Monoclonal antibody for identifying EB (Epstein-Barr) virus gp350 protein and application thereof

Abstract

The invention belongs to the technical field of antibodies, and discloses a monoclonal antibody for recognizing EB (Epstein-Barr) virus gp350 protein and application of the monoclonal antibody. The invention provides an anti-EB virus gp350 protein monoclonal antibody or an antigen binding fragment thereof, the anti-EB virus gp350 protein monoclonal antibody comprises 1A12 and 2E4, the 1A12 has high affinity for the gp350 protein (KD (M) = 3.415 E-10), the 2E4 has high affinity for the gp350 protein (KD (M) = 4.851 E-9), the two monoclonal antibodies or the antigen binding fragments thereof can significantly inhibit infection of EBV on epithelial cells and B cells, and the anti-EB virus gp350 protein monoclonal antibody or the antigen binding fragment thereof can be used for preparing anti-EB virus gp350 protein anti-EB virus gp350 protein anti-EB virus gp350 protein anti-EB virus gp350 protein anti-EB virus gp350 protein anti-EB virus. The kit can be used for detecting the existence or level of the gp350 protein in a sample, detecting EB virus, diagnosing diseases caused by EB virus infection, preventing EB virus infection and / or treating and / or preventing diseases caused by EB virus infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and in particular relates to a monoclonal antibody for recognizing Epstein-Barr virus gp350 protein and an application thereof. Background Art

[0002] Epstein-Barr virus (EBV), also known as human herpesvirus type 4, is a known DNA oncogenic virus. It was first isolated and established by Epstein and Barr from Burkitt lymphoma cells through in vitro suspension culture in 1964. EBV belongs to the lymphotropic virus of the γ-herpesvirus subfamily. The virus particle consists of four parts: core protein, capsid, capsid and envelope. The core protein carries the entangled DNA.

[0003] EB virus mainly exists in the form of latent infection, and more than 90% of infected people remain in this state for life. The virus can be activated under certain conditions, which in turn causes carcinogenic effects. Current studies have shown that EB virus is closely related to the occurrence of a variety of malignant tumors, including nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and a variety of epithelial cell cancers including gastric cancer. The initial replication of EB virus usually occurs in the oropharynx, and it grows and multiplies in B lymphocytes and oral epithelial cells, then infects B lymphocytes. These infected cells enter the blood circulation, causing systemic infection. When the host's immune function is low, the latent EB virus can be activated, causing recurrent infection.

[0004] Despite the widespread health threats posed by EBV infection, there is currently no effective vaccine, and specific treatments for EBV-related diseases are also lacking. Treatment of infectious mononucleosis usually relies on antiviral drugs such as acyclovir. Although these drugs can relieve symptoms to a certain extent, they cannot completely eliminate EBV in B lymphocytes and throat epithelium. For EBV-related tumors, chemotherapy and radiotherapy remain the main treatment methods, but the efficacy is often unsatisfactory when dealing with metastasis or recurrence.

[0005] gp350 is one of the most important envelope glycoproteins of Epstein-Barr virus and plays a key role in the process of viral infection. As the most abundant protein on the surface of Epstein-Barr virus, gp350 mainly mediates the binding of the virus to host cell surface receptors (such as CD21, also known as CR2), initiating the process of viral infection of B lymphocytes. Due to the key function of gp350 in viral invasion, it has become an ideal target for vaccine development and antibody therapy. The gp350 antibody specifically recognizes and binds to gp350, effectively blocking the binding of Epstein-Barr virus to B lymphocytes, thereby preventing the spread of viral infection. This blocking effect provides a theoretical basis for gp350 antibodies in preventing primary infection and controlling the reactivation of latent viruses.

[0006] Although there are many studies on gp350, no monoclonal antibodies have been widely used in clinical practice. The main research direction is focused on developing highly effective gp350 antibodies to inhibit EB virus infection and the occurrence of related diseases. Through genetic engineering technology, high-affinity and high-specificity gp350 antibodies can be developed to enhance their antiviral efficacy. These antibodies can not only be used to treat acute infections, but also have the potential to be used as preventive treatments, especially in organ transplant patients or individuals in immunosuppressed states.

[0007] Monoclonal antibodies, due to their controllability, high affinity and high specificity in production, significantly reduce adverse reactions in clinical applications and become a promising means in the treatment of infectious diseases. Through molecular modification, the antiviral efficacy of these antibodies can be further improved. However, there is still no monoclonal antibody listing for the Epstein-Barr virus envelope glycoprotein. Therefore, the development of monoclonal antibodies for Epstein-Barr virus will provide more effective means for the prevention and treatment of diseases associated with Epstein-Barr virus. Summary of the invention

[0008] The first aspect of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0009] The second aspect of the present invention aims to provide a recombinant protein.

[0010] The third aspect of the present invention aims to provide biological materials related to the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention or the recombinant protein of the second aspect.

[0011] The fourth aspect of the present invention aims to provide a conjugate.

[0012] The purpose of the fifth aspect of the present invention is to provide the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fourth aspect in the preparation of a product.

[0013] The sixth aspect of the present invention aims to provide a kit.

[0014] The seventh aspect of the present invention aims to provide a medicine.

[0015] The purpose of the eighth aspect of the present invention is to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0016] In order to achieve the above object, the technical solution adopted by the present invention is:

[0017] The first aspect of the present invention provides an anti-EBV gp350 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain.

[0018] In some embodiments of the present invention, the monoclonal antibody or antigen-binding fragment thereof comprises any one of A) or B).

[0019] A) designated as monoclonal antibody or antigen-binding fragment thereof 1A12;

[0020] B) is designated as the monoclonal antibody or antigen-binding fragment thereof 2E4.

[0021] A) in the monoclonal antibody or antigen-binding fragment thereof 1A12; the heavy chain comprises:

[0022] a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 14;

[0023] The light chain comprises:

[0024] A light chain variable region, comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 28.

[0025] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 30 respectively, and the CDRs are defined according to the Kabat definition scheme.

[0026] Preferably, the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 are shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29 and SEQ ID NO: 30 respectively, the amino acid sequence of the CDR-L2 is: GAS, and the CDR is defined according to the IMGT definition scheme.

[0027] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 30, respectively, and the CDRs are defined according to the Chothia definition scheme.

[0028] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35 respectively, and the CDRs are defined according to the Contact definition scheme.

[0029] Preferably, the amino acid sequence of the heavy chain variable region comprises:

[0030] a1-A) SEQ ID NO: 14; or

[0031] a2-A) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 14 and which has the same function as the protein shown in SEQ ID NO. 14; or

[0032] a3-A) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO: 14 and has an amino acid sequence with the same function as the protein shown in SEQ ID NO. 14.

[0033] The amino acid sequence of the light chain variable region comprises:

[0034] b1-A) SEQ ID NO.28; or

[0035] b2-A) an amino acid sequence in which one or more amino acids of SEQ ID NO.28 are substituted and / or deleted and / or added and which has the same function as the protein shown in SEQ ID NO.28; or

[0036] b3-A) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.28 and has an amino acid sequence with the same function as the protein shown in SEQ ID NO.28.

[0037] Preferably, the heavy chain further comprises a heavy chain constant region; and the light chain further comprises a light chain constant region.

[0038] Preferably, the amino acid sequence of the heavy chain constant region comprises:

[0039] c1-A) an amino acid sequence consisting of amino acids 144 to 473 of SEQ ID NO: 13; or

[0040] c2-A) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in c1-A) and which has the same function as the protein of the amino acid sequence described in c1); or

[0041] c3-A) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in c1-A) and has the same function as the protein of the amino acid sequence described in c1-A).

