Anti-HBeAg antibody or functional fragment thereof and application thereof

Abstract

The invention discloses an anti-hepatitis B E antigen (HBeAg) antibody or a functional fragment thereof and application thereof. The anti-HBeAg antibody or the functional fragment thereof disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, and the antibody or the functional fragment thereof has relatively good specificity and sensitivity to HBeAg, and can be used for detecting the HBeAg and diagnosing or assisting in diagnosing related diseases taking the HBeAg as a marker.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an anti-HBeAg antibody or its functional fragment and its application. Background Art

[0002] Hepatitis B (HBV) is a viral hepatitis caused by the hepatitis B virus, which can be transmitted through unprotected sexual contact, blood transfusion, reuse of contaminated needles and syringes, mother-to-child transmission during childbirth, etc. Due to its extremely strong infectivity, there are approximately 350 million HBV carriers worldwide. There are about 93 million HBV carriers and 30 million hepatitis B patients in China, and the total infection rate is as high as 35.5%-61.1%. Establishing the detection and prevention of hepatitis B virus is one of the primary tasks in China currently.

[0003] The main markers for current hepatitis B diagnosis are the "five hepatitis B markers", including hepatitis B virus surface antigen, hepatitis B virus surface antibody, hepatitis B virus e antigen, hepatitis B virus e antibody, and hepatitis B virus core antigen, that is, the commonly said "two pairs and a half of hepatitis B". Hepatitis B e antigen (HBeAg) is a soluble protein in the core particles of the hepatitis B virus. The detection rate of HBeAg increases during the active stage of hepatitis B, indicating severe damage to liver cells and strong infectivity of the patient.

[0004] Common detection methods for HBeAg include enzyme-linked immunosorbent assay, colloidal gold method, chemiluminescence method, etc., and the reaction principle is based on the sandwich method of antigen-antibody reaction. Currently, there are few commercial antibody sources for detecting HBeAg, and most detection kits use imported kits from abroad. However, the demand for hepatitis B detection in China is very large, so preparing antibodies against it can effectively improve the domestic detection ability for this marker. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-HBeAg antibody or its functional fragment, as well as a reagent for detecting HBeAg. This antibody or its functional fragment has good affinity for hepatitis B e antigen, and using this antibody or its functional fragment to detect HBeAg has good sensitivity and specificity.

[0006] Specifically as follows:

[0007] On the one hand, the present invention provides an anti-HBeAg antibody or its functional fragment, and the anti-HBeAg antibody or its functional fragment has the following complementarity-determining regions:

[0008] CDR-VH1: X1-Y-H-M-X2, where X1 is E or D, and X2 is Y or T;

[0009] CDR-VH2: Y-I-S-I-G-X1-G-S-I-A-Y-S-X2-T-V-X3-G, where X1 is G or A, X2 is D or E, and X3 is E or D;

[0010] CDR-VH3: H-D-X1-Y-G-D-Y-X2-M-D-Y, where X1 is T or Y, and X2 is A or G;

[0011] CDR-VL1: R-A-X1-Q-E-I-S-X2-Y-L-T, where X1 is T or S, and X2 is G or A;

[0012] CDR-VL2: V-X1-S-T-L-X2-S, where X1 is G or A, and X2 is D or W;

[0013] CDR-VL3: L-Q-X1-A-S-S-X2-Y-T, where X1 is Y or T, and X2 is P or V.

[0014] Furthermore, X2 of CDR-VH1 of the complementarity determining region is Y, X2 of CDR-VH2 is D, X1 of CDR-VH3 is T, X2 of CDR-VL1 is G, X1 of CDR-VL2 is A, and X1 of CDR-VL3 is Y.

[0015] The CDR segment division of the present invention adopts the Kabat algorithm.

[0016] "CDR" is used herein to refer to the "complementarity determining region" within the antibody variable sequence. Each of the variable regions of the heavy chain and the light chain has 3 CDRs, starting from the N-terminus of the heavy chain or the light chain.

[0017] The antigen binding site may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 in the present invention). A polypeptide containing a single CDR (such as CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, or CDR-VL3) may be referred to as a "molecular recognition unit". Crystallographic analysis of the antigen-antibody complex has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, and the most extensive antigen contacts are with the heavy chain CDR3. Therefore, the molecular recognition unit may be mainly responsible for the specificity of the antigen binding site. Generally, CDR residues directly and most substantially participate in affecting antigen binding.

