A neutralizing monoclonal antibody against Epstein-Barr virus gB antigen and its application

Abstract

The present invention discloses a neutralizing monoclonal antibody against Epstein-Barr virus gB antigen and its application. The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises heavy chain CDR-H1 shown in SEQ ID NO:15; heavy chain CDR-H2 shown in SEQ ID NO:16; and heavy chain CDR-H3 shown in SEQ ID NO:17. The light chain variable region comprises light chain CDR-L1 shown in SEQ ID NO:18; light chain CDR-L2 shown in SEQ ID NO:19; and light chain CDR-L3 shown in SEQ ID NO:20. The antibody in the present invention can specifically recognize Epstein-Barr virus gB antigen and has a good neutralizing effect, so that various expression vectors and transformants can be developed based on the antibody, and thus can be effectively used for the detection, prevention and treatment of Epstein-Barr virus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a neutralizing monoclonal antibody against Epstein-Barr virus (EBV) gB antigen and its application. Background Art

[0002] Epstein-Barr virus (EBV), also known as human herpesvirus 4, was first isolated from Burkitt lymphoma cells by Epstein and Barr through in vitro suspension culture and cell line establishment in 1964. It is a DNA tumor virus belonging to the lymphotropic virus genus of the γ-subfamily. The EBV particle consists of four structural components: core protein, capsid, tegument, and envelope. The core is the core protein wrapped with DNA.

[0003] The most common way of EBV infection is latent infection. More than 90% of EBV-infected individuals have lifelong latent infection, and the virus can be activated and initiate carcinogenesis under certain conditions. Current research has found that human EBV is closely related to the occurrence of malignant tumors such as nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin and non-Hodgkin lymphoma, Burkitt lymphoma, and epithelial cell carcinoma including gastric cancer. The initial replication site of EBV is the oropharynx, where it grows and multiplies in B lymphocytes and oral epithelial cells, and then infects B lymphocytes. These cells enter the blood circulation in large numbers, causing systemic infection. When the body's immune function is low, latent EBV is activated to form recurrent infection.

[0004] Currently, there is no effective vaccine against EBV, and there is also a lack of specific treatment methods for diseases caused by EBV infection. The treatment of infectious mononucleosis mostly uses antiviral drugs such as acyclovir. Although these drugs can relieve symptoms to a certain extent, they cannot eliminate EBV in B lymphocytes and pharyngeal epithelial cells. The treatment of EBV-related tumors is mainly chemotherapy and radiotherapy, but the curative effect is poor for metastatic or recurrent patients.

[0005] Monoclonal antibodies can be mass-produced. Based on their high affinity and high specificity for antigen binding, the 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 in the treatment of infectious diseases due to their specificity and flexibility in use. However, there is still no humanized monoclonal antibody against EBV envelope glycoprotein on the market. Therefore, the development of human monoclonal antibodies against EBV will provide more effective prevention and treatment means for EBV infection-related diseases. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a monoclonal antibody against Epstein-Barr virus (EBV) gB antigen and its application. This monoclonal antibody can bind to EBV gB antigen with high specificity and high affinity, and its neutralization effect and affinity are higher than those of conventional EBV antibodies, having extremely high application value.

[0007] In the first aspect of the present invention, there is provided an antibody that specifically binds to EBV gB protein, and this antibody comprises a heavy-chain variable region and a light-chain variable region. The heavy-chain variable region comprises heavy-chain CDR-H1 shown in SEQ ID NO:15; heavy-chain CDR-H2 shown in SEQ ID NO:16; and heavy-chain CDR-H3 shown in SEQ ID NO:17. The light-chain variable region comprises light-chain CDR-L1 shown in SEQ ID NO:18; light-chain CDR-L2 shown in SEQ ID NO:19; and light-chain CDR-L3 shown in SEQ ID NO:20.

[0008] Among them, CDR-H1 is GFTFDDYAMH (SEQ ID NO:15); CDR-H2 is GISWNAENIAYADSVKG (SEQ ID NO:16); CDR-H3 is DRAHYFGSGSYFDSS (SEQ ID NO:17).

[0009] CDR-L1 is RASQSISSYLN (SEQ ID NO:18); CDR-L2 is AASSLQS (SEQ ID NO:19); CDR-L3 is QQSYSTPFT (SEQ ID NO:20).

[0010] In some embodiments of the present invention, the amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:21.

[0011] In some embodiments of the present invention, the amino acid sequence shown in SEQ ID NO:21 is: EVQLLESGGGLVQPGRSLRLSCATSGFTFDDYAMHWVRQAPGKGLEWVSGISWNAENIAYADSVKGRFTISRDNAKNSLFLHMNSLRAEDTAFYYCARDRAHYFGSGSYFDSSGQGTLVTVSSASTKGPS (SEQ ID NO:21).

[0012] In some embodiments of the present invention, in addition to the heavy-chain variable region, the heavy chain of the antibody further comprises other sequences, including but not limited to a signal peptide.

[0013] In some embodiments of the present invention, the full-length sequence of the heavy chain is as shown in SEQ ID NO: 11.

[0014] In some embodiments of the present invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 22.

[0015] In some embodiments of the present invention, the amino acid sequence shown in SEQ ID NO: 22 is: EIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK (SEQ ID NO: 22).

[0016] In some embodiments of the present invention, in addition to the light chain variable region, the light chain of the antibody further includes other sequences, including but not limited to a signal peptide.

[0017] In some embodiments of the present invention, the full-length sequence of the light chain is as shown in SEQ ID NO: 13.

[0018] The second aspect of the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect of the present invention.

[0019] In some embodiments of the present invention, the nucleic acid molecule encoding CDR-H1 is GGATTCACCTTTGATGATTATGCCATGCAC (SEQ ID NO: 25); the nucleic acid molecule encoding CDR-H2 is GGTATTAGTTGGAATGCTGAAAATATAGCCTATGCGGACTCTGTGAAGGGC (SEQ ID NO: 26); the nucleic acid molecule encoding CDR-H3 is GATAGGGCCCATTACTTTGGTTCGGGGAGTTATTTTGACTCTTCG (SEQ ID NO: 27).

