Monoclonal antibody recognizing the gp350 protein of epstein-barr virus and use thereof

Abstract

This invention belongs to the field of antibody technology and discloses a monoclonal antibody that recognizes the Epstein-Barr virus (EBV) gp350 protein and its applications. This invention provides a monoclonal antibody against the EBV gp350 protein or its antigen-binding fragment, comprising 1A12 and 2E4, wherein 1A12 has a high affinity for the gp350 protein (KD(M) = 3.415E). ‑10 ), 2E4 has a high 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. They can be used to detect the presence or level of gp350 protein in samples, detect EBV, diagnose diseases caused by EBV infection, prevent EBV infection and / or treat and / or prevent diseases caused by EBV infection.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a monoclonal antibody that recognizes the gp350 protein of EB virus and its application. Background Technology

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

[0003] Epstein-Barr virus (EBV) primarily exists as a latent infection, with over 90% of infected individuals remaining in this state throughout their lives. Under specific conditions, the virus can be activated, triggering carcinogenic effects. Current research indicates that EBV is closely associated with the development of various malignant tumors, including nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and various epithelial cell carcinomas, including gastric cancer. Initial replication of EBV typically occurs in the oropharynx, where it grows and multiplies in B lymphocytes and oral epithelial cells, subsequently infecting B lymphocytes. These infected cells enter the bloodstream, leading to systemic infection. When the host's immune function is weakened, latent EBV can be activated, triggering recurrent infections.

[0004] Despite the widespread health threats posed by Epstein-Barr virus (EBV) infection, there is currently no effective vaccine, and specific treatments for EBV-related diseases are also lacking. Treatment for infectious mononucleosis typically relies on antiviral drugs such as acyclovir; while these drugs can alleviate symptoms to some extent, they cannot completely eliminate EBV from B lymphocytes and the pharyngeal epithelium. For EBV-related tumors, chemotherapy and radiotherapy remain the primary treatment methods, but their effectiveness is often unsatisfactory when dealing with metastasis or recurrence.

[0005] gp350 is one of the most important envelope glycoproteins of the Epstein-Barr virus (EBV) and plays a crucial role in viral infection. As the most abundant protein on the EBV surface, gp350 primarily 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 its critical function in viral invasion, gp350 has become an ideal target for vaccine development and antibody therapy. gp350 antibodies, by specifically recognizing and binding to gp350, effectively block the binding of EBV to B lymphocytes, thereby preventing viral infection and transmission. This blocking effect provides a theoretical basis for the use of gp350 antibodies in preventing primary infection and controlling latent viral reactivation.

[0006] Despite numerous studies on gp350, no related monoclonal antibodies have yet been widely used clinically. The main research focus is on developing highly effective gp350 antibodies to inhibit EBV infection and related diseases. Through genetic engineering, high-affinity and high-specificity gp350 antibodies can be developed, thereby enhancing their antiviral efficacy. These antibodies not only have the potential to treat acute infections but also hold promise as a preventative treatment, particularly in organ transplant recipients or immunosuppressed individuals.

[0007] Monoclonal antibodies, due to their controllable production, high affinity, and high specificity, have significantly reduced adverse reactions in clinical applications, making them a promising approach for treating infectious diseases. Molecular modification can further enhance the antiviral efficacy of these antibodies. However, to date, no monoclonal antibodies targeting the Epstein-Barr virus (EBV) envelope glycoprotein are commercially available. Therefore, developing monoclonal antibodies against EBV will provide a more effective means of preventing and treating EBV-related diseases. 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] A second aspect of the present invention is to provide a recombinant protein.

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

[0011] A fourth aspect of the present invention is to provide a coupling agent.

[0012] The fifth aspect of this invention aims 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 products.

[0013] The sixth aspect of this invention is to provide a reagent kit.

[0014] The seventh aspect of this invention is to provide a medicine.

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

[0016] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0017] In a first aspect, the present invention provides a monoclonal antibody against EBV gp350 or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment comprising a heavy chain and a light chain.

[0018] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment includes either A) or B).

[0019] A) Name it a monoclonal antibody or its antigen-binding fragment 1A12;

[0020] B) Name it as a monoclonal antibody or its antigen-binding fragment 2E4.

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

[0022] The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 14;

[0023] The light chain comprises:

[0024] The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having 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 as 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 CDR is defined using the Kabat definition scheme.

[0026] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are as shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, and the amino acid sequence of CDR-L2 is GAS. The CDR is defined using 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 as 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 CDR is 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 as 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 CDR is defined using 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 of SEQ ID NO: 14 with one or more amino acid substitutions and / or deletions and / or additions that have 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 the same amino acid sequence as the protein shown in SEQ ID NO: 14.

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

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

[0035] b2-A) An amino acid sequence of SEQ ID NO. 28 with one or more amino acid substitutions and / or deletions and / or additions that have 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 the same amino acid sequence as the protein shown in SEQ ID NO. 28.

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

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

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

[0040] c2-A) An amino acid sequence having the same function as the amino acid sequence described in c1-A) by substitution and / or deletion and / or addition of one or more amino acids; or

[0041] The amino acid sequences described in c3-A) and c1-A) have 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology and have the same function as the proteins described in c1-A).

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

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

[0044] d2-A) An amino acid sequence having the same function as the amino acid sequence described in d1-A) by substitution and / or deletion and / or addition of one or more amino acids; or

[0045] The amino acid sequence described in d3-A) 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 described in d1-A).

[0046] Preferably, the heavy chain further comprises a heavy chain signal peptide; 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) The amino acid sequence consisting of amino acids 1 to 19 in SEQ ID NO: 13; or

[0049] e2-A) An amino acid sequence having the same function as the amino acid sequence described in e1-A) by substitution and / or deletion and / or addition of one or more amino acids; or

[0050] The amino acid sequences described in e3-A) and e1-A) have 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology and have the same function as the proteins described in e1-A).

