Anti-herpes zoster virus glycoprotein E monoclonal antibody, binding epitope analysis method

Abstract

The present invention belongs to the field of antibody technology, and specifically relates to anti-herpes zoster virus glycoprotein E monoclonal antibodies and binding epitope analysis methods. VZV gE protein is used as an immunogen to immunize mice, determine the protein titer, perform fusion experiments, and subclone experiments to obtain monoclonal antibodies. The analysis method is as follows: determine that the antibody recognition epitopes are all conformational epitopes; analyze the complete structure of the VZV gE protein, predict the disulfide bond position and antigenic epitope; mutate and identify the cysteine ​​residues in the VZV gE sequence predicted to form disulfide bonds or affect the formation of disulfide bonds one by one; transfect and express the recombinant mutant plasmid, detect the expression of mutant and wild-type proteins; detect the activity of monoclonal antibodies; detect the activity of monoclonal antibodies corresponding to the mutant proteins with the same expression amount; and detect the affinity of monoclonal antibodies. Application: pharmaceutical composition, reagent, test strip or kit, biological product. The present invention fills the gap in the current analysis of conformational epitopes of VZV gE protein antigens.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibodies, and in particular relates to an anti-herpes zoster virus glycoprotein E monoclonal antibody and a binding epitope analysis method. Background Art

[0002] Antigenic epitopes are special chemical groups that exist in antigen molecules and determine antigen specificity. In protein molecule antigens, due to their relatively complex structures, they often contain a variety of different antigenic epitopes. Antigenic epitopes composed of short peptides composed of amino acid residues arranged continuously and linearly are linear antigenic epitopes; although some amino acids are not arranged continuously in sequence, they form a specific conformation in space, which is also called spatial epitope. Antigenic epitopes are the basis of protein resistance. Therefore, in-depth research on protein antigenic epitopes is of great significance for the diagnosis and prognosis of diseases, the site-specific modification of protein molecules to reduce the immunogenicity of protein drugs, or the design of highly immunogenic recombinant protein vaccines without toxic side effects.

[0003] Furthermore, obtaining antigen fragments by screening immune effector target molecules has become a key technology for new vaccine design, that is, antigen epitopes have become the basic starting point for vaccine design. The antigenic specificity of proteins is mainly limited to specific antigenic determinants. For immune responses that mainly induce humoral immune responses, it is necessary to consider the B-cell antigen sites in the antigenic components of pathogens, namely B-cell epitopes. Most B-cell epitopes are recognized through the spatial conformation of antibodies and epitopes.

[0004] Varicella-zoster virus (VZV) is a very complex virus that expresses more than 70 different proteins, including 9 trans-capsule glycoproteins. These glycoproteins and internal proteins are likely to be used as antigens. Among the 9 glycoproteins of VZV, gE protein has the highest expression in the viral envelope and on the surface of infected cells. It is not only the first glycoprotein recognized by the immune system, but also can induce the production of neutralizing antibodies and cell-mediated immune responses, making it a major candidate protein for the preparation of viral subunit vaccines.

[0005] The existing technical solutions for detecting antibody binding epitopes are mainly for analyzing linear epitopes. The present invention uses bioinformatics and amino acid site-directed mutagenesis to analyze the antigenic epitopes recognized by 5 monoclonal antibodies specific for the conformational epitopes of the VZV gE protein, mainly the effect of the lack of disulfide bonds on antibody recognition, filling the current gap in the analysis of the conformational epitopes of the VZV gE protein antigen. Summary of the invention

[0006] The first object of the present invention is to provide an anti-herpes zoster virus glycoprotein E monoclonal antibody, the second object of the present invention is to provide a method for preparing the anti-herpes zoster virus glycoprotein E monoclonal antibody, the third object of the present invention is to provide an analysis method for the binding epitope of the anti-herpes zoster virus glycoprotein E monoclonal antibody, and the fourth object of the present invention is to provide an application of the anti-herpes zoster virus glycoprotein E monoclonal antibody. The present invention fills the gap in the current analysis of the conformational epitope of the VZV gE protein antigen.

[0007] In order to achieve the above-mentioned object and other related objects, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing an anti-herpes zoster virus glycoprotein E monoclonal antibody, comprising the following steps: using VZV gE protein as an immunogen, immunizing BALB / c mice, determining the titer of the antiserum against VZVgE protein by an indirect ELISA method, screening mice that meet the requirements for fusion experiments and subcloning experiments, and obtaining a monoclonal cell line and a monoclonal antibody corresponding to the monoclonal cell line.

[0009] In one embodiment of the present invention, the VZV gE protein includes a recombinant VZ protein, and the recombinant VZ protein includes at least one of a recombinant VZV gE protein expressed eukaryotically using a VZV gE protein amino acid sequence and a recombinant VZV gE protein expressed prokaryotically using a VZV gE protein amino acid sequence.

[0010] In one embodiment of the present invention, the VZV gE protein includes recombinant VZ protein, and the recombinant VZ protein includes at least one of a recombinant VZV gE protein expressed in eukaryotes using a partial amino acid sequence of the VZV gE protein, a recombinant VZV gE protein expressed in prokaryotes using a partial amino acid sequence of the VZV gE protein, a recombinant VZV gE protein expressed in eukaryotes using the complete amino acid sequence of the VZV gE protein, and a recombinant VZV gE protein expressed in prokaryotes using the complete amino acid sequence of the VZV gE protein; the titer of the antiserum against the VZV gE protein includes at least one of 364.5K, 1093.5K, and 3280.5K.

[0011] In a second aspect, the present invention provides an anti-herpes zoster virus glycoprotein E monoclonal antibody prepared by the aforementioned preparation method.