[0020] In some embodiments of the present invention, the nucleic acid molecule encoding CDR-L1 is CGGGCAAGTCAGAGCATTAGCAGCTATTTAAAT (SEQ ID NO: 28); the nucleic acid molecule encoding CDR-L2 is GCTGCATCCAGTTTGCAAAGT (SEQ ID NO: 29); the nucleic acid molecule encoding CDR-L3 is CAACAGAGTTACAGTACCCCATTCACT (SEQ ID NO: 30).

[0021] In some embodiments of the present invention, the nucleic acid molecule contains the nucleotide sequence shown in SEQ ID NO: 23 and / or SEQ ID NO: 24.

[0022] In some embodiments of the present invention, SEQ ID NO: 23 is the nucleotide sequence encoding the variable region of the heavy chain of the antibody, specifically: 5’-GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAACCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATGCTGAAAATATAGCCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCACATGAACAGTCTGAGAGCTGAGGACACGGCCTTCTATTACTGTGCAAGAGATAGGGCCCATTACTTTGGTTCGGGGAGTTATTTTGACTCTTCGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCC-3’ (SEQ ID NO: 23).

[0023] In some embodiments of the present invention, SEQ ID NO: 24 is the nucleotide sequence encoding the variable region of the light chain of the antibody, specifically: 5’-GAAATTGTGCTGACGCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA-3’ (SEQ ID NO: 24).

[0024] In some embodiments of the present invention, in addition to the variable regions, the heavy chain and / or light chain of the antibody further comprises other sequences, including but not limited to signal peptides.

[0025] In some embodiments of the present invention, the full-length nucleotide sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 12.

[0026] In some embodiments of the present invention, the full-length nucleotide sequence of the light chain of the antibody is as shown in SEQ ID NO: 14.

[0027] The third aspect of the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect of the present invention.

[0028] In some embodiments of the present invention, the expression vector further contains sequences for auxiliary purposes such as tag sequences and antibiotic sequences. Of course, those skilled in the art should understand that the sequences for auxiliary purposes include but are not limited to the above-mentioned tag sequences and antibiotic sequences.

[0029] The fourth aspect of the present invention provides a transformant containing the nucleic acid molecule described in the second aspect of the present invention and / or the expression vector described in the third aspect of the present invention.

[0030] In some embodiments of the present invention, the transformant includes viruses, bacteria, fungi, and cells.

[0031] Of course, those skilled in the art can reasonably select other transformants for the preservation, purification, expression, secretion, or other necessary uses of the target protein or target fragment according to actual usage requirements.

[0032] In some embodiments of the present invention, the transformant is a cell, a bacterium, and a phage.

[0033] The fifth aspect of the present invention provides an EB virus detection product, which contains at least one of the following (1) to (4):

[0034] (1) The antibody described in the first aspect of the present invention;

[0035] (2) The nucleic acid molecule described in the second aspect of the present invention;

[0036] (3) The expression vector described in the third aspect of the present invention;

[0037] (4) The transformant described in the fourth aspect of the present invention.

[0038] In a specific embodiment of the present invention, the inventors verified through experiments that based on at least one of the above-mentioned antibody, nucleic acid molecule, expression vector or transformant, it can effectively play a role in the detection of Epstein-Barr virus, and the detection effect is accurate. Compared with conventional general Epstein-Barr virus antibodies, it has better affinity, and the KD(M) can reach 1.122E-09.

[0039] The sixth aspect of the present invention provides an Epstein-Barr virus treatment or prevention product, which contains at least one of the following (1) to (4):

[0040] (1) The antibody described in the first aspect of the present invention;

[0041] (2) The nucleic acid molecule described in the second aspect of the present invention;

[0042] (3) The expression vector described in the third aspect of the present invention;

[0043] (4) The transformant described in the fourth aspect of the present invention.

[0044] In a specific embodiment of the present invention, the inventors verified through experiments that based on at least one of the above-mentioned antibody, nucleic acid molecule, expression vector or transformant, it can effectively play a neutralizing effect against Epstein-Barr virus, and the neutralizing effect is stronger than that of conventional general Epstein-Barr virus antibodies, and has excellent preventive or therapeutic efficacy.

[0045] The seventh aspect of the present invention provides the use of the antibody described in the first aspect of the present invention in the preparation of Epstein-Barr virus detection, treatment or prevention products.

[0046] In a specific embodiment of the present invention, the Epstein-Barr virus detection, treatment or prevention products include: detection reagents, detection kits, detection chips, drugs, vaccines.

[0047] The beneficial effects of the present invention are:

[0048] The present invention provides a monoclonal antibody against Epstein-Barr virus gB antigen. This antibody can specifically recognize Epstein-Barr virus gB antigen and has a good neutralizing effect. Therefore, various expression vectors and transformants can be developed based on this antibody, and it can be effectively used for the detection, prevention and treatment of Epstein-Barr virus. Moreover, this monoclonal antibody has high affinity for Epstein-Barr virus, the KD(M) can reach 1.122E-09, and the neutralizing effect is also significantly improved compared with general Epstein-Barr virus antibodies, and it has better use effects and technical advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is the affinity curve of 4H2 antibody with different concentrations measured by BLI method.

[0050] Figure 2 Western Blotting electrophoresis diagram of gB protein based on 4H2 antibody.

[0051] Figure 3 Western Blotting electrophoresis diagram of 293T cells transiently transfected with gB recombinant plasmid based on 4H2 antibody.

[0052] Figure 4 Flow cytometry diagram based on 4H2 antibody.

[0053] Figure 5 Neutralization activity test results of 4H2 antibody. Detailed implementation manners

[0054] In order to make the invention object, technical solutions and their technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with specific implementation manners. It should be understood that the specific implementation manners described in this specification are only for explaining the present invention and not for limiting the present invention.

[0055] The experimental materials and reagents used, unless otherwise specified, are all conventional consumables and reagents that can be obtained commercially.

[0056] Preparation of EBV gB recombinant protein

[0057] gB protein plays an important role in the process of EBV invading epithelial cells and B cells. In the embodiments of the present invention, the inventor selects gB as a bait protein to screen specific antibodies.