[0042] Preferably, the amino acid sequence of the light chain constant region comprises:

[0043] d1-A) an amino acid sequence consisting of amino acids 128 to 233 of SEQ ID NO: 27; or

[0044] d2-A) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in d1-A) and which has the same function as the protein of the amino acid sequence described in d1-A); or

[0045] d3-A) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in d1-A) and has the same function as the protein of the amino acid sequence described in d1-A).

[0046] Preferably, the heavy chain further comprises a heavy chain signal peptide; and the light chain further comprises a light chain signal peptide.

[0047] Preferably, the amino acid sequence of the heavy chain signal peptide comprises:

[0048] e1-A) an amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 13; or

[0049] e2-A) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in e1-A) and which has the same function as the protein of the amino acid sequence described in e1); or

[0050] e3-A) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in e1-A) and has the same function as the protein of the amino acid sequence described in e1-A).

[0051] Preferably, the amino acid sequence of the light chain signal peptide comprises:

[0052] f1-A) an amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 27; or

[0053] f2-A) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in f1-A) and which has the same function as the protein of the amino acid sequence described in f1-A); or

[0054] f3-A) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in f1-A) and has the same function as the protein of the amino acid sequence described in f1-A).

[0055] B) In the monoclonal antibody or antigen-binding fragment thereof 2E4, the heavy chain comprises:

[0056] a heavy chain variable region comprising CDR-H4, CDR-H5 and CDR-H6 of the heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 38;

[0057] The light chain comprises:

[0058] A light chain variable region, comprising CDR-L4, CDR-L5 and CDR-L6 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 52.

[0059] Preferably, the amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, CDR-L5 and CDR-L6 are shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:54 respectively, and the CDRs are defined according to the Kabat definition scheme.

[0060] Preferably, the amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4 and CDR-L6 are shown in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 53 and SEQ ID NO: 54 respectively, the amino acid sequence of CDR-L5 is: RNN, and the CDRs are defined according to the IMGT definition scheme.

[0061] Preferably, the amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, CDR-L5, and CDR-L6 are shown in SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 44, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 54, respectively, and the CDRs are defined according to the Chothia definition scheme.

[0062] Preferably, the amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, CDR-L5 and CDR-L6 are shown in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 respectively, and the CDRs are defined according to the Contact definition scheme.

[0063] Preferably, the amino acid sequence of the heavy chain variable region comprises:

[0064] a1-B) SEQ ID NO: 38; or

[0065] a2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 38 and which has the same function as the protein shown in SEQ ID NO: 38; or

[0066] a3-B) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology to SEQ ID NO: 38 and has the same function as the protein shown in SEQ ID NO: 38;

[0067] The amino acid sequence of the light chain variable region comprises:

[0068] b1-B) SEQ ID NO:52; or

[0069] b2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 52 and which has the same function as the protein shown in SEQ ID NO: 52; or

[0070] b3-B) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology to SEQ ID NO: 52 and has the same function as the protein shown in SEQ ID NO: 52.

[0071] Preferably, the heavy chain further comprises a heavy chain constant region; and the light chain further comprises a light chain constant region.

[0072] Preferably, the amino acid sequence of the heavy chain constant region comprises:

[0073] c1-B) an amino acid sequence consisting of amino acids 139 to 468 of SEQ ID NO: 37; or

[0074] c2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in c1) and which has the same function as the protein of the amino acid sequence described in c1-B); or

[0075] c3-B) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in c1-B) and has the same function as the protein of the amino acid sequence described in c1-B).

[0076] Preferably, the amino acid sequence of the light chain constant region comprises:

[0077] d1-B) an amino acid sequence consisting of amino acids 130 to 235 of SEQ ID NO: 51; or

[0078] d2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in d1-B) and which has the same function as the protein of the amino acid sequence described in d1-B); or

[0079] d3-B) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in d1-B) and has the same function as the protein of the amino acid sequence described in d1-B).

[0080] Preferably, the heavy chain further comprises a heavy chain signal peptide; and the light chain further comprises a light chain signal peptide.

[0081] Preferably, the amino acid sequence of the heavy chain signal peptide comprises:

[0082] e1-B) an amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 37; or

[0083] e2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in e1-B) and which has the same function as the protein of the amino acid sequence described in e1-B); or

[0084] e3-B) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in e1-B) and has the same function as the protein of the amino acid sequence described in e1-B).

[0085] Preferably, the amino acid sequence of the light chain signal peptide comprises:

[0086] f1-B) an amino acid sequence consisting of amino acids 1 to 19 of SEQ ID NO: 51; or

[0087] f2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in f1-B) and which has the same function as the protein of the amino acid sequence described in f1-B); or

[0088] f3-B) is an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in f1-B) and has the same function as the protein of the amino acid sequence described in f1-B).

[0089] In some embodiments of the present invention, the monoclonal antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, a bispecific antibody, and a multispecific antibody.

[0090] In some embodiments of the present invention, the amino acid sequence of EBV gp350 comprises:

[0091] g1) an amino acid sequence consisting of amino acids 11 to 817 of SEQ ID NO: 4; or

[0092] g2) an amino acid sequence obtained by replacing and / or deleting and / or adding one or more amino acids to the amino acid sequence described in g1) and having the same function as the protein of the amino acid sequence described in g1); or

[0093] g3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in g1) and has the same function as the protein of the amino acid sequence described in g1).

[0094] The second aspect of the present invention provides a recombinant protein comprising: the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and an optional tag sequence for facilitating expression and / or purification.

[0095] Preferably, the tag sequence is selected from at least one of the following groups: a His tag, a GGGS sequence, a FLAG tag; further a His tag; and further a 6×His tag.

[0096] The third aspect of the present invention provides a biological material related to the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the recombinant protein of the second aspect of the present invention, wherein the biological material comprises at least one of h1) to h16):

[0097] h1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the first aspect, or the recombinant protein of the second aspect of the present invention;

[0098] h2) an expression cassette comprising the nucleic acid molecule described in h1);

[0099] h3) a vector comprising the nucleic acid molecule described in h1);

[0100] h4) a vector comprising the expression cassette described in h2);

[0101] h5) a transgenic cell line comprising the nucleic acid molecule described in h1);

[0102] h6) a transgenic cell line comprising the expression cassette described in h2);

[0103] h7) a transgenic cell line comprising the vector described in h3);

[0104] h8) a transgenic cell line comprising the vector described in h4);

[0105] h9) a microorganism comprising the nucleic acid molecule described in h1);

[0106] h10) A microorganism comprising the expression cassette described in h2);

[0107] h11) a microorganism comprising the vector described in h3);

[0108] h12) a microorganism comprising the vector described in h4);

[0109] h13) A virus comprising the nucleic acid molecule described in h1);

[0110] h14) A virus comprising the expression cassette described in h2);

[0111] h15) A virus comprising the vector described in h3);

[0112] h16) A virus comprising the vector described in h4).

[0113] Preferably, the transgenic cell line comprises no reproductive material.

[0114] Preferably, the nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the present invention and a nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0115] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises:

[0116] A) Monoclonal Antibody or Antigen Binding Fragment thereof 1A12 Related:

[0117] a211-A) the nucleotide sequence shown in SEQ ID NO: 26; or

[0118] a212-A) a nucleotide sequence in which one or more nucleotides of SEQ ID NO: 26 are substituted and / or deleted and / or added, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 26; or

[0119] a213-A) has 80%, 85% or 90% or more homology with SEQ ID NO: 26, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 26.

