Monoclonal antibody targeting EB virus gB antigen and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a monoclonal antibody targeting Epstein-Barr virus gB antigen and application thereof. Background Art

[0002] Epstein-Barr virus (EBV) is classified as a Class I carcinogen by the World Health Organization and is the first confirmed human oncogenic virus, with an infection rate of over 95% in the population. Studies estimate that tumors caused by EBV infection account for 1%-2% of all human tumors, and approximately 200,000 new cancers are associated with EBV each year. EBV can infect epithelial cells and lymphocytes and is closely associated with a variety of tumors, including nasopharyngeal carcinoma, gastric cancer, Hodgkin lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, NKT-cell lymphoma, and primary effusion lymphoma. In addition, EBV reactivation sometimes leads to 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 a lack of understanding of immunity to EBV infection and related diseases.

[0003] EBV encodes multiple envelope glycoproteins that determine cell tropism and complete host cell attachment and entry. Glycoprotein B (gB) is functionally conserved in all herpes viruses and is a fusion protein required for viral entry into cells. EBV gB forms the core membrane fusion machinery with glycoprotein H and glycoprotein L heterodimers (gHgL) and drives membrane fusion through conformational changes. EBV gB directly interacts with neuropilin 1 (NRP1) and promotes EBV infection of epithelial cells through internalization and fusion promoted by NRP1. In nanovaccines targeting gB, studies have shown that it can significantly inhibit EBV infection in humanized mice and non-human primates. Therefore, gB has become a hot target for monoclonal antibody research and development and vaccine development. However, research on gB humanized monoclonal antibodies is still very scarce.

[0004] Monoclonal antibodies can be mass-produced, and based on their high affinity and high specificity for binding to antigens, adverse reactions during clinical application can be greatly reduced. These antibody molecules can also be modified to increase their antiviral efficacy. Antibodies have become a very promising means of treating infectious diseases due to their specificity and flexibility of use, but there are still no humanized monoclonal antibodies targeting EBV envelope glycoproteins on the market. Therefore, the development of humanized monoclonal antibodies against EBV will provide more effective prevention and treatment methods for EBV infection-related diseases. Summary of the invention

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

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

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

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

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

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

[0011] The seventh aspect of the present invention aims to provide a medicine.

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

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

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

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

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

[0017] B) is designated as the 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, wherein the heavy chain variable region has the 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, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 26.

[0022] 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: 27 and SEQ ID NO: 28 respectively, 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 in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO.14 and which has the same function as the protein shown in SEQ ID NO.14; or

[0026] a3-A) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology with SEQ ID NO.14 and has an amino acid sequence with the same function as the protein shown in SEQ ID NO.14.

[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 in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 26 and which has the same function as the protein shown in SEQ ID NO. 26; or

[0030] 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.26 and has an amino acid sequence with 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 the heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 36;

[0034] The light chain comprises:

[0035] A light chain variable region, comprising CDR-L4, CDR-L5 and CDR-L6 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 46.

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

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

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

[0039] a2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 36 and which has the same function as the protein shown in SEQ ID NO: 36; or

[0040] a3-B) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology to SEQ ID NO: 36 and has the same function as the protein shown in SEQ ID NO: 36;

[0041] The amino acid sequence of the light chain variable region comprises:

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

[0043] b2-B) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 46 and which has the same function as the protein shown in SEQ ID NO: 46; or

[0044] b3-B) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology to SEQ ID NO: 46 and has the same function as the protein shown in SEQ ID NO: 46.

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

[0046] In some embodiments of the present invention, the heavy chain further comprises a heavy chain signal peptide; and the light chain further comprises a light chain signal peptide.

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

[0048] c1) SEQ ID NO: 18; or

[0049] c2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in c1) and which has the same function as the protein of the amino acid sequence described in c1); or

[0050] c3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in c1) and has the same function as the protein of the amino acid sequence described in 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 in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in d2) and which has the same function as the protein of the amino acid sequence described in d1); or

[0054] d3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in d1) and has the same function as the protein of the amino acid sequence described in d1).

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

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

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

[0058] e2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to the amino acid sequence described in e1) and which has the same function as the protein of the amino acid sequence described in e1); or

[0059] e3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with the amino acid sequence described in e1) and has the same function as the protein of the amino acid sequence described in e1).

[0060] The second aspect of the present invention provides a recombinant protein comprising: the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and an optional tag sequence for facilitating expression and / or purification.

[0061] Preferably, the tag sequence is selected from at least one of the following groups: a His tag, a GGGS sequence, a FLAG tag; further a His tag; and further a 6×His tag.

[0062] The third aspect of the present invention provides a biological material related to the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the recombinant protein of the second aspect of the present invention, wherein the biological material comprises at least one of h1) to h16):

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

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

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

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

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

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

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

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

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

[0072] f10) A microorganism comprising the expression cassette described in f2);

[0073] f11) a microorganism comprising the carrier described in f3);

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

[0075] f13) A virus comprising the nucleic acid molecule described in f1);

[0076] f14) A virus comprising the expression cassette described in f2);

[0077] f15) A virus comprising the vector described in f3);

[0078] f16) A virus comprising the vector described in f4).

[0079] Preferably, the transgenic cell line comprises no reproductive material.