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

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

[0053] f2-A) An amino acid sequence having the same function as the amino acid sequence described in f1-A) by substitution and / or deletion and / or addition of one or more amino acids; or

[0054] The amino acid sequences described in f3-A) and f1-A) have 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology and have the same function as the proteins described in f1-A).

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

[0056] The heavy chain variable region includes CDR-H4, CDR-H5 and CDR-H6 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38;

[0057] The light chain comprises:

[0058] The light chain variable region includes CDR-L4, CDR-L5 and CDR-L6 of the light chain variable region, the light chain variable region having 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 as 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 CDR is defined using the Kabat definition scheme.

[0060] Preferably, the amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4, and CDR-L6 are as 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 CDR is defined using 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 as 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 CDR is 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 as 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 CDR is defined using 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 of SEQ ID NO: 38 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 38; or

[0066] a3-B) 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: 38 and has the same amino acid sequence as the protein shown in SEQ ID NO: 38;

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

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

[0069] b2-B) An amino acid sequence of SEQ ID NO: 52 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 52; or

[0070] b3-B) 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: 52 and has the same amino acid sequence as the protein shown in SEQ ID NO: 52.

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

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

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

[0074] c2-B) An amino acid sequence having the same function as the amino acid sequence described in c1) by substitution and / or deletion and / or addition of one or more amino acids; or

[0075] The amino acid sequences described in c3-B) and c1-B) have 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology and have the same function as the proteins with the amino acid sequences described in c1-B).

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

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

[0078] d2-B) An amino acid sequence having the same function as the amino acid sequence described in d1-B) by substitution and / or deletion and / or addition of one or more amino acids; or

[0079] The amino acid sequence described in d3-B) 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 described in d1-B).

[0080] Preferably, the heavy chain further comprises a heavy chain signal peptide; 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) The amino acid sequence consisting of amino acids 1 to 19 in SEQ ID NO: 37; or

[0083] e2-B) An amino acid sequence having the same function as the amino acid sequence described in e1-B) by substitution and / or deletion and / or addition of one or more amino acids; or

[0084] The amino acid sequences described in e3-B) and e1-B) have 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology and have the same function as the proteins with the amino acid sequences described in e1-B).

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

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

[0087] f2-B) An amino acid sequence having the same function as the amino acid sequence described in f1-B) by substitution and / or deletion and / or addition of one or more amino acids; or

[0088] The amino acid sequences described in f3-B) and f1-B) have 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology and have the same function as the proteins with the amino acid sequences described in f1-B).

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

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

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

[0092] g2) An amino acid sequence having the same function as the amino acid sequence described in g1) by substitution and / or deletion and / or addition of one or more amino acids; or

[0093] The amino acid sequence described in g3) 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 described in g1).

[0094] A second aspect of the invention provides a recombinant protein comprising: a monoclonal antibody or an antigen-binding fragment thereof of the first aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

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

[0096] A third aspect of the invention provides biological materials relating to the monoclonal antibody or its antigen-binding fragment of the first aspect of the invention, or the recombinant protein of the second aspect of the invention, said biological material comprising at least one of h1) to h16):

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

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

[0099] h3) A carrier containing the nucleic acid molecule described in h1);

[0100] h4) A carrier containing the expression box described in h2);

[0101] h5) A transgenic cell line containing the nucleic acid molecules described in h1);

[0102] h6) Transgenic cell lines containing the expression cassette described in h2);

[0103] h7) A transgenic cell line containing the vector described in h3);

[0104] h8) A transgenic cell line containing the vector described in h4);

[0105] h9) Microorganisms containing the nucleic acid molecules described in h1);

[0106] h10) contains microorganisms containing the expression cassette described in h2);

[0107] h11) contains microorganisms containing the carrier described in h3);

[0108] h12) contains microorganisms containing the carrier described in h4);

[0109] h13) is a virus containing the nucleic acid molecule described in h1);

[0110] h14) contains the expression cassette described in h2);

[0111] h15) contains a virus with the vector described in h3);

[0112] h16) contains a virus with the vector described in h4).

[0113] Preferably, the transgenic cell line does not contain propagation material.

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

[0116] A) Related to monoclonal antibodies or their antigen-binding fragment 1A12:

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

[0118] a212-A) A nucleotide sequence of SEQ ID NO: 26 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and 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 antibodies or their antigen-binding fragment 2E4 related:

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

[0122] a212-B) A nucleotide sequence of SEQ ID NO: 50 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 50; or

[0123] a213-B) 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 its antigen-binding fragment of the first aspect of the present invention comprises:

[0125] A) Related to monoclonal antibodies or their antigen-binding fragment 1A12:

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

[0127] a222) A nucleotide sequence of SEQ ID NO: 36 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and 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 antibodies or their antigen-binding fragment 2E4 related:

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

[0131] a222-B) A nucleotide sequence of SEQ ID NO: 60 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 60; or

[0132] a223-B) 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] A fourth aspect of the present invention provides a conjugate comprising at least one of a monoclonal antibody or an antigen-binding fragment thereof from the first aspect of the present invention and a recombinant protein from the second aspect of the present invention.

[0134] And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0135] Preferably, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, 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 (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0137] A fifth aspect of the present invention provides 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;

[0138] The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

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

[0140] i1) Prevention of EB virus infection;

[0141] i2) Treatment and / or prevention of diseases caused by EB virus infection.

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

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

[0144] j2) Detection of EB virus;

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

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

[0147] Preferably, the drug comprises a vaccine.

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

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

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

[0151] k3) The coupling of the fourth aspect of the present invention;

[0152] The product includes at least one of reagents, test plates, reagent kits, and test chips.

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

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

[0155] j2) Detection of EB virus;

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

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

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

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

[0160] l2) The recombinant protein of the second aspect of the present invention;

[0161] l3) Benzene invented the third aspect of biomaterials;

[0162] l4) The coupling of the fourth aspect of the present invention.

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

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

[0165] i1) Prevention of EB virus infection;

[0166] i2) Treatment and / or prevention of diseases caused by EB virus infection.

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

[0168] Preferably, the drug comprises a vaccine.