[0120] B) Monoclonal Antibody or Antigen Binding Fragment thereof 2E4 related:

[0121] a211-B) the nucleotide sequence shown in SEQ ID NO: 50; or

[0122] a212-B) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 50 and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 50; or

[0123] a213-B) is a nucleotide sequence that has 80%, 85% or 90% or more homology with SEQ ID NO: 50 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 50.

[0124] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention comprises:

[0125] A) Monoclonal Antibody or Antigen Binding Fragment thereof 1A12 Related:

[0126] a221-A) the nucleotide sequence shown in SEQ ID NO: 36; or

[0127] a222) a nucleotide sequence in which one or more nucleotides of SEQ ID NO: 36 are substituted and / or deleted and / or added, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 36; or

[0128] a223) has 80%, 85% or 90% or more homology with SEQ ID NO: 36, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 36.

[0129] B) Monoclonal Antibody or Antigen Binding Fragment thereof 2E4 related:

[0130] a221-B) the nucleotide sequence shown in SEQ ID NO: 60; or

[0131] a222-B) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 60 and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 60; or

[0132] a223-B) is a nucleotide sequence that has 80%, 85% or 90% or more homology with SEQ ID NO: 60 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 60.

[0133] The fourth aspect of the present invention provides a conjugate comprising: at least one of the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention and the recombinant protein according to the second aspect of the present invention;

[0134] and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0135] Preferably, the detectable marker is selected from a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0136] Preferably, the conjugate is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0137] The fifth aspect of the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the invention, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fourth aspect in the preparation of a product;

[0138] The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0139] Preferably, the drug has at least one of the functions i1) to i2):

[0140] i1) Prevent EB virus infection;

[0141] i2) Treating and / or preventing diseases caused by EB virus infection.

[0142] Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3):

[0143] j1) detecting the presence or level of gp350 protein in a sample;

[0144] j2) Detection of Epstein-Barr virus;

[0145] j3) Diagnosis of diseases caused by EB virus infection.

[0146] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0147] Preferably, the medicament comprises a vaccine.

[0148] A sixth aspect of the present invention provides a product, comprising at least one of k1) to k3):

[0149] k1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0150] k2) the recombinant protein according to the second aspect of the present invention;

[0151] k3) the conjugate according to the fourth aspect of the invention;

[0152] The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

[0153] Preferably, the product has at least one of the functions j1) to j3):

[0154] j1) detecting the presence or level of gp350 protein in a sample;

[0155] j2) Detection of Epstein-Barr virus;

[0156] j3) Diagnosis of diseases caused by EB virus infection.

[0157] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0158] A seventh aspect of the present invention provides a drug, comprising at least one of 11) to 14):

[0159] l1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0160] 12) The recombinant protein according to the second aspect of the present invention;

[0161] l3) benzene The biomaterial of the third aspect of the invention;

[0162] 14) The conjugate according to the fourth aspect of the present invention.

[0163] Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

[0164] Preferably, the drug has at least one of the functions i1) to i2):

[0165] i1) Prevent EB virus infection;

[0166] i2) Treating and / or preventing diseases caused by EB virus infection.

[0167] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, and infectious mononucleosis.

[0168] Preferably, the medicament comprises a vaccine.

[0169] Preferably, a vaccine comprises at least one of 11) to 14) and an adjuvant:

[0170] l1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0171] 12) The recombinant protein according to the second aspect of the present invention;

[0172] l3) benzene The biomaterial of the third aspect of the invention;

[0173] 14) The conjugate according to the fourth aspect of the present invention.

[0174] The eighth aspect of the present invention is to provide a method for preparing the monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention or the recombinant protein of the second aspect, which is obtained by culturing the transgenic cell line, microorganism or virus of the third aspect of the present invention.

[0175] The beneficial effects of the present invention are:

[0176] The present invention provides a monoclonal antibody or antigen-binding fragment thereof against EBV gp350, including 1A12 and 2E4, wherein 1A12 has a high affinity for gp350 protein (KD(M)=3.415E -10 ), 2E4 has a higher affinity for gp350 protein (KD(M) = 4.851E -9 ), these two monoclonal antibodies or their antigen-binding fragments can significantly inhibit EBV infection of epithelial cells and B cells, and can be used to detect the presence or level of gp350 protein in a sample, detect EB virus, diagnose diseases caused by EB virus infection, prevent EB virus infection and / or treat and / or prevent diseases caused by EB virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] Figure 1 This is a graph showing the affinity test results between monoclonal antibody 1A12 and gp350 protein.

[0178] Figure 2 This is a graph showing the results of monoclonal antibody 1A12 blocking EBV infection of epithelial cells.

[0179] Figure 3This is a graph showing the results of monoclonal antibody 1A12 blocking EBV infection of B cells.

[0180] Figure 4 This is a graph showing the affinity test results between monoclonal antibody 2E4 and gp350 protein.

[0181] Figure 5 The figure shows the result of monoclonal antibody 2E4 blocking EBV infection of epithelial cells.

[0182] Figure 6 This is a graph showing the results of monoclonal antibody 2E4 blocking EBV infection of B cells. DETAILED DESCRIPTION

[0183] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0184] Example 1 Preparation of monoclonal antibodies (mAbs) against Epstein-Barr virus gp350 protein (EBV gp350)

[0185] 1.1 Preparation of EBV gp350 recombinant protein

[0186] The gp350 protein plays an important role in the process of EBV invading epithelial cells and B cells. In the examples of the present invention, the inventors selected gp350 as a bait protein to screen specific antibodies.

[0187]

[0188] The above gp350 original sequence was connected to the mammalian cell expression vector pcDNA3.1+ (Invitrogen) as follows:

[0189] (1) Amplification of gp350 protein gene:

[0190] Use 50 μL of NEB The PCR amplification reaction system (Table 1) was used to amplify the gp350 original sequence, wherein the upstream primer was: 5'-GAGGCAGCCTTGCTTGTGTG-3' (SEQ ID NO: 2); the downstream primer was: 5'-GAGGTTTGAGAATCTGGGCTGG-3' (SEQ ID NO: 3).

[0191] The amplified target fragment was analyzed by agarose gel electrophoresis, the band of the correct molecular weight was cut under ultraviolet light, and the PCR product was recovered according to the instructions of the commercially available kit.

[0192] Table 1 gp350 original sequence PCR amplification reaction system

[0193]

[0194] (2) Enzyme digestion and ligation of target fragment and vector:

[0195] The eukaryotic expression plasmid pcDNA3.1+ was used as the vector. The target fragment and the vector were both digested with NotⅠ and BglII (50 μL of the digestion reaction system (Table 2) was used for digestion).

[0196] Table 2 Enzyme digestion reaction system

[0197]

[0198] The enzyme digestion was performed at 37°C for 2 to 3 hours. The digested vector was run on an agarose gel and then recovered using a gel recovery kit to obtain a linearized vector. The inserted fragment was directly recovered using a DNA purification kit. After recovery, 10 μL of the ligation reaction system (Table 3) was used for ligation. The reaction conditions were 37°C for 30 minutes. The ligation product was obtained.

[0199] Table 3 Ligation reaction system

[0200]

[0201]

[0202] (3) Transformation of ligation products and screening of positive clones:

[0203] Add the ligation product to the thawed DH5α competent cell suspension, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and return to ice for 5 minutes. Add 200 μL LB medium and slowly shake at 30°C for 40 minutes. Pipette the culture solution and spread it on an ampicillin-resistant LB plate, and culture at 37°C overnight.

[0204] Pick a single clone colony for sequencing verification, and when the sequencing result is correct, the target recombinant plasmid is obtained. Extract the plasmid in large quantities.