[0012] In a third aspect, the present invention provides an analysis method for the binding epitope of an anti-herpes zoster virus glycoprotein E monoclonal antibody, comprising the following steps: determining by immunoblotting that the antibody recognition epitopes are all conformational epitopes rather than linear epitopes; the antibody comprises an anti-herpes zoster virus glycoprotein E monoclonal antibody; the anti-herpes zoster virus glycoprotein E monoclonal antibody comprises a monoclonal antibody prepared by the aforementioned method for preparing the anti-herpes zoster virus glycoprotein E monoclonal antibody; using bioinformatics to analyze the complete structure of the VZV gE protein to predict the position of disulfide bonds and antigenic epitopes; using amino acid site-directed mutagenesis technology to predict the VZV gE protein that may form disulfide bonds or affect the formation of disulfide bonds The cysteine ​​residues in the gE sequence were site-directed mutated one by one and the PCR products were identified; the recombinant mutant plasmid was transfected and expressed, and the double antibody sandwich ELISA method was used to detect the expression of mutant and wild-type proteins in cells; the double antibody sandwich ELISA method was used to detect the activity of monoclonal antibodies corresponding to the recombinant VZV mutant and wild-type proteins respectively; the double antibody sandwich ELISA method was used to detect the activity of monoclonal antibodies corresponding to the mutant proteins with the same expression amount; the ELISA saturation concentration determination method was used to detect the affinity of the monoclonal antibodies corresponding to the VZV mutant proteins.

[0013] In one embodiment of the present invention, in the immunoblotting method, the sample is subjected to reduction electrophoresis and non-reduction electrophoresis respectively to determine that the epitope recognized by the monoclonal antibody is a conformational epitope. In the reduction electrophoresis, the sample is mixed with the reduced Loading Buffer and then heated in a metal bath at 80-120°C for 3-10 minutes, and in the non-reduction electrophoresis, the sample is mixed with the non-reduced Loading Buffer and then not heated. Preferably, the sample is mixed with the reduced Loading Buffer and then heated in a metal bath at 100°C for 5 minutes.

[0014] In one embodiment of the present invention, the bioinformatics analysis includes the following steps: using the Uniprot database and applying BlastUniprot software to analyze the secondary structure and disulfide bond sites of the VZV gE protein; analyzing the secondary structure model of the VZV gE protein by SWISS-MODEL software, and analyzing the secondary structure model, physicochemical properties and disulfide bond sites of the VZV gE protein by Alphafold software; and performing immunogenicity analysis on the VZV sequence using PREDICTED ANTIGENIC PEPTIDES software.

[0015] In one embodiment of the present invention, it is predicted that the VZV gE protein has 3 pairs of disulfide bonds, namely C387-C413, C396-C405, and C432-C442; or it is predicted that the VZV gE protein has 5 pairs of disulfide bonds, namely C208-C236, C220-C226, C387-C413, C396-C405, and C432-C442.

[0016] In one embodiment of the present invention, the VZV sequence was predicted to be subjected to immunogenicity analysis, and the antigenic epitopes predicted to be immunogenic were obtained, and the antigenic determinants overlapping with the cysteines constituting the disulfide bonds were aa200-aa210, aa212-236, aa346-407, aa413-432, and aa437-445, respectively.

[0017] In one embodiment of the present invention, the site-directed mutations are: C208, C236, C387, C405, C413, C432, C442, H419, and L420.

[0018] In a fourth aspect, the present invention provides the use of anti-herpes zoster virus glycoprotein E monoclonal antibodies.

[0019] A pharmaceutical composition, a reagent, a test strip or a kit comprises the monoclonal antibody.

[0020] The monoclonal antibody is used in preparing a drug for treating VZV-related diseases or a biological product for preventing VZV-related diseases, wherein the biological product includes a vaccine.

[0021] It should be noted that the present invention selects monoclonal antibodies with high affinity to VZV gE protein, 1G6E1 and 3E8G9. Epitope analysis of monoclonal antibodies can promote the development of antibody therapy, guide vaccine design, evaluate the response of protective antibodies in immunized individuals, or locate the target of antimicrobial agents acting on pathogenic microorganisms.

[0022] The beneficial effects of the present invention are:

[0023] (1) Existing mature antigen epitope analysis technologies are often expensive and based on linear epitopes. The scheme proposed in this invention uses low-cost classical molecular biological experimental methods to conduct specific disulfide bond analysis on conformational epitopes in antigens, and preliminarily obtains the disulfide bond positions that may affect the specific binding of antibodies. At the same time, two monoclonal antibodies with high affinity to VZV gE protein, 1G6E1 and 3E8G9, were selected. Epitope analysis of monoclonal antibodies can promote the development of antibody therapy, guide vaccine design, evaluate the response of protective antibodies in immune recipients, or locate the target of antimicrobial agents acting on pathogenic microorganisms.

[0024] (2) This application provides a specific analysis method, which fills the gap in the current analysis of conformational epitopes of VZV gE protein antigens. Specifically, the immunoblotting method determined that the five monoclonal antibodies were all conformational epitopes rather than linear epitopes; bioinformatics analysis of the complete structure of the VZV gE protein was used to predict the possible disulfide bond positions; amino acid site-directed mutagenesis technology was used to predict the VZV that may form disulfide bonds or have an important impact on the disulfide bond structure. The cysteine ​​residues in the gE sequence were site-directed mutated one by one, namely: C208, C236, C387, C405, C413, C432, C442, H419, L420; the mutated protein sequence was cloned and expressed in CHO-S cells; the total expression of mutant proteins and wild proteins in each group was detected by double antibody sandwich ELISA; under the same addition amount, the binding activity was compared with the wild-type protein to analyze the changes in the binding strength of the 5 monoclonal antibodies to the mutant proteins; the ELISA saturation concentration method was used to analyze the affinity of the 5 monoclonal antibodies to different antigens; the results showed that the 1G6E1 monoclonal antibody recognized The antigenic epitope recognized by the 3F3H10 monoclonal antibody is related to the disulfide bond formed by the cysteines at positions C432-C442; the antigenic epitope recognized by the 3F10G4 monoclonal antibody is related to the disulfide bond formed by the cysteines at positions C432-C442; the antigenic epitope recognized by the 3E8G9 monoclonal antibody is related to the disulfide bond formed by the cysteines at positions C432-C442; the antigenic epitope recognized by the 4E2F8 monoclonal antibody is related to the two pairs of disulfide bonds formed by the cysteines at positions C396-C405 and C432-C442. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 It is a schematic diagram of the reaction results of 1G6E1, 3F3H10, 3F10G4 monoclonal antibodies of the present invention and wild-type protein (WT) treated with reduced and non-reduced loading buffers; wherein the lane characterization information is: 1G6E1 monoclonal antibody, lane 1: WT-reduced, lane 2: WT-non-reduced; 3F3H10 monoclonal antibody, lane 3: WT-reduced, lane 4: WT-non-reduced; 3F10G4 monoclonal antibody, lane 5: WT-reduced, lane 6: WT-non-reduced; M is Marker (reference);