[0058] Among them, the selected original gB sequence (including KOZAK sequence and CD5 signal peptide) is:

[0059]

[0060] The above-mentioned gB gene fragment was ligated into the mammalian cell expression vector pcDNA3.1+ (Invitrogen) by the following specific method:

[0061] (1) Amplification of the gB protein gene:

[0062] Select 50 μL of NEB The above-mentioned gB protein gene was amplified using the PCR amplification reaction system (Table 1).

[0063] Table 1 PCR amplification reaction system for the gB protein gene

[0064] Component Content 5×Reaction Buffer 10 μL dNTPs 1 μL Forward primer 2.5 μL Reverse primer 2.5 μL DNA template 1 μL DNA polymerase 0.5 μL High GC Enhancer (from NEB's Q5 kit) 10 μL <![CDATA[ddH2O]]> Make up to 50 μL

[0065] Among them, the upstream primer is: 5’-TGGTGGAATTCGCCACCATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCTGCCTCGGACAGACCCCAGAGCAGCCC-3’ (SEQ ID NO: 2); the downstream primer is 5’-TCGAGCGGCCGCTTAGTGGTGATGGTGATGATGTGACACTGCATTGTCCAAATCC-3’ (SEQ ID NO: 3).

[0066] The amplified target fragment was analyzed by agarose gel electrophoresis. The band with the correct molecular weight was cut under ultraviolet light, and the PCR product was recovered according to the instructions of the commercially available kit.

[0067] (2) Restriction enzyme digestion and ligation of the target fragment and the vector:

[0068] The vector used was the eukaryotic expression plasmid pcDNA3.1+. Both the target fragment and the vector were digested with EcoRⅠ and NotⅠ (using a 50 μL restriction enzyme digestion reaction system (Table 2) for digestion).

[0069] Table 2 Restriction enzyme digestion reaction system

[0070] Component Content 10×CutSmart Buffer 5 μL EcoRⅠ-HF 1 μL NotⅠ-HF 1 μL Target fragment or vector 5 μg <![CDATA[ddH2O]]> Make up to 50 μL

[0071] The restriction enzyme digestion was carried out at 37 °C for 2 - 3 h. After the digested vector was run on agarose gel, it was recovered using a gel recovery kit to obtain a linearized vector. The inserted fragment was directly recovered using a DNA purification kit. After recovery, a 10 μL ligation reaction system (Table 3) was used for ligation.

[0072] Table 3 Ligation reaction system

[0073] Component Content 5×CEⅡ Buffer 2 μL CEⅡ 1 μL Linearized vector 100 ng Insert fragment 100 ng <![CDATA[ddH2O]]> Make up to 10 μL

[0074] The reaction was carried out at 37 °C for 30 min to obtain the ligation product.

[0075] (3) Transformation of the ligation product and screening of positive clones:

[0076] Add the ligation product to the freshly thawed DH5α competent cell suspension, place it on ice for 30 min, heat shock at 42 °C for 90 s, and then put it back on ice for 5 min. Add 200 μL of LB medium and incubate with slow shaking at 30 °C for 40 min. Pipette the culture solution and spread it on an LB plate with ampicillin resistance, and culture overnight at 37 °C.

[0077] Pick single clone colonies for sequencing verification. After the sequencing results are correct, the target recombinant plasmid is obtained. Extract the plasmid in large quantities.

[0078] (4) Expression and extraction of recombinant protein:

[0079] Take human kidney epithelial cells 293F cells for culture to obtain a cell suspension with a cell density of 1.5×10 6 and use the PEI transfection reagent to transfect the recombinant plasmid obtained in step (3). The specific operation is as follows: dilute 2 mg of the recombinant plasmid with 25 mL of Union-293 medium; add 6 mL of 1 mg / mL PEI to 25 mL of Union-293 medium. Vigorously shake and mix the plasmid and PEI, and add it to the 293F cell suspension after standing at room temperature for 20 min. Collect the cell supernatant after culturing for 5 days, centrifuge at 6000 rpm for 1 h at 4 °C, and discard the cell pellet to obtain the supernatant containing the target protein.

[0080] The target protein was purified by affinity chromatography.

[0081] Since the obtained gB recombinant protein has a 6×His tag at the C-terminus, a nickel column can be used for its affinity purification.

[0082] The specific operation is as follows: filter the supernatant containing the target protein obtained in the above example with a 0.65 μm filter paper, bind it to the nickel column beads 3 times, wash the beads 3 times with 30 mM imidazole, and elute the target protein with 500 mM imidazole. Then further purify it by gel filtration chromatography. The specific operation is as follows: concentrate the protein eluted from the nickel column using a 30 kD concentrator tube until the volume is less than 1 mL. Then purify it using Superdex 200 Increase 10 / 300GL.

[0083] The amino acid sequence of the finally obtained target protein is:

[0084] QTPEQPAPPATTVQPTATRQQTSFPFRVCELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKIVTNILIYNGWYADSVTNRHEEKFSVESYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGWLIWTYRTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNTETFHERADSFHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQHWQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKTMHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSPPAPPAARGSTSAAVLRRRRRNAGNATTPVPPAAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETMCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIAGLRKDLDNAVSHHHHHH(SEQ ID NO: 4).

[0085] Construction of phage antibody library

[0086] (1) Total RNA extraction:

[0087] Dilute the blood of nasopharyngeal carcinoma (NPC) patients with PBS at a ratio of 1:1 (10 mL of blood + 10 mL of PBS). Then, carefully cover every 20 mL of the diluted solution on 15 mL of lymphocyte separation solution, maintaining the layered interface. Centrifuge at 2000 rpm at room temperature for 20 min, slowly reduce the speed, aspirate the middle monocyte layer into a new 15 mL centrifuge tube, and add PBS to make up to 15 mL. Centrifuge at 300 g at room temperature for 20 min, transfer the supernatant to a new 50 mL centrifuge tube, and centrifuge at 300 g at room temperature for 20 min. Discard the supernatant. Resuspend the cell pellets in the 15 mL and 50 mL centrifuge tubes with 1 mL of Trizol each. (That is, resuspend with 2 mL of Trizol per 10 mL of blood).