[0080] Preferably, the nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect of the present invention comprises a nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the present invention and a nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0081] In some embodiments of the present invention, the nucleotide sequence of 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 invention comprises:

[0082] A) Monoclonal Antibody or Antigen Binding Fragment thereof 4C1 Related:

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

[0084] a212-A) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 20 and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 20; or

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

[0086] B) Monoclonal Antibody or Antigen Binding Fragment thereof 4B2 Related:

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

[0088] a212-B) a nucleotide sequence in which one or more nucleotides in SEQ ID NO: 41 are substituted and / or deleted and / or added, and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 41; or

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

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

[0091] A) Monoclonal Antibody or Antigen Binding Fragment thereof 4C1 Related:

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

[0093] a222) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 31 and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 31; or

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

[0095] B) Monoclonal Antibodies or Antigen-binding Fragments thereof 4B1 Related:

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

[0097] a222-B) a nucleotide sequence in which one or more nucleotides are substituted and / or deleted and / or added to SEQ ID NO: 50 and which has the same function as the nucleic acid molecule shown in SEQ ID NO: 50; or

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

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

[0100] and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

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

[0102] Preferably, the conjugate is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0103] The fifth aspect of the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the invention, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fourth aspect in the preparation of a product;

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

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

[0106] g1) Prevention of EB virus infection;

[0107] g2) Treating and / or preventing diseases caused by EB virus infection.

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

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

[0110] h2) Detection of Epstein-Barr virus;

[0111] h3) Diagnosis of diseases caused by Epstein-Barr 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, and infectious mononucleosis.

[0113] Preferably, the medicament comprises a vaccine.

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

[0115] i1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0116] i2) the recombinant protein according to the second aspect of the present invention;

[0117] i3) the conjugate according to the fourth aspect of the present invention;

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

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

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

[0121] j2) Detection of Epstein-Barr virus;

[0122] j3) Diagnosis of diseases caused by EB 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, and infectious mononucleosis.

[0124] The seventh aspect of the present invention provides a drug, which comprises at least one of k1) to k4):

[0125] k1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0126] k2) the recombinant protein according to the second aspect of the present invention;

[0127] k3) benzene The biomaterial according to the third aspect of the invention;

[0128] k4) The conjugate according to the fourth aspect of the present invention.

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

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

[0131] g1) Prevention of EB virus infection;

[0132] g2) Treating and / or preventing diseases caused by EB 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, and infectious mononucleosis.

[0134] Preferably, the medicament comprises a vaccine.

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

[0136] k1) the monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0137] k2) the recombinant protein according to the second aspect of the present invention;

[0138] k3) benzene The biomaterial according to the third aspect of the invention;

[0139] k4) The conjugate according to the fourth aspect of the present invention.

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

[0141] The beneficial effects of the present invention are as follows: the present invention provides a monoclonal antibody or antigen-binding fragment thereof against EB virus gB protein, including 4C1 and 4B3, wherein 4C1 has a high affinity for gB protein (KD(M)=1.0E -9 ), 4B3 has a higher affinity for gB protein (KD(M) = 3.968E -9 ), these two monoclonal antibodies or their antigen-binding fragments can be used to detect the presence or level of gB protein in a sample, detect Epstein-Barr virus, diagnose diseases caused by Epstein-Barr virus infection, prevent Epstein-Barr virus infection and / or treat and / or prevent diseases caused by Epstein-Barr virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 This is the result of Coomassie Brilliant Blue staining after denaturing gel run with 4C1 antibody.

[0143] Figure 2 This is the result of Coomassie Brilliant Blue staining after denaturing gel run with 4B3 antibody.

[0144] Figure 3 The affinity results of gB protein and 4C1 antibody were detected by BLI.

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

[0146] Figure 5 This is the application effect of 4C1 antibody in flow cytometry detection.

[0147] Figure 6 This is the application effect of 4C1 antibody in immunohistochemistry detection.

[0148] Figure 7 This is the application effect of 4C1 antibody in tumor targeting.

[0149] Figure 8 The affinity results of gB protein and 4B3 antibody were detected by BLI.

[0150] Fig. 9 This is the detection effect of 4B3 antibody in Western Blot.

[0151] Fig.10 This is the application effect of 4B3 antibody in flow cytometry detection.

[0152] Fig.11 This is the application effect of 4B3 antibody in immunohistochemistry detection.

[0153] Fig.12 This is the application effect of 4B3 antibody in tumor targeting. DETAILED DESCRIPTION

[0154] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0155] Example 1 Preparation of EBV gB recombinant protein

[0156] First, in this example, a plasmid of gB was constructed for protein purification, wherein the N-terminus contained a KOZAK sequence and a CD5 signal peptide sequence, and the C-terminus contained a His tag and a stop codon. The plasmid expression fragment sequence is as follows:

[0157]

[0158] The gB gene fragment was connected to the mammalian cell expression vector pcDNA3.1+ (Invitrogen) as follows:

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

[0160] Use 50 μL of NEB The PCR amplification reaction system (Table 1) was used to amplify the gB protein gene.

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

[0162] Components content 5×Reaction Buffer 10μL dNTPs 1μL Upstream primer 2.5μL Downstream primer 2.5μL DNA template 1μL DNA polymerase 0.5μL High GC Enhancer (Q5 kit from NEB) 10μL <![CDATA[ddH2O]]> Make up to 50 μL

[0163] The upstream primer is: 5'-TGGTGGAATTCGCCACCATGCCCATGGGGTCTCTGCAACCGC TGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCTGCCTCGGACAGACCCCAGAG CAGCCC-3' (SEQ ID NO: 2); the 5'-downstream primer is TCGAGCGGCCGCTTAGTGGTGATGGTGAT GATGTGACACTGCATTGTCCAAATCC-3' (SEQ ID NO: 3).

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

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

[0166] The eukaryotic expression plasmid pcDNA3.1+ was used as the vector. The target fragment and the vector were both digested with EcoRⅠ and NotⅠ (50 μL of the digestion reaction system (Table 2) was used for digestion).

[0167] Table 2 Enzyme digestion reaction system

[0168] Components content 10×CutSmart Buffer 5μL EcoRⅠ-HF 1μL NotⅠ-HF 1μL Target fragment or vector 5μg <![CDATA[ddH2O]]> Make up to 50 μL

[0169] The enzyme digestion was performed at 37°C for 2 to 3 hours. The digested vector was run on an agarose gel and then recovered using a gel recovery kit to obtain a linearized vector. The inserted fragment was directly recovered using a DNA purification kit. After recovery, 10 μL of the ligation reaction system (Table 3) was used for ligation.