[0205] (4) Expression and extraction of recombinant proteins:

[0206] Human renal epithelial 293F cells were cultured to obtain a cell density of 1.5×10 6 Cell suspension. Use PEI transfection reagent to transfect the recombinant plasmid obtained in step (3). The specific operation is as follows: dilute 2 mg of recombinant plasmid with 25 mL of Union-293 medium; add 6 mL of 1 mg / mL PEI to 25 mL of Union-293 medium. Vortex the plasmid and PEI thoroughly, place them at room temperature for 20 minutes, and then add them to the 293F cell suspension. After culturing for 5 days, collect the cell supernatant, centrifuge at 6000 rpm for 1 hour at 4°C, discard the cell precipitate, and obtain the supernatant containing the target protein.

[0207] The target protein was purified by affinity chromatography.

[0208] Since the obtained gp350 recombinant protein carries a 10×His tag, it can be affinity purified using a nickel column. The specific operation is: filter the supernatant containing the target protein obtained above using 0.65μm filter paper, bind to the nickel column beads 3 times, elute the beads 3 times with 30mM imidazole, and elute the target protein with 500mM imidazole. Then use gel filtration chromatography for further purification. The specific operation is: use a 3kD concentrator tube to concentrate the protein eluted from the nickel column to a volume of less than 1mL. Then use Superdex 200Increase 10 / 300GL for purification.

[0209] The final amino acid sequence of the target protein is: HHHHHHHHHHHEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELAL T(SEQ ID NO: 4); wherein, the sequence consisting of amino acid residues at positions 11 to 817 in SEQ ID NO: 4 is the extracellular segment sequence of the gp350 protein, and the sequence consisting of amino acid residues at positions 1 to 10 and 818 to 827 is the His tag sequence.

[0210] 1.2 Construction of phage antibody library

[0211] (1) Total RNA extraction:

[0212] The blood of nasopharyngeal carcinoma (NPC) patients was diluted with PBS 1:1 (10mL blood + 10mL PBS), and then 20mL of the dilution was carefully covered on 15mL of lymphocyte separation solution, the stratified interface was maintained, and centrifuged at 2000rpm for 20min at room temperature. The speed was slowly reduced, and the middle mononuclear cell layer was aspirated into a new 15mL centrifuge tube, and the volume was fixed to 15mL with PBS, and centrifuged at 300g for 20min at room temperature. The supernatant was transferred to a new 50mL centrifuge tube, and centrifuged at 300g for 20min at room temperature. The supernatant was removed, and the cell pellets in the 15mL and 50mL centrifuge tubes were each resuspended with 1mL Trizol (i.e., 2mL Trizol was used to resuspend every 10mL of blood).

[0213] Take 20mL Trizol-cell pellet resuspension and add 4mL chloroform. After shaking on a shaker for 15s, let it stand at room temperature for 5min. Centrifuge at 4000g for 30min at 4℃. After centrifugation, stratification will appear. Use a pipette to carefully transfer the top transparent layer to a new 50mL centrifuge tube without RNase and DNase. Add 1:1 volume of isopropanol to each tube, mix it upside down several times, and let it stand at room temperature for 10min. Centrifuge at 4000g for 30min to remove the supernatant and retain the precipitate. Add 1mL 75% ethanol to the precipitate and transfer it to a 1.5mL centrifuge tube. Flip the precipitate several times to fully contact the liquid. Centrifuge at 7500g for 5min, remove the supernatant and retain the precipitate. Keep the centrifuge tube open, let it dry at room temperature for 10min, add 400μL enzyme-free water, and incubate at 55℃ for 10min to ensure that the RNA is completely dissolved, and then obtain total RNA.

[0214] Take 1 μL and use a nucleic acid concentration meter to detect the RNA concentration and A260 / A280 and record them.

[0215] (2) RNA reverse transcription:

[0216] Reverse transcription kit (Promega GoScript TM Reverse transcription kit) was used to reverse transcribe the total RNA obtained in the above step (1).

[0217] The specific operations are:

[0218] The total RNA sample was divided into two parts, one using the Oligo dT Primer in the kit as a primer, and the other using the Random 6-mers in the kit as a primer. The reaction solution was prepared in a 1.5 mL centrifuge tube according to the system shown in Table 4, and the reaction system was expanded in the same proportion according to the amount of RNA for amplification.

[0219] The reaction solution after the above reaction was divided into eight PCR tubes and placed in a PCR instrument. Centrifuge briefly, react at 70℃ for 5 minutes to denature the RNA, and cool quickly on ice after the reaction. Then, add the remaining reaction solution to the eight PCR tubes according to the ratio in Table 5. Mix the reaction solution in Table 5 and centrifuge briefly. Place the centrifuge tube in a PCR instrument, react at 45℃ for 60 minutes, then at 75℃ for 15 minutes, and cool on ice to obtain cDNA after total RNA reverse transcription, which is stored at 4℃.

[0220] Table 4 5 μL reverse transcription reaction system

[0221]

[0222] Table 5 Other reaction liquid ratios

[0223]

[0224]

[0225] (3) PCR amplification:

[0226] Using the cDNA obtained in step (2) as a template, NEB Q5 high-fidelity DNA polymerase was used to amplify the single-chain antibody fragment (ScFv) through two rounds of PCR, and the constructed structure was VL-linker-VH (light chain-linker-heavy chain).

[0227] The first round of PCR was performed using cDNA as a template. The first round of PCR reaction system is shown in Table 6. The PCR reaction used a three-temperature point method: each cycle was denatured at 95°C for 10 seconds, annealed at 60°C for 30 seconds, heated to 72°C for 1 minute, and cycled 35 times. The primers in the first round of PCR included:

[0228] Vλ forward primer: 5′-CCTTTCTATGCGGCCCAGCCGGCCgagctcCAGTCTGTSB TGACGCAGCCGCC-3′ (SEQ ID NO: 5);

[0229] Vλ-linker reverse primer: 5′-GGAAGATCTAGAGGAACCACCTAGGACGGTSASCTTGGTCC-3′ (SEQ ID NO: 6);

[0230] Vκ forward primer: 5′-CCTTTCTATGCGGCCCAGCCGGCCgagctcGACATCCRGD TGACCCAGTCTCC-3′ (SEQ ID NO: 7);

[0231] Vκ-linker reverse primer: 5′-GGAAGATCTAGAGGAACCACCTTTGATTTCCACCTTGGTCC-3′ (SEQ ID NO: 8);

[0232] linker-VH forward primer: 5'-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCT CGGCGGTGGTGGGCAGGTGCAGCTGCAGGAGTCSG-3' (SEQ ID NO: 9);

[0233] Linker-VH reverse primer: 5'-CAGTCATTCTCGACTTactagtTGAGGAGACRGT GACCAGGGTG-3' (SEQ ID NO: 10).

[0234] Among them, the lowercase part in each primer is the restriction site; the Vλ forward primer is used in pairs with the Vλ-linker reverse primer, the Vκ forward primer is used in pairs with the Vκ-linker reverse primer, and the linker-VH forward primer is used in pairs with the linker-VH reverse primer.

[0235] Table 6 First round PCR reaction system

[0236]

[0237] After the reaction, all PCR products were subjected to 1.5% agarose gel electrophoresis, and the target fragments of about 320 bp (corresponding to VL) and 350 bp (corresponding to VH) were recovered by gel electrophoresis. DNA was recovered using NEB DNAGEL purification kit according to the kit instructions. The collected DNA solution was the first round of PCR amplification product, and was stored at 4°C after measuring the concentration.