[0018] In an alternative embodiment, X1 in the CDR-VH1 is E;

[0019] In an alternative embodiment, X1 in the CDR-VH1 is D;

[0020] In an alternative embodiment, X2 in the CDR-VH1 is Y;

[0021] In an alternative embodiment, X2 in the CDR-VH1 is T;

[0022] In an alternative embodiment, X1 in the CDR-VH2 is G;

[0023] In an alternative embodiment, X1 in the CDR-VH2 is A;

[0024] In an alternative embodiment, X2 in the CDR-VH2 is D;

[0025] In an alternative embodiment, X2 in the CDR-VH2 is E;

[0026] In an alternative embodiment, X3 in the CDR-VH2 is E;

[0027] In an alternative embodiment, X3 in the CDR-VH2 is D;

[0028] In an alternative embodiment, X1 in the CDR-VH3 is T;

[0029] In an alternative embodiment, X1 in the CDR-VH3 is Y;

[0030] In an alternative embodiment, X2 in the CDR-VH3 is A;

[0031] In an alternative embodiment, X2 in the CDR-VH3 is G;

[0032] In an alternative embodiment, X1 in the CDR-VL1 is T;

[0033] In an alternative embodiment, X1 in the CDR-VL1 is S;

[0034] In an alternative embodiment, X2 in the CDR-VL1 is G;

[0035] In an alternative embodiment, X2 in the CDR-VL1 is A;

[0036] In an alternative embodiment, X1 in the CDR-VL2 is G;

[0037] In an alternative embodiment, X1 in the CDR-VL2 is A;

[0038] In an alternative embodiment, X2 in the CDR-VL2 is D;

[0039] In an alternative embodiment, X2 in the CDR-VL2 is W;

[0040] In an alternative embodiment, X1 in the CDR-VL3 is Y;

[0041] In an alternative embodiment, X1 in the CDR-VL3 is T;

[0042] In an alternative embodiment, X2 in the CDR-VL3 is P;

[0043] In an alternative embodiment, X2 in the CDR-VL3 is V;

[0044] Preferably, each of the complementarity-determining regions is selected from any one of the following mutation combinations:

[0045]

[0046]

[0047]

[0048] Furthermore, the antibody includes a light-chain framework region FR1-L, FR2-L, FR3-L, and FR4-L and a heavy-chain framework region FR1-H, FR2-H, FR3-H, and FR4-H; the heavy-chain framework regions FR1-H, FR2-H, FR3-H, and FR4-H are sequentially selected from SEQ ID NO: 1-4; the light-chain framework regions FR1-L, FR2-L, FR3-L, and FR4-L are sequentially selected from SEQ ID NO: 5-8.

[0049] Furthermore, the antibody further comprises a constant region.

[0050] Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; preferably, the species source of the constant region is bovine, equine, dairy cow, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, gamecock, or human.

[0051] Preferably, the functional fragment is selected from any one of VHH, F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody.

[0052] Furthermore, the antibody is a coating antibody.

[0053] The "coating antibody" as described in the present invention is a coating that can capture an antigen.

[0054] The term "antibody" includes various forms of antibody structures, including but not limited to intact antibodies and antibody fragments. The antibodies according to the present invention are preferably goat, sheep, mouse, rabbit or rat antibodies, chimeric antibodies or further genetically engineered antibodies, as long as the characteristic properties according to the present invention are retained. "Antibody fragment" includes a part of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies, single-chain antibody molecules and multispecific antibodies formed by antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; diabodies; and multispecific antibodies formed by antibody fragments.

[0055] Preferably, the coating carrier coated with the antibody includes but is not limited to nitrocellulose membranes, ELISA plates, magnetic beads, latex microspheres, etc.

[0056] Preferably, the magnetic beads include but are not limited to carboxyl magnetic beads, methylacrylamide magnetic beads, streptavidin magnetic beads, etc.

[0057] Preferably, the latex microspheres are often polyvinyl alcohol toluene, polystyrene, or microspheres formed by copolymerizing these two substances as the main components and adding other monomers.

[0058] Preferably, the latex microspheres include but are not limited to carboxyl latex microspheres, amino latex microspheres, etc.

[0059] On the other hand, the present invention discloses a vector, which contains a nucleic acid fragment encoding the above-mentioned antibody or its functional fragment.

[0060] On the other hand, the present invention discloses a recombinant cell, which contains the above-mentioned vector.