[0170] Table 3 Ligation reaction system

[0171] Components content 5×CEⅡBuffer 2μL CEⅡ 1μL Linearized vector 100ng Insert 100ng <![CDATA[ddH2O]]> Make up to 10 μL

[0172] The reaction conditions were 37°C for 30 min.

[0173] (3) Transformation of ligation products and screening of positive clones:

[0174] Add the ligation product to the thawed DH5α competent cell suspension, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and return to ice for 5 minutes. Add 200 μL LB medium and slowly shake at 30°C for 40 minutes. Pipette the culture solution and spread it on an ampicillin-resistant LB plate, and culture at 37°C overnight.

[0175] Pick a single clone colony for sequencing verification, and once the sequencing result is correct, the target recombinant plasmid is obtained. Extract the plasmid in large quantities.

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

[0177] Human renal epithelial 293F cells were cultured to obtain a cell density of 1.5×10 6 Cell suspension. Use PEI transfection reagent to transfect the recombinant plasmid obtained in step (3). The specific operation is as follows: dilute 2 mg of recombinant plasmid with 25 mL of Union-293 medium; add 6 mL of 1 mg / mL PEI to 25 mL of Union-293 medium. Vortex the plasmid and PEI thoroughly, place them at room temperature for 20 minutes, and then add them to the 293F cell suspension. After culturing for 5 days, collect the cell supernatant, centrifuge at 6000 rpm for 1 hour at 4°C, discard the cell precipitate, and obtain the supernatant containing the target protein.

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

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

[0180] The specific operation is as follows: the supernatant containing the target protein obtained in the above example is filtered using 0.65 μm filter paper, bound to the nickel column beads 3 times, eluted with 30 mM imidazole beads 3 times, and eluted the target protein with 500 mM imidazole. Then, gel filtration chromatography is used for further purification, and the specific operation is as follows: the protein eluted from the nickel column is concentrated using a 30 kD concentrator tube to a volume of less than 1 mL. Then, Superdex 200 Increase 10 / 300 GL is used for purification.

[0181] The final amino acid sequence of the target protein is:

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

[0183] Construction of the phage antibody library in Example 2

[0184] (1) Total RNA extraction:

[0185] Dilute the blood of NPC patients with PBS 1:1 (10mL blood + 10mL PBS), then carefully cover each 20mL of dilution on 15mL of lymphocyte separation solution, maintain the layered interface, centrifuge at 2000rpm for 20min at room temperature, slowly decelerate, draw the middle mononuclear cell layer into a new 15mL centrifuge tube, add PBS to make up to 15mL. Centrifuge at 300g for 20min at room temperature, transfer the supernatant to a new 50mL centrifuge tube, and centrifuge at 300g for 20min at room temperature. Remove the supernatant. Resuspend the cell pellets in the 15mL and 50mL centrifuge tubes with 1mL Trizol each. (i.e., resuspend with 2mL Trizol for every 10mL of blood).

[0186] Take 4 mL of chloroform for every 20 mL of Trizol-cell pellet resuspension, shake on a shaker for 15 seconds, and let it stand at room temperature for 5 minutes. Centrifuge at 4000g for 30 minutes at 4°C. After centrifugation, stratification will appear. Use a pipette to carefully transfer the top transparent layer to a new 50 mL centrifuge tube without RNase and DNase. Add 1:1 volume of isopropanol to each tube, mix it upside down several times, and let it stand at room temperature for 10 minutes. Centrifuge at 4000g for 30 minutes to remove the supernatant and retain the precipitate. Add 1 mL of 75% ethanol to the precipitate and transfer it to a 1.5 mL centrifuge tube. Flip the precipitate several times to fully contact the liquid. Centrifuge at 7500g for 5 minutes, remove the supernatant and retain the precipitate. Keep the centrifuge tube open, let it dry at room temperature for 10 minutes, add 400uL of enzyme-free water, and incubate at 55°C for 10 minutes to ensure that the RNA is completely dissolved, and then obtain total RNA.

[0187] Take 1 μL and use a nucleic acid concentration meter to detect the RNA concentration and A260 / A280 and record them.

[0188] (2) RNA reverse transcription:

[0189] Reverse transcription kit (Promega GoScript TM Reverse transcription kit) was used to reverse transcribe the total RNA obtained in the above step (1).

[0190] The specific operations are:

[0191] The total RNA sample was divided into two parts, one using the Oligo dT Primer in the kit as a primer, and the other using the Random 6-mers in the kit as a primer. The reaction solution was prepared in a 1.5 ml centrifuge tube according to the system shown in Table 4, and the reaction system was expanded in the same proportion according to the amount of RNA for amplification.

[0192] Table 4 5 μL reverse transcription reaction system

[0193] Components content Oligo dT Primer(50μM) / Random 6-mers(50μM) 1μL Total RNA 2μg <![CDATA[ddH2O]]> Make up to 5 μL

[0194] The above reaction solution was divided into eight PCR tubes and placed in a PCR instrument. Centrifuged briefly, reacted at 70°C for 5 minutes to denature the RNA, and quickly cooled on ice after the reaction. Then, the remaining reaction solution was added to the eight PCR tubes according to the ratio in Table 5.

[0195] Table 5 Other reaction liquid ratios

[0196] Components content Table 4 System after reaction 5μL 5×Reaction Buffer 4μL <![CDATA[MgCl2]]> 2uL dNTP mix 1uL RNase inhibitors 0.4μL RTase 1μL <![CDATA[ddH2O]]> Make up to 20 μL

[0197] Mix the reaction solution in Table 5 and centrifuge briefly. Place the centrifuge tube in a PCR instrument, react 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, and store at 4°C.

