Monoclonal antibody targeting ebv gB antigen and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a monoclonal antibody targeting an EB virus gB antigen and application thereof. The application provides a monoclonal antibody or antigen-binding fragment thereof against an EB virus gB protein, including 4C1 and 4B3, wherein the 4C1 has a higher affinity (KD(M)=1.0E ‑9 ) to the gB protein, and the 4B3 has a higher affinity (KD(M)=3.968E ‑9 ) to the gB protein, and the two monoclonal antibodies or antigen-binding fragments thereof can be used for detecting the presence or level of the gB protein in a sample, detecting the EB virus, diagnosing a disease caused by the EB virus infection, preventing the EB virus infection, and / or treating and / or preventing a disease caused by the EB virus infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a monoclonal antibody targeting EB virus gB antigen and application thereof. BACKGROUND

[0002] Epstein-Barr virus (EBV) is listed as a class I carcinogen by the World Health Organization, and is the first human carcinogenic virus that has been confirmed. Its infection rate in the population is more than 95%. Studies estimate that tumors caused by EBV infection account for 1-2% of all human tumors, and about 200,000 new cases of cancer are associated with EBV each year. EBV can infect epithelial cells and lymphocytes and is closely related to a variety of tumors, including nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphoma, NKT cell lymphoma, and primary effusion lymphoma. In addition, reactivation of EBV sometimes causes fatal lymphoproliferative disease (LPD) in immunocompromised AIDS patients or post-transplant patients. However, there is currently no specific therapy or effective vaccine for EBV infection, mainly due to the lack of understanding of the immunity to EBV infection and related diseases.

[0003] EBV encodes a variety of envelope glycoproteins that determine cell tropism and complete attachment and entry of host cells. Glycoprotein B (gB) is conserved in all herpes viruses and is a fusion protein necessary for viral entry into cells. EBV gB forms a core membrane fusion mechanism with glycoprotein H and glycoprotein L heterodimer (gHgL) and drives membrane fusion through conformational changes. EBV gB directly interacts with neuropilin 1 (NRP1), which promotes EBV infection of epithelial cells through NRP1-promoted internalization and fusion. In a nanovaccine targeting gB, studies have shown that it can significantly inhibit EBV infection in humanized mice and non-human primates. Therefore, gB has become a popular target for the development of monoclonal antibodies and vaccines. However, to date, there has been a lack of research on humanized monoclonal antibodies against gB.

[0004] Monoclonal antibodies can be produced in large quantities, and based on their high affinity and high specificity in binding to antigens, they can greatly reduce adverse reactions in clinical applications. These antibody molecules can also be modified to increase their antiviral efficacy. Antibodies are very promising means in the treatment of infectious diseases due to their specificity and flexibility of use, but to date, no humanized monoclonal antibody against EBV envelope glycoprotein has been marketed. Therefore, the development of humanized monoclonal antibodies against EBV will provide more effective prevention and treatment means for diseases related to EBV infection. SUMMARY

[0005] The first aspect of the present application aims to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0006] The second aspect of the present application aims to provide a recombinant protein.

[0007] The third aspect of the present application aims to provide a biological material related to the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0008] The fourth aspect of the present application aims to provide a conjugate.

[0009] The fifth aspect of the present application aims to provide use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the biological material of the third aspect of the present application, and / or the conjugate of the fourth aspect of the present application in the preparation of a product.

[0010] The sixth aspect of the present application aims to provide a kit.

[0011] The seventh aspect of the present application aims to provide a medicament.

[0012] The eighth aspect of the present application aims to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application.

[0013] In order to achieve the above-mentioned objects, the technical solutions adopted by the present application are as follows:

[0014] The first aspect of the present application provides a monoclonal antibody or antigen-binding fragment thereof against EBV gB, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain.

[0015] In some embodiments of the present application, the monoclonal antibody or antigen-binding fragment thereof comprises any one of A) or B).

[0016] A) is designated as monoclonal antibody or antigen-binding fragment thereof 4C1;

[0017] B) is designated as monoclonal antibody or antigen-binding fragment thereof 4B2.

[0018] A) in the monoclonal antibody or antigen-binding fragment thereof 4C1; the heavy chain comprises:

[0019] a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region, the heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 14;

[0020] the light chain comprises:

[0021] a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, the light chain variable region having an amino acid sequence shown in SEQ ID NO: 26.

[0022] The amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L3 are shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 28, respectively, and the amino acid sequence of CDR-L2 is DNH, and the CDRs are defined according to the IMGT definition scheme.

[0023] Preferably, the amino acid sequence of the heavy chain variable region comprises:

[0024] a1-A) SEQ ID NO. 14; or

[0025] a2-A) an amino acid sequence which is derived from SEQ ID NO. 14 by substitution and / or deletion and / or addition of one or several amino acids and which has the same function as the protein shown in SEQ ID NO. 14; or

[0026] a3-A) an amino acid sequence which 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 which has the same function as the protein shown in SEQ ID NO. 14.

[0027] The amino acid sequence of the light chain variable region comprises:

[0028] b1-A) SEQ ID NO: 26; or

[0029] b2-A) an amino acid sequence which is derived from SEQ ID NO: 26 by substitution and / or deletion and / or addition of one or several amino acids and which has the same function as the protein shown in SEQ ID NO. 26; or

[0030] b3-A) an amino acid sequence which 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. 26 and which has the same function as the protein shown in SEQ ID NO. 26.

[0031] Preferably, the heavy chain further comprises a heavy chain constant region; and the light chain further comprises a light chain constant region.

[0032] B) In the monoclonal antibody or antigen-binding fragment thereof 4B2, the heavy chain comprises:

[0033] a heavy chain variable region comprising CDR-H4, CDR-H5 and CDR-H6 of said heavy chain variable region, said heavy chain variable region having the amino acid sequence of SEQ ID NO: 36;

[0034] said light chain comprises:

[0035] a light chain variable region comprising CDR-L4, CDR-L5 and CDR-L6 of said light chain variable region, said light chain variable region having the amino acid sequence of SEQ ID NO: 46.

[0036] Preferably, the amino acid sequences of CDR-H4, CDR-H5, CDR-H6, CDR-L4, CDR-L6 are in the order of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 47, SEQ ID NO: 48, respectively, and CDR-L5 is KVS, said CDRs are defined in the IMGT definition scheme.

[0037] Preferably, the amino acid sequence of said heavy chain variable region comprises:

[0038] a1-B) SEQ ID NO: 36; or

[0039] a2-B) an amino acid sequence which is derived from SEQ ID NO: 36 by one or several substitutions and / or deletions and / or additions of amino acids and which has the same function as the protein of SEQ ID NO: 36; or

[0040] a3-B) an amino acid sequence which 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: 36 and which has the same function as the protein of SEQ ID NO: 36;

[0041] Preferably, the amino acid sequence of said light chain variable region comprises:

[0042] b1-B) SEQ ID NO: 46; or

[0043] b2-B) an amino acid sequence which is derived from SEQ ID NO: 46 by one or several substitutions and / or deletions and / or additions of amino acids and which has the same function as the protein of SEQ ID NO: 46; or

[0044] b3-B) an amino acid sequence having 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: 46 and having the same function as the protein shown in SEQ ID NO: 46.

[0045] Preferably, the heavy chain further comprises a heavy chain constant region; the light chain further comprises a light chain constant region.

[0046] In some embodiments of the application, 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] c1) SEQ ID NO: 18; or

[0049] c2) an amino acid sequence of c1) having one or several substitutions and / or deletions and / or additions of amino acid(s) and having the same function as the protein of the amino acid sequence of c1); or

[0050] c3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology to the amino acid sequence of c1) and having the same function as the protein of the amino acid sequence of c1).

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

[0052] d1) SEQ ID NO: 29; or

[0053] d2) an amino acid sequence of d2) having one or several substitutions and / or deletions and / or additions of amino acid(s) and having the same function as the protein of the amino acid sequence of d1); or

[0054] d3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology to the amino acid sequence of d1) and having the same function as the protein of the amino acid sequence of d1).

