Monoclonal antibody aiming at hepatitis B virus and preparation method thereof

Abstract

The invention belongs to the field of biological medicine, and provides a monoclonal antibody aiming at hepatitis B virus, and the monoclonal antibody aiming at the hepatitis B virus comprises a heavy chain variable region as shown in SEQ ID NO.7 and a light chain variable region as shown in SEQ ID NO.8. The monoclonal antibody aiming at the hepatitis B virus provided by the invention has good biological activity, strong specificity, high titer and high affinity, has important application value in preparation of detection products of the hepatitis B virus, and has wide application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for treating hepatitis B virus ( hepatitis B virus, HBV) monoclonal antibody and preparation method thereof. Background Art

[0002] Hepatitis B virus (HBV) is 42nm in diameter, and its particles are divided into two parts: the shell and the core. The HBV genome is about 3200bp long, which is a relatively small virus. It is a partially double-stranded circular DNA. It is the pathogen that causes hepatitis B (abbreviated as HBV) and belongs to the Hepadnaviridae family. The viruses in this family include the Orthohepadnavirus genus and the Avian Hepadnavirus genus. The Orthohepadnavirus genus causes human infection.

[0003] Hepatitis B, caused by the hepatitis B virus, is considered a significant public health problem. The disease is mainly transmitted through blood, mother-to-child and sexual contact. According to the World Health Organization, about 1.5 million people are infected with hepatitis B virus each year, and about 296 million people were infected with hepatitis B virus in 2019. The hepatitis B virus infection rate in my country is about 60%-70%. Individuals infected with hepatitis B virus are at higher risk of complications such as cirrhosis, hepatocellular carcinoma and liver decompensation than the general population. About 25% of cirrhosis cases and 30% of hepatocellular carcinoma cases are caused by hepatitis B virus. New data from 187 countries show that the estimated number of deaths from viral hepatitis has increased from 1.1 million in 2019 to 1.3 million in 2022. Of these, 83% are caused by hepatitis B, making viral hepatitis the second leading cause of death from infectious diseases and the seventh leading cause of death worldwide.

[0004] Monoclonal antibodies are produced by artificially prepared hybridoma cells, which are formed by the fusion of an antigen-activated B cell and a myeloma cell. Monoclonal antibodies have the advantages of high purity, high sensitivity, strong specificity, less cross-reaction, and low preparation cost. They are usually prepared using hybridoma technology. The main steps to obtain monoclonal antibodies are: fusion of immune animal spleen cells and myeloma cells to obtain hybridoma cells → screening hybridoma cells to obtain positive monoclonal hybridoma cells that secrete target antibodies → expanding the culture of positive monoclonal hybridoma cells → in vitro culture or in vivo ascites induction method to obtain a large number of monoclonal antibodies.

[0005] The global incidence of hepatitis B is declining, but the mortality rate is rising, and long-term management is still needed. Although diagnostic and treatment tools are currently available on the market, the coverage of testing and treatment has stagnated. Overall, the number of people who receive a standard dose of hepatitis B vaccine at birth is still relatively limited worldwide. Therefore, antibodies with excellent performance that can be used for the prevention and treatment of hepatitis B virus have important application significance and translational value. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a monoclonal antibody against hepatitis B virus and a preparation method thereof.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a monoclonal antibody, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3; The light chain variable region includes CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6; SEQ ID NO.1: DYYIH SEQ ID NO.2:WINPENGHTFFDPSFQG SEQ ID NO.3: YNYDDWYFDV SEQ ID NO.4: RASSNVKYMY SEQ ID NO.5: YTSDLAS SEQ ID NO.6: QQFTGSPSMCT Furthermore, the amino acid sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO.7; SEQ ID NO.7: QVQLQQSGAELVRPGALVKLSCKASGFNIEDYYIHWVKQRPKQGLEWIGWINPENGHTFFDPSFQGKASIATDTSSNTAFLQLSSLTSEDSVVYYCVRYNYDDWYFDVWGAGTTVTVSS Furthermore, the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.8; SEQ ID NO.8: ENVLTQSPAIMSATLGERVTMTCRASSNVKYMYWFQQKSGTSPKLWIYYTSDLASGVPPRFSGSGSGTSYSLTISSVEAEDAATYYCQQFTGSPSMCTFGTGTKLEIN Furthermore, the monoclonal antibody against hepatitis B virus also includes any one of mouse IgG1, IgG2, IgG3, and IgG4 constant regions or a combination of at least two of them, preferably the mouse IgG1 constant region.