[0198] (3) PCR amplification:

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

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

[0201] Table 6 First round PCR reaction system

[0202]

[0203]

[0204] The PCR reaction used a three-temperature method: each cycle was denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, and the cycle was repeated 35 times.

[0205] The primers in the first PCR included:

[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] The lowercase part in each primer is the restriction site.

[0213] Among them, 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.

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

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

[0216] Table 7 Second round 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 were the same as above. After the reaction, the PCR product was subjected to 1.5% agarose gel electrophoresis at 160V for 20min, and the target band with a fragment size of 750bp was finally selected. 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, which was stored at 4°C after the concentration was measured.

[0222] (4) Enzyme ligation of vector and PCR amplification product:

[0223] The second round PCR product was connected to the phage plasmid pComb3XSS by restriction digestion, thereby constructing a phage plasmid library containing the amplified target fragment (ScFv fragment). The restriction endonucleases SpeI and SacI were used for the digestion.

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

[0225] Table 8 Vector restriction enzyme digestion system

[0226] Components content Vector (pComb3XSS) 20 μg SpeI 10μL SacI 10μL 10×CutSmart Buffer 50μL Quick CIP 5μL <![CDATA[ddH2O]]> Make up to 500 μL

[0227] Table 9 Target fragment (ScFv fragment) enzyme digestion system:

[0228] Components content scFv 5μg SpeI 5μL SacI 5μL CutSmart 10×buffer 25μL <![CDATA[ddH2O]]> Make up to 250 μL

[0229] Reaction conditions: incubate at 37℃ for 2h, 80℃ for 3min. After the digestion, the vector digestion product is run on gel and then recovered using the NEB DNA purification and recovery kit (do not irradiate with UV); the ScFv digestion product does not need to be run on gel and can be directly recovered.

[0230] After the enzyme digestion reaction and purification, the concentration of the recovered product was measured, and then the digested vector and the ScFv digestion product were ligated according to the ligation reaction system shown in Table 10.

[0231] Table 10 Ligation reaction system

[0232]

[0233]

[0234] The above ligation system was reacted at 37°C overnight (16-24h). After the ligation was completed, the ligation product was recovered using the NEB DNA purification recovery kit. The concentration of the recovered ligation product was detected and recorded, and stored at 4°C.

[0235] Example 3 Construction of bacterial library

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

[0237] Take the TG1 strain and streak a single colony on 2×YT solid medium, culture overnight at 37℃, pick a single colony from the single colony plate to 10ml 2×YT medium, culture overnight at 37℃, 220rpm. Inoculate the bacteria into 100mL 2×YT medium at a dilution ratio of 1:100, culture at 37℃250rpm for 40min, measure the OD value, and then measure it every 20min until OD600 = 0.3-0.35. Collect the bacterial solution, centrifuge at 3200g, 0-4℃ for 10min, discard the supernatant, place on ice, add 40mL pre-cooled ddH2O to resuspend, centrifuge at 3200g, 0-4℃ for 10min, discard the supernatant, place on ice, add 1mL pre-cooled ddH2O to resuspend, transfer to a 1.5mL pre-cooled EP tube, centrifuge at 10000g, 4℃ for 30s, and repeat once. The supernatant was discarded, and the cell suspension was placed on ice. 400 μL of pre-cooled ddH2O was added to resuspend the cell suspension to obtain a TG1 E. coli competent cell suspension.

[0238] Take the ScFv fragment obtained in the above example, and use the electroporation method to construct an E. coli library. The specific method is: use a pre-cooled gun tip to add 100ng of ScFv fragment to 50μL of the above TG1 E. coli competent cell suspension, blow gently, transfer to a pre-cooled 1mm electroporation cup, confirm that the mixture is at the bottom of the electroporation cup and there are no bubbles, set 1800V, 1mm spacing, and perform electroporation. Immediately after completion, add 1000μL of 37℃ SOC culture medium, take the mixed solution out of the electroporation cup, and shake at 37℃, 180rpm for 90min to recover. Use 2×YT liquid culture medium for 10-fold gradient dilution, a total of 6 gradient dilutions (10 dilutions each 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 5 μL of each gradient was evenly added to 2×YT-GA solid medium, dried and incubated at 37°C overnight. The number of colonies on the gradient dilution plate was counted and the ligation efficiency was calculated. The ligation efficiency formula is:

[0239] E(pfu / ug)=N×D×10

[0240] Wherein, E is the competent efficiency (unit: pfu / ug), D is the dilution factor, and N is the number of monoclones on the plate with the corresponding dilution factor.

[0241] Repeat 100 electroporation transformation reactions according to the above method, and evenly spread the revived bacterial solution on 100 2×YT-GA245mm square culture medium plates, dry them, and invert them for overnight culture at 37°C. Take the square plates cultured overnight, add 6mL 2×YT liquid culture medium to the surface of each culture plate, gently scrape off the 100 square plate colonies with a spreading stick and collect the bacterial solution into a 50mL centrifuge tube, add glycerol with a final concentration of 20%, and it is the bacterial library.

[0242] Take 10 μL of bacterial solution into 990 μL of 2×YT liquid medium and measure OD600 using NanoDrop. Calculate and record the total OD600 of the bacterial library.

[0243] T OD600 =M OD600 ×100

[0244] Among them, T OD600 is the total bacterial OD600, M OD600 is the measured OD600.

[0245] Example 4 Phage library construction

[0246] Take an appropriate amount from the above bacterial library into a 1.5ml EP tube. The calculation formula for the bacterial liquid volume is:

[0247]

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

[0249] Transfer it to 100mL 2×YT-GA liquid medium to make the initial OD600 0.1. Cultivate in a constant temperature shaker at 37°C, 250rpm until the bacterial solution OD600 reaches 0.5-0.55. Calculate and add helper phage M13K07 according to the following formula to make the number of bacteria: phage 1:20. The formula for calculating the amount of phage added is:

[0250]

[0251] Where V is the volume of helper phage added (in mL), T helper-phage is the titer of the helper phage used, and OD600 is the OD600 value of the bacterial solution.