[0088] Add 4 mL of chloroform to every 20 mL of the Trizol-cell pellet resuspension. After shaking on a shaker for 15 s, let it stand at room temperature for 5 min. Centrifuge at 4000 g at 4 °C for 30 min. After centrifugation, a layered structure appears. Carefully transfer the top transparent layer to a new 50 mL centrifuge tube free of RNase and DNase using a pipette. Add isopropanol with a volume ratio of 1:1 to each tube, mix well by inverting up and down several times, and let it stand at room temperature for 10 min. Centrifuge at 4000 g for 30 min to remove the supernatant and retain the precipitate. Add 1 mL of 75% ethanol to the precipitate, transfer it to a 1.5 mL centrifuge tube, and resuspend the precipitate by flicking it up and down several times to ensure full contact with the liquid. Centrifuge at 7500 g for 5 min, remove the supernatant and retain the precipitate. Keep the centrifuge tube open and let it dry at room temperature for 10 min. Add 400 μL of enzyme-free water and incubate at 55 °C for 10 min to ensure complete dissolution of RNA, thus obtaining total RNA.

[0089] Take 1 μL and use a nucleic acid concentration measuring instrument to detect the concentration of RNA and A260 / A280 and record the results.

[0090] (2) RNA reverse transcription:

[0091] Use a reverse transcription kit (Promega GoScript TM Reverse Transcription Kit) to perform reverse transcription on the total RNA obtained in the above step (1).

[0092] The specific operation is as follows:

[0093] Divide the above total RNA samples into two parts. Use the Oligo dT Primer in the kit as the primer for one part and the Random 6-mers in the kit as the primer for the other part. Configure the reaction solution in a 1.5 ml centrifuge tube according to the system shown in Table 4, and amplify by scaling up the reaction system proportionally according to the amount of RNA.

[0094] Table 4 5 μL reverse transcription reaction system

[0095] Component Content Oligo dT Primer (50 μM) / Random 6-mers (50 μM) 1 μL Total RNA 2 μg <![CDATA[ddH2O]]> Make up to 5 μL

[0096] 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.

[0097] Table 5 Other reaction liquid ratios

[0098] Component Content System after reaction in Table 4 5 μL 5×Reaction Buffer 4 μL <![CDATA[MgCl2]]> 2 μL dNTP mix 1 μL RNase inhibitor 0.4 μL RTase 1 μL <![CDATA[ddH2O]]> Make up to 20 μL

[0099] 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.

[0100] (3) PCR amplification:

[0101] 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).

[0102] The first round of PCR was performed using cDNA as a template. The first round of PCR reaction system is shown in Table 6.

[0103] Table 6 First round PCR reaction system

[0104] Component Content cDNA 5 μL Forward primer 2.5 μL Reverse primer 2.5 μL dNTP Mix 1 μL 5×Reaction Buffer 10 μL High GC Enhancer 10 μL Polymerase 0.5 μL <![CDATA[ddH2O]]> Make up to 50 μL

[0105] 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.

[0106] The primers in the first PCR included:

[0107] Vλ forward primer: 5′-CCTTTCTATGCGGCCCAGCCGGCCgagctcCAGTCTGTSBTGACGCAGCCGCC-3′ (SEQ ID NO: 5).

[0108] Vλ-linker reverse primer: 5′-GGAAGATCTAGAGGAACCACCTAGGACGGTSASCTTGGTCC-3′ (SEQ ID NO: 6).

[0109] Vκ forward primer: 5’-CCTTTCTATGCGGCCCAGCCGGCCgagctcGACATCCRGDTGACCCAGTCTCC-3’ (SEQ ID NO: 7).

[0110] Vκ-linker reverse primer: 5’-GGAAGATCTAGAGGAACCACCTTTGATTTCCACCTTGGTCC-3’ (SEQ ID NO: 8).

[0111] linker-VH forward primer: 5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGCAGGAGTCSG-3’ (SEQ ID NO: 9).

[0112] linker-VH reverse primer: 5’-CAGTCATTCTCGACTTactagtTGAGGAGACRGTGACCAGGGTG-3’ (SEQ ID NO: 10).

[0113] Among them, the lowercase parts in each primer are restriction enzyme sites.

[0114] Among them, the Vλ forward primer and the Vλ-linker reverse primer are used in pair, the Vκ forward primer and the Vκ-linker reverse primer are used in pair, and the linker-VH forward primer and the linker-VH reverse primer are used in pair.

[0115] After the reaction, all PCR products are subjected to 1.5% agarose gel electrophoresis, and the gel is cut to recover the bands with the target fragment sizes around 320 bp (corresponding to VL) and 350 bp (corresponding to VH). The DNA is recovered using the NEB DNA GEL purification kit according to the kit instructions. The collected DNA solution is the first-round PCR amplification product, and after measuring the concentration, it is stored at 4°C.

[0116] The second-round PCR is carried out using the first-round PCR amplification product as a template, and the second-round PCR reaction system is shown in Table 7.

[0117] Table 7 Second-round PCR reaction system

[0118] Component Content VH / VL gel recovery product 80 ng VH + 80 ng VL dNTP Mix 1 μL 5×Reaction Buffer 10 μL High GC Enhancer 10 μL Forward primer OF 2.5 μL Reverse primer OR 2.5 μL Polymerase 0.5 μL <![CDATA[ddH2O]]> Make up to 50 μL

[0119] OF: 5’-CCTTTCTATGCGGCCCAGCCGGCCgagctc-3’ (SEQ ID NO: 31).

[0120] OR: 5’-CAGTCATTCTCGACTTactagt-3’ (SEQ ID NO:32).

[0121] The reaction conditions were the same as above. After the reaction, the PCR products were subjected to 1.5% agarose gel electrophoresis at 160 V for 20 min. Finally, the band with a target fragment size of 750 bp in the electrophoresis result was selected, and the DNA was recovered using the NEB DNA Gel purification kit according to the kit instructions. The collected DNA solution was the product of the second-round PCR amplification. After measuring the concentration, it was stored at 4°C.

[0122] (4) Enzymatic digestion and ligation of the vector and the PCR amplification product:

[0123] The second-round PCR products were enzymatically digested and ligated into the phage plasmid pComb3XSS to construct a phage plasmid library containing the amplified target fragment (ScFv fragment). Among them, the restriction endonucleases SpeI and SacI were used for enzymatic digestion.