[0169] Preferably, a vaccine comprises at least one of l1) to l4) and an adjuvant:

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

[0171] l2) The recombinant protein of the second aspect of the present invention;

[0172] l3) Benzene invented the third aspect of biomaterials;

[0173] l4) The coupling of the fourth aspect of the present invention.

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

[0175] The beneficial effects of this invention are:

[0176] This invention provides a monoclonal antibody against EBV gp350 or its antigen-binding fragment, comprising 1A12 and 2E4, wherein 1A12 has a high affinity for gp350 protein (KD(M) = 3.415E). -10 ), 2E4 has a high 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. They can be used to detect the presence or level of gp350 protein in samples, detect EBV, diagnose diseases caused by EBV infection, prevent EBV infection and / or treat and / or prevent diseases caused by EBV infection. Attached Figure Description

[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 diagram showing the results of monoclonal antibody 1A12 blocking EBV infection of epithelial cells.

[0179] Figure 3This is a diagram 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 This is a diagram showing the results of monoclonal antibody 2E4 blocking EBV infection of epithelial cells.

[0182] Figure 6 This is a diagram showing the results of monoclonal antibody 2E4 blocking EBV infection of B cells. Detailed Implementation

[0183] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0184] Example 1: Preparation of monoclonal antibody (mAb) against EBV 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 embodiments of this invention, the inventors selected gp350 as a bait protein to screen for specific antibodies.

[0187]

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

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

[0190] 50 μL of NEB was selected. The PCR amplification reaction system (Table 1) was used to amplify the above gp350 original sequence. 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 with the correct molecular weight was cut under UV light, and the PCR product was recovered according to the instructions of the commercially available kit.

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

[0193]

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

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

[0196] Table 2 Enzyme digestion reaction system

[0197]

[0198] Enzyme digestion was performed at 37°C for 2–3 hours. After running on an agarose gel, the digested vector was recovered using a gel extraction kit to obtain a linearized vector. The insert fragment was directly recovered using a DNA purification kit. Ligation was then performed using a 10 μL ligation reaction mixture (Table 3). The reaction was carried out at 37°C for 30 minutes. The ligation product was obtained.

[0199] Table 3 Connection Reaction System

[0200]

[0201]

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

[0203] Add the ligation product to freshly thawed DH5α competent cell suspension, incubate on ice for 30 min, heat shock at 42°C for 90 s, and return to ice for 5 min. Add 200 μL of LB medium and incubate at 30°C with gentle shaking for 40 min. Spread the culture medium onto ampicillin-resistant LB agar plates and incubate overnight at 37°C.

[0204] Select a single cloned colony for sequencing verification. Once the sequencing results are correct, the target recombinant plasmid is obtained. Extract plasmids in large quantities.

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

[0206] Human renal epithelial cells 293F were cultured, and the cell density of 1 L was 1.5 × 10⁻⁶ cells / mL. 6 Cell suspension. The recombinant plasmid obtained in step (3) was transfected using PEI transfection reagent. The specific operation was as follows: 2 mg of recombinant plasmid was diluted with 25 mL of Union-293 medium; 6 mL of 1 mg / mL PEI was added to 25 mL of Union-293 medium. The plasmid and PEI were thoroughly vortexed and mixed, and after being placed at room temperature for 20 min, it was added to the 293F cell suspension. After culturing for 5 days, the cell supernatant was collected, centrifuged at 6000 rpm for 1 h at 4℃, and the cell pellet was discarded to obtain the supernatant containing the target protein.

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

[0208] Since the obtained gp350 recombinant protein carries a 10×His tag, it can be purified using a nickel column affinity chromatography. The specific procedure is as follows: filter the supernatant containing the target protein through 0.65 μm filter paper, pass it through a nickel column with beads three times, wash the beads three times with 30 mM imidazole, and then elute the target protein with 500 mM imidazole. Further purification is then performed using gel filtration chromatography. Specifically, the protein eluted from the nickel column is concentrated using a 3 kDa concentrator to a volume less than 1 mL. Finally, purification is achieved using a Superdex 200 Increase 10 / 300GL.

[0209] The final amino acid sequence of the target protein is: HHHHHHHHHHEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELAL T(SEQ ID NO: 4); wherein, the sequence consisting of amino acid residues 11 to 817 in SEQ ID NO: 4 is the extracellular segment sequence of gp350 protein, and the sequence consisting of amino acid residues 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] Blood from nasopharyngeal carcinoma (NPC) patients was diluted 1:1 with PBS (10 mL blood + 10 mL PBS). 20 mL of the diluted solution was carefully spread over 15 mL of lymphocyte separation medium, maintaining the separation interface. The mixture was centrifuged at 2000 rpm for 20 min at room temperature. The centrifugation speed was then slowly reduced, and the intermediate mononuclear cell layer was transferred to a new 15 mL centrifuge tube. PBS was added to bring the volume to 15 mL, and the tube was centrifuged at 300 g for 20 min at room temperature. The supernatant was transferred to a new 50 mL centrifuge tube, centrifuged at 300 g for 20 min at room temperature, and the supernatant was discarded. The cell pellets in both the 15 mL and 50 mL centrifuge tubes were resuspended with 1 mL of Trizol (i.e., 2 mL Trizol for every 10 mL of blood).

[0213] Add 4 mL of chloroform to 20 mL of Trizol-resuspended cell pellet. Vortex for 15 seconds and incubate at room temperature for 5 minutes. Centrifuge at 4000 g for 30 minutes at 4°C. After centrifugation, the pellet will separate into layers. Carefully transfer the top clear layer to a new 50 mL centrifuge tube free of RNase and DNase using a pipette. Add isopropanol in a 1:1 ratio to each tube, mix thoroughly by inverting several times, and incubate at room temperature for 10 minutes. Centrifuge at 4000 g for 30 minutes, remove the supernatant, and retain the pellet. Add 1 mL of 75% ethanol to the pellet, transfer to a 1.5 mL centrifuge tube, and gently tap the pellet several times to ensure thorough contact with the liquid. Centrifuge at 7500 g for 5 minutes, remove the supernatant, and retain the pellet. Keep the centrifuge tube open, incubate at room temperature for 10 minutes to dry, add 400 μL of enzyme-free water, and incubate at 55°C for 10 minutes to ensure complete RNA dissolution. This yields the total RNA.