[0055] In some embodiments of the application, the monoclonal antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a bispecific antibody, a multispecific antibody.

[0056] In some embodiments of the application, the amino acid sequence of the EBV gB comprises:

[0057] e1) an amino acid sequence consisting of amino acids 1-661 of SEQ ID NO: 4; or

[0058] e2) an amino acid sequence having one or several substitutions and / or deletions and / or additions of amino acids to the amino acid sequence of e1) and having the same function as the protein of the amino acid sequence of e1); or

[0059] e3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology to the amino acid sequence of e1) and having the same function as the protein of the amino acid sequence of e1).

[0060] In a second aspect of the present application, a recombinant protein is provided, comprising: the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application; and optionally a tag sequence to facilitate expression and / or purification.

[0061] Preferably, the tag sequence is selected from at least one of the group consisting of: His tag, GGGS sequence, FLAG tag; further His tag; and more further 6xHis tag.

[0062] In a third aspect of the present application, a biological material related to the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application, or the recombinant protein of the second aspect of the present application is provided, the biological material comprising at least one of f1) to f16):

[0063] f1) a nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application, or the recombinant protein of the second aspect of the present application;

[0064] f2) an expression cassette comprising the nucleic acid molecule of f1);

[0065] f3) a vector comprising the nucleic acid molecule of f1);

[0066] f4) a vector comprising the expression cassette of f2);

[0067] f5) a transgenic cell line comprising the nucleic acid molecule of f1);

[0068] f6) a transgenic cell line comprising the expression cassette of f2);

[0069] f7) a transgenic cell line comprising the vector of f3);

[0070] f8) a transgenic cell line comprising the vector of f4);

[0071] f9) a microorganism comprising the nucleic acid molecule of f1);

[0072] f10) a microorganism comprising the expression cassette of f2);

[0073] f11) a microorganism comprising the vector of f3);

[0074] f12) a microorganism comprising the vector of f4);

[0075] f13) a virus comprising the nucleic acid molecule of f1);

[0076] f14) a virus comprising the expression cassette of f2);

[0077] f15) a virus comprising the vector of f3);

[0078] f16) a virus comprising the vector of f4).

[0079] Preferably, the transgenic cell line does not comprise reproductive material.

[0080] Preferably, the nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment thereof according to the first aspect of the application comprises a nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen binding fragment thereof according to the first aspect of the application and a nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen binding fragment thereof according to the first aspect of the application.

[0081] In some embodiments of the application, the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody or antigen binding fragment thereof according to the first aspect of the application comprises a nucleotide sequence selected from the group consisting of:

[0082] A) Monoclonal antibody or antigen binding fragment 4C1 related:

[0083] a211-A) the nucleotide sequence as shown in SEQ ID NO: 20; or

[0084] a212-A) a nucleotide sequence which is identical to the nucleic acid molecule as shown in SEQ ID NO: 20, except for one or several nucleotide substitutions and / or deletions and / or additions, and which has the same function as the nucleic acid molecule as shown in SEQ ID NO: 20; or

[0085] a213-A) a nucleotide sequence which has 80%, 85% or 90% or more homology with SEQ ID NO: 20 and which has the same function as the nucleic acid molecule as shown in SEQ ID NO: 20.

[0086] B) Monoclonal antibody or antigen binding fragment 4B2 related:

[0087] a211-B) the nucleotide sequence as shown in SEQ ID NO: 41; or

[0088] a212-B) a nucleotide sequence that differs from SEQ ID NO: 41 by one or several nucleotide substitutions and / or deletions and / or additions, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 41; or

[0089] a213-B) a nucleotide sequence that has 80%, 85% or 90% or more homology with SEQ ID NO: 41, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 41.

[0090] In some embodiments of the present application, the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application comprises a nucleotide sequence of:

[0091] A) Monoclonal antibody or antigen-binding fragment thereof 4C1 related:

[0092] a221-A) a nucleotide sequence as shown in SEQ ID NO: 31; or

[0093] a222) a nucleotide sequence that differs from SEQ ID NO: 31 by one or several nucleotide substitutions and / or deletions and / or additions, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 31; or

[0094] a223) a nucleotide sequence that has 80%, 85% or 90% or more homology with SEQ ID NO: 31, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 31.

[0095] B) Monoclonal antibody or antigen-binding fragment thereof 4B1 related:

[0096] a221-B) a nucleotide sequence as shown in SEQ ID NO: 50; or

[0097] a222-B) a nucleotide sequence that differs from SEQ ID NO: 50 by one or several nucleotide substitutions and / or deletions and / or additions, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 50; or

[0098] a223-B) a nucleotide sequence that has 80%, 85% or 90% or more homology with SEQ ID NO: 50, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 50.

[0099] The fourth aspect of the present application provides a conjugate comprising at least one of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application and the recombinant protein of the second aspect of the present application;

[0100] and a coupling moiety comprising at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme.

[0101] Preferably, the detectable label is selected from a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0102] Preferably, the conjugate is selected from a fluorescent substance, a chemiluminescent label, a colored substance, a radioisotope, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme, a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0103] In a fifth aspect of the present application, 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 is provided.

[0104] The product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip.

[0105] Preferably, the drug has at least one of the following functions g1) to g2):

[0106] g1) preventing EB virus infection;

[0107] g2) treating and / or preventing a disease caused by EB virus infection.

[0108] Preferably, the reagent, detection plate, detection chip, or kit has at least one of the following functions h1) to h3):

[0109] h1) detecting the presence or level of gB protein in a sample;

[0110] h2) detecting EB virus;

[0111] h3) diagnosing a disease caused by EB virus infection.

[0112] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disease, infectious mononucleosis.

[0113] Preferably, the drug comprises a vaccine.

[0114] In a sixth aspect, the present application provides a product comprising at least one of i1) to i3):

[0115] i1) a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application;

[0116] i2) a recombinant protein of the second aspect of the present application;

[0117] i3) a conjugate of the fourth aspect of the present application.

[0118] The product comprises at least one of a reagent, a detection plate, a kit, a detection chip.

[0119] Preferably, the product has at least one of j1) to j3) functions:

[0120] j1) detecting the presence or level of gB protein in a sample;

[0121] j2) detecting Epstein-Barr virus;

[0122] j3) diagnosing a disease caused by Epstein-Barr virus infection.

[0123] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disease, infectious mononucleosis.

[0124] In a seventh aspect, the present application provides a medicament comprising at least one of k1) to k4):

[0125] k1) a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application;

[0126] k2) a recombinant protein of the second aspect of the present application;

[0127] k3) a biomaterial of the third aspect of the present application;

[0128] k4) a conjugate of the fourth aspect of the present application.

[0129] Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

[0130] Preferably, the medicament has at least one of g1) to g2) functions:

[0131] g1) preventing Epstein-Barr virus infection;

[0132] g2) treating and / or preventing a disease caused by Epstein-Barr virus infection.

[0133] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disease, infectious mononucleosis.

[0134] Preferably, the drug comprises a vaccine.

[0135] Preferably, a vaccine comprises at least one of k1) to k4) and an adjuvant:

[0136] k1) a monoclonal antibody or an antigen-binding fragment thereof of the first aspect of the present application;

[0137] k2) a recombinant protein of the second aspect of the present application;

[0138] k3) a biomaterial of the third aspect of the present application;

[0139] k4) a conjugate of the fourth aspect of the present application.

[0140] The eighth aspect of the present application is to provide a method for preparing the monoclonal antibody or the antigen-binding fragment thereof of the first aspect of the present application or the recombinant protein of the second aspect of the present application, which is obtained by culturing the transgenic cell line, microorganism or virus of the third aspect of the present application.