[0252] The culture was continued for 30 min at 37°C and 220 rpm in a constant temperature shaker. The TG1 bacteria were collected by centrifugation at 3200 g for 5 min, the supernatant was removed, and the pellet was resuspended and transferred to 100 mL of 2×YT-AK liquid culture medium and cultured overnight at 30°C and 250 rpm in a constant temperature shaker.

[0253] Transfer the overnight culture to a new 50mL centrifuge tube and centrifuge at 4000g for 30min at 4℃. Take the supernatant, add 1 / 4 volume of 20% PEG / 2.5M NaCl precooled at 4℃, mix thoroughly and place on ice for 30min. Centrifuge at 4000rpm at 4℃ for 30min, discard the supernatant, and invert on paper for 2min. Add 1mL PBS to resuspend the precipitate, centrifuge at 12000rpm at 4℃ for 20min. Take the supernatant, add 1 / 4 volume of precooled 20% PEG / 2.5M NaCl solution, mix well and place on ice for 10min. Centrifuge at 12000rpm at 4℃ for 10min, discard the supernatant, add 1ml PBS to resuspend the precipitate. Centrifuge at 12000rpm at 4℃ for 2min, take the supernatant, which is the phage library, add glycerol to a final concentration of 20%, and store at -80℃.

[0254] The titer of the phage library was tested. The specific operation was as follows: add the phage library to 10mL 2×YT liquid culture medium, and culture at 37°C and 250rpm for about 45min-60min until the OD600 is 0.5-0.55. Take 10μL of the cultured phage library and dilute it 10 times (13 dilution gradients in total). Add 90μL of untreated TG1 bacterial solution to each dilution gradient, mix well, and incubate at 37°C for 20min. Then take 5μL from each dilution gradient and add it dropwise to 2×YT-GA solid culture medium, dry it, and culture it overnight at 37°C. Count it, and calculate the number of phage particles per milliliter of phage solution according to the phage library titer formula.

[0255] T(pfu / ml)=N×D×400

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

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

[0258] Antibodies that specifically bind to gB protein were screened using antigen solid phase adsorption screening method.

[0259] The specific operations are as follows:

[0260] Dissolve 50 μg of the gB protein obtained in the above example in 2 mL PBS and coat the immunotube overnight at 4°C. Coat the negative control protein (50 μg BSA) as a negative control at the same time. Discard the coated protein, rinse with 2 mL PBS 3 times, and then block with 2 mL 3% BSA (dissolved in PBST) at room temperature for 2 hours. Discard the blocking solution, take 100 μL of the phage library in the above example, dilute with 2 mL PBS, and incubate in the immunotube for 1 hour. Discard the liquid, wash with 2 mL PBST 5 times, 5 minutes each time. Then wash with 2 mL PBS 5 times, 5 minutes each time. While washing, take 1 mL of the overnight TG1 saturated bacterial solution to 100 mL 2×YT liquid culture medium, and culture at 37°C 150 rpm until OD600 = 0.5 (about 1.5 hours). Discard the washing solution and elute with 1 mL 0.1 mg / mL Trypsin at room temperature for 30 minutes. Add 1 mL of the eluate to 10 mL of the cultured TG1 bacterial solution (OD = 0.5), infect at 37°C, 150 rpm for 30 min; centrifuge at 4°C, 3000 g for 10 min. Then resuspend the precipitate in 1 mL of 2×YT liquid culture medium. Evenly spread 1 mL of the resuspended product eluted from the positive immune tube on a 245 mm × 245 mm 2×YT solid culture dish, and culture at 37°C overnight (16 to 20 h). Determine the phage titer of the positive tube and the negative control tube respectively, using the same determination method as in the above example.

[0261] According to the phage titer comparison results, 5 mL of 2×YT liquid culture medium was added to the culture dish with high phage titer after overnight culture, all colonies were scraped off with a coating rod, and a daughter phage library was prepared according to the method in the above embodiment. The above operation was repeated until the antibody with the highest affinity to 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 highest affinity antibodies were tested using ELISA.

[0264] Take 10 μL of the culture solution of the antibody with the highest affinity to gB, and dilute the solution with 1 ml of 2×YT liquid medium. Perform clone streaking on 2×YT-GA solid culture plate, and culture overnight at 37°C for 16 to 20 hours. Pick 192 monoclonal clones to 96-well plate (each well contains 200 μL 2×YT-GA liquid), and culture at 37°C until saturation. Take 2 μL of saturated bacterial solution to a new 96-well plate (each well contains 200 μL 2×YT-A liquid), so that the initial OD is about 0.03, and let it stand at 37°C for 2.5h to 3h until the OD is about 0.5. Add 0.1 μL of the above helper phage to each well, and infect at 37°C for 30 minutes. Add 0.2 μL Kana (kanamycin) to each well, and culture overnight at 30°C. Protein (- / +) coated ELISA plate overnight. Centrifuge the 96-well plate cultured overnight at 4°C and 3400g for 5 minutes. Discard the liquid in the ELISA plate, wash once with 350μL PBS. Block with 350μL 3% BSA (PBST) at 37℃ for 1h. Discard the blocking solution, wash once with 350μL PBST, and tap the plate to remove the liquid. Add 140μL 3% BSA (PBST) to each well, and then add 60μL of the above phage library expressed antibody (the antibody with the highest affinity, as the primary antibody), and incubate at 37℃ for 1h. Discard the liquid (primary antibody), wash 5 times with 350μL PBST, and tap the plate. Add 100μL M13Antibody (HRP) as the secondary antibody (1:8000 addition ratio, relative to the blocking solution), incubate at 37℃ for 1h, discard the liquid (secondary antibody), wash 5 times with 350μL PBST, and tap the plate. 100μL TMB is protected from light for 2-3min, and 100μL dilute hydrochloric acid (concentrated hydrochloric acid: water = 1:12) is used to terminate the reaction. Read the OD450 and OD630 of the ELISA plate.