[0027] Figure 2It is a schematic diagram of the reaction results of 3E8G9 and 4E2F8 monoclonal antibodies of the present invention with wild-type proteins treated with reduced and non-reduced loading buffers; wherein the lane characterization information is: 3E8G9 monoclonal antibody, lane 7: WT-reduced, lane 8: WT-non-reduced; 4E2F8 monoclonal antibody, lane 9: WT-reduced, lane 10: WT-non-reduced; M is Marker (reference);

[0028] Figure 3 It is a schematic diagram of the positions of three pairs of disulfide bonds in the VZV gE protein structure simulated by the SWISS-MODEL software of the present invention;

[0029] Figure 4 It is the position map of 5 pairs of disulfide bonds of VZV gE protein simulated by Alphafold software of the present invention;

[0030] Figure 5 It is the agarose gel electrophoresis identification of the mutant gene PCR amplification product of the present invention; wherein, Figure 5 Include Figure 5 a and Figure 5 b; among which, Figure 5 b Lane characterization information is: lane 1: 419 (GXL enzyme), lane 2: 420 (GXL enzyme); Figure 5 aThe lane information is shown in the figure; M is Marker (reference);

[0031] Figure 6 It is a bar graph of the expression of VZV gE mutant protein and wild-type protein detected by the double antibody sandwich method of the present invention;

[0032] Figure 7 The Logistic curve model of the present invention when using 5 kinds of monoclonal antibodies to measure the activity of recombinant VZV mutant and wild-type proteins;

[0033] Figure 8 It is a bar graph of the activity concentration of VZV gE mutant protein and wild type detected by five monoclonal antibodies using the double antibody sandwich method of the present invention;

[0034] Fig. 9 It is the Logistic curve model when the double antibody sandwich ELISA method of the present invention detects recombinant VZV mutant and wild-type proteins under the same His activity;

[0035] Fig.10 The double antibody sandwich method of the present invention uses five monoclonal antibodies to detect the activity bar graph of recombinant mutant and wild-type proteins under the same His activity;

[0036] Fig.11 The C208S mutant protein of the present invention corresponds to a four-parameter ELISA logistic curve model of different antibody affinities;

[0037] Fig.12 The C236S mutant protein of the present invention corresponds to a four-parameter ELISA logistic curve model of different antibody affinities;

[0038] Fig.13 The C387S mutant protein of the present invention corresponds to a four-parameter ELISA logistic curve model of different antibody affinities;

[0039] Fig.14 The four-parameter logistic curve model of the C405S mutant protein of the present invention corresponding to different antibody affinities in ELISA;

[0040] Fig.15 The C413S mutant protein of the present invention corresponds to a four-parameter ELISA logistic curve model of different antibody affinities;

[0041] Fig.16 The four-parameter logistic curve model of the C432S mutant protein of the present invention corresponding to different antibody affinities in ELISA;

[0042] Fig.17 The C442S mutant protein of the present invention corresponds to a four-parameter ELISA logistic curve model of different antibody affinities;

[0043] Fig.18 The four-parameter logistic curve model of the H419A mutant protein of the present invention corresponding to different antibody affinities in ELISA;

[0044] Fig.19 The four-parameter logistic curve model of the L420A mutant protein of the present invention corresponding to different antibody affinities in ELISA;

[0045] Fig. 20 It is a four-parameter logistic curve model of ELISA corresponding to different antibody affinities of the WT mutant protein of the present invention;

[0046] Fig.21 It is a four-parameter Logistic curve model of 1G6E1 antibody affinity ELISA corresponding to different mutant proteins of the present invention;

[0047] Fig. 22 It is a four-parameter logistic curve model of 3F3H10 antibody affinity ELISA corresponding to different mutant proteins of the present invention;

[0048] Fig.23 It is a four-parameter Logistic curve model of 3F10G4 antibody affinity ELISA corresponding to different mutant proteins of the present invention;

[0049] Fig.24 It is a four-parameter logistic curve model of 3E8G9 antibody affinity ELISA corresponding to different mutant proteins of the present invention;

[0050] Fig.25 It is a four-parameter Logistic curve model of 4E2F8 antibody affinity ELISA corresponding to different mutant proteins of the present invention. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The equipment and materials used in the embodiments can be easily obtained from commercial companies unless otherwise specified. In the following, the manufacturer / model is noted after the key features that appear for the first time in this application, and no repeated description is given. In addition, in Example 1, VZV gE protein is used as an immunogen, and VZV gE protein can also be a recombinant VZV gE protein of Jianda Biopharmaceutical (Nanjing) Co., Ltd. This protein is a recombinant VZV gE protein expressed in eukaryotic cells using the complete amino acid sequence of VZV gE protein, wherein the eukaryotic expression system is a CHO cell expression system.

[0052] Example 1

[0053] After immunizing mice with the recombinant expressed purified VZV gE protein, spleen cells were taken and fused with mouse myeloma cells for hybridoma cell screening. A total of 5 monoclonal cell lines were obtained, named 1G6E1, 3F3H10, 3F10G4, 3E8G9, and 4E2F8. The antibodies prepared by the 5 monoclonal cell lines were named after the corresponding cell lines, and ELISA and WB analysis were performed. The specific steps are as follows:

[0054] 1. VZV gE protein was used as an immunogen to immunize 8 BALB / c mice. Blood was collected after 3-4 immunizations. The titer of antiserum against VZV gE protein was determined by indirect ELISA method. Mice that met the requirements were screened for fusion experiments and subcloning experiments. VZV gE protein is VZV-gE antigen, manufacturer: Sino Biological, catalog number: 40907-VNAS.

[0055] 1. Animal Immunization Experiment

[0056] Reagents and consumables: BALB / c mice 18±2g (Yangzhou University); antigen: VZV gE protein; Freund's adjuvant (purchased from Sigma); secondary antibody: goat anti-mouse-HRP (Nanjing Zhongding Biotechnology); 0.22μm sterile filter and dialysis bag (purchased from Millipore); high-glucose DMEM culture medium (purchased from Gibco); red blood cell lysis solution (purchased from Sigma); cell electrofusion solution (purchased from BTX, USA); other reagents are all domestic analytical grade.