[0061] On the other hand, the present invention discloses a reagent for detecting HBeAg, which includes the above-mentioned antibody or its functional fragment.

[0062] On the other hand, the present invention discloses the application of the above-mentioned antibody and its antigen-binding fragment in a chemiluminescent detection kit for HBeAg.

[0063] Beneficial effects:

[0064] At present, there are few raw materials of HBeAg-related antibodies on the market, and their performances are also uneven. The present invention not only provides a group of monoclonal antibodies with high affinity and specificity for binding to HBeAg, which can well meet the clinical detection needs, especially when applied in chemiluminescent detection reagents, it can significantly improve the detection performance of the reagents and provide multiple references for immunoassay reactions. Description of the drawings

[0065] Figure 1The calibration curve of the HBeAg chemiluminescence detection reagent prepared in Example 3. Detailed implementation mode

[0066] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein. Unless otherwise noted, the techniques employed herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0068] As used herein, the terms "comprising," "including," "having," "may," and variations thereof are intended to be transitional phrases, terms, or words that are open-ended and do not exclude the possibility of additional acts or structures.

[0069] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir and C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0070] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0071] Example 1 Preparation of Monoclonal Antibodies

[0072] 1. Mouse Immunization and Antibody Detection

[0073] Select 5 SPF female BALB / c mice aged 6 - 8 weeks. Mix Freund's complete adjuvant and HBs protein at a concentration of 2 mg / ml in equal volume and emulsify. Immunize the 6 - 8 - week - old SPF female BALB / c mice with the emulsified antigen. Inject 50 μg of antigen protein into each mouse by plantar injection or subcutaneous injection in the back. Two weeks after the primary immunization, mix the antigen protein with Freund's incomplete adjuvant and emulsify, and then inject 50 μg of antigen protein into each mouse again by plantar injection or subcutaneous injection in the back. Two weeks later, collect blood from the tail vein, centrifuge to collect the supernatant, and detect the serum titer by ELISA. Immunize once every two weeks and detect the serum titer. After 2 immunizations, the serum titer after million - fold dilution is as high as 2.0 or more. Screen the mice with a serum titer of 10 6 or more, and take lymph to isolate lymphocytes for cell fusion.

[0074] 2. Cell fusion, screening and sub - cloning of positive hybridoma cells

[0075] Isolate lymphocytes from immunized mice and fuse them with cultured SP2 / 0 cells by PEG1500 - mediated fusion or electro - fusion. The fused cells are cultured and screened in HAT - 1640 medium containing 20% FBS serum. After one week, change the medium. After culturing for another 4 days, take the culture supernatant for screening of positive clones. Use HBs protein for screening positive wells. Select the wells with a higher ratio of ELISA positive value to cell number for multiple sub - cloning. Coat the ELISA plate with HBs protein. Take the culture supernatant of sub - cloning and screen for monoclonal antibodies that can show affinity under the condition of antigen coating, and select the monoclonal hybridoma cell with the highest affinity from them. Finally, obtain a hybridoma cell line with a relatively high antibody titer that can secrete HBs monoclonal antibody, named 15#, and it has good stability.

[0076] 3. Production and purification of monoclonal antibody

[0077] Select two groups of BALB / c mice aged 6 - 8 weeks, and intraperitoneally inject 500 μL of paraffin oil to inhibit the immune response of mice. One week after the injection, inject 0.5 ml of cell 15# into the peritoneal cavity of the mice, and the cell number is about 1×10 6 . Two weeks later, start collecting ascites. The collected ascites is subjected to ammonium sulfate precipitation and affinity purification with protein A to obtain the target antibody 15#.

[0078] 4. Subtype identification and gene sequence cloning of monoclonal antibody

[0079] Use the SBAClonotyping System - HRP kit from Southern Biothech company to identify the subtypes of the heavy and light chains of monoclonal antibody according to the operation instructions of the manual. The specific operation is as follows:

[0080] 1) Dilute the HBs antigen with coating buffer (0.05 M carbonate and bicarbonate buffer, pH 9.5) to 1 μg / mL, add 100 μL per well to the ELISA plate, and coat overnight at 4°C. Wash the plate 3 times with PBS buffer (washing buffer) containing 0.05%

[0081] Tween-20.

[0082] 2) Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with dilution buffer (1% BSA, 0.1% PBST), add 100 μL per well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) 1:3000 with dilution buffer.