[0265] The complete heavy chain fragment can be expressed by connecting the upstream of the antibody heavy chain variable region with the CMV fragment and the downstream with the constant region of human IgG1 and the ployA fragment; and the complete light chain fragment can be expressed by connecting the upstream of the antibody light chain variable region with the CMV fragment and the downstream with the constant region of the light chain κ / λ and the ploy A fragment. The expression of the antibody can be achieved by co-transfecting the plasmids carrying the full-length sequences of the heavy and light chains of the above antibodies into 293T cells, and the antibody can be purified using protein A beads.

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

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

[0268] The 4C1 full-length heavy chain has a total of 467 amino acid residues (excluding *), 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 bold parts are the amino acid sequences of the three complementary 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). * indicates a stop codon.

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

[0272]

[0273]

[0274] Among them, the underlined part in the sequence is the nucleotide sequence of the heavy chain variable region (SEQ ID NO: 20). The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region 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 4C1 full-length light chain has a total of 235 amino acid residues (excluding *), specifically:

[0276]

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

[0278] The coding gene of 4C1 full-length light chain has a total of 708 bases, specifically:

[0279]

[0280]

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

[0282] Coomassie Brilliant Blue staining was used to identify the expression of light and heavy chains of 4C1 antibody.

[0283] The specific operation steps are as follows: After preparing 10% separation gel according to the table below, quickly add 7mL to the gel plate mold after mixing, and add 2mL of anhydrous ethanol to seal. After the lower layer of separation gel solidifies, discard the anhydrous ethanol and let it stand to evaporate. Prepare concentrated gel, add 2mL to the upper layer of separation gel, and quickly insert the comb. Use the BCA protein quantitative analysis kit for protein quantification. Add 10ug 4C1 protein sample to 5×SDS loading buffer and 95℃ metal bath for 10min. After the gel is dry, put the mold into the electrophoresis tank, add electrophoresis solution, and unplug the comb. Add the treated protein sample to the sample well and add marker. First use 80V low voltage for protein concentration, and then change the voltage to 120V for protein separation. The electrophoresis time is determined according to the marker position. Put the gel into Coomassie Brilliant Blue Stain (Novozyme) and shake it in a shaker until the band appears, discard the color developing solution, add double distilled water and wash the gel in a shaker, repeat several times until the gel background is clean.

[0284] The results are as follows Figure 1 As shown, 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 4B3 full-length heavy chain has a total of 467 amino acid residues (excluding *), specifically:

[0289]

[0290]

[0291] Among them, the underlined part in the sequence is the amino acid sequence of the heavy chain variable region (SEQ ID NO: 36). The underlined and bold parts are the amino acid sequences of the three complementary 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). The italic part is the signal peptide (SEQ ID NO: 18). * indicates the stop codon.

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

[0293]

[0294] Among them, the underlined part in the sequence is the nucleotide sequence of the heavy chain variable region (SEQ ID NO: 41). The underlined and bold parts are the nucleotide sequences of the three complementary 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). The three bases at the very beginning of the 5' end and the very end of the 3' end are the start codon and the stop codon, respectively. The italic part is the signal peptide (SEQ ID NO: 24).

[0295] The 4B3 full-length light chain has a total of 238 amino acid residues (excluding *), specifically:

[0296]

[0297]

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

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

[0300]

[0301] The underlined part of the sequence is the nucleotide sequence of the light chain variable region (SEQ ID NO: 50). The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-L4 and CDR-L6 in the light chain variable region defined by the IMGT scheme (SEQ ID NO: 51, 52, respectively, and the nucleotide sequence of CDR-L5 is ). The first three bases at the 5' end and the last three bases at the 3' end are the start codon and the stop codon, respectively. The italic part 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. The experimental operation method was the same as that in Example 6. Figure 2 shown.

[0303] Effect Example 1 4C1 Antibody Affinity Determination

[0304] The affinity of antibody 4C1 was determined by biofilm interferometry (BLI).

[0305] BLI can be performed according to the conventional method in the art. In this embodiment, the specific operation is as follows: a biosensor (Sartorius, Germany) is placed in the SA probe) is immersed in a buffer solution (a mixture of KB buffer, 0.1% BSA and 0.02% Tween 20) for equilibrium. Then it is taken out and immersed in a solution containing 10μg / mL gB-Biotin (biotin-labeled gB protein). The gB antigen in the solution will bind to the surface of the SA (streptavidin) biological probe, increasing the thickness of the surface film layer. Then the biosensor with a solidified antigen of known concentration is immersed in a buffer solution as a baseline. By immersing the solidified biosensor with a known antigen concentration in a sample solution containing 0-1000nM 4C1 antibody, the specific binding between antigen and antibody will lead to an increase in the film thickness. The biosensor bound to the 4C1 antibody is immersed in a buffer solution for dissociation, and the antibody to be tested (4C1 antibody) will fall off from the surface of the biosensor, resulting in a decrease in the film thickness. By real-time monitoring of the biosensor biofilm thickness during the experiment, the kinetic constant of the sample to be tested (4C1 antibody) can be obtained.

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

[0307] After testing, the KD (M) of the 4C1 antibody of the present invention is 1.0E -9 This indicates that the 4C1 antibody has a clear affinity with the gB antigen.

[0308] Effect Example 2 Related Applications of 4C1 Antibody

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

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

[0311] In this embodiment, the inventors used gB protein for Western Blot as an example to verify its practical application effect.