[0124] The enzymatic digestion systems of the pComb3XSS vector and the second-round PCR amplification products are shown in Table 8 and Table 9, respectively.

[0125] Table 8 Vector enzymatic digestion system

[0126] Component Content Vector (pComb3XSS) 20 μg SpeI 10 μL SacI 10 μL 10×CutSmart Buffer 50 μL Quick CIP 5 μL <![CDATA[ddH2O]]> Make up to 500 μL

[0127] Table 9 Target fragment (ScFv fragment) enzymatic digestion system:

[0128] Component Content scFv 5 μg SpeI 5 μL SacI 5 μL CutSmart 10×buffer 25 μL <![CDATA[ddH2O]]> Make up to 250 μL

[0129] Enzymatic digestion reaction conditions: Incubate at 37°C for 2 h and at 80°C for 3 min. After the enzymatic digestion, the vector enzymatic digestion products were run on a gel, and the gel was cut and the DNA was recovered using the NEB DNA purification and recovery kit (do not irradiate with ultraviolet light); the ScFv enzymatic digestion products did not need to be run on a gel and could be directly recovered.

[0130] After the purification of the enzymatic digestion reaction, the concentration of the recovered product was measured, and then the digested vector and the ScFv enzymatic digestion products were ligated according to the ligation reaction system shown in Table 10.

[0131] Table 10 Ligation reaction system

[0132] Component Content 10×T4 reaction Buffer 2 μL ScFv fragment (after digestion) 68 ng Vector (after digestion) 100 ng T4 ligase 1 μL <![CDATA[ddH2O]]> Make up to 20 μL

[0133] The above ligation system was reacted at 37°C overnight (16 - 24 h). After the ligation, the ligation products were recovered using the NEB DNA purification and recovery kit. The concentration of the recovered ligation products was detected and recorded, and it was stored at 4°C.

[0134] Construction of the bacterial library

[0135] (1) Preparation of TG1 Escherichia coli competent cells:

[0136] Inoculate a single colony of the TG1 strain on a 2×YT solid medium by streaking, and culture it overnight at 37°C. Pick a single colony from the single colony plate into 10 ml of 2×YT medium, and culture it overnight at 37°C with shaking at 220 rpm. Inoculate the bacteria into 100 mL of 2×YT medium at a dilution ratio of 1:100, and culture it at 37°C with shaking at 250 rpm for 40 min, then measure the OD value. Thereafter, measure it every 20 min until OD600 = 0.3 - 0.35. Collect the bacterial solution, centrifuge it at 3200 g for 10 min at 0 - 4°C, discard the supernatant, place it on ice, add 40 mL of pre-cooled ddH2O to resuspend, centrifuge it at 3200 g for 10 min at 0 - 4°C, discard the supernatant, place it on ice, add 1 mL of pre-cooled ddH2O to resuspend, transfer it to a pre-cooled 1.5 mL EP tube, centrifuge it at 10000 g for 30 s at 4°C, and repeat once. Discard the supernatant, place it on ice, add 400 μL of pre-cooled ddH2O to resuspend, and obtain the TG1 Escherichia coli competent cell suspension.

[0137] Take the ScFv fragment obtained in the above example and construct an Escherichia coli library by electroporation. The specific method is as follows: Use a pre-cooled pipette tip to add 100 ng of the ScFv fragment to 50 μL of the above TG1 Escherichia coli competent cell suspension, gently blow it evenly, transfer it to a pre-cooled 1 mm electroporation cuvette. After confirming that the mixture is at the bottom of the electroporation cuvette and there are no bubbles, set 1800 V and 1 mm spacing for electroporation. Immediately add 1000 μL of SOC medium at 37°C after completion, take out the mixture from the electroporation cuvette, and resuscitate it by shaking at 37°C and 180 rpm for 90 min. Dilute it with 2×YT liquid medium in 10-fold gradients, a total of 6 gradients (diluted 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 times). Take 5 μL of each gradient and evenly drop it onto a 2×YT-GA solid medium. After air-drying, culture it statically at 37°C overnight. Count the number of colonies on the gradient dilution plate and calculate the ligation efficiency. The ligation efficiency formula is:

[0138] E(pfu / ug) = N × D × 10

[0139] where E is the competent efficiency (unit pfu / ug), D is the dilution factor, and N is the number of monoclonal colonies on the plate with the corresponding dilution factor.

[0140] Repeat the above method 100 times for electrotransformation reactions. Spread the resuscitated bacterial solution evenly onto 100 square plates of 2×YT-GA 245 mm culture medium. After air drying, incubate the plates upside down overnight at 37°C. Take the overnight-cultured square plates, add 6 mL of 2×YT liquid medium to the surface of each culture plate, gently scrape the colonies from the 100 square plates with a spreader and collect the bacterial solution into a 50 mL centrifuge tube, and add glycerol with a final concentration of 20%, which is the bacterial library.

[0141] Take 10 μL of the bacterial solution into 990 μL of 2×YT liquid medium and measure the OD600 using a NanoDrop. Calculate and record the total OD600 of the bacterial library.

[0142] T OD600 = M OD600 ×100

[0143] Among them, T OD600 is the total OD600 of the bacteria, and M OD600 is the measured OD600.

[0144] Phage library construction

[0145] Take an appropriate amount from the above bacterial library into a 1.5 ml EP tube. The calculation formula for the amount of bacterial solution is:

[0146]

[0147] Among them, V is the volume of the transferred bacterial solution (unit: μL), and OD600 is the total OD600 of the constructed bacterial library.

[0148] Transfer it to 100 mL of 2×YT-GA liquid medium to make the initial OD600 0.1. Incubate in a constant temperature shaker at 37°C and 250 rpm until the OD600 of the bacterial solution reaches 0.5 - 0.55. Calculate and add the helper phage M13K07 according to the following formula to make the ratio of bacteria to phage 1:20. The calculation formula for the amount of phage added is:

[0149]

[0150] Among them, V is the volume of the added helper phage (unit: mL), T helper-phage is the titer of the used helper phage, and OD600 is the OD600 value of the bacterial solution.