[0083] 3) After washing the plate 3 times with the washing buffer, add 100 μL of the diluted enzyme-labeled antibody to each well, and incubate at 37°C for 30 minutes. Wash the plate 3 times again, then add the chromogenic solution. After about 5 minutes (depending on the strength of the reaction), add 2 M sulfuric acid to terminate the reaction, and read the OD450 absorbance value. After identification, the heavy chain subtype of antibody 15# is IgG1, and the light chain is Kappa.

[0084] According to the antibody subtype results, clone the antibody gene sequence using the method based on the RACE technical route. Collect hybridoma cells in good growth state, obtain the total RNA of hybridoma cells using a total RNA extraction kit, reverse transcribe mRNA into cDNA according to the operation method of the SMARTer RACE instruction manual of Takara Company, and amplify the full-length sequence of the target antibody.

[0085] 5. In vitro expression of antibody

[0086] According to the full-length antibody sequence, synthesize it into the pcDNA3.1(+) vector separately for the heavy and light chains, transfect it into HEK293 for expression verification, and obtain the antibody after purification of the fermentation supernatant with protein G.

[0087] Example 2 Verification of antibody performance

[0088] 1. ELISA affinity test

[0089] Dilute goat anti-mouse to 1 μg / mL with coating solution (0.05 M carbonate and bicarbonate buffer, pH = 9.5), add 100 μL per well to a 96-well ELISA plate, and coat overnight at 4°C. Wash the plate 3 times with PBS buffer containing 0.05% Tween-20. Dilute the antibody to 1 μg / mL with dilution solution (1% BSA, 0.1% PBST), add 100 μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. Dilute biotin-labeled HBs protein to 500 ng / mL with dilution solution. Take out the ELISA plate incubated at 37°C, wash it 3 times, add the prepared biotin-labeled HBs protein dilution at 150 μL per well to row A of the 96-well ELISA plate, and add the dilution solution at 100 μL per well to rows B - H of the 96-well ELISA plate. Take 50 μL of the antibody dilution in row A wells, perform 3-fold serial dilution successively from row B, and incubate at 37°C for 30 minutes. Dilute avidin labeled with horseradish peroxidase 1:5000 with dilution solution. Take out the ELISA plate, wash it 3 times, add it at 100 μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. Take out the ELISA plate, wash it 3 times, add the chromogenic solution, develop color at room temperature for 3 minutes, add 0.5 M sulfuric acid to terminate the reaction, and read the OD450 value on a microplate reader.

[0090] The heavy chain sequence of antibody 24# in Example 1 is SEQ ID NO:11, and the light chain sequence is SEQ ID NO:12. The heavy chain variable region is as shown in SEQ ID NO:9, and the amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows:

[0091] CDR-VH1: E(X1)-Y-H-M-Y(X2);

[0092] CDR-VH2: Y-I-S-I-G-A(X1)-G-S-I-A-Y-S-E(X2)-T-V-D(X3)-G;

[0093] CDR-VH3: H-D-T(X1)-Y-G-D-Y-A(X2)-M-D-Y;

[0094] The light chain variable region is as shown in SEQ ID NO:10, and the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0095] CDR-VL1: R-A-T(X1)-Q-E-I-S-A(X2)-Y-L-T;

[0096] CDR-VL2: V-A(X1)-S-T-L-W(X2)-S;

[0097] CDR-VL3: L-Q-Y(X1)-A-S-S-P(X2)-Y-T;

[0098] Based on antibody 24#, mutations were made at the sites related to antibody activity in the complementary determining regions, where X1, X2, and X3 are all mutation sites.

[0099] Table 1 Mutation sites related to antibody activity

[0100]

[0101] Table 2 Antibody activity analysis data

[0102]

[0103] As can be seen from the above table, the activity effect of mutation 1 is the best. Therefore, mutation 1 was used as the backbone sequence to screen other mutation sites with better affinity. Some of the results are as follows:

[0104] Table 3 Mutation sites related to antibody affinity

[0105]

[0106]

[0107] For HBe protein at 1000 ng / ml, the absorbance value of OD450 for each mutation was measured. The results are as follows, and it can be seen that the affinity of each mutation is relatively high.

