform a four-parameter nonlinear fit on the absorbance value at each concentration of the sample and calculate its IC50 as the serum neutralization titer.

[0210] Finally, as shown in the serum neutralization titer Table 8, the GFP positive ratio of Raji cells was Figure 5 The results showed that the neutralization effect of the three-molecule combination was significantly higher than that of the two-complex combination.

[0211] Table 8 IC50 of neutralization experiment of mixed immunization of mice with vaccines

[0212] gp350 gh / gl / gp42 B gp350+gB gp350+gh / gl / gp42 gB+gh / gl / gp42 gp350+gh / gl / gp42+gB 7014 8192 138.4 9134 8738.5 12739.4 16772 8916 7283 488.98 9203.3 10985 11630 18522 6314 7678 101.6 8493 9339 12111 19714 9793 8451 303.7 9544.6 10811.9 9555.4 15802 9073 8683.2 477.6 8148 9536.5 8916.7 17746

[0213] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0214] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0215] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises: (1) EBV gp350 polypeptide variants, gh / gl / gp42 complex or gB polypeptide variants; (2) Tetrahydropterin synthase.

2. The fusion protein according to claim 1, characterized in that The protein sequence of the polypeptide variant includes modifications, and the modifications include extension of the amino acid sequence. Preferably, the extension of the amino acid sequence includes adding 1-20 amino acids. Further preferably, the modifications are located at the N-terminus or C-terminus of the polypeptide. More preferably, the modifications include adding positions 451-464 of SEQ ID No: 1, positions 431-435 of SEQ ID No: 5 and / or positions 675-688 of SEQ ID No:

5.

3. The fusion protein according to claim 1 or 2, characterized in that The protein sequence of the polypeptide variant or complex includes: (A), 26-464 of SEQ ID No: 1, 26-1023 of SEQ ID No: 3 or 26-688 of SEQ ID No: 5, (B) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of (A); (C) A protein having 80% or more identity with any of (A) to (B) and having the same function.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The fusion protein further comprises (3) a trimerization-promoting tag. Preferably, the tag comprises adiponectin or its collagen-like domain, T4 fibrin trimer domain or the trimer structure of human collagen.

5. A mixed protein, characterized in that The mixed protein comprises a mixture of any two or three of EBV gp350 fusion protein, gh / gl / gp42 fusion protein and gB fusion protein, and the fusion protein is selected from any one of claims 1-4.

6. A biomaterial, characterized in that The biological material comprises: (I) a nucleotide molecule encoding the fusion protein according to any one of claims 1 to 4 or the mixed protein according to claim 5; (II) a vector, the vector comprising any nucleotide molecule described in (I); (III) A host cell, comprising the nucleotide molecule described in any one of (I) or the vector described in (II).

7. The biomaterial according to claim 6, characterized in that The nucleotide molecule includes DNA and / or RNA. Preferably, the nucleotide molecule includes: (G1), SEQ ID No: 2, 4 or 6, (G2), complementary, degenerate or transcribed sequence of (G1), (G3) A DNA molecule or mRNA that has 75% or more identity with the DNA molecule or mRNA defined in (G1) or (G2) and encodes the corresponding protein in the fusion protein.

8. A method for preparing the fusion protein according to any one of claims 1 to 4, characterized in that: The preparation method comprises introducing the nucleotide molecules or vectors in the biomaterial according to claim 7 into host cells and culturing the host cells.

9. A use of the fusion protein according to any one of claims 1 to 4, the mixed protein according to claim 5 or the biomaterial according to claim 7, wherein the use comprises any one of the following: (1) Use in the preparation of products for preventing and / or treating diseases caused by EBV virus infection; (2) Application in the preparation of products for inducing immune response to EBV virus antigens; (3) Use in the prevention and / or treatment of diseases caused by EBV infection; (4) Application in inducing immune response to EBV virus antigens.

10. A drug, characterized in that The drug comprises the fusion protein according to any one of claims 1 to 4, the mixed protein according to claim 5 or the biomaterial according to claim 7. Preferably, the drug is a vaccine. More preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

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