[0238] The second round of PCR was performed using the amplified product of the first round of PCR as a template, and the second round of PCR reaction system is shown in Table 7. The reaction conditions of the second round of PCR were the same as those of the first round of PCR. The primers in the second round of PCR included: OF: 5'-CCTTTCTATGCGGCCCAGCCGGCCgagctc-3' (SEQ ID NO: 11); OR: 5'-CAGTCATTCTCGACTTactagt-3' (SEQ ID NO: 12).

[0239] Table 7 Second round PCR reaction system

[0240]

[0241] After the reaction, the PCR product was subjected to 1.5% agarose gel electrophoresis at 160 V for 20 min. Finally, the band with a target band fragment size of 750 bp in the electrophoresis result was selected and DNA was recovered using NEB DNA GEL purification kit according to the kit instructions. The collected DNA solution was the second round of PCR amplification product and stored at 4°C after concentration measurement.

[0242] (4) Enzyme ligation of vector and PCR amplification product:

[0243] The second round PCR product (ScFv fragment) was connected to the phage plasmid pComb3XSS by restriction digestion, thereby constructing a phage plasmid library containing the amplified target fragment (ScFv fragment). The restriction endonucleases SpeI and SacI were used for the digestion.

[0244] The restriction enzyme digestion systems of the pComb3XSS vector and the second-round PCR amplification product (amplified target fragment (ScFv fragment)) are shown in Tables 8 and 9, respectively.

[0245] Table 8 Vector restriction enzyme digestion system

[0246]

[0247] Table 9 Target fragment (ScFv fragment) enzyme digestion system

[0248]

[0249]

[0250] Reaction conditions for enzyme digestion: incubate at 37℃ for 2h, 80℃ for 3min. After the digestion, the vector digestion product is run on a gel and then cut off the gel and recovered using the NEB DNA purification recovery kit (do not irradiate with UV); the ScFv fragment digestion product does not need to be run on a gel and can be directly recovered.

[0251] After the enzyme digestion reaction is purified, the concentration of the recovered product is measured, and then the digested vector and the ScFv digestion product are connected according to the connection reaction system shown in Table 10. The above connection system is reacted at 37°C overnight (16-24h). After the connection is completed, the connection product is recovered using the NEB DNA purification and recovery kit. The concentration of the recovered connection product is detected and recorded, and stored at 4°C.

[0252] Table 10 Ligation reaction system

[0253]

[0254] 1.3 Construction of bacterial library:

[0255] (1) Preparation of TG1 E. coli competent cells:

[0256] Take the TG1 strain and streak a single colony on 2×YT solid medium, culture overnight at 37℃, pick a single colony from the single colony plate to 10mL 2×YT medium, culture overnight at 37℃, 220rpm. Inoculate the bacteria into 100mL 2×YT medium at a dilution ratio of 1:100, culture at 37℃250rpm for 40min, measure the OD value, and then measure it every 20min until OD600 = 0.3-0.35. Collect the bacterial solution, centrifuge at 3200g, 0-4℃ for 10min, discard the supernatant, place on ice, add 40mL pre-cooled ddH2O to resuspend, centrifuge at 3200g, 0-4℃ for 10min, discard the supernatant, place on ice, add 1mL pre-cooled ddH2O to resuspend, transfer to a 1.5mL pre-cooled EP tube, centrifuge at 10000g, 4℃ for 30s, and repeat once. The supernatant was discarded, and the cell suspension was placed on ice. 400 μL of pre-cooled ddH2O was added to resuspend the cell suspension to obtain a TG1 E. coli competent cell suspension.

[0257] Take the Comb3XSS recombinant plasmid containing the ScFv fragment obtained in the above embodiment, and use the electroporation method to construct an Escherichia coli library. The specific method is: use a pre-cooled gun tip to add 100ng of the Comb3XSS recombinant plasmid containing the ScFv fragment to 50μL of the above-mentioned TG1 Escherichia coli competent cell suspension, blow gently, transfer to a pre-cooled 1mm electroporation cup, and after confirming that the mixture is at the bottom of the electroporation cup and there are no bubbles, set 1800V, 1mm spacing, and perform electroporation. Immediately after completion, add 1000μL of 37℃ SOC culture medium, take the mixed solution out of the electroporation cup, and shake and recover at 37℃, 180rpm for 90min. Use 2×YT liquid culture medium to perform a 10-fold gradient dilution, for a total of 6 gradient dilutions (10 dilutions each 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 5 μL of each gradient was evenly added to 2×YT-GA solid medium, dried and incubated at 37°C overnight. The number of colonies on the gradient dilution plate was counted and the ligation efficiency was calculated. The ligation efficiency formula is:

[0258] E (pfu / μg) = N × D × 10;

[0259] Wherein, E is the competent efficiency (unit: pfu / μg), D is the dilution factor, and N is the number of monoclones on the plate with the corresponding dilution factor.

[0260] Repeat 100 electroporation transformation reactions according to the above method, and evenly spread the revived bacterial solution on 100 2×YT-GA245mm square culture medium plates, dry them, and invert them for overnight culture at 37°C. Take the square plates cultured overnight, add 6mL 2×YT liquid culture medium to the surface of each culture plate, gently scrape off the 100 square plate colonies with a spreading stick and collect the bacterial solution into a 50mL centrifuge tube, add glycerol with a final concentration of 20%, and it is the bacterial library.

[0261] Take 10 μL of bacterial solution into 990 μL of 2×YT liquid medium and measure OD600 using NanoDrop, calculate and record the total OD600 of the bacterial library.

[0262] T OD600 =M OD600 ×100;

[0263] Among them, T OD600 is the total bacterial OD600, M OD600 is the measured OD600.

[0264] 1.4 Construction of phage library:

[0265] Take an appropriate amount from the above bacterial library into a 1.5mL EP tube. The calculation formula for the bacterial liquid volume is:

[0266]

[0267] Wherein, V is the volume of the transferred bacterial solution (unit: μL), and OD600 is the total OD600 of the constructed bacterial library.

[0268] Transfer it to 100mL 2×YT-GA liquid medium to make the initial OD600 0.1. Cultivate in a constant temperature shaker at 37°C, 250rpm until the bacterial solution OD600 reaches 0.5-0.55. Calculate and add helper phage M13K07 according to the following formula to make the ratio of bacteria to phage 1:20. The formula for calculating the amount of phage added is:

[0269]

[0270] Where V is the volume of helper phage added (in mL), T helper-phage is the titer of the helper phage used, and OD600 is the OD600 value of the bacterial solution.

[0271] The culture was continued for 30 min at 37°C and 220 rpm in a constant temperature shaker. The TG1 bacteria were collected by centrifugation at 3200 g for 5 min, the supernatant was removed, and the pellet was resuspended and transferred to 100 mL of 2×YT-AK liquid culture medium and cultured overnight at 30°C and 250 rpm in a constant temperature shaker.

[0272] Transfer the overnight culture to a new 50mL centrifuge tube and centrifuge at 4000g for 30min at 4℃. Take the supernatant, add 1 / 4 volume of 20% PEG / 2.5M NaCl precooled at 4℃, mix thoroughly and place on ice for 30min. Centrifuge at 4000rpm at 4℃ for 30min, discard the supernatant, and invert on paper for 2min. Add 1mL PBS to resuspend the precipitate, centrifuge at 12000rpm at 4℃ for 20min. Take the supernatant, add 1 / 4 volume of precooled 20% PEG / 2.5M NaCl solution, mix well and place on ice for 10min. Centrifuge at 12000rpm at 4℃ for 10min, discard the supernatant, add 1ml PBS to resuspend the precipitate. Centrifuge at 12000rpm at 4℃ for 2min, take the supernatant, which is the phage library, add glycerol to a final concentration of 20%, and store at -80℃.