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

[0215] (2) RNA reverse transcription:

[0216] Using a reverse transcription kit (Promega GoScript) TM The total RNA obtained in step (1) above was reverse transcribed using a reverse transcription kit.

[0217] The specific steps are as follows:

[0218] The total RNA sample was divided into two portions. One portion used Oligo dT Primer from the kit as a primer, and the other portion used Random 6-mers from the kit as a primer. The reaction solution was prepared in 1.5 mL centrifuge tubes according to the system shown in Table 4. The reaction system was scaled up proportionally according to the amount of RNA for amplification.

[0219] Aliquot the reaction solution from the above reaction into 8-tube PCR instruments and place them in a PCR instrument. Briefly centrifuge and incubate at 70°C for 5 min to denature the RNA. After the reaction, cool rapidly on ice. Then, prepare the remaining reaction solution into the 8-tube PCR instruments according to the proportions in Table 5. Mix the reaction solutions in Table 5, briefly centrifuge, and place the centrifuge tubes into a PCR instrument. Incubate at 45°C for 60 min, then at 75°C for 15 min. Cool on ice to obtain cDNA after reverse transcription of total RNA. Store at 4°C.

[0220] Table 4. 5μL reverse transcription reaction system

[0221]

[0222] Table 5 Other reaction solution ratios

[0223]

[0224]

[0225] (3) PCR amplification:

[0226] Using the cDNA obtained in step (2) as a template, a single-chain antibody fragment (ScFv) was amplified by two rounds of PCR using NEB Q5 high-fidelity DNA polymerase. 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, and the reaction system is shown in Table 6. The PCR reaction used a three-temperature method: denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute per cycle, for a total of 35 cycles. The primers used 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'-GGAAGATCTAGAGGAACCACCTAGGACGGTSASC TTGGTCC-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] In each primer, the lowercase part represents the restriction enzyme 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 bands with target fragment sizes of approximately 320 bp (corresponding to VL) and 350 bp (corresponding to VH) were excised and recovered. DNA was recovered using the NEB DNAGEL purification kit according to the kit instructions. The collected DNA solution was the first-round PCR amplification product; after concentration determination, it was stored at 4°C.

[0238] The second round of PCR was performed using the amplified products from the first round of PCR as templates. The reaction system for the second round of PCR is shown in Table 7. The reaction conditions for the second round of PCR were the same as those for the first round of PCR. The primers used 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 products were subjected to 1.5% agarose gel electrophoresis at 160V for 20 minutes. The target band with a size of 750bp was selected from the electrophoresis results. The DNA was recovered using the NEB DNA GEL purification kit according to the kit instructions. The collected DNA solution was the second round of PCR amplification product. After the concentration was measured, it was stored at 4℃.

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

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

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

[0245] Table 8. Vector Enzyme Digestion System

[0246]

[0247] Table 9 Enzyme digestion system for the target fragment (ScFv fragment)

[0248]

[0249]

[0250] Enzyme digestion conditions: incubation at 37℃ for 2 hours, followed by incubation at 80℃ for 3 minutes. After digestion, the vector digestion products were run on a gel and then excised using the NEB DNA purification and recovery kit (do not expose to UV light); the ScFv fragment digestion products did not require gel running and could be directly recovered.

[0251] After purification via enzymatic digestion, the concentration of the recovered product was measured. Then, the digested vector and ScFv digested product were ligated according to the ligation reaction system shown in Table 10. The ligation system was incubated overnight (16–24 h) at 37°C. After ligation, the ligation product was recovered using a NEBDNA purification and recovery kit. The concentration of the recovered ligation product was measured and recorded, and stored at 4°C.

[0252] Table 10 Connection Reaction System

[0253]

[0254] 1.3 Construction of bacterial libraries:

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

[0256] Strawberry strain TG1 was streaked onto 2×YT solid medium and incubated overnight at 37°C. A single colony was picked from the streak plate and transferred to 10 mL of 2×YT medium, incubated overnight at 37°C and 220 rpm. The bacteria were then inoculated into 100 mL of 2×YT medium at a 1:100 dilution and incubated at 37°C and 250 rpm for 40 min. The OD value was measured, and then measured every 20 min until OD600 = 0.3–0.35. The bacterial suspension was collected, centrifuged at 3200 g at 0–4°C for 10 min, the supernatant was discarded, and the suspension was placed on ice. 40 mL of pre-chilled ddH2O was added for resuspending, and the suspension was centrifuged again at 3200 g at 0–4°C for 10 min, the supernatant was discarded, and the suspension was placed on ice. 1 mL of pre-chilled ddH2O was added for resuspending, and the suspension was transferred to a 1.5 mL pre-chilled EP tube. The suspension was centrifuged at 10000 g at 4°C for 30 s. This process was repeated once. Discard the supernatant, place on ice, add 400 μL of pre-cooled ddH2O to resuspend, and obtain TG1 Escherichia coli competent cell suspension.

[0257] The Comb3XSS recombinant plasmid containing the ScFv fragment obtained in the above examples was used to construct an E. coli library using electroporation. Specifically, 100 ng of the Comb3XSS recombinant plasmid containing the ScFv fragment was added to 50 μL of the above TG1 E. coli competent cell suspension using a pre-chilled pipette tip. The mixture was gently blown to mix and transferred to a pre-chilled 1 mm electroporation cuvette. After confirming the mixture was at the bottom of the cuvette and free of air bubbles, electroporation was performed at 1800 V and 1 mm spacing. Immediately after completion, 1000 μL of 37°C SOC medium was added. The mixture was removed from the cuvette and incubated at 37°C with shaking at 180 rpm for 90 min. A 10-fold serial dilution was performed using 2×YT liquid medium, resulting in six dilutions (each diluted 10^6 times). 1 10 2 10 3 10 4 10 5 10 6 (Multiple times). For each gradient, 5 μL was evenly added dropwise to 2×YT-GA solid medium, air-dried, and then incubated overnight at 37°C. The number of colonies on the gradient dilution plates was counted, and the ligation efficiency was calculated. The ligation efficiency formula is:

[0258] E(pfu / μg)=N×D×10;

[0259] Where E is the competent cell efficiency (unit: pfu / μg), D is the dilution factor, and N is the number of single clones on the plate at the corresponding dilution factor.