[0008] In a second aspect, a base sequence encoding a monoclonal antibody against hepatitis B virus according to the first aspect is provided, and a nucleic acid molecule is provided; Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody against hepatitis B virus includes the sequence shown in SEQ ID NO.9; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody against hepatitis B virus includes the sequence shown in SEQ ID NO.10.

[0009] SEQ ID NO.9: CAGGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGGCCCGGCGCCCTGGTGAAGCTGAGCTGCAAGGCCAGCGGCTTCAACATCGAGGACTACTACATCCACTGGGTGAAGCAGAGGCCCAAGCAGGGCCTGGAGTGGATCGGCTGGATCAACCCGAGAACGGCCACACCTTCT TCGACCCCAGCTTCCAGGGCAAGGCCAGCATCGCCACCGACACCAGCAGCAACACCGCCTTCCTGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGTGGTGTACTACTGCGTGAGGTACAACTACGACGACTGGTACTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGC SEQ ID NO.10: GAGAACGTGCTGACCCAGAGCCCCGCCATCATGAGCGCCACCCTGGGCGAGAGGGTGACCATGACCTGCAGGGCCAGCAGCAACGTGAAGTACATGTACTGGTTCCAGCAGAAGAGCGGCACCAGCCCCAAGCTGTGGATCTACTACACCAGCGACCTGGCC AGCGGCGTGCCCCCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCGTGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTTCACCGGCAGCCCCAGCATGTGCACCTTCGGCACCGGCACCAAGCTGGAGATCAAC In a third aspect, the present invention provides a host cell, wherein the nucleic acid molecule according to the second aspect is integrated into the genome of the host cell; Furthermore, the host cell includes 293F cell or CHO-K1 cell.

[0010] In a fourth aspect, the present invention provides a method for preparing a monoclonal antibody against hepatitis B virus according to the first aspect, the method steps are as follows: (1) Preparation of antigens: Preparation of hepatitis B virus antigens by transient transfection; (2) Animal immunization: using the prepared antigen as an immunogen to immunize Balb / c female mice, obtaining immunized mice, and detecting the titer of mouse serum by indirect ELISA; (3) Preparation of monoclonal antibodies: immune spleen cells were obtained from immunized mice and fused with mouse myeloma cells SP2 / 0. Positive hybridoma cells secreting monoclonal antibodies were obtained after screening. Total RNA was extracted from the hybridoma cells, and the cDNA chains were synthesized by reverse transcription and the variable regions of the light and heavy chains were amplified. The monoclonal antibody expression vector was constructed and purified for expression; (4) The EC50 value of the antibody is used to indicate the affinity of the monoclonal antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the SDS-PAGE electrophoresis result of the monoclonal antibody against hepatitis B virus obtained by affinity purification with Protein A Beads in specific example 3 of the present invention.

[0012] Figure 2 This is the affinity test result of the monoclonal antibody in specific example 4. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the technical solutions of the present invention are further described below through specific implementation methods. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0014] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0015] Example 1 Antigen preparation The preparation method of hepatitis B virus antigen is as follows: (1) Construction of recombinant plasmid According to the coding gene sequence published by NCBI, a His tag sequence was added, and based on the pcDNA3.4 map, Anshengda Biotechnology Co., Ltd. performed gene synthesis to construct the eukaryotic expression recombinant plasmid pcDNA3.4-HBV.

[0016] (2) Endotoxin-free plasmid extraction According to the instructions, use the endotoxin-free plasmid extraction kit to extract plasmid DNA, and perform cell transfection after concentration determination and purity verification.