[0141] The beneficial effects of the present application are that the present application provides a monoclonal antibody or an antigen-binding fragment thereof against the gB protein of Epstein-Barr virus, including 4C1 and 4B3, wherein 4C1 has a higher affinity (KD(M) = 1.0E -9 ) to the gB protein and 4B3 has a higher affinity (KD(M) = 3.968E -9 ) to the gB protein, and the two monoclonal antibodies or antigen-binding fragments thereof can be used for detecting the presence or level of the gB protein in a sample, detecting Epstein-Barr virus, diagnosing diseases caused by Epstein-Barr virus infection, preventing Epstein-Barr virus infection and / or treating and / or preventing diseases caused by Epstein-Barr virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0142] Figure 1 It is the result of Coomassie brilliant blue staining after denaturation and running of 4C1 antibody.

[0143] Figure 2 It is the result of Coomassie brilliant blue staining after denaturation and running of 4B3 antibody.

[0144] Figure 3 It is the result of BLI detection of the affinity of gB protein and 4C1 antibody.

[0145] Figure 4 It is the detection effect of 4C1 antibody in Western Blot.

[0146] Figure 5 Application effect of 4C1 antibody in flow detection.

[0147] Figure 6 Application effect of 4C1 antibody in immunohistochemical detection.

[0148] Figure 7 Application effect of 4C1 antibody in tumor targeting.

[0149] Figure 8 BLI detection of gB protein and 4B3 antibody affinity results.

[0150] Figure 9 Detection effect of 4B3 antibody in Western Blot.

[0151] Figure 10 Application effect of 4B3 antibody in flow detection.

[0152] Figure 11 Application effect of 4B3 antibody in immunohistochemical detection.

[0153] Figure 12 Application effect of 4B3 antibody in tumor targeting. DETAILED DESCRIPTION

[0154] The concept and the technical effects produced by the present application will be described below in conjunction with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0155] Example 1 Preparation of EBV gB recombinant protein

[0156] Firstly, the plasmid of gB was constructed for protein purification in this embodiment, wherein the N terminal contains KOZAK sequence and CD5 signal peptide sequence, and the C terminal contains His tag and stop codon. The sequence of the plasmid expression fragment is as follows:

[0157]

[0158] The gB gene fragment is connected in the mammalian cell expression vector pcDNA3.1+(Invitrogen), and the specific method is as follows:

[0159] (1) Amplification of the gB protein gene:

[0160] 50 μL of NEB The gB protein gene is amplified by the PCR amplification reaction system (Table 1).

[0161] Table 1 PCR amplification reaction system of gB protein gene

[0162] Component Amount 5x Reaction Buffer 10 μL dNTPs 1 μL Upstream Primer 2.5 μL Downstream Primer 2.5 μL DNA Template 1 μL DNA Polymerase 0.5 μL High GC Enhancer (from Q5 kit from NEB) 10 μL ddH2O q.s. to 50 μL

[0163] The upstream primer is 5'-TGGTGGAATTCGCCACCATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCTGCCTCGGACAGACCCCAGAGCAGCCC-3'(SEQ ID NO: 2); and the downstream primer is 5'-TCGAGCGGCCGCTTAGTGGTGATGGTGATGATGTGACACTGCATTGTCCAAATCC-3'(SEQ ID NO: 3).

[0164] The amplified target fragment is analyzed by agarose gel electrophoresis, and the band with the correct molecular weight is cut under the ultraviolet lamp. The PCR product is recovered according to the instructions of the commercially available kit.

[0165] (2) Enzymatic digestion and ligation of the target fragment and the vector:

[0166] The vector uses the eukaryotic expression plasmid pcDNA3.1+. The target fragment and the vector are both digested by EcoR I and Not I (50 μL of the enzyme digestion reaction system (Table 2) is used for enzyme digestion).

[0167] Table 2 Enzyme digestion reaction system

[0168] Component Amount 10x CutSmart Buffer 5 μL EcoRI-HF 1 μL NotI-HF 1 μL Fragment or Vector of Interest 5 μg ddH2O q.s. to 50 μL

[0169] The enzyme digestion is carried out at 37°C, and the enzyme digestion time is 2-3 h. After the enzyme-digested vector is run on agarose gel, the gel recovery kit is used for recovery to obtain the linearized vector. The inserted fragment is directly recovered by the DNA purification kit. After recovery, 10 μL of the ligation reaction system (Table 3) is used for ligation.

[0170] Table 3 Ligation reaction system

[0171] Component Amount 5x CEII Buffer 2 μL CEII 1 μL Linearized Vector 100 ng Insert Fragment 100 ng ddH2O q.s. to 10 μL

[0172] The reaction condition is 37°C for 30 min. The ligation product is obtained

[0173] (3) Conversion of the ligation product and screening of positive clones:

[0174] The ligation product is added to a suspension of just-melted DH5a competent cells, which is placed on ice for 30 min, heated at 42°C for 90 s, and then placed back on ice for 5 min. 200 μL of LB medium is added, and the cells are recovered at 30°C for 40 min. The culture solution is aspirated and spread on an ampicillin-resistant LB plate, which is incubated at 37°C overnight.

[0175] A single clone is picked for sequencing verification. After the sequencing result is correct, the target recombinant plasmid is obtained. The plasmid is extracted in a large amount.

[0176] (4) Expression and extraction of the recombinant protein:

[0177] Human kidney epithelial cells 293F are cultured to obtain a cell suspension with a cell density of 1.5 x 10 6 The recombinant plasmid obtained in step (3) is transfected using PEI transfection reagent. Specifically, 2 mg of the recombinant plasmid is diluted with 25 mL of Union-293 medium; 6 mL of 1 mg / mL PEI is added to 25 mL of Union-293 medium. The plasmid and PEI are mixed well by shaking, and then placed at room temperature for 20 min before being added to the 293F cell suspension. After 5 days of culture, the cell supernatant is collected, centrifuged at 6000 rpm for 1 h at 4°C, and the cell pellet is discarded, thereby obtaining the supernatant containing the target protein.

[0178] The target protein is purified by affinity chromatography.

[0179] Since the obtained gB recombinant protein has a 6 x His tag at the C terminus, it can be affinity purified using a nickel column.

[0180] Specifically, the supernatant containing the target protein obtained in the above examples is filtered using 0.65 μm filter paper, combined with nickel column beads for 3 times, washed with 30 mM imidazole for 3 times, and eluted with 500 mM imidazole. Then, the target protein is further purified by gel filtration chromatography. Specifically, the protein eluted from the nickel column is concentrated to a volume of less than 1 mL using a 30 kD concentration tube. Then, the protein is purified using Superdex 200 Increase 10 / 300 GL.

[0181] The amino acid sequence of the finally obtained target protein is as follows:

[0182] QTPEQPAPPATTVQPTATRQQTSFPFRVCELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKIVTNILIYNGWYADSVTNRHEEKFSVESYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGWLIWTYRTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNTETFHERADSFHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQHWQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKTMHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSPPAPPAARGSTSAAVLRRRRRNAGNATTPVPPAAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETMCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIAGLRKDLDNAVSHHHHHH* (SEQ ID NO: 4).

[0183] Example 2 Construction of a phage antibody library

[0184] (1) Total RNA extraction:

[0185] The blood of nasopharyngeal carcinoma (NPC) patients is diluted with PBS at 1:1 (10 mL blood + 10 mL PBS), and then carefully overlaid on 15 mL lymphocyte separation medium per 20 mL diluent, keeping the layered interface, 2000 rpm at room temperature for 20 min, slow speed reduction, and the middle mononuclear cell layer is aspirated into a new 15 mL centrifuge tube, and PBS is added to 15 mL. 300g at room temperature for 20 min, the supernatant is transferred to a new 50 mL centrifuge tube, 300g at room temperature for 20 min. Remove the supernatant. Resuspend the cell pellets in the 15 mL and 50 mL centrifuge tubes with 1 mL Trizol, respectively. (That is, resuspend 2 mL Trizol per 10 mL blood)

[0186] Take 4 mL chloroform per 20 mL Trizol-cell pellet resuspension, shake on a shaker for 15 s, and stand at room temperature for 5 min. Centrifuge at 4000g at 4°C for 30 min, and after centrifugation, the layers are separated, and the transparent layer at the top is carefully transferred to a new 50 mL centrifuge tube without RNase and DNase, 1:1 volume of isopropanol is added to each tube, and the mixture is mixed several times by inverting up and down, then stand at room temperature for 10 min. 4000g centrifuge for 30 min to remove the supernatant and reserve the precipitate. Add 1 mL of 75% ethanol to the precipitate and transfer it to a 1.5 mL centrifuge tube. The precipitate is repeatedly bounced up and down to fully contact with the liquid. 7500g centrifuge for 5 min, remove the supernatant and reserve the precipitate. Keep the centrifuge tube opening, dry at room temperature for 10 min, add 400 uL of enzyme-free water, incubate at 55°C for 10 min to ensure that the RNA is completely dissolved, and the total RNA is obtained.

