Monoclonal antibody cAbD4 of hepatitis B virus core protein and preparation and application thereof

Abstract

The invention discloses a monoclonal antibody cAbD4 of a hepatitis B virus core protein and preparation and application thereof, the cAbD4 recognizes the core protein HBc of the hepatitis B virus HBV, amino acid sequences of CDR1-H, CDR2-H and CDR3-H in a heavy chain variable region of the cAbD4 are respectively SEQ ID NO.1-3, and amino acid sequences of CDR1-L, CDR2-L and CDR3-L in a light chain variable region of the cAbD4 are respectively SEQ ID NO.4, 'LGS' and SEQ ID NO.5. The monoclonal antibody obtained through screening is high in HBc binding capacity. According to the embodiment of the invention, the application of cAbD4 in various biochemical methods is verified, and the ability of cAbD4 to detect different HBV genotypes is verified. The monoclonal antibody cAbD4 disclosed by the invention has the characteristics of high efficiency, broad spectrum and multiple purposes, and has a wide application prospect in the field of HBV infection diagnosis.

Description

Technical Field

[0001] The present invention belongs to the medical technology field of molecular biology and cellular immunology, and specifically relates to a monoclonal antibody cAbD4 of hepatitis B virus core protein and the preparation and application thereof. Background Art

[0002] Hepatitis B virus (HBV) infection remains a major public health problem worldwide. Currently, although nucleoside (acid) analogs (NAs) and interferon-α (IFN-α) are antiviral drugs for chronic hepatitis B, they can effectively inhibit HBV DNA replication and disease progression, but the functional cure rate is very low. Therefore, there is still an urgent need to develop more effective strategies for the detection and treatment of HBV infection.

[0003] HBV is a hepatotropic DNA virus with a diameter of about 42 nm. Its internal capsid is assembled by the core protein (HBc). In most HBV genotypes, the core protein consists of 183 amino acids (aa), including an N-terminal assembly domain (1-149a.a.) for capsid formation and an arginine-rich C-terminal domain (150-183a.a.) for nucleic acid binding and nuclear localization. HBc first forms dimers, and usually 120 dimers self-assemble into a capsid with T=4 icosahedral symmetry. The capsid envelops the viral RNA and polymerase, which not only provides a closed environment for the reverse transcription process of viral RNA, but also promotes nucleocapsid packaging and the formation of viral particles. In addition, HBc can regulate the transport of capsids into the nucleus and is associated with the formation of cccDNA during infection. In addition to being a structural protein and playing a regulatory role in multiple stages of the viral life cycle, HBc can also play an important role in the pathogenesis of HBV-related diseases by inhibiting host immune responses or activating multiple signaling pathways such as the MAPK pathway and the Wnt / β-catenin pathway. These functions of HBc suggest that it can become an important target for HBV detection and intervention.

[0004] HBV-related research mainly indirectly evaluates HBV infection titers by detecting surface antigens, e-antigens and HBV DNA in cell supernatants. However, there are very few anti-HBc monoclonal antibodies that can be used for the detection of multiple HBV genotypes and various biochemical experiments such as enzyme-linked immunosorbent assay (ELISA), Western blot, immunofluorescence assay (IFA), flow cytometry, immunospot assay, immunohistochemistry (IHC), etc., which makes it difficult to directly detect HBV-infected cells. Therefore, the development of more sensitive and broad-spectrum anti-HBc monoclonal antibodies is very important for the development of HBV-related research. Summary of the invention

[0005] In order to be applicable to the detection of multiple HBV genotypes and multiple biochemical experimental methods, the present invention screened and obtained a monoclonal antibody against HBV core protein, which has the characteristics of high efficiency, broad spectrum and multi-purpose. The present invention further provides the use of monoclonal antibodies against HBc for multiple methods to detect HBV virus infection. The above monoclonal antibodies have strong specific binding ability to HBV core protein and can detect HBV core protein through multiple biochemical experiments. The above monoclonal antibodies are expected to become detection antibodies for HBV live virus infection, providing a powerful tool for antigen detection of multiple HBV genotypes.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide a monoclonal antibody cAbD4 against the core protein of hepatitis B virus, wherein the monoclonal antibody recognizes the core protein HBc of hepatitis B virus HBV.

[0008] Furthermore, the monoclonal antibody is a broad-spectrum human monoclonal antibody that can recognize HBc of all HBV genotypes, including HBV genotypes A, B, C, D, E, F, G, H, I, and J.