[0017] (3) The cell transfection steps are as follows: (a) Preheat the thermostatic water bath to 37°C. When the temperature reaches 37°C, take out a frozen cell from the liquid nitrogen tank and quickly put it into the thermostatic water bath. After the cells thaw, put them into the biosafety cabinet. Transfer the cells to a sterile centrifuge tube and centrifuge at 800 rpm for 5 min. Discard the supernatant and resuspend the cell pellet with fresh preheated Transpro CD 01 medium. Transfer the cell pellet to a cell culture bottle and culture the cells in a carbon dioxide cell shaker at 37°C, 120 rpm, 37°C, and 8% carbon dioxide.

[0018] (b) Take out the cell culture flask from the CO2 cell culture shaker and place it in a biosafety cabinet. Take out 20 uL of the cell suspension and use an automatic cell counter to measure the cell density and cell viability. The cell viability is about 95~99% and the cell density needs to be adjusted to 3×106 cells / mL using fresh preheated Transpro CD 01 culture medium before cell transfection can be performed.

[0019] (c) According to the ratio of plasmid DNA 1.6 mg and plasmid DNA: PEI=1:3.2, the plasmid DNA and PEI were diluted using TransproCD 01 medium respectively. The prepared PEI was added to the plasmid DNA. After the transfection system was configured, it was slowly inverted to mix and allowed to stand for 10-15 minutes.

[0020] (d) Add the above transfection system to the cell culture flask to be transfected, and slowly shake the cell suspension while adding. After the addition is completed, place it in a carbon dioxide cell shaker at 37°C, 120 rpm, 37°C, 8% carbon dioxide to continue culturing the cells.

[0021] (e) 18-22 h after transfection, add Transpro feed 01 at a 5% supplement.

[0022] (4) The steps for purifying the antigen protein by affinity chromatography are as follows: (a) Select a gravity chromatography column of appropriate specifications, rinse it once with deionized water and then rinse it again with 0.01M PBS solution, and then fill the column with column material.

[0023] (b) The loaded column material is equilibrated with 10-15 column volumes of 0.01M PBS solution. After equilibration, the sample is loaded 2-3 times, washed with 10-15 column volumes of 0.01M PBS solution, and then eluted with 3-5 column volumes of 0.3M imidazole solution, and the eluate is collected.

[0024] (c) The eluate was placed in a 15 ml 30 kDa ultrafiltration tube and centrifuged at 6000 rpm at 4°C for 30 min. After centrifugation, 0.01 M PBS solution was added to the eluate and the solution was replaced 10,000 times.

[0025] (d) Pipette some protein from the ultrafiltration tube, detect the protein concentration using an ultra-micro-volume UV spectrophotometer, and run a gel to verify the band size.

[0026] Example 2 Animal Immunization (1) Select 6-8 weeks old female Balb / c mice and divide them into 5 experimental groups. Inject the prepared hepatitis B virus protein antigen solution subcutaneously at the back of the mice at multiple points. The animal immunization steps are as follows: (a) Week 1, primary immunization: The hepatitis B virus protein antigen solution was completely emulsified with an equal volume of Freund's complete adjuvant in a 1:1 ratio, and the immunization dose was 50 ug / mouse, and multiple subcutaneous injections were performed on the back of mice.

[0027] (b) Two weeks later, boost immunization: emulsify the HBV protein antigen solution with Freund's incomplete adjuvant in a 1:1 ratio and inject subcutaneously at multiple points on the back of mice at an immunization dose of 50 ug / mouse.

[0028] (c) After an interval of two weeks, repeat step (b) above.

[0029] (d) After an interval of two weeks, repeat step (b) above.

[0030] (e) After an interval of two weeks, shock immunization was performed again: 50 ug of hepatitis B virus protein antigen solution was directly injected into the abdominal cavity of mice without using any adjuvant. The immunization lasted for 3 days. Blood was collected after immunization and allowed to stand at room temperature for 2 hours. The blood was then placed in a 4°C refrigerator for 3-4 hours. After the blood coagulated and the clot shrank, it was centrifuged at 4000 rpm for 10 minutes. The serum was collected and dispensed into sterile EP tubes for the indirect ELISA method to determine the serum titer.