[0187] Take 1 μL to detect the concentration and A260 / A280 of RNA with a nucleic acid concentration measuring instrument and record.

[0188] (2) RNA reverse transcription:

[0189] The total RNA obtained in step (1) above is reverse transcribed using a reverse transcription kit (GoScript Reverse Transcription Kit, Promega Company). TM Reverse transcription kit).

[0190] The specific operation is as follows:

[0191] The total RNA sample is divided into two parts, one uses Oligo dT Primer in the kit as primer, and the other uses Random 6-mers in the kit as primer, and the reaction solution is configured in a 1.5 ml centrifuge tube according to the system shown in Table 4, and the reaction system is enlarged according to the RNA amount to amplify.

[0192] Table 4 5 μL reverse transcription reaction system

[0193] Component Amount Oligo dT Primer (50 μM) / Random 6-mers (50 μM) 1 μL Total RNA 2 μg ddH2O q.s. to 5 μL

[0194] The reaction solution was divided into PCR octuple tubes, centrifuged briefly, and denatured at 70°C for 5 min to make the RNA denatured. After the reaction, the PCR octuple tubes were placed on ice to cool quickly. Then, the remaining reaction solution was prepared according to the proportions in Table 5.

[0195] Table 5 Proportions of the remaining reaction solution

[0196] Component Amount Table 4 Post-Reaction System 5 μL 5x Reaction Buffer 4 μL MgCl2 2uL dNTP mix 1 uL RNase Inhibitor 0.4 μL RTase 1 μL ddH2O q.s. to 20 μL

[0197] The reaction solution in Table 5 was mixed and centrifuged briefly, and the centrifuge tube was placed in a PCR instrument for reaction at 45°C for 60 min and then at 75°C for 15 min. The tube was cooled on ice to obtain the cDNA after total RNA reverse transcription, which was stored at 4°C.

[0198] (3) PCR amplification:

[0199] The cDNA obtained in step (2) was used as a template, and NEB Q5 high-fidelity DNA polymerase was used to amplify the single-chain antibody fragment (ScFv) through two rounds of PCR, and the structure was VL-linker-VH (light chain-linker-heavy chain).

[0200] The first round of PCR was performed using cDNA as a template, and the first round of PCR reaction system is shown in Table 6.

[0201] Table 6 First round of PCR reaction system

[0202]

[0203]

[0204] The PCR reaction used a three-temperature point 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 35 cycles.

[0205] The primers in the first PCR include:

[0206] Vλ forward primer: 5'-CCTTTCTATGCGGCCCAGCCGGCCgagctcCAGTCTGTSBTGACGCAG CCGCC-3' (SEQ ID NO: 5).

[0207] Vλ-linker reverse primer: 5'-GGAAGATCTAGAGGAACCACCTAGGACGGTSASCTTGGTCC-3' (SEQ ID NO: 6).

[0208] Vκ forward primer: 5'-CCTTTCTATGCGGCCCAGCCGGCCgagctcGACATCCRGDTGACCCAG TCTCC-3' (SEQ ID NO: 7).

[0209] Vκ-linker reverse primer: 5'-GGAAGATCTAGAGGAACCACCTTTGATTTCCACCTTGGTCC-3' (SEQ ID NO: 8).

[0210] linker-VH forward primer: 5'-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCG GGCGGTGGTGGGCAGGTGCAGCTGCAGGAGTCSG-3' (SEQ ID NO: 9).

[0211] linker-VH reverse primer: 5'-CAGTCATTCTCGACTTactagtTGAGGAGACRGTGACCAGGGT G-3' (SEQ ID NO: 10).

[0212] wherein the lowercase part in each primer is a restriction site.

[0213] wherein the Vλ forward primer and the Vλ-linker reverse primer are used in pairs, the Vκ forward primer and the Vκ-linker reverse primer are used in pairs, and the linker-VH forward primer and the linker-VH reverse primer are used in pairs.

[0214] After the reaction, all the PCR products were subjected to 1.5% agarose gel electrophoresis, and the bands with a size of about 320 bp (corresponding to VL) and 350 bp (corresponding to VH) were cut and recovered. DNA was recovered using NEB DNA GEL purification kit according to the kit instructions. The DNA solution was the first round of PCR amplification product, which was stored at 4°C after concentration measurement.

[0215] The second round of PCR was performed using the first round of PCR amplification product as a template, and the second round of PCR reaction system is shown in Table 7.

[0216] Table 7 Second round of PCR reaction system

[0217]

[0218]

[0219] OF: 5'-CCTTTCTATGCGGCCCAGCCGGCCgagctc-3' (SEQ ID NO: 11).

[0220] OR: 5'-CAGTCATTCTCGACTTactagt-3' (SEQ ID NO: 12).

[0221] The reaction conditions are the same as above. After the reaction, the PCR product is subjected to 1.5% agarose gel electrophoresis at 160V for 20min. The band with a size of 750bp in the electrophoresis result is selected, and the DNA is recovered using the NEB DNA GEL purification kit according to the kit instructions. The DNA solution is the second round of PCR amplification product, which is stored at 4°C after concentration measurement.

[0222] (4) Enzymatic digestion and ligation of the vector and the PCR amplification product:

[0223] The second round of PCR product is ligated to the phage plasmid pComb3XSS by enzymatic digestion, thereby constructing a phage plasmid library containing the amplified target fragment (ScFv fragment). The restriction enzymes SpeI and SacI are used for enzymatic digestion.

[0224] The enzymatic digestion systems of the pComb3XSS vector and the second round of PCR amplification product are shown in Table 8 and Table 9, respectively.

[0225] Table 8 Enzymatic digestion system of the vector

[0226] Component Amount Vector (pComb3XSS) 20 μg SpeI 10 μL SacI 10 μL 10x CutSmart Buffer 50 μL Quick CIP 5 μL ddH2O q.s. to 500 μL

[0227] Table 9 Enzymatic digestion system of the target fragment (ScFv fragment):

[0228] Component Amount scFv 5 μg SpeI 5 μL SacI 5 μL CutSmart 10x buffer 25 μL ddH2O q.s. to 250 μL

[0229] The enzymatic digestion reaction conditions are incubation at 37°C for 2h and 80°C for 3min. After the enzymatic digestion, the vector digestion product is recovered using the NEB DNA purification recovery kit after gel cutting (without UV irradiation); the ScFv digestion product does not need to be run on the gel and can be directly recovered.

[0230] After the enzymatic digestion reaction is purified, the concentration of the recovered product is measured, and then the vector and the ScFv digestion product after enzymatic digestion are ligated according to the ligation reaction system shown in Table 10.

[0231] Table 10 Ligation reaction system

[0232]

[0233]

[0234] The above connection system is reacted overnight (16-24 h) at 37°C. After the end of the connection, the NEB DNA purification recovery kit is used to recover the connection product. The concentration of the recovered connection product is detected and recorded, and it is stored at 4°C.