[0057] 1.1 Animal immunization: 8 BALB / c mice were selected, and the antigen was mixed with Freund's adjuvant for immunization. 50 μg / mouse was injected subcutaneously, and booster immunization was performed once every 2-3 weeks. Blood was collected for testing, and the titer of antiserum against JD01 protein was determined by indirect ELISA method.

[0058] 1.2 Antiserum titer detection and screening

[0059] 1.2.1 Design the coated plates according to the experimental requirements and mark the strips.

[0060] 1.2.2 Dilute VZV gE protein to the required concentration of 1 μg / mL with PBS coating solution, mix the coating solution and add it to the strips, 100 μL / well, and keep in a refrigerator at 4°C overnight. Coating antigen: VZV gE protein; coating concentration: 1 μg / mL, 100 μL / well; coating buffer: phosphate buffered saline (PBS, pH 7.4).

[0061] 1.2.3 After coating, discard the coating solution, wash the plate 3 times, add 200 μL / well of blocking solution, and incubate at 37℃ for 1 hour. Take out the ELISA plate, discard the inner solution, and wash the plate once.

[0062] 1.2.4 Antiserum was diluted 3-fold at 1:500, 100 μL / well, and incubated at 37°C for 1 h.

[0063] 1.2.5 Take out the ELISA plate, discard the internal solution, wash the plate 3 times, add 100 μL / well of the diluted ELISA secondary antibody, the ELISA secondary antibody: goat anti-mouse-HRP is 1:20000. Incubate at 37℃ for 1 hour.

[0064] 1.2.6 Take out the ELISA plate, discard the internal solution, wash the plate 4 times, add 100 μL / well of TMB color development solution, and determine the color development time according to the depth of the color, generally 37℃, 15 minutes.

[0065] 1.2.7 Add 100μL 1 M HCL solution to each well to terminate the reaction. Immediately read the sample at 450nm on the microplate reader, and the dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample. Coating antigen: VZV gE protein; coating concentration: 1μg / mL, 100μL / well; coating buffer: phosphate buffer (PBS, pH7.4); secondary antibody: goat anti-mouse-HRP, 1:20000 (dilution multiple); the specific results are shown in Table 1 below:

[0066] Table 1

[0067]

[0068] 1.3 Results discussion and analysis: Referring to Table 1, through the detection of mouse antiserum and VZV gE protein titer after 4 immunizations, the antiserum titer of 1# and 7# mice against VZV gE protein was about 364.5K, the antiserum titer of 2#, 3#, and 8# mice against VZV gE protein was about 1093.5K, and the antiserum titer of 4#, 5#, and 6# mice against JD01 protein was about 3280.5K.

[0069] 2. Cell Electrofusion Experiment

[0070] 2.1 Preparation of myeloma cells: One week before fusion, expand SP2 / 0 cells with DMEM medium containing 10% FBS. At the time of fusion, the cells have grown to fill about 6 bottles of T75 cell culture flasks. On the day of fusion, collect SP2 / 0 cells into a 50mL centrifuge tube and centrifuge at 1500rpm for 5min. Discard the supernatant, then add 20mL of DMEM basal medium, blow off the cells and count them.

[0071] 2.2 Preparation of spleen cells: Mice with serum ELISA titers that meet the requirements after immunization are immunized 3 days before fusion and 100μg of antigen is injected intraperitoneally. On the day of fusion, the mice to be fused are euthanized by cervical dislocation. Soak in 75% alcohol for 5 minutes. Take the spleen aseptically and put it in a culture dish with 10mL DMEM basal culture. Take the sieve and put it in another plate, transfer the spleen to the sieve, and grind the spleen with a syringe. Add DMEM to the sieve and rinse the sieve to collect more spleen cells in the plate. Transfer the cells to a 50mL centrifuge tube, wash the spleen cells once with serum-free DMEM, and centrifuge at 1500rpm for 5min. Collect spleen cells and remove red blood cells with red blood cell lysis buffer, wash the spleen cells once with serum-free DMEM, and centrifuge at 1500rpm for 5min. Collect spleen cells and count them.

[0072] 2.3 Cell fusion: Mix myeloma cells and spleen cells so that the ratio of myeloma cells to spleen cells is 1:2. Place the cells in a 50mL centrifuge tube, dilute with DMEM basal medium, and centrifuge at 1500rpm for 5min. Discard the supernatant. Shake the centrifuge tube to make the cells uniform, wash the cells once with electrofusion solution, and then centrifuge at 1500rpm for 5min. Discard the supernatant. Dilute the cells to 2×10 7 / mL, then add 2mL of cell suspension to the fusion pool for electrofusion experiment, and let it stand for 10 minutes after electroporation. Then add HAT DMEM medium. Spread the fused cells into a 96-well plate, 100μL / well. Then put the cell culture plate in a CO2 incubator for culture. Check 6 days after fusion, the hybridoma cell cloning rate is above 50%, there is a small amount of cell debris, and the cells are in good growth state. Screening detection begins 10 days after fusion.

[0073] 2.4 Fusion screening: One day before the test, coat the ELISA plate with 1μg / mL antigen with PBS overnight. The next day, take 100μL / well of cell supernatant for ELISA test. According to the ELISA results, judge the positive wells (OD value of sample well / OD value of negative well ≥ 2.1, then it is judged as a positive well). Select the positive wells detected in the whole plate and conduct a second confirmation test to further confirm the positive wells. The confirmed positive wells are subcloned.

[0074] 2.5 Fusion screening results: BALB / c mouse 5# was selected for cell fusion, and the results of ELISA screening of positive clones were as follows: coating antigen: VZV gE protein; coating concentration: 1.0 μg / mL, 100 μL / well; coating buffer: phosphate buffer (PBS, pH7.4); secondary antibody: goat anti-mouse-HRP, 1:20000; see 1.2 for specific steps, and refer to Tables 2 to 5 for specific results.

[0075] Table 2: Fusion screening results of plate 1

[0076]

[0077] Table 3: Fusion screening results of plate 2

[0078]

[0079] Table 4: Fusion screening results of plate 3

[0080]

[0081] Table 5: Results of fusion screening on plate 4

[0082]

[0083] 2.6 Experimental conclusion: Referring to Tables 2 to 5, after cell fusion, 4 96-well cell culture plates were plated, and 10 positive fusion cell lines specific to VZV gE protein (1F8, 1G6, 2F11, 2G5, 3E8, 3F3, 3F6, 3F10, 4E2, 4G2) were obtained through ELISA screening. Among them, 1F8 represents row F and column 8 of plate 1 (plate 1 is Table 2), and the rest are analogous. It should be noted that 4H11 is the negative control data and 4H12 is the positive control data.