[0031] (f) The serum titer was determined by indirect ELISA, and the protein concentration was measured by ultra-micro UV spectrophotometer. The optimal antiserum was screened out based on the titer and protein concentration.

[0032] (2) Indirect ELISA method for detecting serum titer, the steps are as follows: (a) Hepatitis B virus protein was used as the coating antigen and added to a 96-well plate at a coating concentration of 2 ng / uL at 4°C overnight.

[0033] (b) Add 200 μL PBST to each well and wash three times. After each wash, gently shake the PBST to mix well and then discard the PBST.

[0034] (c) Add 200 μL of blocking solution to each well and block at 37°C for 90 min.

[0035] (d) After blocking, discard the blocking solution and repeat step (b) above. Place in a 4°C refrigerator for later use.

[0036] (e) Dilute the prepared mouse antibody serum in a 2-fold gradient and add 100 uL per well to the ELISA plate. Incubate at 37°C for 60 min, and then repeat the above step (b).

[0037] (f) HRP-labeled goat anti-mouse IgG was used as the enzyme-labeled antibody, diluted 1:5000, 100 μL was added to each well, incubated at 37°C for 60 min, and then the above step (b) was repeated.

[0038] (g) Add 100 μL of TMB color development solution to each well and incubate at 37°C for 15 min.

[0039] (f) Add 100 μL of stop solution to each well to terminate the reaction, and read the OD450 value on a microplate reader.

[0040] Example 3 Preparation of monoclonal antibodies (1) Hybridoma cell fusion and screening, the steps are as follows: (a) Mice with high serum titers were killed by cervical dislocation and then disinfected by soaking in 75% (v / v) ethanol for 10 min.

[0041] (b) Under sterile conditions, mice are dissected, and their immune spleen cells are obtained and prepared into a cell suspension.

[0042] (c) Mouse spleen cells were fused with myeloma cells SP2 / 0 and hybridoma cells were prepared using the PEG fusion method.

[0043] (d) The fused hybridoma cells are then screened using HAT medium (H: hypoxanthine, A: aminopterin, T: thymidine) to selectively retain hybridoma cells with good growth status.

[0044] (e) The cell supernatant on day 10-14 after cell fusion was collected and antibody screening was performed using the indirect ELISA (enzyme-linked immunosorbent assay) method to select hybridoma cell lines that secrete positive antibodies.

[0045] (f) The positive cell lines were continuously expanded and cultured using the limiting dilution method. On the 10th day after cloning, the cell pools with high antibody titers, monoclonal growth and good morphology were cloned and expanded and cryopreserved using the same method.

[0046] (2) Extract total RNA using the Trizol method. The steps are as follows: (a) After all hybridoma cells were homogenized, 500 μL TRIzol reagent was added and allowed to stand at room temperature for 5 min to allow them to be fully lysed.

[0047] (b) Transfer all the lysate to a sterile eppendorf tube, add 300 μL of chloroform, let stand at room temperature for 5 min, and then centrifuge at 12,000 rpm and 4°C for 10 min.

[0048] (c) Pipette the supernatant into a new sterile EP tube, add 500 μL of isopropanol to precipitate RNA, place at -20°C for 5 min, and centrifuge at 12,000 rpm at 4°C for 10 min.

[0049] (d) Discard the supernatant, add 600 μL of 70% ethanol solution, rinse twice, and centrifuge to remove the supernatant.

[0050] (e) Dissolve the obtained RNA in RNase-free water, measure its concentration and set aside.

[0051] (3) Reverse transcription to synthesize cDNA chains and amplify light and heavy chain variable regions The extracted total RNA was used as a template and the total RNA was reverse transcribed into cDNA. The variable regions of the heavy chain and light chain were amplified according to the standard operating procedure of rapid amplification of cDNA ends (RACE). The amplified antibody gene fragments were cloned into standard cloning vectors, respectively. Colony PCR was performed to screen the target fragments with the correct size and sequencing was performed for verification. Finally, the sequence SEQ ID NO.9 of the heavy chain variable region encoding the anti-hepatitis B virus monoclonal antibody and the light chain variable region sequence SEQ ID NO.10 were obtained.