[0273] The titer of the phage library was tested. The specific operation was as follows: add the phage library to 10 mL of 2×YT liquid culture medium, and culture at 37°C and 250 rpm for about 45 min to 60 min until the OD600 was 0.5 to 0.55. Take 10 μL of the cultured phage library and dilute it 10 times (a total of 13 dilutions). Add 90 μL of untreated TG1 bacterial solution to each dilution gradient, mix well, and incubate at 37°C for 20 min. Then take 5 μL of each dilution gradient and add it dropwise to 2×YT-GA solid culture medium, dry it, and culture it overnight at 37°C, count it, and calculate the number of phage particles per milliliter of phage solution according to the phage library titer formula.

[0274] T (pfu / ml) = N × D × 400;

[0275] Wherein, T is the phage titer (unit: pfu / mL), D is the dilution multiple, and N is the number of single colonies at the corresponding dilution multiple.

[0276] 1.5 Screening of gp350 protein-specific antibodies

[0277] Antibody specific binding to gp350 protein was screened using antigen solid phase adsorption screening method.

[0278] The specific operations are as follows:

[0279] Dissolve 50 μg of the gp350 protein obtained above in 2 mL PBS and coat the immunotube overnight at 4°C (positive tube). At the same time, coat the negative control protein (50 μg BSA) as a negative control. Discard the coated protein, rinse with 2 mL PBS 3 times, and then block with 2 mL 3% BSA (dissolved in PBST) at room temperature for 2 hours. Discard the blocking solution, take 100 μL of the obtained phage library, dilute with 2 mL PBS, and incubate in the immunotube for 1 hour. Discard the liquid, wash with 2 mL PBST 5 times, 5 minutes each time. Then wash with 2 mL PBS 5 times, 5 minutes each time. While washing, take 1 mL of the overnight TG1 saturated bacterial solution to 100 mL 2×YT liquid culture medium, and culture at 37°C 150 rpm until OD600 = 0.5 (about 1.5 hours). Discard the washing solution and elute with 1 mL 0.1 mg / mL Trypsin at room temperature for 30 minutes. Add 1 mL of the eluate to 10 mL of the cultured TG1 bacterial solution (OD = 0.5), infect at 37°C, 150 rpm for 30 min; centrifuge at 4°C, 3000 g for 10 min. Then resuspend the precipitate in 1 mL of 2×YT liquid culture medium. Evenly spread 1 mL of the resuspended product eluted from the positive immune tube on a 245 mm × 245 mm 2×YT solid culture dish, and culture at 37°C overnight (16 to 20 h). Determine the phage titer of the positive tube and the negative control tube respectively, using the same determination method as in the above example.

[0280] According to the phage titer comparison results, 5 mL of 2×YT liquid culture medium was added to the culture dish with high phage titer after overnight culture, all colonies were scraped off with a coating rod, and a daughter phage library was prepared according to the method in the above embodiment. The above operation was repeated until the antibody with the highest affinity to gp350 was screened (compared with the negative control).

[0281] In the antigen coating, blocking, incubation, washing and elution processes, the rotation speed was 15 rpm unless otherwise specified.

[0282] The highest affinity antibodies were tested using ELISA.

[0283] Take 10 μL of the culture solution of the antibody with the highest affinity for gp350 and dilute it with 1 ml of 2×YT liquid medium. Perform clone streaking on 2×YT-GA solid culture plates and culture overnight at 37°C for 16 to 20 hours. Pick 192 monoclonal clones to a 96-well plate (each well contains 200 μL of 2×YT-GA liquid) and culture at 37°C until saturation. Take 2 μL of saturated bacterial solution to a new 96-well plate (each well contains 200 μL of 2×YT-A liquid) to make the initial OD about 0.03, and let it stand at 37°C for 2.5 to 3 hours until the OD is about 0.5. Add 0.1 μL of the above helper phage to each well and infect at 37°C for 30 minutes. Add 0.2 μL of Kana (kanamycin) to each well and culture overnight at 30°C. Coat the ELISA plate with protein (- / +) overnight. Centrifuge the 96-well plate incubated overnight at 4°C and 3400g for 5 minutes. Discard the liquid in the ELISA plate and wash once with 350μL PBS. Block with 350μL 3% BSA (PBST) at 37°C for 1h. Discard the blocking solution, wash once with 350μL PBST, and tap the plate to remove the liquid. Add 140μL 3% BSA (PBST) to each well, and then add 60μL of the above phage library expressed antibody (the antibody with the highest affinity, as the primary antibody) and incubate at 37°C for 1h. Discard the liquid (primary antibody), wash 5 times with 350μL PBST, and tap the plate. Add 100μL M13 Antibody (HRP) as the secondary antibody (1:8000 addition ratio, relative to the blocking solution), incubate at 37°C for 1h, discard the liquid (secondary antibody), wash 5 times with 350μL PBST, and tap the plate. 100 μL TMB was protected from light for 2-3 min, and 100 μL dilute hydrochloric acid (concentrated hydrochloric acid: water = 1:12) was used to terminate the reaction. The OD450 and OD630 of the ELISA plate were read. In this embodiment, the inventors finally obtained two antibodies with the best effects, which were named 1A12 and 2E4 respectively.

[0284] Example 2 Expression and purification of monoclonal antibody 1A12

[0285] The complete heavy chain fragment can be expressed by connecting the upstream of the antibody heavy chain variable region with the CMV fragment and the downstream with the constant region of human IgG1 and the ployA fragment; and the complete light chain fragment can be expressed by connecting the upstream of the antibody light chain variable region with the CMV fragment and the downstream with the constant region of the light chain κ / λ and the ploy A fragment. The expression of the antibody can be achieved by co-transfecting the plasmids carrying the full-length sequences of the heavy and light chains of the above antibodies into 293T cells, and the antibody can be purified using protein A beads.

[0286] The 1A12 full-length heavy chain has a total of 473 amino acid residues (excluding *), specifically:

[0287]

[0288]

[0289] Among them, the underlined part in the sequence is the amino acid sequence of the heavy chain variable region (SEQ ID NO: 14). The underlined and bold parts are the amino acid sequences of the three complementary regions CDR-H1 (SEQ ID NO: 15), CDR-H2 (SEQ ID NO: 16) and CDR-H3 (SEQ ID NO: 17) in the heavy chain variable region (IMGT definition scheme). The italic part is the signal peptide. The amino acids 144 to 473 in SEQ ID NO: 13 are the heavy chain constant region. * indicates a stop codon.

[0290] CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region using other CDR definition schemes are shown in Table 11.

[0291] Table 11 CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region with other CDR definition schemes

[0292]

[0293] The coding gene of the 1A12 full-length heavy chain has a total of 1422 bases, specifically:

[0294]

[0295]

[0296] Among them, the underlined part in the sequence is the nucleotide sequence of the heavy chain variable region. The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region (IMGT definition scheme). The three bases at the 5' end and the 3' end are the start codon and the stop codon respectively. The italic part is the signal peptide.

[0297] The 1A12 full-length light chain has a total of 233 amino acid residues (excluding *), specifically:

[0298]

[0299] Among them, the underlined part in the sequence is the amino acid sequence of the light chain variable region (SEQ ID NO: 28). The underlined and bold parts are the amino acid sequences of the three complementary determining regions CDR-L1 (SEQ ID NO: 29), CDR-L2 (GAS) and CDR-L3 (SEQ ID NO: 30) in the light chain variable region (IMGT definition scheme). The italic part is the signal peptide. The amino acids 128 to 233 in SEQ ID NO: 27 are the light chain constant region. * indicates a stop codon.