[0235] Construction of bacterial library

[0236] (1) Preparation of TG1 E. coli competent cells:

[0237] The TG1 strain is streaked on a 2xYT solid medium and incubated overnight at 37°C. A single colony is picked from the single colony plate and inoculated into 10 ml of 2xYT medium, which is incubated overnight at 37°C at 220 rpm. The bacteria are inoculated into 100 mL of 2xYT medium at a dilution ratio of 1:100, and the OD value is measured after incubation at 37°C at 250 rpm for 40 min. Then, the OD value is measured every 20 min until the OD600=0.3-0.35. The bacterial solution is collected and centrifuged at 3200g at 0-4°C for 10 min. The supernatant is discarded and placed on ice. 40 mL of pre-cooled ddH2O is added to resuspend the bacteria, which are then centrifuged at 3200g at 0-4°C for 10 min. The supernatant is discarded and placed on ice. 1 mL of pre-cooled ddH2O is added to resuspend the bacteria, which are then transferred to a 1.5 mL pre-cooled EP tube. The mixture is centrifuged at 10000g at 4°C for 30 s, and the operation is repeated once. The supernatant is discarded and placed on ice. 400 μL of pre-cooled ddH2O is added to resuspend the bacteria, and the TG1 E. coli competent cell suspension is obtained.

[0238] The ScFv fragment obtained in the above example is used to construct an E. coli library by electroporation. Specifically, 100 ng of the ScFv fragment is added to 50 μL of the above TG1 E. coli competent cell suspension using a pre-cooled gun tip. The mixture is gently blown and transferred to a pre-cooled 1 mm electroporation cup. After confirming that the mixture is at the bottom of the cup and there are no air bubbles, the mixture is subjected to electroporation at 1800 V and 1 mm spacing. Immediately after completion, 1000 μL of 37°C SOC medium is added, and the mixture is taken out of the cup and incubated at 37°C at 180 rpm for 90 min. The 2xYT liquid medium is used for 10-fold gradient dilution, and a total of 6 gradients are diluted (diluted by 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 times). 5 μL of each gradient is uniformly added to 2xYT-GA solid medium, which is then dried and incubated at 37°C overnight. The number of colonies on the gradient dilution plate is counted, and the connection efficiency is calculated. The connection efficiency formula is:

[0239] E (pfu / μg) = N x D x 10

[0240] Wherein, E is the competence efficiency (unit pfu / μg), D is the dilution fold, N is the number of monoclonal colonies on the plate of the corresponding dilution fold.

[0241] The 100 electroporation reactions were repeated according to the above method, and the recovered bacterial liquid was uniformly coated on 100 2xYT-GA245mm square culture medium plates, and after air drying, it was cultured at 37°C overnight. The square plate of overnight culture was taken, 6mL of 2xYT liquid medium was added to the surface of each culture plate, and the 100 square plate colonies were scraped with a coating rod and the bacterial liquid was collected into a 50mL centrifuge tube, and 20% glycerol was added to a final concentration, which was the bacterial library.

[0242] 10μL of bacterial liquid was taken into 990μL of 2xYT liquid medium and the OD600 was measured using NanoDrop, and the total OD600 of the bacterial library was calculated and recorded.

[0243] T OD600 = M OD600 x 100

[0244] Wherein, T OD600 is the total OD600 of bacteria, M OD600 is the measured OD600.

[0245] Example 4 Phage library construction

[0246] An appropriate amount of the above bacterial library was taken into a 1.5ml EP tube, and the calculation formula for the amount of bacterial liquid was:

[0247]

[0248] Wherein, V is the volume of the transferred bacterial liquid (unit μL), and OD600 is the total OD600 of the constructed bacterial library.

[0249] It was transferred to 100mL of 2xYT-GA liquid medium to make the initial OD600 0.1. It was cultured at 37°C, 250rpm in a constant temperature shaker until the OD600 of the bacterial liquid reached 0.5-0.55. The auxiliary phage M13K07 was calculated and added according to the following formula to make the number of bacteria:phage 1:20. The calculation formula for the amount of phage added was:

[0250]

[0251] Wherein, V is the volume of the added auxiliary phage (unit mL), T helper-phage is the titer of the auxiliary phage used, and OD600 is the OD600 value of the bacterial liquid.

[0252] The TG1 bacteria were collected by centrifugation at 3200 g for 5 min, and the supernatant was removed. The precipitate was resuspended and transferred into 100 mL of 2xYT-AK liquid medium, and incubated at 30°C, 250 rpm overnight.

[0253] The bacteria in the overnight culture were transferred into a new 50 mL centrifuge tube, and centrifuged at 4000 g, 4°C for 30 min. The supernatant was removed, and 1 / 4 volume of 4°C pre-cooled 20% PEG / 2.5 M NaCl was added. After mixing well, the mixture was placed on ice for 30 min. The mixture was centrifuged at 4000 rpm, 4°C for 30 min, and the supernatant was discarded. The precipitate was resuspended in 1 mL of PBS, and centrifuged at 12000 rpm, 4°C for 20 min. The supernatant was removed, and 1 / 4 volume of pre-cooled 20% PEG / 2.5 M NaCl solution was added. After mixing well, the mixture was placed on ice for 10 min. The mixture was centrifuged at 12000 rpm, 4°C for 10 min, and the supernatant was discarded. The precipitate was resuspended in 1 mL of PBS. The mixture was centrifuged at 12000 rpm, 4°C for 2 min, and the supernatant was removed. The phage library was obtained, and glycerol was added to a final concentration of 20%. The mixture was stored at -80°C.

[0254] The titer of the phage library was detected as follows. The phage library was added to 10 mL of 2xYT liquid medium, and incubated at 37°C, 250 rpm for about 45 min to 60 min, until the OD600 was 0.5 to 0.55. 10 μL of the incubated phage library was diluted by 10-fold in gradient (a total of 13 gradients). 90 μL of untreated TG1 bacteria was added to each dilution gradient, and mixed well. The mixture was incubated at 37°C for 20 min. Then, 5 μL of the mixture in each dilution gradient was added to 2xYT-GA solid medium, and incubated at 37°C overnight. The number of colonies was counted, and the number of phagemids in 1 mL of the phage solution was calculated according to the formula for the titer of the phage library

[0255] T (pfu / mL) = N x D x 400

[0256] Wherein, T is the titer of the phage (unit pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0257] Example 5 Screening of antibodies specific to gB protein

[0258] Antibodies specifically binding to gB protein were screened by using antigen solid-phase adsorption screening method.

[0259] The specific operation is as follows:

[0260] The gB protein (50 μg) obtained in the above example was dissolved in 2 mL PBS and coated on an immunotube at 4°C overnight. A negative control protein (50 μg BSA) was coated at the same time as a negative control. The coated protein was discarded and washed with 2 mL PBS for 3 times, and then blocked with 2 mL 3% BSA (dissolved in PBS) at room temperature for 2 h. The blocking solution was discarded, and 100 μL of the phage library in the above example was diluted with 2 mL PBS and incubated in the immunotube for 1 h. The solution was discarded, and the immunotube was washed with 2 mL PBST for 5 times, each for 5 min. Then the immunotube was washed with 2 mL PBS for 5 times, each for 5 min. At the same time, 1 mL of saturated TG1 bacteria solution was added to 100 mL 2 x YT liquid medium and incubated at 37°C and 150 rpm until OD600=0.5 (about 1.5 h). The washing solution was discarded, and 1 mL of 0.1 mg / mL trypsin was used to elute the phage at room temperature for 30 min. 1 mL of the eluted solution was added to 10 mL of the incubated TG1 bacteria solution (OD=0.5) and incubated at 37°C and 150 rpm for 30 min. The bacteria were then centrifuged at 4°C and 3000 g for 10 min. The precipitate was resuspended with 1 mL of 2 x YT liquid medium. The resuspended product from the positive immunotube was evenly spread on a 245 mm x 245 mm 2 x YT solid culture dish and incubated at 37°C overnight (16-20 h). The phage titers of the positive tube and the negative control tube were determined by the method described in the above example.

[0261] According to the comparison of the phage titers, 5 mL of 2 x YT liquid medium was added to the culture dish with the highest phage titer after overnight incubation, and all the colonies were scraped off with a spreader. The sub-phage library was prepared according to the method described in the above example. The above operation was repeated until the antibody with the highest affinity to gB was screened (compared with the negative control).