[0312] The steps are:

[0313] Denature 10ug of gB protein and perform electrophoresis (the electrophoresis steps are the same as the staining). After completion, carefully open the gel plate and cut off the concentrated gel. Use methanol to activate the 0.22μm PVDF membrane. Place 2 layers of filter paper, PVDF membrane, separation gel, and 2 layers of filter paper in order from bottom to top and fix them in the transfer splint. Put the splint into the transfer instrument, add the transfer solution, and transfer the membrane at 100mA constant current for 2h. Transfer the PVDF membrane to 5% milk blocking solution and incubate on a shaker at room temperature for 1h. Transfer the PVDF membrane to the above 4C1 antibody solution (1μg / mL) and incubate at 4℃ overnight. Rinse with TBST 3 times, 5min each time. Transfer the PVDF membrane to Rabbit Anti-HumanIgG H&L (HRP) antibody (abcam, 1:10000 dilution) and incubate at room temperature for 1h. Rinse with TBST 3 times, 5min each time. Evenly drop the chemiluminescent solution on the surface of the PVDF membrane and react for several minutes in the dark. Use a chemiluminescent imager to image the PVDF membrane. Set up a blank control.

[0314] The results are as follows Figure 4 shown.

[0315] It can be found that when using 4C1 antibody as the primary antibody for Western Blot, the gB protein sample will show a black band at 35kD~40kD, indicating that 4C1 antibody can be used for Western Blot detection.

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

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

[0318] The specific steps are as follows: 293T cells were cultured in a 12-well plate with a cell density of about 40%. After the cells adhered to the wall, the gB-Flag recombinant plasmid was transiently transfected (the preparation method was the same as the above-mentioned gB recombinant plasmid), and a negative control was set up at the same time, and the culture was continued for 24 hours. The culture medium was replaced, and the 4C1 antibody labeled with 403 fluorescence was incubated at 4°C for 30 minutes. Anti-Flag-488 was incubated at 4°C for 30 minutes. The 2G4 antibody labeled with 403 fluorescence was used as a negative control antibody. Among them, the 2G4 antibody is a general gB antibody with a non-highest affinity that was eliminated in the above embodiment. The cells were digested with 1mL of trypsin for 3min, centrifuged at 200g for 5min, and resuspended and washed with PBS for 3 times. The cells were resuspended in 500μL PBS and filtered using a 40μm cell mesh. Cell fluorescence was detected using a flow cytometer. 293T cells without any treatment were used as blank controls.

[0319] The results are as follows Figure 5 As shown. The 4C1 antibody labeled with 403 fluorescence was incubated at 4°C for 30 minutes, and then Anti-Flag-488 was used to incubate at 4°C for 30 minutes, and the 2G4 antibody labeled with 403 fluorescence was used as a negative control antibody (2G4 antibody is a control antibody against Ebola virus). The results showed that the positive rate of the 4C1 antibody group was 11.77%, and the negative control group was 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 used for flow cytometry.

[0320] It can be found that the screening effect using 4C1 antibody is significantly better than that of general gB antibody, indicating that 4C1 antibody has better specific binding and detection effect for general gB antibody.

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

[0322] In this embodiment, sections of nasopharyngeal carcinoma patients were used as test objects to test the actual application effect of 4C1 antibody in immunohistochemistry.

[0323] The specific steps are as follows: baking: 65 degrees for 1 hour; dewaxing: soaking in xylene 3 times; hydration: soaking in 100% ethanol for 10 minutes twice, soaking in 95%, 90%, 80%, 70%, 60% ethanol for 5 minutes, soaking in double distilled water for 5 minutes twice; removing endogenous peroxidase: treating with 3% hydrogen peroxide for 10 minutes; high-pressure repair: adding 1L repair solution to the pressure cooker and boiling at 2100W, then adjusting to 1000W and timing for 2 minutes and 30 seconds; natural cooling and drawing circles, washing with PBST for 5 minutes 3 times; blocking with 5% BSA at 37℃ for 30 minutes; shaking off BSA and directly adding primary antibody 4C1 (150ng / ul) and incubating at 4℃ overnight; washing primary antibody: washing with PBST for 5 minutes 3 times, adding 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; DAB color development for 1 minute; hematoxylin nucleus staining for 1 minute; rinsed with running water for 2 hours; after drying the slides, they were sealed with neutral resin.

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

[0325] The results showed that brown tissue cell staining was visible in NPC sections, indicating that 4C1 antibody can be used for immunohistochemical staining and gB expression in NPC sections.

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

[0327] In this embodiment, C666 nasopharyngeal carcinoma cells were used as test subjects after forming tumors in mice to test the actual application effect of 4C1 antibody in tumor tissues.

[0328] The specific steps are as follows: nude mice were subcutaneously injected with 2*10 ^6 nasopharyngeal carcinoma C666 cells were allowed to form tumors. TM AbFluor TM 4C1 antibody (100ug / mouse) was labeled with 647 coupling kit (Abkine), and an equal amount of 2G4 antibody was also labeled as a control. The labeled antibody was injected into the mouse through the tail vein. After 48 hours, the mouse was imaged using PE IVISSpectrum small animal in vivo imager.

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

[0330] The results showed that mice injected with 4C1 antibody had fluorescence at the tumor site, while mice injected with the control group 2G4 antibody did not have fluorescence accumulated at the tumor site, suggesting that 4C1 antibody can be used as a tumor targeting marker.

[0331] Effect Example 3 4B3 Antibody Affinity Determination

[0332] The affinity of antibody 4B3 was determined by biofilm interferometry (BLI).

[0333] BLI can be performed according to the conventional method in the art, and the specific operation is the same as that in Example 1.

[0334] The results are as follows Figure 8 shown.

[0335] After testing, the KD (M) of the 4B3 antibody of the present invention is 3.968E -9 This indicates that the 4B3 antibody has a clear affinity with the gB antigen.

[0336] Effect Example 4 Related Applications of 4B3 Antibody

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

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

[0339] In this embodiment, the inventors used gB protein for Western Blot as an example to verify its practical application effect.

[0340] The operation steps are the same as those of Example 2.

[0341] The results are as follows Fig. 9 shown.