[0151] Continue to incubate in a constant temperature shaker at 37°C and 220 rpm for 30 min. Centrifuge the TG1 bacteria at 3200 g for 5 min, discard the supernatant, resuspend the precipitate and transfer it to 100 mL of 2×YT-AK liquid medium, and incubate overnight at 30°C and 250 rpm in a constant temperature shaker.

[0152] Transfer the overnight culture broth to a new 50 mL centrifuge tube and centrifuge at 4000 g for 30 min at 4°C. Take the supernatant, add 1 / 4 volume of 20% PEG / 2.5 M NaCl pre-cooled at 4°C, mix well, and place on ice for 30 min. Centrifuge at 4000 rpm for 30 min at 4°C, discard the supernatant, and invert on paper for 2 min. Add 1 mL of PBS to resuspend the precipitate, and centrifuge at 12000 rpm for 20 min at 4°C. Take the supernatant, add 1 / 4 volume of pre-cooled 20% PEG / 2.5 M NaCl solution, mix well, and place on ice for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, add 1 ml of PBS to resuspend the precipitate. Centrifuge at 12000 rpm for 2 min at 4°C, take the supernatant, which is the phage library, add glycerol with a final concentration of 20%, and store at -80°C.

[0153] Detect the titer of the phage library. The specific operation is as follows: Take the phage library and add it to 10 mL of 2×YT liquid medium, culture at 37°C and 250 rpm for about 45 min - 60 min until the OD600 is 0.5 - 0.55. Take 10 μL of the cultured phage library and dilute it 10-fold in gradient (a total of 13 gradients). Add 90 μL of untreated TG1 bacterial solution to each dilution gradient, mix well, and incubate at 37°C for 20 min. Then take 5 μL from each dilution gradient and drop it onto the 2×YT-GA solid medium, air-dry, and culture overnight at 37°C, count, and calculate the number of phagemids in each milliliter of the phage solution according to the phage library titer formula.

[0154] T (pfu / ml) = N × D × 400

[0155] Among them, T is the phage titer (unit pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0156] Screening of gB protein-specific antibodies

[0157] Use the antigen solid-phase adsorption screening method to screen for antibodies that specifically bind to the gB protein.

[0158] The specific operation is as follows:

[0159] Dissolve 50 μg of the gB protein obtained in the above example in 2 mL of PBS and coat the immunization tube overnight at 4°C. At the same time, coat the negative control protein (50 μg BSA) as a negative control. Discard the coated protein, rinse the tube 3 times with 2 mL of PBS, and then block it with 2 mL of 3% BSA (dissolved in PBST) at room temperature for 2 h. Discard the blocking solution, take 100 μL of the phage library in the above example, dilute it with 2 mL of PBS, and incubate it in the immunization tube for 1 h. Discard the liquid, wash it 5 times with 2 mL of PBST for 5 min each time. Then wash it 5 times with 2 mL of PBS for 5 min each time. While washing, take 1 mL of the overnight TG1 saturated bacterial solution into 100 mL of 2×YT liquid medium and culture it at 37°C and 150 rpm until OD600 = 0.5 (about 1.5 h). Discard the washing solution, elute it with 1 mL of 0.1 mg / mL Trypsin at room temperature for 30 min. Add 1 mL of the eluate to 10 mL of the cultured TG1 bacterial solution (OD = 0.5) and infect it at 37°C and 150 rpm for 30 min; centrifuge at 4°C and 3000 g for 10 min. Then resuspend the pellet with 1 mL of 2×YT liquid medium. Spread the 1 mL of the resuspended product eluted from the positive immunization tube evenly on a 245 mm × 245 mm 2×YT solid culture dish and culture it overnight at 37°C (16 - 20 h). Measure the phage titers of the positive tube and the negative control tube respectively, and the measurement method is the same as that in the above example.

[0160] According to the comparison results of the phage titers, add 5 mL of 2×YT liquid medium to the culture dish with the higher phage titer after overnight culture, scrape off all the colonies with a spreader, and prepare the sub - phage library according to the method in the above example. Repeat the above operation until the antibody with the highest affinity for gB (compared with the negative control) is screened out.

[0161] Among them, during the processes of antigen coating, blocking, incubation, washing, and elution, unless otherwise specified, the rotation speed is 15 rpm.

[0162] Use ELISA to detect the antibody with the highest affinity.

[0163] Take 10 μL of the culture broth of the antibody with the highest affinity for gB, and dilute the broth with 1 mL of 2×YT liquid medium. Perform cloning streaking on a 2×YT-GA solid culture plate and incubate overnight at 37°C for 16 - 20 h. Pick 192 monoclonal colonies into a 96-well plate (each well contains 200 μL of 2×YT-GA liquid), and incubate statically at 37°C until saturated. Take 2 μL of the saturated broth into a new 96-well plate (each well contains 200 μL of 2×YT-A liquid) to make the initial OD approximately 0.03, and incubate statically at 37°C for 2.5 h - 3 h until the OD is approximately 0.5. Add 0.1 μL of the above-mentioned helper phage to each well and infect at 37°C for 30 min. Add 0.2 μL of Kana (kanamycin) to each well and culture overnight at 30°C. Protein (- / +) coat the ELISA plate overnight. Centrifuge the overnight-cultured 96-well plate at 4°C and 3400 g for 5 min. Discard the liquid in the ELISA plate and wash once with 350 μL of PBS. Block with 350 μL of 3% BSA (PBST) at 37°C for 1 h. Discard the blocking solution, wash once with 350 μL of PBST, and tap the plate to remove the liquid. Add 140 μL of 3% BSA (PBST) to each well, and then add 60 μL of the antibody expressed by the above phage library (the antibody with the highest affinity, as the primary antibody) respectively, and incubate at 37°C for 1 h. Discard the liquid (primary antibody), wash 5 times with 350 μL of PBST, and tap the plate. Add 100 μL of M13 Antibody (HRP) as the secondary antibody (added at a ratio of 1:8000 relative to the blocking solution), incubate at 37°C for 1 h, discard the liquid (secondary antibody), wash 5 times with 350 μL of PBST, and tap the plate. Add 100 μL of TMB and avoid light for 2 - 3 min, and then add 100 μL of dilute hydrochloric acid (concentrated hydrochloric acid: water = 1:12) to terminate the reaction. Read the OD450 and OD630 of the ELISA plate.