[0262] In the antigen coating, blocking, incubation, washing and elution processes, the rotation speed was 15 rpm unless otherwise specified.

[0263] The antibody with the highest affinity was detected by ELISA.

[0264] Take 10 μL of the highest affinity antibody with gB culture bacteria, dilute the bacteria with 1 ml of 2 x YT liquid medium. Cloning streak on 2 x YT-GA solid culture plate, 37°C overnight culture for 16-20h. Pick 192 single colonies into 96-well plates (200 μL of 2 x YT-GA liquid per well), 37°C static culture to saturation. Take 2 μL of saturated bacteria to a new 96-well plate (200 μL of 2 x YT-A liquid per well), the initial OD is about 0.03, 37°C for 2.5h-3h to OD about 0.5. Add 0.1 μL of the above helper phage to each well, 37°C for 30 min. Add 0.2 μL of Kana (kanamycin) to each well, 30°C overnight culture. Protein (- / +) coated ELISA plate overnight. Centrifuge the 96-well plate at 4°C, 3400g for 5 min. Discard the liquid in the ELISA plate, wash once with 350 μL of PBS. Block with 350 μL of 3% BSA (PBST) at 37°C for 1h. Discard the blocking solution, wash once with 350 μL of PBST, tap the plate to remove the liquid. Add 140 μL of 3% BSA (PBST) to each well, then add 60 μL of the above phage library expressed antibody (the highest affinity antibody as the primary antibody) respectively, 37°C for 1h. Discard the liquid (primary antibody), wash 5 times with 350 μL of PBST, tap the plate. Add 100 μL of M13Antibody (HRP) as the secondary antibody (1:8000 ratio, relative to the blocking solution), 37°C for 1h, discard the liquid (secondary antibody), wash 5 times with 350 μL of PBST, tap the plate. Add 100 μL of TMB for 2-3 min in the dark, add 100 μL of dilute hydrochloric acid (concentrated hydrochloric acid: water = 1:12) to stop the reaction. Read the OD450, OD630 of the ELISA plate.

[0265] The antibody heavy chain variable region is connected to the CMV fragment upstream and the constant region of human IgG1 downstream, as well as the ploy A fragment, which can express the full-length heavy chain fragment; while the antibody light chain variable region is connected to the CMV fragment upstream and the constant region of light chain κ / λ downstream, as well as the ploy A fragment, which can express the full-length light chain fragment. Co-transfect the plasmid with the full-length sequence of the above antibody heavy chain and light chain into 293T cells to realize the expression of the antibody, and use protein A beads to realize the purification of the antibody.

[0266] In this embodiment, the inventors finally obtained two antibodies with the best effect, named 4C1 and 4B3.

[0267] Example 6 Expression and purification of monoclonal antibody 4C1

[0268] The full-length heavy chain of 4C1 has 467 amino acid residues (not counting *), specifically:

[0269]

[0270] The underlined part in the sequence is the amino acid sequence of the heavy chain variable region (SEQ ID NO: 14). The underlined and bolded part is the amino acid sequence of the three complementarity regions CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region defined by the IMGT scheme (SEQ ID NO: 15, 16, 17 respectively), and the italic part is the signal peptide (SEQ ID NO: 18). The * represents the stop codon.

[0271] The coding gene of the full-length heavy chain of 4C1 is 1404 bases in length, specifically:

[0272]

[0273]

[0274] The underlined part in the sequence is the nucleotide sequence of the heavy chain variable region (SEQ ID NO: 20). The underlined and bolded part is the nucleotide sequence of the three complementarity regions CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region defined by the IMGT scheme (SEQ ID NO: 21, 22, 23 respectively). 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 (SEQ ID NO: 24).

[0275] The full-length light chain of 4C1 is 235 amino acid residues in length (not counting *), specifically:

[0276]

[0277] The underlined part in the sequence is the amino acid sequence of the light chain variable region (SEQ ID NO: 26). The underlined and bolded part is the amino acid sequence of the three complementarity regions CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region defined by the IMGT scheme (CDR-L1, CDR-L3 are SEQ ID NO: 27, 28 respectively; CDR-L2 is DNH). The italic part is the signal peptide (SEQ ID NO: 29), and the * represents the stop codon.

[0278] The coding gene of the full-length light chain of 4C1 is 708 bases in length, specifically:

[0279]

[0280]

[0281] The underlined part in the sequence is the nucleotide sequence of the light chain variable region (SEQ ID NO: 31). The underlined and bolded parts are the nucleotide sequences of the three complementarity regions CDR-L1, CDR-L2 and CDR-L3 in the light chain variable region defined by the IMGT scheme in turn (CDR-L1, CDR-L3 are SEQ ID NO: 32, 33 respectively; CDR-L2 is GACAATCAT). The 3 bases at the 5' end and the 3' end are the start codon and the stop codon respectively. The italicized part is the signal peptide (SEQ ID NO: 34).

[0282] The expression of the light and heavy chains of the 4C1 antibody was identified by Coomassie blue staining.

[0283] The specific operation steps are as follows: according to the following table, 10% separation gel is prepared, mixed and quickly added to the gel plate mold, and 2 mL of anhydrous ethanol is added for sealing. After the lower separation gel solidifies, discard the anhydrous ethanol and let it stand to evaporate. Prepare concentrated gel, add 2 mL to the upper layer of the separation gel, and quickly insert the comb. Use the BCA protein quantification analysis kit for protein quantification. Add 10 μg of 4C1 protein sample to 5x SDS loading buffer and incubate at 95°C for 10 min. After the gel is dry, place the mold in the electrophoresis tank, add the electrophoresis liquid, and pull out the comb. Add the treated protein sample to the sample well and add the marker. First use low voltage of 80V for protein concentration, then change the voltage to 120V for protein separation. The electrophoresis time is determined according to the position of the marker. Place the gel in the Coomassie blue staining agent (Novizen) and shake in the shaker until the bands appear. Discard the color developing solution, add distilled water to wash the gel in the shaker, repeat several times, and the gel background will be clean.

[0284] The results are shown in Table 11. Figure 1 The 55 kDa band is the heavy chain and the 25 kDa band is the light chain.

[0285] Table 11 Composition of protein gel related reagents

[0286]

[0287] Example 7 Expression and purification of monoclonal antibody 4B3

[0288] The full-length heavy chain of 4B3 has 467 amino acid residues (not counting *), which are as follows:

[0289]

[0290]

[0291] The underlined part in the sequence is the amino acid sequence of the heavy chain variable region (SEQ ID NO: 36). The underlined and bolded parts are the amino acid sequences of the three complementarity regions CDR-H4, CDR-H5 and CDR-H6 in the heavy chain variable region defined by the IMGT scheme (SEQ ID NO: 37, 38, 39) in turn. The italic part is the signal peptide (SEQ ID NO: 18). The * represents the stop codon.

[0292] The coding gene of the full-length heavy chain of 4B3 has 1404 bases, specifically:

[0293]

[0294] The underlined part in the sequence is the nucleotide sequence of the heavy chain variable region (SEQ ID NO: 41). The underlined and bolded parts are the nucleotide sequences of the three complementarity regions CDR-H4, CDR-H5 and CDR-H6 in the heavy chain variable region defined by the IMGT scheme (SEQ ID NO: 42, 43, 44) in turn. 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 (SEQ ID NO: 24).

[0295] The full-length light chain of 4B3 has 238 amino acid residues (not counting *), specifically:

[0296]

[0297]

[0298] The underlined part in the sequence is the amino acid sequence of the light chain variable region (SEQ ID NO: 46). The underlined and bolded parts are the amino acid sequences of the three complementarity regions CDR-L4 and CDR-L6 in the light chain variable region defined by the IMGT scheme (SEQ ID NO: 47, 48 respectively, CDR-L5 is KVS). The italic part is the signal peptide (SEQ ID NO: 29), and the * represents the stop codon.