[0260] Repeat the above method for 100 electroporation transformation reactions. Spread the revived bacterial culture evenly onto 100 2×YT-GA2 45mm square agar plates, air-dry, and incubate overnight at 37°C upside down. Take one of the overnight incubated square plates, add 6 mL of 2×YT liquid medium to the surface of each plate, and gently scrape off the colonies from all 100 plates with a spreader. Collect the bacterial culture into a 50 mL centrifuge tube, add glycerol to a final concentration of 20%, and the bacterial library is obtained.

[0261] Take 10 μL of bacterial culture 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 Total bacterial OD600, M OD600 This is the measured OD600.

[0264] 1.4 Phage Library Construction:

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

[0266]

[0267] Where V is the volume of the transferred bacterial culture (in μL), and OD600 is the total OD600 of the constructed bacterial library.

[0268] Transfer the culture medium to 100 mL of 2×YT-GA liquid medium to achieve an initial OD600 of 0.1. Incubate at 37°C and 250 rpm in a shaker until the OD600 reaches 0.5–0.55. Calculate and add helper phage M13K07 according to the following formula to achieve a bacteria:phage ratio of 1:20. The formula for calculating the amount of phage to add is:

[0269]

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

[0271] Incubate at 37°C and 220 rpm for 30 min in a constant temperature shaker. Centrifuge at 3200g for 5 min to collect TG1 bacteria, discard the supernatant, resuspend the precipitate and transfer it to 100 mL of 2×YT-AK liquid medium, and incubate overnight at 30°C and 250 rpm in a constant temperature shaker.

[0272] Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 g for 30 min at 4 °C. Collect the supernatant and add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl (4 °C). Mix thoroughly and incubate on ice for 30 min. Centrifuge at 4000 rpm for 30 min at 4 °C, discard the supernatant, and invert the tube on paper for 2 min. Resuspend the precipitate in 1 mL PBS and centrifuge at 12000 rpm for 20 min at 4 °C. Collect the supernatant and add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl solution. Mix well and incubate on ice for 10 min. Centrifuge at 12000 rpm for 10 min at 4 °C, discard the supernatant, and resuspend the precipitate in 1 mL PBS. Centrifuge at 12000 rpm for 2 min at 4 °C, collect the supernatant (this is the phage library), add glycerol to a final concentration of 20%, and store at -80 °C.

[0273] The titer of the phage library was determined as follows: The phage library was added to 10 mL of 2×YT liquid medium and incubated at 37°C and 250 rpm for approximately 45–60 min, until the OD600 reached 0.5–0.55. 10 μL of the cultured phage library was then serially diluted 10-fold (a total of 13 dilutions). For each dilution, 90 μL of untreated TG1 bacterial culture was added, mixed, and incubated at 37°C for 20 min. Then, 5 μL from each dilution was added dropwise to 2×YT-GA solid medium, air-dried, and incubated overnight at 37°C. The phage cells were counted, and the number of phage particles per milliliter of phage solution was calculated according to the phage library titer formula.

[0274] T(pfu / ml) = N × D × 400;

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

[0276] Screening for 1.5gp350 protein-specific antibodies

[0277] Antibodies that specifically bind to the gp350 protein were screened using an antigen solid-phase adsorption screening method.

[0278] The specific steps are as follows:

[0279] Dissolve 50 μg of the gp350 protein obtained above in 2 mL PBS and coat the immunotubes overnight at 4°C (positive tubes). Simultaneously coat the tubes with a negative control protein (50 μg BSA) as a negative control. Discard the coated protein, wash three times with 2 mL PBS, and then block with 2 mL 3% BSA (dissolved in PBST) at room temperature for 2 h. Discard the blocking solution, take 100 μL of the obtained phage library, dilute with 2 mL PBS, and incubate in an immunotube for 1 h. Discard the liquid, wash five times with 2 mL PBST for 5 min each time. Then wash five times with 2 mL PBS for 5 min each time. Simultaneously, take 1 mL of overnight TG1 saturated bacterial culture and add it to 100 mL of 2×YT liquid medium, incubate at 37°C and 150 rpm until OD600 = 0.5 (approximately 1.5 h). Discard the washing solution, and wash with 1 mL of 0.1 mg / mL Trypsin at room temperature for 30 min. Add 1 mL of the eluent to 10 mL of the cultured TG1 bacterial suspension (OD = 0.5), infect at 37°C and 150 rpm for 30 min; then centrifuge at 4°C and 3000 g for 10 min. Resuspend the pellet in 1 mL of 2×YT liquid medium. Spread 1 mL of the resuspended product from the positive immunotherapy tube evenly onto a 245 mm × 245 mm 2×YT solid culture dish and incubate overnight (16–20 h) at 37°C. Measure the phage titer of the positive and negative control tubes using the same method as described in the previous example.

[0280] Based on the phage titer comparison results, 5 mL of 2×YT liquid medium was added to the culture dishes with high phage titers after overnight culture. All colonies were scraped off with a spreader, and a sub-phage library was prepared according to the method described in the above examples. The above operations were repeated until an antibody with the highest affinity for gp350 (compared to the negative control) was screened.

[0281] Unless otherwise specified, the rotation speed for antigen coating, blocking, incubation, washing, and elution processes is 15 rpm.

[0282] Use ELISA to detect the antibody with the highest affinity.