[0108] Table 4 Affinity test of antibody mutations

[0109] Mutation 1-1 2.394 Mutation 1-11 2.33 Mutation 1-21 2.378 Mutation 1-2 2.31 Mutation 1-12 2.456 Mutation 1-22 2.397 Mutation 1-3 2.368 Mutation 1-13 2.207 Mutation 1-23 2.279 Mutation 1-4 2.433 Mutation 1-14 2.253 Mutation 1-24 2.224 Mutation 1-5 2.288 Mutation 1-15 2.306 Mutation 1-25 2.233 Mutation 1-6 2.372 Mutation 1-16 2.381 Mutation 1-26 2.278 Mutation 1-7 2.369 Mutation 1-17 2.354 Mutation 1-27 2.449 Mutation 1-8 2.392 Mutation 1-18 2.274 Mutation 1-28 2.499 Mutation 1-9 2.234 Mutation 1-19 2.397 Mutation 1-29 2.456 Mutation 1-10 2.358 Mutation 1-20 2.392 Mutation 1-30 2.397

[0110] 2. Stability determination

[0111] The antibody was thermally accelerated at 37°C for 7 days in a defined buffer (PBS, 0.05% ProClin TM 300), and the accelerated antibody was evaluated by SDS-PAGE and indirect ELISA, with 4°C as the control to identify the long-term stability of the antibody. In addition, by performing 5 cycles of repeated freezing and thawing at -20°C, the test results were shown as the deviation between the value at 4°C and the value after acceleration. The measurement results are as follows. The concentration of the antibody in the present invention is 1 μg / mL, and the concentration of HBe protein is 1000 ng / mL.

[0112] Table 5 Stability study

[0113]

[0114]

[0115]

[0116] Example 3 Antibody Application Research

[0117] 1. Application of Antibody in HBeAg Luminescence Detection Kit

[0118] The affinities of the mutants are all relatively high, so the effects applied to immunoassays can be predicted to be similar for Mutants 1-1 to 1-30. Given that it is impossible to exhaust all examples in the embodiments, Mutant 1-8 is selected for testing in this embodiment.

[0119] (1) Reagent Preparation

[0120] Reagent R1: Aliquot 7 ml of the special reagent 1 buffer for HBeAg.

[0121] Reagent R2: Dilute the biotin-labeled antibody to 1 μg / ml with the special anti-reagent 2 buffer for HBeAg, prepare 7 mL, label it and place it on a vortex mixer for at least 60 s of mixing, and set aside for use.

[0122] Magnetic Bead Reagent: Dilute the 3-25 antibody labeled with magnetic beads to 1 μg / ml with the special anti-reagent buffer for HBeAg, prepare 7 mL, label it and place it on a vortex mixer for at least 60 s of mixing, and set aside for use.

[0123] (2) Instrument Testing

[0124] Set the instrument according to the "Operation Instruction Manual for the Use and Maintenance of EXI1800 Automatic Chemiluminescence Immunoassay Analyzer" and the HBeAg project parameters, and load Reagents R1, R2, and magnetic bead reagent.

[0125] Test enterprise reference products and clinical samples: Put the HBeAg enterprise reference products and clinical samples into the sample tray of the EXI1800 (Zhongyuan Huiji Biotechnology Co., Ltd.) instrument, and apply for testing according to the "Operation Instruction Manual for the Use and Maintenance of EXI1800 Automatic Chemiluminescence Immunoassay Analyzer".

[0126] (3) Data Results

[0127] a. Calibration Experiment

[0128] Table 6 HBeAg Calibration Test Data

[0129]

[0130]

[0131] Complete the calibration using the method of serial dilution calibration. The calibration results are shown in the above table, and the calibration results meet the requirements: the linear regression equation of the standard curve R 2 ≥0.99 (as attachedFigure 1 as shown

[0132] b. Clinical test

[0133] Table 7 Detection data of positive and negative samples

[0134] Sample Sample Number Mean RLU Concentration Positive Diluted 2-fold S1 17988716 120.98 Positive Diluted 20-fold S2 2487862 15.23 Positive Diluted 200-fold S3 227651 1.59 All-Negative Blood S4 2501 0

[0135] It can be seen therefrom that this strain can be effectively applied to the quantitative detection of the HBeAg project, can effectively distinguish the positive and negative of hepatitis B antigen, and quantitatively determine the positive samples.