[0084] 3. Cell subcloning experiment

[0085] 3.1 Cell subcloning: blow the cells in the positive wells, count them, add N / 4 mL DMEM medium into the centrifuge tube (N is the cell count result), take 100 μL of cell suspension into the centrifuge tube, blow it evenly and leave 1 mL, add DMEM to 4 mL, blow it evenly, and leave 100 μL (about 2 drops) at the bottom of the tube. Add DMEM to 5mL in a centrifuge tube, mix well and add dropwise to the first three rows of a 96-well plate, one drop per well, leaving 1.8-2mL at the bottom of the tube; add DMEM to 5mL, blow evenly and add dropwise to rows D, E, and F of the 96-well plate, one drop per well, leaving 1.5-1.8mL at the bottom of the tube; add DMEM to 2.8-3mL, blow evenly and add dropwise to rows G and H of the 96-well plate, one drop per well; observe under a microscope after 7-10 days, detect wells with clonal growth, mark the wells with monoclonal growth, pick out positive monoclonal cells as much as possible for re-subcloning, and after detecting 100% positivity, pick out the monoclonal wells for expansion and culture to determine the strain.

[0086] 3.2 Subclone screening: One day before the test, coat the ELISA plate with 1μg / mL antigen with PBS overnight. The next day, take 100μL / well of cell supernatant for ELISA test. According to the ELISA results, determine the positive wells (OD value of sample well / OD value of negative well ≥ 2.1, then it is determined as a positive well). Select the positive wells detected in the whole plate and conduct a second confirmation test to further confirm the positive wells. The confirmed positive wells are subcloned.

[0087] 3.3 Fusion screening results: BALB / c mouse 5# was selected for cell fusion, and the results of ELISA screening of positive clones were as follows: coating antigen: VZV gE protein; coating concentration: 1.0 μg / mL, 100 μL / well; coating buffer: phosphate buffer (PBS, pH 7.4); secondary antibody: goat anti-mouse-HRP, 1:20000; specific steps are shown in 1.2; specific results are shown in Table 6:

[0088] Table 6

[0089]

[0090] 3.4 Experimental conclusion: Through subcloning experiments on the above-mentioned five fusion cell lines (1G6, 3E8, 3F3, 3F10, and 4E2), five monoclonal cell lines (1G6E1, 3E8G9, 3F3H10, 3F10G4, and 4E2F8) were obtained, which were specific to the JD01 protein by indirect ELISA detection.

[0091] Second, the antibodies prepared from the five monoclonal cell lines were named after the corresponding cell lines, and ELISA and WB analysis were performed, which will be described in detail in the following examples.

[0092] Example 2

[0093] In this example, the immunoblotting method was used to determine that the epitopes recognized by the five monoclonal antibodies (mAbs) were all conformational epitopes rather than linear epitopes. The specific steps were as follows:

[0094] 1.1 Prepare gel: Prepare 10% gel according to the gel kit (Biyuntian, SDS-PAGE gel rapid preparation kit, P0012AC).

[0095] 1.2 Electrophoresis: Take appropriate samples and add and mix them evenly according to "Sample: Reduced / Non-reduced Loading = 1:4". The Loading buffer (Loading) contains two types of reduced buffer and non-reduced buffer. The samples (monoclonal cell lines) are reduced and non-reduced respectively using different buffers. The treatment methods are as follows: the sample is mixed with the reduced Loading and heated in a 100℃ metal bath for 5 minutes, and is mixed with the non-reduced Loading without heating. Then, the mixed sample is centrifuged instantaneously using a low-speed centrifuge. Install the gel glass plate into the electrophoresis tank, pour 1×SDS-PAGERunning Buffer into the electrophoresis tank, cover the glass plate, pull out the sample tank template, and prepare for sample loading. Add the sample to the well slot according to its concentration, and then add 2μL protein Marker. Cover the electrophoresis tank lid and connect the positive and negative electrodes. Adjust the voltage to 90V first and set the running time to 10min; after the end, adjust the voltage to 300V until the end of the run.

[0096] 1.3 Transfer: After the electrophoresis is completed, remove the gel plate, cut off the concentrated gel, soak the PVDF membrane in anhydrous ethanol until it is translucent, and soak the PVDF membrane, four layers of filter paper and two layers of sponge in transfer solution for 3 minutes. Then clamp from the negative electrode to the positive electrode in the order of "one layer of sponge--two layers of filter paper--gel--PVDF membrane--two layers of filter paper--one layer of sponge", transfer the membrane in an ice bath, and set the transfer condition to 150mA constant current and run for 1h.

[0097] 1.4 Blocking, antibody incubation and chemiluminescence: After the transfer is completed, remove the PVDF membrane and wash it three times in TBST solution. Then, block it with 5% skim milk powder at room temperature for 1 hour, and wash it again with TBST solution three times, each time for 3 minutes. Add diluted monoclonal antibody (antibodies secreted by the above-mentioned 5 hybridoma cells) to cover the PVDF membrane and incubate it in a refrigerator at 4°C overnight; wash it three times with TBST solution the next day, each time for 3 minutes; then add diluted enzyme-labeled secondary antibody (HRP-conjugated Goat Anti-Mouse IgG, Sangon Biotechnology; CAT: D110087) and incubate it at room temperature for 1 hour, wash it three times with PBST, each time for 3 minutes; finally, use ECL color developing solution to develop the color, and use a gel imager to expose the bands. The experimental results are shown in Figure 1 , Figure 2 .

[0098] Result analysis: The five monoclonal antibodies WT had no obvious bands in the reduced electrophoresis, but had obvious bands in the non-reduced electrophoresis. The protein maintained its original three-dimensional structure in the non-reduced electrophoresis, indicating that the five monoclonal antibodies of the present application recognized conformational epitopes.

[0099] Example 3

[0100] In this example, the complete structure of the recombinant VZV gE protein was analyzed using bioinformatics, and the disulfide bond positions and antigenic epitope prediction results were as follows: There are 10 cysteine ​​residues in the complete amino acid sequence of the VZV gE protein, located at C208, C220, C226, C236, C387, C396, C405, C413, C432, and C442, respectively.