[0009] Furthermore, the amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the monoclonal antibody are SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the monoclonal antibody are SEQ ID NO.4, "LGS" and SEQ ID NO.5, respectively.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is as shown in SEQ ID NO.11 or an amino acid sequence having at least 80% identity thereto, and the amino acid sequence of the light chain variable region VL of the monoclonal antibody is as shown in SEQ ID NO.12 or an amino acid sequence having at least 80% identity thereto.

[0011] It is understandable that the heavy chain sequence and the paired light chain sequence constitute the above-mentioned monoclonal antibody, and their variable regions determine the binding recognition and specificity of the antibody to the antigen, and the antibody specificity depends on the interaction between the antibody binding site and the antigenic determining region. The binding sites of the heavy chain and the light chain are mainly composed of the residues of 3 complementary determining regions (CDR), and the CDRs are connected by the framework region (FR). Thus, those skilled in the art can easily determine the framework region after knowing the amino acid sequence of the CDR. That is, when the CDR sequences of the heavy chain and the light chain remain unchanged, the function and application of the monoclonal antibody cAbD4 of the present invention can be basically realized. Therefore, the monoclonal antibody of the present invention, its specific sequence is not limited to the above specific heavy chain sequence variable region and light chain sequence variable region, and the variable region of the above specific sequence is only the monoclonal antibody sequence specifically adopted in a kind of implementation of the present invention.

[0012] A protein consisting of an amino acid sequence that is "at least 80% identical" to a reference sequence may contain mutations such as deletions, insertions and / or substitutions compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from a species different from the reference sequence. "Amino acid substitutions" can be conservative or non-conservative. Preferably, the substitution is a conservative substitution, in which one amino acid is replaced by another amino acid with similar structure and / or chemical properties. Specifically, the sequence of the heavy chain or light chain variable region differs from the reference sequence only by conservative amino acid substitutions.

[0013] The second aspect of the present invention is to provide a biological material related to the monoclonal antibody described in the first aspect of the present invention, wherein the biological material is selected from one of the following (A) to (B):

[0014] (A) a nucleic acid molecule encoding the monoclonal antibody cAbD4 described in the first aspect of the present invention;

[0015] (B) An expression cassette, a recombinant vector, and a recombinant cell line containing the nucleic acid molecule described in (A).

[0016] Furthermore, the expression cassette, recombinant vector, and recombinant cell line can be used to express the above-mentioned heavy chain sequence, light chain sequence, or monoclonal antibody.

[0017] Further, the nucleic acid sequence encoding CDR1-H shown in SEQ ID NO.1 is shown in SEQ ID NO.6;

[0018] The nucleic acid sequence encoding CDR2-H shown in SEQ ID NO.2 is shown in SEQ ID NO.7;

[0019] The nucleic acid sequence encoding CDR3-H shown in SEQ ID NO.3 is shown in SEQ ID NO.8;

[0020] The nucleic acid sequence encoding CDR1-L shown in SEQ ID NO.4 is shown in SEQ ID NO.9;

[0021] The nucleic acid sequence encoding CDR2-L is "TTGGGTTCT";

[0022] The nucleic acid sequence encoding CDR3-L shown in SEQ ID NO.5 is shown in SEQ ID NO.10.

[0023] Further, the nucleic acid sequence encoding the VH shown in SEQ ID NO.11 is shown in SEQ ID NO.13;

[0024] The nucleic acid sequence encoding the VL shown in SEQ ID NO.12 is shown in SEQ ID NO.14.

[0025] It is understandable that the above specific nucleic acid sequence is only a specific nucleic acid sequence used in one implementation of the present invention. According to the degeneracy of codons, while ensuring that the encoded amino acid sequence remains unchanged, in addition to the nucleic acid sequence defined above, several nucleic acid sequences that can encode the same heavy chain sequence or light chain sequence (for example, conservative nucleotide sequence variants are derived from genetic code degeneracy and silent variants, and nucleotide substitutions, deletions and additions are also included), all of which are within the scope of protection of the present invention.

[0026] In the above-mentioned biological materials, the expression cassette containing the nucleic acid molecule encoding the antibody refers to a DNA capable of expressing the antibody in a host cell, and the DNA may include not only a promoter for initiating transcription of the antibody gene, but also a terminator for terminating transcription of the antibody gene.