[0299] The coding gene of the full-length light chain of 4B3 has 717 bases, specifically:

[0300]

[0301] The underlined part in the sequence is the nucleotide sequence of the light chain variable region (SEQ ID NO: 50). The underlined and bolded parts are the nucleotide sequences of the three complementarity regions CDR-L4 and CDR-L6 in the light chain variable region defined by the IMGT scheme (SEQ ID NO: 51, 52 respectively, the nucleotide sequence of CDR-L5 is The initial 5' and final 3' bases are the start and stop codons, respectively. The italicized portion is the signal peptide (SEQ ID NO: 34).

[0302] Coomassie brilliant blue staining was used to identify the expression of the light and heavy chains of the 4B3 antibody, following the same experimental procedure as in Example 6. The results are as follows: Figure 2 As shown.

[0303] Example 1: 4C1 Antibody Affinity Assay

[0304] The affinity of antibody 4C1 was determined using biomembrane interference (BLI) technique.

[0305] BLI can be performed according to conventional practices 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 10 μg / mL gB-Biotin (biotin-labeled gB protein). The gB 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 0–1000 nM 4C1 antibody, the specific binding between antigen and antibody led to an increase in membrane thickness. Immersing the biosensor with the bound 4C1 antibody in the buffer solution caused dissociation, resulting in the detachment of the 4C1 antibody from the biosensor surface and a decrease in membrane thickness. Real-time monitoring of the biosensor biomembrane thickness during the experiment allowed the determination of the kinetic constants of the test sample (4C1 antibody).

[0306] The results are as follows Figure 3 As shown.

[0307] The KD(M) of the 4C1 antibody in this invention was determined to be 1.0E. -9 This indicates that the 4C1 antibody and the gB antigen have a clear affinity.

[0308] Example 2: Applications of the 4C1 antibody

[0309] Based on the high affinity of the 4C1 antibody, it can be effectively used for the detection of gB antigen, such as in qualitative or quantitative detection in Western blotting.

[0310] (1) Application of 4C1 antibody in Western Blot:

[0311] In this embodiment, the inventors take the Western Blot of gB protein as an example to verify the actual application effect.

[0312] The operation steps are as follows:

[0313] After denaturation of 10 μg of gB protein, electrophoresis was performed (the electrophoresis step was the same as the Western Blot), after completion, the gel plate was carefully opened, and the concentrated gel was cut off. Methanol was used to activate 0.22 μm PVDF membrane. Two layers of filter paper, PVDF membrane, separation gel, and two layers of filter paper were placed in the order from bottom to top, and fixed in the transfer clamp plate. The clamp plate was placed in the transfer instrument, and the transfer solution was added, and the constant current transfer was performed at 100 mA for 2 h. The PVDF membrane was transferred to 5% milk blocking solution, and incubated on a shaking bed at room temperature for 1 h. The PVDF membrane was transferred to the above-mentioned 4C1 antibody solution (1 μg / mL), and incubated at 4°C with shaking overnight. TBST was rinsed for 3 times, each for 5 min. The PVDF membrane was transferred to Rabbit Anti-Human IgG H&L (HRP) antibody (abcam, 1:10000 dilution), and incubated at room temperature with shaking for 1 h. TBST was rinsed for 3 times, each for 5 min. The chemiluminescence solution was evenly dropped onto the surface of the PVDF membrane, and reacted in the dark for several minutes. The chemiluminescence imager was used to image the PVDF membrane. The blank control was set.

[0314] The results are shown in Figure 4 .

[0315] It can be found that when the 4C1 antibody is used as the primary antibody for Western Blot, the gB protein sample will present a black band at 35kD-40kD, which indicates that the 4C1 antibody can be applied to Western Blot detection.

[0316] (2) Application of 4C1 antibody in flow cytometry:

[0317] In this embodiment, the 293T cells transiently transfected with gB recombinant plasmid were taken as the test object to test the actual application effect of 4C1 antibody in flow cytometry.

[0318] Specific steps are as follows: 293T cells are cultured in a 12-well plate, and the cell density is about 40%. After the cells adhere, gB-Flag recombinant plasmid (preparation method is the same as that of the above gB recombinant plasmid) is transiently transfected, and a negative control is set, and the culture is continued for 24 h. The culture medium is replaced, and 4C1 antibody labeled with 403 fluorescence is used for incubation at 4°C for 30 min. Anti-Flag-488 is used for incubation at 4°C for 30 min. 2G4 antibody labeled with 403 fluorescence is used as a negative control antibody. Among them, 2G4 antibody is a general gB antibody with the lowest affinity eliminated in the above examples. The cells are digested with 1 mL trypsin for 3 min, centrifuged at 200 g for 5 min, and washed with PBS for 3 times. The cells are resuspended in 500 μL PBS, and filtered with a 40 μm cell screen. The fluorescence of the cells is detected by flow cytometry. The 293T cells without any treatment are used as a blank control.

[0319] The results are shown in Figure 5 The 4C1 antibody labeled with 403 fluorescence is used for incubation at 4°C for 30 min, and then Anti-Flag-488 is used for incubation at 4°C for 30 min, and 2G4 antibody labeled with 403 fluorescence is used as a negative control antibody (2G4 antibody 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 cocktail against Ebola virus. Sci. Rep. 4, 6881, 2014). The results show that the positive rate of the 4C1 antibody group is 11.77%, and the negative control group is 2G4 (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 cocktail against Ebola virus. Sci. Rep. 4, 6881, 2014). It is suggested that the 4C1 antibody can be applied to flow detection.

[0320] It can be found that the screening effect of the 4C1 antibody is obviously better than that of the general gB antibody, which shows that the 4C1 antibody has better specific binding and detection effect than the general gB antibody.

[0321] (3) Application of 4C1 antibody in immunohistochemistry.

[0322] In this embodiment, the section of a nasopharyngeal carcinoma patient is used as a test object to test the actual application effect of the 4C1 antibody in immunohistochemistry.

[0323] The specific steps are as follows: Baking: 65 degrees Celsius for 1 hour; Dewaxing: Immersion in xylene 3 times; Hydration: Immersion in 100% ethanol for 10 minutes twice, immersion in 95%, 90%, 80%, 70%, and 60% ethanol for 5 minutes each, and immersion in double-distilled water for 5 minutes twice; Removal of endogenous peroxidase: Treatment with 3% hydrogen peroxide for 10 minutes; High-pressure retrieval: Add 1L of retrieval solution to a pressure cooker, bring to a boil at 2100W, then adjust to 1000W and time for 2 minutes and 30 seconds; Allow to cool naturally and run in circles, wash with PBST for 5 minutes 3 times; Block with 5% BSA at 37°C for 30 minutes; Discard the BSA and add primary antibody 4C1 (150ng / ul) directly, incubate at 4°C overnight; Wash with primary antibody: Wash with PBST for 5 minutes 3 times, add RabbitAnti-Human IgG. H&L (HRP) antibody (abcam, 1:1000 dilution), incubated at 37 degrees for 30 minutes; washed with PBST for 10 minutes three times; developed with DAB for 1 minute; stained the nucleus with hematoxylin for 1 minute; rinsed with running water for 2 hours; after drying the slides, mounted with neutral resin.

[0324] The results are as follows Figure 6 As shown.

[0325] The results indicated that brown histiocytic staining was visible in the nasopharyngeal carcinoma sections, suggesting that the 4C1 antibody could be used for immunohistochemical staining and that gB was expressed in the nasopharyngeal carcinoma sections.

[0326] (4) Application of 4C1 antibody in tumor targeting

[0327] In this implementation case, C666 nasopharyngeal carcinoma cells were used as test subjects after tumor formation in mice to test the practical application effect of the 4C1 antibody in tumor tissue.

[0328] The specific steps are as follows: Subcutaneous injection of 2*10 into nude mice. ^6 Nasopharyngeal carcinoma C666 cells were allowed to develop into tumors. LinKine was used. TM AbFluor TM The 647 conjugation kit (Abkine) labeled 4C1 antibody (100 μg / mouse), with an equal amount of 2G4 antibody also labeled as a control. The labeled antibodies were injected into mice via the tail vein. Forty-eight hours later, the mice were imaged using the PE IVIS Spectrum small animal in vivo imaging system.