[0101] 2.1 Structural analysis of the complete amino acid sequence of VZV gE protein using Uniprot and SWISS-MODEL software: BlastUniprot (BLAST | UniProt, website: https: / / www.uniprot.org / blast / ) software was used to analyze the secondary structure and disulfide bond sites of VZV gE protein. Uniprot predicted that VZV gE protein has three pairs of disulfide bonds: C387-C413, C396-C405, and C432-C442. Since Q9J3M8 (protein Primary accession number) does not have an existing tertiary structure diagram, the secondary structure model of VZV gE protein was analyzed using SWISS-MODEL (SWISS-MODEL (expasy.org)) software. The results are shown in Figure 3 It should be noted that the additional URL is for obtaining the software more clearly and directly.

[0102] 2.2 Structural analysis of the complete amino acid sequence of VZV gE protein using Alphafold software: Alphafold software (URL: https: / / alphafold.com / ) was used to analyze the secondary structure model, physicochemical properties and disulfide bond sites of VZV gE protein. Alphafold predicted that VZV gE protein has 5 pairs of disulfide bonds: C208-C236, C220-C226, C387-C413, C396-C405, C432-C442. The results are shown in Figure 4 It should be noted that the additional URL is for obtaining the software more clearly and directly. Figure 4 The Chinese character information and site information correspond to the aforementioned disulfide bond information. Combined with the aforementioned content, Figure 4 The information contained is clearly known.

[0103] 2.3 PREDICTED ANTIGENIC PEPTIDES software was used to analyze the immunogenicity of the complete amino acid sequence of VZV gE protein: PREDICTED ANTIGENIC PEPTIDES software (Immunomedicine Group: Tools>> PREDICTED ANTIGENIC PEPTIDES (ucm.es), more specifically http: / / imed.med.ucm.es / Tools / antigenic.pl) was used to analyze the immunogenicity of the VZV sequence. A total of 22 antigenic epitopes that were expected to be immunogenic were obtained, and the antigenic determinants overlapping with the cysteine ​​that constitutes the disulfide bond were aa200-aa210, aa212-236, aa346-407, aa413-432, and aa437-445, respectively. The results are shown in Table 7 below. It should be noted that the additional website is for a clearer and more direct way to obtain the software.

[0104] Table 7

[0105]

[0106] Example 4

[0107] In this example, amino acid site-directed mutagenesis was used to perform site-directed mutagenesis on cysteine ​​residues in the VZV gE sequence that were predicted to form disulfide bonds or affect disulfide bond formation, and the PCR products were identified:

[0108] 3.1 Primer synthesis: Based on Table 7 and the VZV gene sequence of Jianda Biopharmaceuticals (Nanjing) Co., Ltd., 9 pairs of specific mutation primers were designed to mutate the cysteine ​​at the corresponding position to serine to prevent the formation of disulfide bonds at this site. The primers were sent to Qingke Bio for synthesis. The primer sequences are shown in Table 8:

[0109] Table 8 Primer sequences used for recombinant plasmid construction

[0110]

[0111] 3.2 PCR amplification: Using the original plasmid VZVE-HIS in pCDNA3.1 from Jianda Biopharmaceuticals (Nanjing) Co., Ltd. as a template, the target fragment was amplified using the synthesized primers, and the PCR product was verified by agarose gel electrophoresis. The PCR reaction conditions and procedures are shown in Table 9:

[0112] Table 9: GXL enzyme test PCR conditions

[0113]

[0114] 3.3 DpnⅠ enzyme digestion reaction system: Use DpnⅠ enzyme digestion to remove the non-mutated template. Table 10 shows the DpnⅠ enzyme digestion reaction system:

[0115] Table 10: DpnⅠ enzyme digestion reaction system

[0116]

[0117] 3.4TOP10 competent cell transformation, plating, sequencing and plasmid extraction: Take 2μL of digestion product and add it to 75μL TOP10 competent cell suspension, mix well, let it stand in an ice bath for 30 minutes, place the centrifuge tube in a 42℃ water bath for 60-90s, then transfer the centrifuge tube to an ice bath, wait for the cells to cool for 2-3 minutes, then spread on LB-Amp+ plates, mix well, place in a 37℃ incubator for overnight culture, and then select single colonies for sequencing; after sequencing is correct, extract the plasmid according to the instructions of Tiangen's rapid plasmid extraction kit.

[0118] 3.5 The primers carrying the mutant sequence were used to amplify the VZV genome by PCR. The PCR products were identified by agarose gel. Figure 5 , see Figure 5 a and Figure 5 b.

[0119] Example 5

[0120] In this example, the recombinant mutant plasmid was transfected and expressed, and the double antibody sandwich ELISA method was used to detect the expression results of the mutant and wild-type proteins in CHO-S cells:

[0121] 4.1 Transfection and expression of recombinant mutant plasmids: Place the CD06 medium of Qua cell in a 37℃ water bath for rewarming and set aside. Use the correctly sequenced C208S, C236S, C387S, C405S, C413S, C432S, C442S, H419A, L420A, VZVE-HIS in pCDNA3.1 original plasmids to filter and sterilize with a 0.22μm filter for use. Take out the 1000mL shake flask with CHO-S cells from the constant temperature shaking incubator and put it into the biosafety cabinet. Use a 2mL pipette to draw 20μL of cell suspension into a 1.5mL EP tube for counting: draw 20μL of cell suspension and 20μL of trypan blue dye solution, mix well, and then put it into the cell counting plate and use a cell counter for counting. Take 10 125mL triangular shake flasks, add 30mL of fresh CD06 medium and 4 mM glutamine to each shake flask, and set aside. According to the counting results, a cell suspension with a total cell volume of 200X cells was taken in a 50 mL centrifuge tube, that is, 21 mL per cell suspension, a total of 10 cells, and centrifuged at 1200 rpm for 5 min.