[0300] CDR-H1, CDR-H2, and CDR-H3 in the light chain variable region using other CDR definition schemes are shown in Table 12.

[0301] Table 12 CDR-H1, CDR-H2, CDR-H3 in the light chain variable region with other CDR definition schemes

[0302]

[0303] The coding gene of 1A12 full-length light chain has 702 bases in total, specifically:

[0304]

[0305]

[0306] Among them, the underlined part in the sequence is the nucleotide sequence of the light chain variable region. The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region. The three bases at the very beginning of the 5' end and the very end of the 3' end are the start codon and the stop codon respectively. The italic part is the signal peptide.

[0307] Example 3 Expression and purification of monoclonal antibody 2E4

[0308] The complete heavy chain fragment can be expressed by connecting the upstream of the antibody heavy chain variable region with the CMV fragment and the downstream with the constant region of human IgG1 and the ployA fragment; and the complete light chain fragment can be expressed by connecting the upstream of the antibody light chain variable region with the CMV fragment and the downstream with the constant region of the light chain κ / λ and the ploy A fragment. The expression of the antibody can be achieved by co-transfecting the plasmids carrying the full-length sequences of the heavy and light chains of the above antibodies into 293T cells, and the antibody can be purified using protein A beads.

[0309] The 2E4 full-length heavy chain has a total of 468 amino acid residues (excluding *), specifically:

[0310]

[0311] Among them, the underlined part in the sequence is the amino acid sequence of the heavy chain variable region (SEQ ID NO: 38). The underlined and bold parts are the amino acid sequences of the three complementary regions CDR-H4 (SEQ ID NO: 39), CDR-H5 (SEQ ID NO: 40) and CDR-H6 (SEQ ID NO: 41) in the heavy chain variable region (IMGT definition scheme). The italic part is the signal peptide. The amino acids 139 to 468 in SEQ ID NO: 37 are the heavy chain constant region. * indicates a stop codon.

[0312] CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region using other CDR definition schemes are shown in Table 13.

[0313] Table 13 CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region with other CDR definition schemes

[0314]

[0315] The coding gene of 2E4 full-length heavy chain has a total of 1408 bases, specifically:

[0316]

[0317]

[0318] Among them, the underlined part in the sequence is the nucleotide sequence of the heavy chain variable region. The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region (IMGT definition scheme). The three bases at the 5' end and the 3' end are the start codon and the stop codon respectively. The italic part is the signal peptide.

[0319] The 2E4 full-length light chain has a total of 235 amino acid residues (excluding *), specifically:

[0320]

[0321] Among them, the underlined part in the sequence is the amino acid sequence of the light chain variable region (SEQ ID NO: 52). The underlined and bold parts are the amino acid sequences of the three complementary determining regions CDR-L4 (SEQ ID NO: 53), CDR-L5 (RNN) and CDR-L6 (SEQ ID NO: 54) in the light chain variable region (IMGT definition scheme). The italic part is the signal peptide. The amino acids 130 to 235 in SEQ ID NO: 51 are the light chain constant region. * indicates a stop codon.

[0322] CDR-H1, CDR-H2, and CDR-H3 in the light chain variable region using other CDR definition schemes are shown in Table 14.

[0323] Table 12 CDR-H1, CDR-H2, CDR-H3 in the light chain variable region with other CDR definition schemes

[0324]

[0325]

[0326] The coding gene of 2E4 full-length light chain has a total of 708 bases, specifically:

[0327]

[0328] Among them, the underlined part in the sequence is the nucleotide sequence of the light chain variable region. The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region. The three bases at the very beginning of the 5' end and the very end of the 3' end are the start codon and the stop codon respectively. The italic part is the signal peptide.

[0329] Effect Example 1 Effect of monoclonal antibody 1A12

[0330] 1. The affinity of antibody 1A12 was determined by ELISA.

[0331] In order to determine the affinity of antibody 1A12, the present embodiment adopts the ELISA method. First, the gp350 protein is diluted with PBS buffer and added to the enzyme-labeled plate, 100 ng per well, and incubated at 4°C overnight. The next day, the unbound antigen is discarded and the plate is washed 3 times with PBS T buffer, each washing for 3 minutes. Then, 5% BSA solution is added for blocking, and after incubation at 37°C for 1-2 hours, the plate is washed again with PBST. Next, antibody 1A12 is gradiently diluted and added to the corresponding wells, incubated at 37°C for 1-2 hours to promote the binding of the antibody to the coated gp350 protein (BSA solution is used as a negative control). After incubation, the plate is washed 3 times with PBST, and then an HRP-labeled enzyme-labeled secondary antibody (such as an anti-human IgG secondary antibody) is added, and incubated at 37°C for 1 hour. Subsequently, the plate is washed 3 times with PBST, TMB color substrate is added, and incubated at room temperature for 5-15 minutes until the color reaches the expected intensity, and the reaction is terminated with dilute hydrochloric acid. Finally, the absorbance value (OD value) of each well was measured at a wavelength of 450 nm, and a curve between the antibody concentration and the OD value was drawn. Figure 1 : IC50 of 1A12 antibody = 0.017 μg / mL, indicating that 1A12 antibody has a high affinity with gp350 antigen.

[0332] 2. The affinity of antibody 1A12 was determined using biofilm interferometry (BLI).

[0333] BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: a biosensor (Sartorius, Germany) SA probe) is immersed in a buffer solution (a mixture of KB buffer, 0.1% BSA and 0.02% Tween 20) for equilibrium. It is then taken out and immersed in a solution containing 5μg / mL gp350-Biotin (biotin-labeled gp350 protein). The gp350 antigen in the solution will bind to the surface of the SA (streptavidin) biological probe, increasing the thickness of the surface film. The biosensor with a solidified antigen of known concentration is then immersed in a buffer solution as a baseline. By immersing the solidified biosensor with a known antigen concentration in a sample solution containing 1A12 antibody for about 100 seconds, the specific binding between the antigen and the antibody will lead to an increase in the thickness of the film layer. The biosensor bound to the 1A12 antibody is immersed in a buffer solution for dissociation, and the antibody to be tested (1A12 antibody) will fall off the surface of the biosensor, resulting in a decrease in the thickness of the film layer. By real-time monitoring of the biosensor biofilm thickness during the experiment, the kinetic constant of the sample to be tested (1A12 antibody) can be obtained. The results are shown in Figure 2 As shown: KD(M) of 1A12 antibody = 3.415E-10, indicating that 1A12 antibody has a high affinity with gp350 antigen.

[0334] 3 Neutralizing activity of 1A12 antibody

[0335] (1) Preparation of EBV virus:

[0336] 1) CNE2 cells infected with EBV-GFP (the virus has been disclosed in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus) were cultured in RPMI1640 + 5% FBS in a 37 degree incubator (5% CO2). When the cells grew to 90% density (10 cm dish), TPA with a final concentration of 20 ng / mL and NaB (sodium butyrate) with a final concentration of 2.5 mM were added for induction, and the medium was changed after 12 hours.

[0337] 2) 48-72 hours after the medium is replaced, the culture supernatant is collected to separate and purify the virus. The supernatant is directly aspirated, centrifuged and filtered with a 0.45um filter. After concentration, the supernatant is resuspended in serum-free RPMI1640 and immediately used for infection or stored at -80 degrees.