[0283] Take 10 μL of the bacterial culture containing the antibody with the highest affinity for gp350 and dilute it with 1 ml of 2×YT liquid medium. Streak the colonies onto a 2×YT-GA solid culture plate and incubate overnight at 37°C for 16–20 h. Pick 192 single colonies and transfer them to a 96-well plate (each well containing 200 μL of 2×YT-GA liquid), and incubate at 37°C until saturation. Transfer 2 μL of the saturated bacterial culture to a new 96-well plate (each well containing 200 μL of 2×YT-A liquid), adjusting the initial OD to approximately 0.03, and incubate at 37°C for 2.5–3 h until the OD reaches approximately 0.5. Add 0.1 μL of the above helper phage to each well and infect at 37°C for 30 min. Add 0.2 μL of kanamycin to each well and incubate overnight at 30°C. Coat the protein (- / +) plate overnight. Centrifuge the overnight cultured 96-well plate at 4°C and 3400g for 5 min. Discard the liquid in the ELISA plate and wash once with 350 μL PBS. Block with 350 μL 3% BSA (PBST) at 37°C for 1 h. Discard the blocking solution, wash once with 350 μL PBST, and tap the plate to remove the liquid. Add 140 μL 3% BSA (PBST) to each well, then add 60 μL of the above phage library expression antibody (the antibody with the highest affinity, as the primary antibody), and incubate at 37°C for 1 h. Discard the liquid (primary antibody), wash 5 times with 350 μL PBST, and tap the plate. Add 100 μL M13 Antibody (HRP) as the secondary antibody (1:8000 ratio relative to the blocking solution), incubate at 37°C for 1 h, discard the liquid (secondary antibody), wash 5 times with 350 μL PBST, and tap the plate. Add 100 μL of TMB to the light-protected solution for 2-3 minutes, then terminate the reaction with 100 μL of dilute hydrochloric acid (concentrated hydrochloric acid: water = 1:12). Read the OD450 and OD630 values ​​of the ELISA plate. In this embodiment, the inventors ultimately obtained two antibodies with the best performance, which were named 1A12 and 2E4, respectively.

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

[0285] By linking the variable region of the antibody heavy chain upstream to the CMV fragment, downstream to the constant region of human IgG1, and the polyA fragment, the complete heavy chain fragment can be expressed. Similarly, by linking the variable region of the antibody light chain upstream to the CMV fragment, downstream to the constant region of the light chain κ / λ, and the polyA fragment, the complete light chain fragment can be expressed. Co-transfection of plasmids containing the full-length sequences of both the antibody heavy and light chains into 293T cells achieves antibody expression, and protein A beads can be used for antibody purification.

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

[0287]

[0288]

[0289] The underlined portion of the sequence represents the amino acid sequence of the heavy chain variable region (SEQ ID NO: 14). The underlined and bolded portions represent 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) within the heavy chain variable region (IMGT definition scheme). The italicized portion represents the signal peptide. Amino acids 144–473 in SEQ ID NO: 13 constitute the heavy chain constant region. * indicates a stop codon.

[0290] Table 11 shows CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain, which are defined by other CDR schemes.

[0291] Table 11 shows CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region, defined by other CDR schemes.

[0292]

[0293] The gene encoding the full-length heavy chain 1A12 consists of 1422 bases, specifically:

[0294]

[0295]

[0296] The underlined portion of the sequence represents the nucleotide sequence of the heavy chain variable region. The underlined and bolded portions represent the nucleotide sequences of the three complementary regions CDR-H1, CDR-H2, and CDR-H3 within the heavy chain variable region (IMGT definition scheme). The first three bases at the 5' end and the last three bases at the 3' end are the start and stop codons, respectively. The italicized portion represents the signal peptide.

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

[0298]

[0299] In this sequence, the underlined portion represents the amino acid sequence of the light chain variable region (SEQ ID NO: 28). The underlined and bolded portions represent the amino acid sequences (IMGT definition scheme) of the three complementarity-determining regions CDR-L1 (SEQ ID NO: 29), CDR-L2 (GAS), and CDR-L3 (SEQ ID NO: 30) within the light chain variable region. The italicized portion represents the signal peptide. Amino acids 128–233 in SEQ ID NO: 27 constitute the light chain constant region. * indicates a stop codon.

[0300] Table 12 shows CDR-H1, CDR-H2, and CDR-H3 in the variable region of this light chain, which are defined by other CDR schemes.

[0301] Table 12 CDR-H1, CDR-H2, and CDR-H3 in the variable region of light chains, defined by other CDR schemes.

[0302]

[0303] The gene encoding the full-length light chain 1A12 consists of 702 bases, specifically:

[0304]

[0305]

[0306] The underlined portion of the sequence represents the nucleotide sequence of the light chain variable region. The underlined and bolded portions represent the nucleotide sequences of the three complementary regions CDR-L1, CDR-L2, and CDR-L3 within the light chain variable region. The first three bases at the 5' end and the last three bases at the 3' end are the start and stop codons, respectively. The italicized portion represents the signal peptide.

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

[0308] By linking the variable region of the antibody heavy chain upstream to the CMV fragment, downstream to the constant region of human IgG1, and the polyA fragment, the complete heavy chain fragment can be expressed. Similarly, by linking the variable region of the antibody light chain upstream to the CMV fragment, downstream to the constant region of the light chain κ / λ, and the polyA fragment, the complete light chain fragment can be expressed. Co-transfection of plasmids containing the full-length sequences of both the antibody heavy and light chains into 293T cells achieves antibody expression, and protein A beads can be used for antibody purification.

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

[0310]

[0311] The underlined portion of the sequence represents the amino acid sequence of the heavy chain variable region (SEQ ID NO: 38). The underlined and bolded portions represent 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) within the heavy chain variable region (IMGT definition scheme). The italicized portion represents the signal peptide. Amino acids 139–468 in SEQ ID NO: 37 constitute the heavy chain constant region. * indicates a stop codon.

[0312] Table 13 shows CDR-H1, CDR-H2, and CDR-H3, which define CDR schemes in the variable region of the heavy chain.

[0313] Table 13 shows CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region, defined by other CDR schemes.