[0122] Take one portion of the centrifuged cells and discard the supernatant, aspirate the residual liquid, add 400μL of the prepared electroporation solution, and add 100μg of C208S plasmid suspension, and gently blow with a 1mL gun tip to mix the cells and plasmid thoroughly while avoiding bubbles during the blowing process. Then gently add the mixed liquid to the 1mL electroporation tube, and avoid bubbles during the sample addition process. Add the sleeve used for the 1mL electroporation tube to the electroporation instrument, add the electroporation tube to the sleeve, and perform electroporation at +8V voltage. After the electroporation is completed, take out the sample, aspirate the sample and slowly add it to the 125mL shake bottle prepared in advance, and then put it in a static incubator at 37℃, 5% CO2 for 1h, and then transfer it to a constant temperature shaking incubator at 110rpm, 37℃, 5% CO2 for culture. The remaining 9 plasmids were electroporated in the same way as above. Feed was added 24 hours after electroporation. 3% Feed 04A and 0.3% Feed B03 (QuaCell® CHO feed) were added to each bottle of electroporated cells according to the culture volume. Three bottles were sampled for counting. After the feed was added, the cells were placed in the incubator for continued culture. Feed was added every other day, and samples were collected and tested on D4, D5, and D6 of the culture.

[0123] 4.2 Double antibody sandwich ELISA method was used to determine the expression levels of recombinant VZV wild-type and mutant proteins using Anti-His monoclonal antibody.

[0124] 4.2.1 Coating: Dilute rabbit anti-VZV polyclonal antibody (Jianda Biopharmaceuticals (Nanjing) Co., Ltd., batch number: ZD2021D012717) to 10 μg / mL with coating solution, add 100 μL / well to the ELISA plate, refrigerate overnight or incubate at 37°C for 2 hours. Quickly shake off the solution in the plate.

[0125] 4.2.2 Blocking: Add blocking solution to the ELISA plate, 200 μL / well, and incubate in a 37°C incubator for 1 hour.

[0126] 4.2.3 Washing: Shake off the solution in the plate, add washing solution, 200 μL per well, let stand for 1 min, repeat this step three times, and pat dry.

[0127] 4.2.4 Sample addition: Add standard solution (0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 ng / mL) and test solution to the ELISA plate in order, with the addition amount of 100 μL / well. Add the standard solution in duplicate wells in parallel and incubate in a 37°C incubator for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0128] 4.2.5 Add mouse anti-His tag monoclonal antibody: dilute mouse anti-His tag monoclonal antibody (manufacturer: Huading Biotechnology; catalog number: 10007) with antibody diluent, add the diluted primary antibody to the coated plate, 100 μL / well, incubate at 37°C for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0129] 4.2.6 Add enzyme-labeled antibody: Use antibody diluent to dilute HRP conjugated, rabbit-anti-mouse IgG (manufacturer: Sangon Biotechnology; catalog number: D110098) to obtain enzyme-labeled antibody. Add the diluted enzyme-labeled antibody to the coated plate at 100 μL / well and incubate at 37°C for 1.5 hours. Quickly shake off the solution in the plate and wash the plate 3 times. During the incubation, remove the TMB colorimetric solution and return it to room temperature for later use.

[0130] 4.2.7 Color development reaction: Add TMB color development solution to the coated plate in sequence, 100 μL / well, gently shake to mix, and incubate at 37°C in the dark for 10 min.

[0131] 4.2.8 Reaction termination: Add 3M phosphoric acid stop solution to the coated plate, 50 μL / well, and gently shake to mix. Use an ELISA reader to shake the coated plate to mix, and detect at a wavelength of 450 nm to record the absorbance value. The results of the analysis using GraphPadPrism8 software are shown in Table 11. Figure 6 .

[0132] Table 11 Double antibody sandwich ELISA standard curve concentration and its corresponding OD450 value

[0133]

[0134] Example 6

[0135] In this embodiment, the double antibody sandwich ELISA method detects the activities of 5 monoclonal antibodies corresponding to the recombinant VZV mutant and wild-type proteins respectively and the results are as follows: Steps include 5.1 to 5.8, which are as follows:

[0136] Steps 5.1 to 5.3 refer to the methods 4.2.1 to 4.2.3 in Example 5, wherein 5.1 is coating; 5.2 is blocking; and 5.3 is washing.

[0137] 5.4 Sample addition: Add standard solution (0, 5, 10, 20, 40, 60, 80 ng / mL) and test solution to the ELISA plate in sequence, with 100 μL added to each well. Add the standard solution to duplicate wells in parallel and incubate in a 37°C incubator for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0138] 5.5 Add primary antibody: Dilute 1G6E1, 3F3H10, 3F10G4, 3E8G9, 4E2F8 monoclonal antibodies respectively with antibody diluent, add the diluted primary antibody to the coated plate, 100 μL / well, incubate at 37°C for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0139] Steps 5.6 to 5.8 refer to the methods of 4.2.6 to 4.2.8 in Example 5. In particular, step 5.6 adds enzyme-labeled antibody. Step 5.7 develops color. Step 5.8 terminates the reaction. The results of the analysis using GraphPadPrism 8 software are shown in Tables 12, 13, Figure 7~Figure 8 .

[0140] Table 12 Double antibody sandwich ELISA standard curve concentration and its corresponding OD450 values ​​for 5 monoclonal antibody detections

[0141]

[0142] Table 13 Double antibody sandwich ELISA 5 antibodies detect VZV gE mutant and wild-type protein activity

[0143]

[0144] Example 7

[0145] In this embodiment, the double antibody sandwich ELISA method detects the activities of 5 monoclonal antibodies corresponding to the mutant proteins with the same expression amount, and the steps include 6.1 to 6.8, which are specifically as follows:

[0146] Steps 6.1 to 6.3 refer to the methods of 4.2.1 to 4.2.3 in Example 5, wherein 6.1 is coating; 6.2 is blocking; and 6.3 is washing.

[0147] 6.4 Sample addition: Add standard solutions (0, 10, 20, 40, 60, 80, 120 ng / mL) to the ELISA plate in sequence. Dilute the test solution to 400 ng / mL according to the His monoclonal antibody test results. Add 100 μL / well. Add the standard solution in duplicate wells in parallel and incubate in a 37°C incubator for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0148] 6.5 Add primary antibody: Use antibody diluent to dilute 1G6E1, 3F3H10, 3F10G4, 3E8G9, 4E2F8 monoclonal antibodies respectively by the corresponding multiples, add the diluted primary antibody to the coated plate, 100 μL / well, incubate at 37℃ for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0149] Steps 6.6 to 6.8 refer to the methods of 4.2.6 to 4.2.8 in Example 5. In particular, 6.6 adds enzyme-labeled antibodies. 6.7 develops color. 6.8 terminates the reaction. The results of the analysis using GraphPadPrism 8 software are shown in Tables 14 to 15 below. Figures 9 and 10 .