[0027] Among the above-mentioned biological materials, a vector is a nucleic acid carrier that can insert a polynucleotide encoding a certain protein and make the protein expressed. A vector can transform, transduce or transfect host cells so that the genetic material elements it carries can be expressed in the host cells. The vector can be a plasmid, cosmid, phage or virus vector.

[0028] In the above biological materials, host cells refer to cells into which vectors are introduced, including prokaryotic cells, fungal cells, insect cells, animal cells, etc., such as Escherichia coli, yeast cells, S2 fruit fly cells, BHK cells, CHO cells, HEK293 cells. The above expression cassettes, recombinant vectors, and recombinant cell lines can all be prepared by conventional methods in the art.

[0029] The third aspect of the present invention is to provide a method for preparing the monoclonal antibody cAbD4 described in the first aspect of the present invention, the method comprising: transfecting a host cell with a recombinant vector containing a nucleic acid molecule encoding the monoclonal antibody cAbD4; culturing the transfected host cells and collecting the supernatant, and purifying to obtain the monoclonal antibody cAbD4.

[0030] Furthermore, the recombinant vector is a pCDNA3.4 plasmid, and the host cell is a 293F cell.

[0031] The fourth aspect of the present invention is to provide the use of the monoclonal antibody cAbD4 described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the monoclonal antibody cAbD4 prepared by the preparation method described in the third aspect of the present invention in the preparation of products for detecting or diagnosing HBV.

[0032] The fifth aspect of the present invention is to provide a product for detecting or diagnosing HBV, which comprises the monoclonal antibody cAbD4 described in the first aspect of the present invention, the biological material described in the second aspect of the present invention, or the monoclonal antibody cAbD4 prepared by the preparation method described in the third aspect of the present invention.

[0033] Furthermore, the above product can be a diagnostic reagent (diagnostic kit), a detection reagent (detection kit), etc., which can also include other reaction reagents. The above product can be applied to ELISA, Western blot, flow cytometry, IFA, immunospot and immunohistochemistry methods to detect HBV core protein HBc.

[0034] The sixth aspect of the present invention is to provide a method for detecting HBV or its HBc for non-diagnostic purposes, the method comprising: using the product described in the fifth aspect of the present invention to mix with a sample to be tested to detect the core protein HBc of HBV.

[0035] The above-mentioned samples to be tested cover various sample types obtained from subjects and can be used in diagnosis or detection, including but not limited to blood and other liquid samples of biological origin, solid tissue samples, clinical samples, cells in culture medium, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples, etc.

[0036] Furthermore, the detection methods include: ELISA, Western blot, flow cytometry, IFA, immunoblot and immunohistochemistry.

[0037] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0038] The monoclonal antibody cAbD4 obtained by screening for the core protein of hepatitis B virus has a strong ability to bind to HBc. The present invention verifies the application of the monoclonal antibody cAbD4 in various biochemical methods through examples, and verifies its ability to detect different HBV genotypes. The monoclonal antibody cAbD4 has the characteristics of high efficiency, broad spectrum and multi-purpose, and has broad application prospects in the field of diagnosis of HBV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0040] Figure 1 This is a schematic diagram of the ELISA results of the activity of the monoclonal antibody cAbD4 binding to the C genotype HBV core protein in Example 2 of the present invention;

[0041] Figure 2 This is a schematic diagram of the results of the monoclonal antibody cAbD4 identifying HBc of different genotypes of HBV in Example 3 of the present invention;

[0042] Figure 3 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 4 of the present invention to identify HBc in HBV-infected cells using the Western blot method;

[0043] Figure 4 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 5 of the present invention to identify HBc in HBV-infected cells using the IFA method;

[0044] Figure 5 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 6 of the present invention to detect HBc in HBV-infected cells by flow cytometry;

[0045] Figure 6 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 7 of the present invention to detect HBc in HBV-infected cells using the immunospot method;

[0046] Figure 7 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 8 of the present invention to detect intracellular HBc in HBV-infected liver tissue using the immunohistochemical method. DETAILED DESCRIPTION

[0047] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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 experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0048] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0049] Example 1: Screening, expression and purification of antibodies

[0050] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (approval number: 2021-030). All participants in the samples provided written informed consent for subsequent sample processing and analysis. The required peripheral blood mononuclear cell (PBMC) samples and liver tissue sections were all from patients with chronic HBV infection. PBMCs were stored in liquid nitrogen, and liver tissue was embedded in wax blocks for storage.