[0314]

[0315] The gene encoding the full-length heavy chain 2E4 consists of 1408 bases, specifically:

[0316]

[0317]

[0318] The underlined portion of the sequence represents the nucleotide sequence of the heavy chain variable region. The underlined and bolded portions represent the nucleotide sequences of the three complementary regions CDR-H1, CDR-H2, and CDR-H3 within the heavy chain variable region (IMGT definition scheme). The first three bases at the 5' end and the last three bases at the 3' end are the start and stop codons, respectively. The italicized portion represents the signal peptide.

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

[0320]

[0321] In this sequence, the underlined portion represents the amino acid sequence of the light chain variable region (SEQ ID NO: 52). The underlined and bolded portions represent the amino acid sequences (IMGT definition scheme) of the three complementarity-determining regions CDR-L4 (SEQ ID NO: 53), CDR-L5 (RNN), and CDR-L6 (SEQ ID NO: 54) within the light chain variable region. The italicized portion represents the signal peptide. Amino acids 130–235 in SEQ ID NO: 51 constitute the light chain constant region. * indicates a stop codon.

[0322] Table 14 shows CDR-H1, CDR-H2, and CDR-H3, which define CDR schemes in the variable region of the light chain.

[0323] Table 12 CDR-H1, CDR-H2, and CDR-H3 in the variable region of light chains, defined by other CDR schemes.

[0324]

[0325]

[0326] The gene encoding the full-length light chain 2E4 consists of 708 bases, specifically:

[0327]

[0328] The underlined portion of the sequence represents the nucleotide sequence of the light chain variable region. The underlined and bolded portions represent the nucleotide sequences of the three complementary regions CDR-L1, CDR-L2, and CDR-L3 within the light chain variable region. The first three bases at the 5' end and the last three bases at the 3' end are the start and stop codons, respectively. The italicized portion represents the signal peptide.

[0329] Effect Example 1: Effect of Monoclonal Antibody 1A12

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

[0331] To determine the affinity of antibody 1A12, this effective example uses the ELISA method. First, gp350 protein was diluted with PBS buffer and added to the microplate at 100 ng per well, incubated overnight at 4°C. The next day, unbound antigen was discarded, and the plate was washed three times with PBS T buffer, 3 minutes each time. Then, 5% BSA solution was added for blocking, and the plate was incubated at 37°C for 1-2 hours, followed by another wash with PBST. Next, antibody 1A12 was serially diluted and added to the corresponding wells, incubated at 37°C for 1-2 hours to promote antibody binding to the coated gp350 protein (BSA solution served as a negative control). After incubation, the plate was washed three times with PBST, and HRP-labeled enzyme-labeled secondary antibody (such as anti-human IgG secondary antibody) was added, incubated at 37°C for 1 hour. Subsequently, the plate was washed three times with PBST, TMB chromogenic substrate was added, and the plate was incubated at room temperature for 5-15 minutes until the color reached the expected intensity, at which point the reaction was terminated with dilute hydrochloric acid. Finally, the absorbance (OD value) of each well was measured at a wavelength of 450 nm, and the results were plotted as follows: Figure 1 The IC50 of the 1A12 antibody was 0.017 μg / mL, indicating that the 1A12 antibody has a high affinity for the gp350 antigen.

[0332] 2. The affinity of antibody 1A12 was determined using biomembrane interference (BLI) technique.

[0333] BLI can be performed according to conventional methods in the art. In this embodiment, the specific operation is as follows: the biosensor (Sartorius, Germany) is used... The SA probe was equilibrated in a buffer solution (a mixture of KB buffer, 0.1% BSA, and 0.02% Tween 20). Then, it was removed and immersed in a solution containing 5 μg / mL gp350-Biotin (biotin-labeled gp350 protein). The gp350 antigen in the solution bound to the SA (streptavidin) bioprobe surface, increasing its surface membrane thickness. A baseline was then established by immersing a biosensor with a known concentration of cured antigen in the buffer solution. By immersing the biosensor with the cured known concentration of antigen in a sample solution containing 1A12 antibody for approximately 100 seconds, the specific binding between antigen and antibody led to an increase in membrane thickness. Immersing the biosensor with the bound 1A12 antibody in the buffer further dissociated it, causing the test antibody (1A12 antibody) to detach from the biosensor surface, resulting in a decrease in membrane thickness. The kinetic constants of the test sample (1A12 antibody) were obtained by real-time monitoring of the biosensor biomembrane thickness during the experiment. Results are as follows: Figure 2 As shown: the KD(M) of the 1A12 antibody is 3.415E-10, indicating that the 1A12 antibody has a high affinity for the gp350 antigen.

[0334] 3. Neutralizing activity of 1A12 antibody

[0335] (1) Preparation of EBV virus:

[0336] 1) CNE2 cells infected with EBV-GFP were cultured in an incubator at 37°C (5% CO2) using RPMI 1640 + 5% FBS. (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). When the cells reached 90% confluence (10cm dish), TPA was added to a final concentration of 20 ng / mL and NaB (sodium butyrate) to induce induction. The medium was changed after 12 hours.

[0337] 2) Collect the culture supernatant 48–72 h after changing the medium to separate and purify the virus. Directly aspirate the supernatant, centrifuge it, and filter it through a 0.45 μm filter. After concentration, resuspend it in serum-free RPMI 1640. Immediately use the resuspended virus for infection or store it at -80°C.

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

[0339] 1) Take the 1A12 monoclonal antibody from the above examples and adjust its concentration to 2 mg / mL. Add 60 μL of RPMI 1640 medium to each well of a new 96-well plate. Add 90 μL of 100 μg / mL 1A12 antibody diluted with RPMI 1640 to the first well (the first well does not contain RPMI 1640 medium).

[0340] 2) After a 3-fold serial dilution (for serial dilution, 30 μL will be aspirated from the first well and added to the second well, and so on, with 30 μL aspirated from the last well and discarded, resulting in a final volume of 60 μL per well); add 60 μL of virus diluent to each well (the virus is diluted with DMEM medium to a titer of approximately 4*10). 6 After incubating at 37°C for 2 hours, add 1*10 mL to each well. 6 Raji cells were incubated at 37 degrees Celsius for 48 hours before being analyzed.