[0329] The results are as follows Figure 7 As shown.

[0330] The results showed that mice injected with 4C1 antibody exhibited fluorescence at the tumor site, while mice injected with the control group 2G4 antibody did not show fluorescence accumulation at the tumor site, suggesting that 4C1 antibody can serve as a tumor-targeting marker.

[0331] Example 3: Determination of Affinity for 4B3 Antibody

[0332] The affinity of the antibody 4B3 was determined by using the bio-layer interferometry (BLI).

[0333] The BLI can be performed according to the routine in the art, and the specific operation is the same as that in the effect example 1.

[0334] The results are shown in the following table. Figure 8

[0335] It was detected that the KD (M) of the 4B3 antibody in the application was 3.968E -9 , which indicated that the 4B3 antibody and the gB antigen had clear affinity.

[0336] Effect example 4: Related application of the 4B3 antibody

[0337] Based on the high affinity of the 4B3 antibody, it can be effectively used for related detection of the gB antigen, such as qualitative or quantitative detection in Western Blot.

[0338] (1) Application of the 4B3 antibody in Western Blot:

[0339] In this embodiment, the inventors took Western Blot of the gB protein as an example to verify the actual application effect.

[0340] The operation steps are the same as those in the effect example 2.

[0341] The results are shown in the following table. Figure 9

[0342] It can be found that the gB protein sample will present a black band at 35kD-40kD when the 4B3 antibody is used as a primary antibody for Western Blot, which indicates that the 4B3 antibody can be applied to Western Blot detection.

[0343] (2) Application of the 4B3 antibody in flow cytometry:

[0344] In this embodiment, the 293T cells transiently transfected with the gB recombinant plasmid were taken as the test object to test the actual application effect of the 4B3 antibody in flow cytometry.

[0345] The specific steps are the same as those in the effect example 2.

[0346] The results are shown in the following table. Figure 10

[0347] It can be found that the screening effect of the 4B3 antibody is obviously better than that of the general gB antibody, which indicates that the 4B3 antibody has better specific binding and detection effect than the general gB antibody.

[0348] ​​​(3) Application of 4B3 antibody in immunohistochemistry.

[0349] In this implementation case, NKT lymphoma slides were used as the test subject to test the practical application effect of the 4B3 antibody in immunohistochemistry.

[0350] The specific steps are as follows: Baking: 65 degrees Celsius for 1 hour; Dewaxing: Immersion in xylene 3 times; Hydration: Immersion in 100% ethanol for 10 minutes twice, 95%, 90%, 80%, 70%, and 60% ethanol for 5 minutes each, and double-distilled water for 5 minutes twice; Removal of endogenous peroxidase: Treatment with 3% hydrogen peroxide for 10 minutes; High-pressure retrieval: Add 1L of retrieval solution to a pressure cooker, bring to a boil at 2100W, then reduce to 1000W and time for 2 minutes and 30 seconds; Allow to cool naturally and run in circles, wash with PBST for 5 minutes 3 times; Block with 5% BSA at 37°C for 30 minutes; Discard the BSA and add primary antibody 4B3 (150ng / ul) directly, incubate at 4°C overnight; Wash with primary antibody: Wash with PBST for 5 minutes 3 times, add RabbitAnti-Human IgG. H&L (HRP) antibody (abcam, 1:1000 dilution), incubated at 37 degrees for 30 minutes; washed with PBST for 10 minutes three times; developed with DAB for 1 minute; stained the nucleus with hematoxylin for 1 minute; rinsed with running water for 2 hours; after drying the slides, mounted with neutral resin.

[0351] The results are as follows Figure 11 As shown.

[0352] The results indicated that brown histocyte staining was visible in NKT lymphoma sections, suggesting that 4B3 antibody can be used for immunohistochemical staining and that gB is expressed in nasopharyngeal carcinoma sections.

[0353] (4) Application of 4B3 antibody in tumor targeting

[0354] In this implementation case, C666 nasopharyngeal carcinoma cells were used as test subjects after tumor formation in mice to test the practical application effect of the 4B3 antibody in tumor tissue.

[0355] The specific steps are as follows: Subcutaneous injection of 2*10 into nude mice. ^6 Nasopharyngeal carcinoma C666 cells were allowed to develop into tumors. LinKine was used. TM AbFluor TM The 647 conjugation kit (Abkine) labeled 4B3 antibody (100 μg / mouse), with an equal amount of 2G4 antibody also labeled as a control. The labeled antibodies were injected into mice via the tail vein. Forty-eight hours later, the mice were imaged using the PE IVIS Spectrum small animal in vivo imaging system.

[0356] The results are as follows Figure 12 As shown.

[0357] The results showed that the mice injected with 4B3 antibody had fluorescence in the tumor site, while the mice injected with control 2G4 antibody had no fluorescence gathered in the tumor site, suggesting that 4B3 antibody can be used as a tumor targeting marker.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof against an Epstein-Barr virus gB protein, characterized in that: the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain; the monoclonal antibody is any one of A) or B) below; the heavy chain comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 14; the light chain comprises: a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 26; the heavy chain comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 36; the light chain comprises: a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

46. 2.The monoclonal antibody or the antigen-binding fragment thereof according to claim 1, characterized in that: the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 are shown as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 27 and SEQ ID NO: 28 in sequence, and the amino acid sequence of the CDR-L2 is DNH, the CDRs are defined according to the IMGT definition scheme; or the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 are shown as SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 47 and SEQ ID NO: 48 in sequence, and the amino acid sequence of the CDR-L2 is KVS, the CDRs are defined according to the IMGT definition scheme. 3.The monoclonal antibody or the antigen-binding fragment thereof according to claim 1 or 2, characterized in that: the monoclonal antibody or the antigen-binding fragment thereof is at least one of a full-length antibody, a Fab, a Fab’, a F(ab’) 2, a Fv, and a multispecific antibody. A） 4.The monoclonal antibody or the antigen-binding fragment thereof according to claim 3, characterized in that: the Fv comprises a scFV; and the multispecific antibody comprises a bispecific antibody. 5.A recombinant protein comprising: the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4; and optionally a tag sequence for facilitating expression and / or purification. 6.A biological material associated with the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, or the recombinant protein according to claim 5, the biological material being at least one of f1) to f12): f1) a nucleic acid molecule encoding the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, or the recombinant protein according to claim 5; ​ ​ ​ B） ​ ​ ​ ​ ​ ​ ​ A） ​ B） ​ ​ ​ ​ ​ ​ ​ ​ ​ f2) an expression cassette comprising the nucleic acid molecule of f1); f3) a vector comprising the nucleic acid molecule of f1); f4) a vector comprising the expression cassette of f2); f5) a transgenic cell line comprising the nucleic acid molecule of f1); f6) a transgenic cell line comprising the expression cassette of f2); f7) a transgenic cell line comprising the vector of f3); f8) a transgenic cell line comprising the vector of f4); f9) a microorganism comprising the nucleic acid molecule of f1); f10) a microorganism comprising the expression cassette of f2); f11) a microorganism comprising the vector of f3); f12) a microorganism comprising the vector of f4).

7. A conjugate comprising: the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4 and at least one of the recombinant protein of claim 5; and a detectable label.

8. The conjugate of claim 7, wherein: the detectable label comprises an enzyme, a radionuclide.

9. Use of at least one of (1)-(4) in the manufacture of a product; (1) the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4; (2) the recombinant protein of claim 5; (3) the biomaterial of claim 6; (4) the conjugate of claim 7 or 8; the product is at least one of a reagent, an assay plate, a kit, an assay chip; the reagent, assay plate, assay chip or kit has at least one of the functions of h1)-h3): h1) detecting the presence or level of gB protein in a sample; h2) detecting EBV; h3) diagnosing a disease caused by EBV infection.

10. A product comprising at least one of i1)-i3): i1) the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4; i2) the recombinant protein of claim 5; i3) the conjugate of claim 7 or 8; the product is at least one of a reagent, an assay plate, a kit, an assay chip.

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