[0150] Table 14 Double antibody sandwich ELISA standard curve concentration and its corresponding OD450 values ​​of 5 monoclonal antibody detection

[0151]

[0152] Table 15 Double antibody sandwich ELISA to detect the activities of 5 monoclonal antibodies corresponding to recombinant VZV mutants and wild-type proteins with the same His activity

[0153]

[0154] Example 8

[0155] In this embodiment, the ELISA saturation concentration assay method detects the affinity test results of 5 monoclonal antibodies corresponding to the VZV mutant proteins, and steps 7.1 to 7.8 are as follows:

[0156] Steps 7.1 to 7.3 refer to the methods 4.2.1 to 4.2.3 in Example 5, wherein 7.1 is coating; 7.2 is blocking; and 7.3 is washing.

[0157] 7.4 Sample addition: Add the test solution (dilute to 50 ng / mL according to the His monoclonal antibody test results) to the ELISA plate in sequence, with an addition volume of 100 μL / well, and incubate in a 37°C incubator for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0158] 7.5 Add primary antibody: Use antibody diluent to dilute 1G6E1, 3F3H10, 3F10G4, 3E8G9, 4E2F8 monoclonal antibodies from 20 μg / mL to 5-fold gradient, add the diluted primary antibody to the coated plate, 100 μL / well, incubate at 37℃ for 1 hour. Quickly shake off the solution in the plate and wash the plate 3 times.

[0159] Steps 7.6 to 7.8 refer to the methods of 4.2.6 to 4.2.8 in Example 5. In particular, step 7.6 adds enzyme-labeled antibody. Step 7.7 develops color. Step 7.8 terminates the reaction. The results of the analysis using GraphPadPrism 8 software are shown in Table 16 below. Figure 11 to Figure 25 .

[0160] Table 16 EC50 values ​​of different VZV gE mutant proteins and 5 monoclonal antibodies

[0161]

[0162] EC50 unit: ng / mL

[0163] The results showed that: Comprehensive analysis showed that the antigen epitope recognized by the 1G6E1 monoclonal antibody was related to the disulfide bond formed by the cysteines at positions C432-C442; the antigen epitope recognized by the 3F3H10 monoclonal antibody was related to the two pairs of disulfide bonds formed by the cysteines at positions C396-C405 and C432S-C442S; the antigen epitope recognized by the 3F10G4 monoclonal antibody was related to the disulfide bond formed by the cysteines at positions C432-C442; the antigen epitope recognized by the 3E8G9 monoclonal antibody was related to the disulfide bond formed by the cysteines at positions C432-C442; and the antigen epitope recognized by the 4E2F8 monoclonal antibody was related to the two pairs of disulfide bonds formed by the cysteines at positions C396-C405 and C432-C442. And among the five monoclonal antibodies, the EC50 values ​​of 1G6E1 and 3E8G9 to wild-type VZV gE protein were much smaller than those of the other three, indicating their high affinity.

[0164] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for analyzing the binding epitope of anti-herpes zoster virus glycoprotein E monoclonal antibody, characterized in that: The following steps are involved: The monoclonal antibody recognition epitopes are determined to be conformational epitopes rather than linear epitopes by immunoblotting: In the immunoblotting, the samples are subjected to reduction electrophoresis and non-reduction electrophoresis respectively to determine that the monoclonal antibody recognition epitopes are conformational epitopes; wherein, in the reduction electrophoresis, the sample is mixed with the reduction type Loading Buffer and then heated in a metal bath at 80-120° C. for 3-10 min, and in the non-reduction electrophoresis, the sample is mixed with the non-reduction type Loading Buffer and then not heated; Bioinformatics was used to analyze the complete structure of VZV gE protein and predict the disulfide bond position and antigenic epitope; Amino acid site-directed mutagenesis was used to perform site-directed mutagenesis on cysteine ​​residues in the VZV gE sequence that were predicted to form disulfide bonds or affect disulfide bond formation, and the PCR products were identified; The recombinant mutant plasmid was transfected and expressed, and the double antibody sandwich ELISA method was used to detect the expression of mutant and wild-type proteins in cells; The double antibody sandwich ELISA method was used to detect the activity of monoclonal antibodies corresponding to recombinant VZV mutant and wild-type proteins; The double antibody sandwich ELISA method was used to detect the activities of monoclonal antibodies corresponding to the mutant proteins with the same expression level; The affinity of monoclonal antibodies corresponding to VZV mutant proteins was detected by ELISA saturation concentration assay.

2. The method for analyzing the binding epitope of the anti-herpes zoster virus glycoprotein E monoclonal antibody according to claim 1, characterized in that: The Uniprot database predicted that the VZV gE protein had three pairs of disulfide bonds: C387-C413, C396-C405, and C432-C442; or the Alphafold software predicted that the VZV gE protein had five pairs of disulfide bonds: C208-C236, C220-C226, C387-C413, C396-C405, and C432-C442.

3. The method for analyzing the binding epitope of the anti-herpes zoster virus glycoprotein E monoclonal antibody according to claim 1, characterized in that: The bioinformatics analysis includes the following steps: The secondary structure and disulfide bond sites of VZV gE protein were analyzed using the Uniprot database and BlastUniprot software; The secondary structure model of VZV gE protein was analyzed by SWISS-MODEL software, and the secondary structure model, physicochemical properties and disulfide bond sites of VZV gE protein were analyzed by Alphafold software; The immunogenicity of VZV sequences was analyzed using PREDICTED ANTIGENIC PEPTIDES software.

4. The method for analyzing the binding epitope of the anti-herpes zoster virus glycoprotein E monoclonal antibody according to claim 2, characterized in that: The immunogenicity analysis of the predicted VZV sequence revealed that the antigenic epitopes predicted to be immunogenic were aa200-aa210, aa212-236, aa346-407, aa413-432, and aa437-445, respectively, overlapping with the cysteines constituting the disulfide bonds.

5. The method for analyzing the binding epitope of the anti-herpes zoster virus glycoprotein E monoclonal antibody according to claim 1, characterized in that: The site-directed mutations are: C208, C236, C387, C405, C413, C432, C442, H419, and L420.

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