[0341] 3) Raji cells were aspirated to prepare a cell suspension. 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 in the number of GFP-positive cells in the antibody-treated group compared with the infection control group (with an equal volume of RPMI 1640 added). The IC50 of the 1A12 monoclonal antibody was calculated using Prism. The 2G4 antibody was used as a negative control antibody (2G4 is an anti-Ebola virus antibody, which has been published 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 was 0.27 μg / mL, while the control antibody had no neutralizing activity; monoclonal antibody 1A12 could significantly inhibit EBV infection of B cells.

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

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

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

[0346] See results Figure 4 The IC50 of the 2E4 antibody was 0.026 μg / mL, indicating that the 2E4 antibody has a high affinity for the gp350 antigen.

[0347] 2. The affinity of antibody 2E4 was determined using biomembrane interference (BLI) technique.

[0348] Experimental method and effect are the same as in Example 1

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

[0350] 3.2 Neutralizing activity of E4 antibody

[0351] Preparation of EBV virus:

[0352] Same effect as Example 1.

[0353] 4.2 Assay for the neutralizing activity of E4 monoclonal antibody in B cells:

[0354] 1) Take the 2E4 monoclonal antibody from the above examples and adjust its concentration to 2 mg / mL. Add 60 μL of RPMI 1640 medium to each well of a new 96-well plate. Add 90 μL of 100 μg / mL 2E4 antibody diluted with RPMI 1640 to the first well (the first well does not contain RPMI 1640 medium).

[0355] 2) After a 3-fold serial dilution (for serial dilution, 30 μL will be aspirated from the first well and added to the second well, and so on, with 30 μL aspirated from the last well and discarded, resulting in a final volume of 60 μL per well); add 60 μL of virus diluent to each well (the virus is diluted with DMEM medium to a titer of approximately 4*10). 6 After incubating at 37°C for 2 hours, add 1*10 mL to each well. 6 Raji cells were incubated at 37 degrees Celsius for 48 hours before being analyzed.

[0356] 3) Raji cells were aspirated to prepare a cell suspension. 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 in the number of GFP-positive cells in the antibody-treated group compared to the infection control group (which had an equal volume of RPMI 1640 added). The IC50 of the 2E4 monoclonal antibody was calculated 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 was 0.054 μg / mL, while the control antibody had no neutralizing activity; monoclonal antibody 2E4 could significantly inhibit EBV infection of B cells.

Claims

1. A monoclonal antibody against EB virus gp350 protein or its antigen-binding fragment, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain; The monoclonal antibody is selected from either A) or B); A） The heavy chain includes: The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 14; The light chain comprises: The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having the amino acid sequence shown in SEQ ID NO: 28; B） The heavy chain includes: The heavy chain variable region includes CDR-H4, CDR-H5 and CDR-H6 of the heavy chain variable region, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38; The light chain comprises: The light chain variable region includes CDR-L4, CDR-L5 and CDR-L6 of the light chain variable region, the light chain variable region having the amino acid sequence shown in SEQ ID NO:

52.

2. The monoclonal antibody or its antigen-binding fragment 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. The CDRs are defined using the Kabat scheme; or The amino acid sequences of 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 CDR-L2 is GAS. The CDR is defined according to the IMGT definition scheme. 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. The CDRs are defined according to the Chothia 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. The CDR is defined using 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. The CDRs are defined using the Kabat 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 CDR is 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 CDR is defined according to the Chothia 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. The CDR is defined using the Contact definition scheme.

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

4. A recombinant protein comprising: the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; and Optional tag sequences to assist in expression and / or purification.

5. A biological material relating to the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the recombinant protein as described in claim 4, wherein the biological material comprises at least one of h1) to h16): h1) A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 3, or a recombinant protein of claim 4; h2) contains an expression cassette containing the nucleic acid molecule described in h1); h3) A carrier containing the nucleic acid molecule described in h1); h4) A carrier containing the expression box described in h2); h5) A transgenic cell line containing the nucleic acid molecules described in h1); h6) Transgenic cell lines containing the expression cassette described in h2); h7) A transgenic cell line containing the vector described in h3); h8) A transgenic cell line containing the vector described in h4); h9) Microorganisms containing the nucleic acid molecules described in h1); h10) Microorganisms containing the expression cassette described in h2); h11) contains microorganisms containing the carrier described in h3); h12) contains microorganisms that carry the vector described in h4); h13) contains viruses containing the nucleic acid molecules described in h1); h14) A virus containing the expression cassette described in h2); h15) contains a virus with the vector described in h3); h16) contains the virus of the vector described in h4).

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

7. The application of at least one of (1) to (4) in the preparation of the product; (1) The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; (2) The recombinant protein according to claim 4; (3) The biomaterial as described in claim 5; (4) The coupling compound according to claim 6; The product includes at least one of reagents, detection plates, reagent kits, and detection chips; The reagent, detection plate, detection chip, or kit has at least one function among j1) to j3): j1) Detect the presence or level of gp350 protein in the sample; j2) Detection of EB virus; j3) Diagnose diseases caused by EB virus infection.

8. The use of at least one of (1) to (4) in the preparation of a drug; (1) The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; (2) The recombinant protein according to claim 4; (3) The biomaterial as described in claim 5; (4) The coupling compound according to claim 6; The drug is one of i1) to i2): i1) Medications to prevent EB virus infection; i2) Medications for the treatment and / or prevention of diseases caused by EB virus infection.

9. A product comprising at least one of k1) to k3): k1) The monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; k2) The recombinant protein according to claim 4; k3) The coupling as described in claim 6; The product includes at least one of reagents, test plates, reagent kits, and test chips.

10. A drug comprising at least one of (l1) to (l4): (l1) The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3; (l2) The recombinant protein according to claim 4; (l3) The biomaterial as described in claim 5; (l4) The coupling as described in claim 6; The drug also contains a pharmaceutically acceptable carrier.

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

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