[0052] (4) Construct a monoclonal antibody expression vector and purify the expression. The steps are as follows: (a) According to the sequencing results of the light and heavy chain variable regions, appropriate restriction sites were selected, and the light and heavy chain variable regions were synthesized respectively, and then connected with the constant region of the antibody. The gene connection products were cloned into the pcDNA3.4 plasmid vector for expression.

[0053] (b) Purify the monoclonal antibody using Protein A Beads, following the same purification steps as in Example 1 (4).

[0054] (c) After purification, the concentration was measured using an ultra-micro UV-visible spectrophotometer and the molecular weight of the monoclonal antibody was verified by SDS-PAGE electrophoresis.

[0055] The SDS-PAGE electrophoresis results of the monoclonal antibody are as follows Figure 1 shown.

[0056] Example 4 Detection of monoclonal antibody affinity (1) The affinity of monoclonal antibodies is determined by using the EC50 value of the antibody. The steps are as follows: (a) Hepatitis B virus protein was used as the coating antigen and added to a 96-well plate at a coating concentration of 2 ng / μL at 4°C overnight.

[0057] (b) Add 200 μL PBST to each well and wash three times. After each wash, gently shake the PBST to mix well and then discard the PBST.

[0058] (c) Add 200 μL of blocking solution containing 2% BSA to each well and block at 37°C for 90 min.

[0059] (d) After blocking, discard the blocking solution and repeat step (b) above. Place in a 4°C refrigerator for later use.

[0060] (e) Dilute the purified anti-HBV monoclonal antibody to a concentration of 10 ng / μl, then perform a 3-fold gradient dilution, add 100 uL per well to the ELISA plate, incubate at 37°C for 60 min, and then repeat the above step (b).

[0061] (f) HRP-labeled goat anti-mouse IgG was used as the enzyme-labeled antibody, diluted 1:5000, 100 μL was added to each well, incubated at 37°C for 60 min, and then the above step (b) was repeated.

[0062] (g) Add 100 μL of TMB color development solution to each well, protect from light, and incubate at 37°C for 15 min.

[0063] (f) Add 100 μL of 10% H2SO4 stop solution to each well to terminate the reaction, and read the OD450 value on a microplate reader.

[0064] The results of the affinity determination of monoclonal antibodies are as follows Figure 2 As shown, EC50 = 0.005576 ng / uL.

[0065] In summary, the present invention provides a method for preparing a monoclonal antibody against hepatitis B virus. The monoclonal antibody is a mouse monoclonal antibody with good specificity and high affinity. The anti-hepatitis B virus monoclonal antibody has important application value in the preparation of hepatitis B virus detection products and has broad application prospects.

[0066] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A monoclonal antibody against hepatitis B virus, characterized in that: The heavy chain variable region includes CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2 and CDR3 shown in SEQ ID NO.3; the light chain variable region includes CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5 and CDR3 shown in SEQ ID NO.

6.

2. The monoclonal antibody against hepatitis B virus according to claim 1, characterized in that: The antibody further comprises a heavy chain variable region amino acid sequence comprising the sequence shown in SEQ ID NO.7 and a light chain variable region amino acid sequence comprising the sequence shown in SEQ ID NO.

8.

3. The monoclonal antibody against hepatitis B virus according to any one of claims 1-2, characterized in that: The monoclonal antibody against hepatitis B virus further comprises any one of mouse IgG1, IgG2, IgG3, and IgG4 constant regions or a combination of at least two of them, preferably mouse IgG1 constant region.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody against hepatitis B virus as described in any one of claims 1-3; preferably, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody against hepatitis B virus includes the sequence shown in SEQ ID NO.9; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody against hepatitis B virus includes the sequence shown in SEQ ID NO.

10.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that The nucleic acid molecule according to claim 4 is integrated into the genome of the host cell.

7. Use of any one of the monoclonal antibody against hepatitis B virus according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, and the host cell according to claim 6, or a combination of at least two thereof, in the preparation of a product for detecting hepatitis B virus infection.

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