[0338] (2) Detection of B cell neutralizing activity of 1A12 monoclonal antibody

[0339] 1) Take the 1A12 monoclonal antibody in the above example and adjust the concentration to 2 mg / mL. Add 60 μL of RPMI1640 medium to each well of a new 96-well plate, and add 90 μL of 100 μg / mL 1A12 antibody diluted with RPMI1640 to the first well (the first well does not contain RPMI1640 medium).

[0340] 2) After 3-fold gradient dilution (gradient dilution will aspirate 30 μL from the first well and add it to the second well, and so on, aspirate 30 μL from the last well and discard it, and the final volume of each well is 60 μL); add 60 μL of virus dilution solution to each well (the virus is diluted with DMEM medium, the titer is about 4*10 6 / mL), and then 1*10 6 Raji cells were placed in a 37°C incubator for 48 hours before testing.

[0341] 3) Raji cells were aspirated to prepare a cell suspension, and the infection rate was detected by flow cytometry. The inhibition rate (neutralization efficiency, %) of the antibody in the Raji B cell infection model was calculated by detecting the reduction ratio of the number of GFP-positive cells in the antibody-treated group compared with the infection control group (adding an equal volume of RPMI1640). The IC50 of the 1A12 monoclonal antibody was calculated and plotted using Prism. The 2G4 antibody was used as a negative control antibody (2G4 is an anti-Ebola virus antibody, which has been disclosed in the literature: Audet, J. et al. Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cock tail against Ebola virus. Sci. Rep. 4, 6881 (2014).).

[0342] The results are as follows Figure 3 As shown: The IC50 of monoclonal antibody 1A12 in the B cell infection model is 0.27 μg / mL, and the control antibody has no neutralizing activity; monoclonal antibody 1A12 can significantly inhibit EBV infection of B cells.

[0343] Effect Example 2 Effect of monoclonal antibody 2E4

[0344] 1. The affinity of antibody 2E4 was determined by ELISA.

[0345] The experimental method is the same as that of Example 1.

[0346] Results Figure 4 : IC50 of 2E4 antibody = 0.026 μg / mL, indicating that 2E4 antibody has a high affinity with gp350 antigen.

[0347] 2. The affinity of antibody 2E4 was determined using biofilm interferometry (BLI).

[0348] Experimental method is the same as that of Example 1

[0349] The results are as follows Figure 5 As shown: KD (M) of 2E4 antibody = 4.851E -9 ; This indicates that the 2E4 antibody has a high affinity with the gp350 antigen.

[0350] Neutralizing activity of 2E4 antibody

[0351] Preparation of EBV virus:

[0352] Same effect as embodiment 1.

[0353] 4.2E4 monoclonal antibody B cell neutralization activity detection:

[0354] 1) Take the 2E4 monoclonal antibody in the above example and adjust the concentration to 2 mg / mL. Add 60 μL of RPMI1640 medium to each well of a new 96-well plate, and add 90 μL of 100 μg / mL 2E4 antibody diluted with RPMI1640 to the first well (the first well does not contain RPMI1640 medium).

[0355] 2) After 3-fold gradient dilution (gradient dilution will aspirate 30 μL from the first well and add it to the second well, and so on, aspirate 30 μL from the last well and discard it, and the final volume of each well is 60 μL); add 60 μL of virus dilution solution to each well (the virus is diluted with DMEM medium, the titer is about 4*10 6 / mL), and then 1*10 6 Raji cells were placed in a 37°C incubator for 48 hours before testing.

[0356] 3) Raji cells were aspirated to prepare cell suspension, and the infection rate was detected by flow cytometry. The inhibition rate (neutralization efficiency, %) of the antibody in the Raji B cell infection model was calculated by detecting the reduction ratio of the number of GFP-positive cells in the antibody-treated group compared with the infection control group (adding an equal volume of RPMI1640). The IC50 of the 2E4 monoclonal antibody was calculated and plotted using Prism. The 2G4 antibody was used as a negative control antibody.

[0357] The results are as follows Figure 6 As shown: The IC50 of monoclonal antibody 2E4 in the B cell infection model is 0.054 μg / mL, and the control antibody has no neutralizing activity; monoclonal antibody 2E4 can significantly inhibit EBV infection of B cells.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against Epstein-Barr virus gp350 protein, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; The monoclonal antibody comprises any one of A) or B); A) The heavy chain comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 14; The light chain comprises: a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 28; B) The heavy chain comprises: a heavy chain variable region comprising CDR-H4, CDR-H5 and CDR-H6 of the heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 38; The light chain comprises: A light chain variable region, comprising CDR-L4, CDR-L5 and CDR-L6 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO:

52.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: A) The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 30, respectively, and the CDRs are defined according to the Kabat definition scheme; or The amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; the amino acid sequence of the CDR-L2 is GAS, and the CDRs are defined according to the IMGT definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 30, respectively, and the CDRs are defined according to the Chothia definition scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively, and the CDRs are defined by the Contact definition scheme; B) The amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, CDR-L5, and CDR-L6 are shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:54, respectively, and the CDRs are defined according to the Kabat definition scheme; or The amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4 and CDR-L6 are shown in SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 53 and SEQ ID NO: 54, respectively; the amino acid sequence of CDR-L5 is: RNN; and the CDRs are defined according to the IMGT definition scheme; or The amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, CDR-L5, and CDR-L6 are shown in SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:44, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:54, respectively, and the CDRs are defined according to the Chothia definition scheme; or The amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, CDR-L5 and CDR-L6 are shown in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 respectively, and the CDRs are defined according to the Contact definition scheme.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, a bispecific antibody, and a multispecific antibody.

4. A recombinant protein comprising: the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and Optional tag sequence to facilitate expression and / or purification.

5. A biological material related to the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4, wherein the biological material comprises at least one of h1) to h16): h1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4; h2) an expression cassette comprising the nucleic acid molecule described in h1); h3) a vector comprising the nucleic acid molecule described in h1); h4) a vector comprising the expression cassette described in h2); h5) a transgenic cell line comprising the nucleic acid molecule described in h1); h6) a transgenic cell line comprising the expression cassette described in h2); h7) a transgenic cell line comprising the vector described in h3); h8) a transgenic cell line comprising the vector described in h4); h9) a microorganism comprising the nucleic acid molecule described in h1); h10) A microorganism comprising the expression cassette described in h2); h11) a microorganism comprising the vector described in h3); h12) a microorganism comprising the vector described in h4); h13) A virus comprising the nucleic acid molecule described in h1); h14) A virus comprising the expression cassette described in h2); h15) A virus comprising the vector described in h3); h16) A virus comprising the vector described in h4).

6. A conjugate comprising: at least one of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and the recombinant protein according to claim 4; and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

7. Use of at least one of (1) to (4) in the preparation of a product; (1) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (2) The recombinant protein according to claim 4; (3) The biomaterial according to claim 5; (4) The conjugate according to claim 6; The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the drug has at least one of the functions i1) to i2): i1) Prevent EB virus infection; i2) Treating and / or preventing diseases caused by EB virus infection; Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions j1) to j3): j1) detecting the presence or level of gp350 protein in a sample; j2) Detection of Epstein-Barr virus; j3) Diagnosis of diseases caused by EB virus infection.

8. A product, comprising at least one of k1) to k3): k1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; k2) The recombinant protein according to claim 4; k3) The conjugate according to claim 6; The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

9. A drug comprising at least one of (11) to (14): (l1) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (12) The recombinant protein according to claim 4; (13) The biomaterial according to claim 5; (14) The conjugate according to claim 6; Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

10. A vaccine comprising at least one of (11) to (14) and an adjuvant: (l1) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (l2) The recombinant protein according to claim 4; (l3) The biomaterial according to claim 5; (l4) The conjugate according to claim 6.

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