[0342] It can be found that when using 4B3 antibody as the primary antibody for Western Blot, the gB protein sample will show a black band at 35kD~40kD, indicating that 4B3 antibody can be used for Western Blot detection.

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

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

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

[0346] The results are as follows Fig.10 shown.

[0347] It can be found that the screening effect using 4B3 antibody is significantly better than that of general gB antibody, indicating that 4B3 antibody has better specific binding and detection effect for general gB antibody.

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

[0349] In this embodiment, NKT lymphoma sections were used as test objects to test the actual application effect of 4B3 antibody in immunohistochemistry.

[0350] The specific steps are as follows: baking: 65 degrees for 1 hour; dewaxing: soaking in xylene 3 times; hydration: soaking in 100% ethanol for 10 minutes twice, soaking in 95%, 90%, 80%, 70%, 60% ethanol for 5 minutes, soaking in double distilled water for 5 minutes twice; removing endogenous peroxidase: treating with 3% hydrogen peroxide for 10 minutes; high-pressure repair: adding 1L repair solution to the pressure cooker and boiling at 2100W, then adjusting to 1000W and timing for 2 minutes and 30 seconds; natural cooling and drawing circles, washing with PBST for 5 minutes 3 times; blocking with 5% BSA at 37℃ for 30 minutes; shaking off BSA and directly adding primary antibody 4B3 (150ng / ul) and incubating at 4℃ overnight; washing primary antibody: washing with PBST for 5 minutes 3 times, adding 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; DAB color development for 1 minute; hematoxylin nucleus staining for 1 minute; rinsed with running water for 2 hours; after drying the slides, they were sealed with neutral resin.

[0351] The results are as follows Fig.11 shown.

[0352] The results showed that brown histiocyte staining was observed in NKT lymphoma sections, indicating that the 4B3 antibody could be used for immunohistochemical staining and gB expression in NPC sections.

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

[0354] In this embodiment, C666 nasopharyngeal carcinoma cells were used as test subjects after forming tumors in mice to test the actual application effect of 4B3 antibody in tumor tissues.

[0355] The specific steps are as follows: nude mice were subcutaneously injected with 2*10 ^6 nasopharyngeal carcinoma C666 cells were allowed to form tumors. TM AbFluor TM 4B3 antibody (100ug / mouse) was labeled with 647 coupling kit (Abkine), and an equal amount of 2G4 antibody was also labeled as a control. The labeled antibody was injected into the mouse through the tail vein. After 48 hours, the mouse was imaged using PE IVISSpectrum small animal in vivo imager.

[0356] The results are as follows Fig.12 shown.

[0357] The results showed that mice injected with 4B3 antibody had fluorescence at the tumor site, while mice injected with the control group 2G4 antibody did not have fluorescence accumulated at the tumor site, suggesting that 4B3 antibody can be used as a tumor targeting marker.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against Epstein-Barr virus gB protein, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; The monoclonal antibody comprises any one of A) or B); A) The heavy chain comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of said heavy chain variable region, The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 14; The light chain comprises: a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 26; B) The heavy chain comprises: a heavy chain variable region comprising CDR-H4, CDR-H5 and CDR-H6 of the heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 36; The light chain comprises: A light chain variable region, comprising CDR-L4, CDR-L5 and CDR-L6 of the light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO:

46.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: A) The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 are shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 27 and SEQ ID NO: 28, respectively; the amino acid sequence of CDR-L2 is DNH; and the CDRs are defined according to the IMGT definition scheme; or B) The amino acid sequences of CDR-H4, CDR-H5, CDR-H6, ​​CDR-L4 and CDR-L6 are shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 47 and SEQ ID NO: 48 respectively, the amino acid sequence of CDR-L5 is KVS, and the CDRs are defined according to the IMGT definition scheme.

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

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

5. A biological material related to the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4, wherein the biological material comprises at least one of f1) to f16): f1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4; f2) an expression cassette comprising the nucleic acid molecule described in f1); f3) a vector comprising the nucleic acid molecule described in f1); f4) a vector comprising the expression cassette described in f2); f5) a transgenic cell line comprising the nucleic acid molecule described in f1); f6) a transgenic cell line comprising the expression cassette described in f2); f7) a transgenic cell line comprising the vector described in f3); f8) a transgenic cell line comprising the vector described in f4); f9) a microorganism comprising the nucleic acid molecule described in f1); f10) A microorganism comprising the expression cassette described in f2); f11) a microorganism comprising the carrier described in f3); f12) a microorganism comprising the vector described in f4); f13) A virus comprising the nucleic acid molecule described in f1); f14) A virus comprising the expression cassette described in f2); f15) A virus comprising the vector described in f3); f16) A virus comprising the vector described in f4).

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

7. Use of at least one of (1) to (4) in the preparation of a product; (1) The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (2) The recombinant protein according to claim 4; (3) The biomaterial according to claim 5; (4) The conjugate according to claim 6; The product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip; Preferably, the drug has at least one of the functions of g1) to g2): g1) Prevention of EB virus infection; g2) Treating and / or preventing diseases caused by EB virus infection; Preferably, the reagent, detection plate, detection chip or kit has at least one of the functions h1) to h3): h1) detecting the presence or level of gB protein in a sample; h2) Detection of Epstein-Barr virus; h3) Diagnosis of diseases caused by Epstein-Barr virus infection.

8. A product, comprising at least one of i1) to i3): i1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; i2) the recombinant protein according to claim 4; i3) The conjugate according to claim 6; The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

9. A drug comprising at least one of k1) to k4): k1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; k2) The recombinant protein according to claim 4; k3) The biological material according to claim 5; k4) the conjugate according to claim 6; Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

10. A vaccine comprising at least one of (k1) to (k4) and an adjuvant: (k1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (k2) the recombinant protein according to claim 4; (k3) The biomaterial according to claim 5; (k4) The conjugate according to claim 6.

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