[0051] This example uses a PBMC sample, and the monoclonal antibody screening and purification steps are as follows:

[0052] (1) Monoclonal antibody screening

[0053] Resuscitation of frozen PBMCs: Wash twice with 10 mL of 1640 medium (72400047, Gibco) containing 10% fetal bovine serum. Take 20 μL of cell suspension, add 20 μL of AO / PI dye (RE010212, Countstar), and count the number of live cells. The number of cells is 1×10 6cells / tube, add 50 μL of staining buffer (PBS + 2% fetal bovine serum) containing Live / Dead dye (L34964, Invitrogen), resuspend the peripheral blood mononuclear cells, and stain for 30 minutes at 4°C. After washing twice with PBS, add CD19-PE-Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD27-APC / Cy7 and IgG-FITC (557835, 558117, 558207, 558121, 560222, 555786, BD Pharmingen) in 50 μL of staining buffer, resuspend the peripheral blood mononuclear cells, and stain for 30 minutes at 4°C. After washing twice with PBS, HBc genotype C (P2301, Shenzhen Innocell Biotechnology Co., Ltd.) labeled with DyLight 550 (84531, Invitrogen) and DyLight 650 (84536, Invitrogen) were added to 50 μL staining buffer, and the peripheral blood mononuclear cells were resuspended and stained at 4°C for 30 minutes. After washing twice with PBS, HBc-specific IgG+ memory B cells were sorted using a BD FACSAria II sorting flow cytometer.

[0054] Single B cells were sorted into 96-well PCR plates containing lysis buffer, and then RT-PCR and nested PCR were performed according to the method of the literature (Liao HX, Levesque MC, Nagel A, Dixon A, Zhang R, Walter E, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. Journal of virological methods. 2009; 158: 171-9.) to amplify the variable regions of heavy and light chains, respectively. After the PCR amplification products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., the obtained antibody heavy chain and light chain variable region sequences were synthesized by GenScript Biotech Co., Ltd. and constructed into full-length IgG1 heavy chain and light chain expression vectors pCDNA3.4 (GenScript Biotech Co., Ltd.), respectively, and finally a large number of antibody heavy chain and light chain plasmids were prepared.

[0055] (2) Antibody expression and purification

[0056] The monoclonal antibody of this example is obtained by expression and purification of 293F cells. Taking 400mL of 293F cells as an example, the specific steps are as follows:

[0057] 1. Prepare expression cells: 400 mL, 1.2 × 10 6 293F cells / mL were cultured in a shaker in an incubator at 8% CO2 and 37°C for 2 hours;

[0058] 2. Prepare transfection plasmid A: Take 200 μg of antibody heavy chain plasmid and antibody light chain plasmid respectively, and dilute them together into 10 mL of opti-MEM (31985070, Gibco);

[0059] 3. Prepare transfection reagent B: add 2 mL of 1 mg / mL PEI transfection reagent (24885-2, Polyscieces) into 10 mL of opti-MEM and let stand for 5 minutes;

[0060] 4. Prepare transfection mixture AB: add solution B to solution A, mix and let stand for 20 minutes;

[0061] 5. Transfection: Add 22 mL of AB mixture dropwise into 293F cells, shaking well while adding;

[0062] 6. Collect cell supernatant: After culturing 293F cells for 5-6 days, centrifuge at 3500g for 30 minutes, collect the supernatant and filter it with a 0.45μm filter membrane;

[0063] 7. Antibody purification: Open the lid of the Protein A gravity column, and add 5 column volumes of PBS solution when the ethanol solution has completely flowed out; then add the filtered cell supernatant, and then wash the gravity column with 3 column volumes of PBS solution, and finally elute with 5 volumes of 0.1M glycine-hydrochloric acid solution (pH=3.0); place the eluate in a 30KD ultrafiltration concentration tube, and centrifuge at 4°C, 3500g until the liquid in the concentration tube is less than 1mL; discard the waste liquid in the collection tube and fill the concentration tube with PBS solution, centrifuge under the same conditions until the liquid in the concentration tube is less than 1mL, and measure the antibody concentration.