[0164] Expression and Purification of Monoclonal Antibody

[0165] After connecting the upstream of the variable region of the heavy chain of the antibody with the CMV fragment, the downstream with the constant region of human IgG1, and the ployA fragment, a fragment that can express the complete heavy chain can be obtained; and after connecting the upstream of the variable region of the light chain of the antibody with the CMV fragment, the downstream with the constant region of light chain κ / λ, and the ploy A fragment, a fragment that can express the complete light chain can be obtained. Co-transfecting the plasmid with the full-length sequences of the above-mentioned heavy chain and light chain of the antibody into 293T cells can achieve the expression of the antibody, and protein Abeads can be used to achieve the purification of the antibody.

[0166] In this example, the antibody with the best effect finally obtained by the inventor is named 4H2.

[0167] The full-length heavy chain of 4H2 has a total of 479 amino acid residues (excluding *), specifically:

[0168]

[0169] Among them, the underlined part in the sequence is the amino acid sequence of the heavy chain variable region. 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 in sequence. The italic part is the signal peptide. * represents the stop codon.

[0170] The coding gene of the full-length heavy chain of 4H2 has a total of 1440 bases, specifically:

[0171]

[0172]

[0173] Among them, the underlined part in the sequence is the nucleotide sequence of the heavy chain variable region. The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region in sequence. The three bases at the 5'-end initial and 3'-end terminal are the start codon and the stop codon respectively. The italic part is the signal peptide.

[0174] The full-length light chain of 4H2 has a total of 232 amino acid residues (excluding *), specifically:

[0175]

[0176] Among them, the underlined part in the sequence is the amino acid sequence of the light chain variable region. 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 in sequence. * represents the stop codon.

[0177] The coding gene of the full-length light chain of 4H2 has a total of 699 bases, specifically:

[0178]

[0179] Among them, the underlined part in the sequence is the nucleotide sequence of the light chain variable region. The underlined and bold parts are the nucleotide sequences of the three complementary regions CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region in sequence. The three bases at the 5'-end initial and 3'-end terminal are the start codon and the stop codon respectively. The italic part is the signal peptide.

[0180] Affinity determination of 4H2 antibody

[0181] The affinity of antibody 4H2 was determined using the biolayer interferometry (BLI) technique.

[0182] BLI can be carried out according to the routine in the art. In this example, the specific operation is as follows: The biosensor (Sartorius, Germany) The SA probe was immersed in a buffer solution (a mixture of KB buffer, 0.1% BSA, and 0.02% Tween 20) for equilibration. Then it was taken out and immersed in a solution containing 5 μg / mL gB-Biotin (biotin-labeled gB protein). The gB antigen in the solution would bind to the surface of the SA (streptavidin) biological probe, increasing the thickness of the surface film layer. Then, the biosensor with the immobilized antigen of known concentration was immersed in the buffer solution as the baseline. By immersing the biosensor with the immobilized antigen of known concentration into a sample solution containing 0 - 200 nM 4H2 antibody, the specific binding between the antigen and the antibody would cause an increase in the film layer thickness. The biosensor with the bound 4H2 antibody was immersed in the buffer solution for dissociation, and the antibody to be detected (4H2 antibody) would fall off from the surface of the biosensor, resulting in a decrease in the film layer thickness. By real-time monitoring of the thickness of the biosensor biofilm layer during the experiment, the kinetic constant of the sample to be detected (4H2 antibody) could be obtained.

[0183] The results are as Figure 1 shown.

[0184] Only for detection, the KD (M) of the 4H2 antibody = 1.122E - 09. This indicates that there is a very high affinity between the 4H2 antibody and the gB antigen.

[0185] Related applications of the 4H2 antibody

[0186] Based on the high affinity of the 4H2 antibody, it can be effectively used for the related detection of the gB antigen, such as in qualitative or quantitative detections like Western Blot.

[0187] (1) Application of the 4H2 antibody in Western Blot:

[0188] In this embodiment, the inventor took Western Blot as an example to verify its actual application effect. Among them, the composition of the related reagents used in Western Blot is shown in Tables 11 - 13.

[0189] Table 11 Composition of related reagents for Western Blot

[0190]

[0191] Table 12 Composition of related reagents for Western Blot

[0192]

[0193] Table 13 Composition of related reagents for Western Blot

[0194]

[0195]

[0196] The operation steps are as follows:

[0197] Prepare 10% separating gel according to the above table. After mixing evenly, quickly add 7 mL to the gel plate mold, and add 2 mL of absolute ethanol for liquid sealing. After the lower-layer separating gel solidifies, discard the absolute ethanol and let it stand to volatilize. Prepare the stacking gel, add 2 mL to the upper layer of the separating gel, and quickly insert the comb. Use the BCA protein quantification analysis kit to perform protein quantification. Add 5×SDS loading buffer to the gB protein sample (in this example, the sample is EBV gB recombinant protein), and place it in a metal bath at 95°C for 10 min. After the gel is dried, place the mold in the electrophoresis tank, add the electrophoresis solution, and remove the comb. Add the processed protein sample to the sample well and add the marker. First, use a low voltage of 80 V for protein concentration, and then change the voltage to 120 V for protein separation. The electrophoresis time is determined according to the position of the marker. After electrophoresis is completed, carefully open the gel plate and cut off the stacking gel. Activate the 0.22 μm PVDF membrane with methanol. Arrange two layers of filter paper, the PVDF membrane, the separating gel, and two layers of filter paper in sequence from bottom to top, and fix them in the transfer membrane splint. Place the splint in the transfer membrane instrument, add the transfer membrane solution, and transfer the membrane at a constant current of 100 mA for 2 h. Transfer the PVDF membrane to 5% milk blocking solution and incubate it on a shaker at room temperature for 1 h. Transfer the PVDF membrane to the above 4H2 antibody solution (1 μg / mL) and incubate it with shaking at 4°C overnight. Rinse it 3 times with TBST, 5 min each time. Transfer the PVDF membrane to Rabbit Anti-Human IgG H&L (HRP) antibody (abcam, diluted 1:10000) and incubate it with shaking at room temperature for 1 h. Rinse it 3 times with TBST, 5 min each time. Drop the chemiluminescence solution evenly on the surface of the PVDF membrane and react in the dark for several minutes. Image the PVDF membrane using a chemiluminescence imager. Set the blank control.