[0136] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. An anti-HBeAg antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment comprises the following complementarity-determining regions: CDR-VH1: X1-Y-H-M-X2, where X1 is E or D, and X2 is Y or T; CDR-VH2: Y-I-S-I-G-X1-G-S-I-A-Y-S-X2-T-V-X3-G, where X1 is G or A, X2 is D or E, and X3 is E or D; CDR-VH3: H-D-X1-Y-G-D-Y-X2-M-D-Y, where X1 is T or Y, and X2 is A or G; CDR-VL1: R-A-X1-Q-E-I-S-X2-Y-L-T, where X1 is T or S, and X2 is G or A; CDR-VL2: V-X1-S-T-L-X2-S, where X1 is G or A, and X2 is D or W; CDR-VL3: L-Q-X1-A-S-S-X2-Y-T, where X1 is Y or T, and X2 is P or V.

2. The anti-HBeAg antibody or functional fragment thereof according to claim 1, characterized in that, In the CDR-VH1 of the complementarity-determining regions, X2 is Y; in the CDR-VH2, X2 is D; in the CDR-VH3, X1 is T; in the CDR-VL1, X2 is G; in the CDR-VL2, X1 is A; and in the CDR-VL3, X1 is Y; The anti-HBeAg antibody or its functional fragment according to claim 1, wherein X1 in the CDR-VH1 is E; Preferably, X1 in the CDR-VH1 is D; Preferably, X2 in the CDR-VH1 is Y; Preferably, X2 in the CDR-VH1 is T; Preferably, X1 in the CDR-VH2 is G; Preferably, X1 in the CDR-VH2 is A; Preferably, X2 in the CDR-VH2 is D; Preferably, X2 in the CDR-VH2 is E; Preferably, X3 in the CDR-VH2 is E; Preferably, X3 in the CDR-VH2 is D; Preferably, X1 in the CDR-VH3 is T; Preferably, X1 in the CDR-VH3 is Y; Preferably, X2 in the CDR-VH3 is A; Preferably, X2 in the CDR-VH3 is G; Preferably, X1 in the CDR-VL1 is T; Preferably, X1 in the CDR-VL1 is S; Preferably, X2 in the CDR-VL1 is G; Preferably, X2 in the CDR-VL1 is A; Preferably, X1 in the CDR-VL2 is G; Preferably, X1 in the CDR-VL2 is A; Preferably, X2 in the CDR-VL2 is D; Preferably, X2 in the CDR-VL2 is W; Preferably, X1 in the CDR-VL3 is Y; Preferably, X1 in the CDR-VL3 is T; Preferably, X2 in the CDR-VL3 is P; Preferably, X2 in the CDR-VL3 is V.

3. The anti-HBeAg antibody or its functional fragment according to any one of claims 1-2, characterized in that, Each of the complementarity-determining regions is selected from any one of the following mutant combinations:

4. The anti-HBeAg antibody or its functional fragment according to any one of claims 1-3, characterized in that, The antibody or its functional fragment further comprises a heavy chain framework region FR1-H, FR2-H, FR3-H and FR4-H with sequences successively shown as SEQ ID NO: 1-4, and / or, a light chain framework region FR1-L, FR2-L, FR3-L and FR4-L with sequences successively shown as SEQ ID NO: 5-8.

5. The anti-HBeAg antibody or functional fragment thereof according to claim 4, wherein The antibody further comprises a constant region; Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. Preferably, the species origin of the constant region is bovine, equine, dairy cattle, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, gamecock or human.

6. The anti-HBeAg antibody or a functional fragment thereof according to claim 5, wherein The antibody is a coating antibody; Preferably, the coating carrier of the coating antibody can be selected from at least one of nitrocellulose membrane, ELISA plate, magnetic beads, and latex microspheres; Preferably, the magnetic beads can be selected from at least one of carboxyl magnetic beads, methylacrylamide magnetic beads, and streptavidin magnetic beads; Preferably, the latex microspheres can be selected from at least one of polyvinyl alcohol toluene microspheres, polystyrene microspheres, or microspheres polymerized mainly from polyvinyl alcohol toluene and polystyrene; Preferably, the latex microspheres can be selected from at least one of carboxyl latex microspheres and amino latex microspheres.

7. A carrier, characterized in that, It contains a nucleic acid fragment encoding the antibody or its functional fragment as described in any one of claims 1-6.

8. A recombinant cell, characterized in that, It contains a vector, and the vector contains the vector described in claim 7.

9. A reagent for detecting HBeAg, characterized in that, It includes the antibody or its functional fragment as described in any one of claims 1-8.

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