[0198] The results are as Figure 2 shown.

[0199] Using the 4H2 antibody as the primary antibody for Western Blotting, the gB protein sample will show a black band at 35 kD - 40 kD.

[0200] Replace the gB protein sample with 293T cells transiently transfected with the gB recombinant plasmid (the transfection method is the same as the above example), and perform detection according to the above Western Blotting method (set the blank control and use β-actin as the internal reference). The results are as Figure 3 shown.

[0201] It can be found that using the 4H2 antibody as the primary antibody for Western Blotting can effectively detect the expression of gB protein in 293T cells transiently transfected with the gB recombinant plasmid.

[0202] (2) Application of the 4H2 antibody in flow cytometry:

[0203] In this example, 293T cells transiently transfected with the gB recombinant plasmid were used as the test object to test the actual application effect of the 4H2 antibody in flow cytometry.

[0204] The specific steps are as follows: Culture 293T cells in a 12-well plate with a cell density of approximately 40%. After the cells adhered, transiently transfect the gB-Flag recombinant plasmid (the preparation method is the same as the above gB recombinant plasmid), and set a negative control at the same time. Continue to culture for 24 h. Replace the medium and incubate with the 4H2 antibody labeled with 403 fluorescence at 4 °C for 30 min. Incubate with Anti-Flag-488 at 4 °C for 30 min. Use the 2G4 antibody labeled with 403 fluorescence as the negative control antibody. Among them, the 2G4 antibody is the general gB antibody with non-highest affinity eliminated in the above example. Digest the cells with 1 mL of trypsin for 3 min, centrifuge at 200 g for 5 min, and resuspend and wash with PBS 3 times. Resuspend the cells with 500 μL of PBS and filter the cells through a 40 μm cell sieve. Detect the cell fluorescence using a flow cytometer. Use untreated 293T cells as the blank control.

[0205] The results are as Figure 4 shown.

[0206] It can be found that the screening effect of using the 4H2 antibody is significantly better than that of the general gB antibody, indicating that the 4H2 antibody has better specific binding and detection effects compared to the general gB antibody.

[0207] Neutralization activity of the 4H2 antibody

[0208] (1) Preparation of EBV virus:

[0209] Culture Akata cells infected with EBV-GFP until the density reaches 2×10 6 cells / mL, transfer them to serum-free 1640 medium containing 8 μg / mL of goat anti-human IgG, and culture in an environment of 37 °C and 5% CO2 for 6 h. After 6 h, change the cell medium to 1640 medium containing 5% FBS and continue to culture in an environment of 37 °C and 5% CO2 for 3 days. After 3 days, collect the cell supernatant, filter it through a 0.45 μm filter membrane, and then centrifuge it at 20,000 rpm and 4 °C for 2 h using an ultra-high-speed centrifuge. Resuspend the virus precipitate with serum-free 1640 medium.

[0210] (2) Detection of neutralizing activity of 4H2 monoclonal antibody

[0211] Take the 4H2 monoclonal antibody in the above-mentioned example, measure the concentration, and incubate it with the EBV virus suspension at 4°C for 3 h after serial dilution. Add the incubated mixture to the HNE1 cell solution and culture the cells for 48 - 72 hours. Prepare the HNE1 cell suspension and use Prism to calculate and plot the IC50 of the 4H2 monoclonal antibody. Use the 2G4 antibody as a negative control antibody. Among them, the 2G4 antibody is a general gB antibody with non-highest affinity eliminated in the above-mentioned example.

[0212] The results are as Figure 5 shown.

[0213] It can be found that the 4H2 monoclonal antibody has a better neutralizing effect compared with the general gB antibody and can be effectively used for the prevention or treatment of the virus.

[0214] The above-mentioned examples are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An antibody that specifically binds to Epstein-Barr virus gB protein, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes a heavy chain CDR-H1 shown in SEQ ID NO: 15; a heavy chain CDR-H2 shown in SEQ ID NO: 16; and a heavy chain CDR-H3 shown in SEQ ID NO: 17; The light chain variable region includes a light chain CDR-L1 shown in SEQ ID NO: 18; a light chain CDR-L2 shown in SEQ ID NO: 19; and a light chain CDR-L3 shown in SEQ ID NO:

20.

2. The antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 21; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

22.

3. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 2.

4. The nucleic acid molecule according to claim 3, wherein The nucleic acid molecule is as shown in SEQ ID NO: 23 and SEQ ID NO:

24.

5. An expression vector containing the nucleic acid molecule according to claim 3 or 4.

6. A transformant containing the nucleic acid molecule according to claim 3 or 4 and / or the expression vector according to claim 5.

7. The transformant according to claim 6, wherein The transformant is a virus, bacterium or fungus.

8. The transformant according to claim 6, wherein The transformant is a cell.

9. An Epstein-Barr virus detection product, characterized in that, The EB virus detection product contains at least one of the following (1) to (4): (1) The antibody according to any one of claims 1 to 2; (2) The nucleic acid molecule according to claim 3 or 4; (3) The expression vector according to claim 5; (4) The transformant according to any one of claims 6 to 8.

10. An Epstein-Barr virus treatment or prevention product, characterized in that, The EB virus treatment or prevention product contains at least one of the following (1) to (4): (1) The antibody according to any one of claims 1 to 2; (2) The nucleic acid molecule according to claim 3 or 4; (3) The expression vector according to claim 5; (4) The transformant according to any one of claims 6 to 8.

11. Use of the antibody according to any one of claims 1 to 2 in the preparation of an EB virus detection, treatment or prevention product.

12. The application according to claim 11, wherein The EB virus detection, treatment or prevention product is: a detection reagent, a detection kit, a detection chip or a drug.

13. The application according to claim 11, wherein The EB virus detection, treatment or prevention product is a vaccine.

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