[0064] Example 2: Enzyme-linked immunosorbent assay (ELISA) to verify the binding ability of monoclonal antibody cAbD4

[0065] An antibody was selected from the antibodies obtained in Example 1 for monoclonal antibody expression and related functional verification, and the monoclonal antibody was named cAbD4. The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of cAbD4 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the nucleic acid sequences encoding SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbD4 are shown in SEQ ID NO.4 and SEQ ID NO.5, CDR2-L is "LGS", the nucleic acid sequences encoding SEQ ID NO.4 and SEQ ID NO.5 are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively, and the nucleic acid sequence encoding CDR2-L is "TTGGGTTCT". The amino acid sequence of the heavy chain variable region VH of cAbD4 is shown as SEQ ID NO.11, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.11 is shown as SEQ ID NO.13; the amino acid sequence of the light chain variable region VL of cAbD4 is shown as SEQ ID NO.12, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.12 is shown as SEQ ID NO.14.

[0066] The specific binding ability of monoclonal antibody cAbD4 to HBV core protein HBc was detected by ELISA experiment. 2μg / mL, 100μL / well of genotype C HBc (P2301, Shenzhen Innosai Biotechnology Co., Ltd.) was coated in a 96-well ELISA plate and coated overnight at 4°C. Prepare PBS solution PBST with 0.05% Tween-20, wash the 96-well plate 5 times. Prepare a blocking solution of 5% skim milk + 2% BSA (prepared in PBS), 200μL / well, and block at room temperature for 1 hour. After washing 3 times, dilute the cAbD4 antibody with blocking solution to the highest concentration of 10μg / mL and gradually dilute it 5 times, with a total of 7 dilutions. 100μL per well was added to the plate washed 3 times with PBST, and incubated at 37°C for 1 hour. The plate was washed 5 times with PBST, HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) was diluted 1:5000 in blocking solution, 100 μL / well, and incubated at 37°C for 1 hour. The plate was washed 5 times with PBST, color development solution A and solution B (E661007, Sangon) were mixed at 1:1, 100 μL / well, and color was developed at room temperature in the dark for 20 minutes. Finally, 50 μL of 2 mol / L H2SO4 was added to terminate the reaction. The optical density was measured at 450 nm (OD) using a VarioskanLUX multimode microplate reader (Thermo Scientific).

[0067] The binding activity of monoclonal antibody cAbD4 to HBV core protein HBc was analyzed by ELISA experiment. Figure 1 As shown in the figure, the monoclonal antibody cAbD4 can specifically bind to HBc of HBV genotype C, and its binding ability is strong, with a half effective concentration EC 50 It is 2.66ng / mL.

[0068] Example 3: Verification of the broad-spectrum recognition ability of monoclonal antibody cAbD4 by flow cytometry

[0069] This example uses flow cytometry experimental technology to verify that the monoclonal antibody cAbD4 recognizes HBc expressed in cells of different genotypes of HBV. The specific experimental steps are as follows:

[0070] First, HBc protein expression plasmids of different HBV genotypes A, B, C, D, E, F, G, H, I and J were transfected into 293T cells respectively, and cells transfected with empty vector were used as controls. After 48 hours, the cells were digested with 0.5% trypsin and terminated by adding complete DMEM medium containing 10% FBS. Gently blow the cells with a pipette and observe under a microscope until more than 90% of the cells are in a single cell state. Pipette the single cell suspension into a 15mL centrifuge tube and centrifuge at 400g for 5 minutes. Discard the supernatant and add 3mL of complete DMEM for culture. Take 20μL of cell suspension, add 20μL of AO / PI dye (RE010212, Countstar), count the number of live cells, and calculate the number of cells as 5×10 5 cells / tube. The cells were fixed and permeabilized using a cell fixation / permeabilization kit (554714, BD Biosciences), centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Then 5 μg / mL cAbD4 was added to each flow tube and incubated at 4°C for 30 minutes; after washing, the cells were stained with Alexa Fluor 647-conjugated goat anti-human secondary antibody (A11013, Life Technologies) and incubated at 4°C for 30 minutes. After washing, the cells were stained with FACSymphony TM The data were collected by A3 flow cytometer (BD Biososciences), and analyzed by FlowJo software V10.9 (BD Biososciences). The experimental results are shown in Figure 2 shown.

[0071] The results showed that the cAbD4 antibody could clearly detect HBc expressed in cells and could widely identify HBc derived from different HBV genotypes including A, B, C, D, E, F, G, H, I and J.

[0072] Example 4: Verification of the function of monoclonal antibody cAbD4 using Western blot experiment

[0073] In this example, the monoclonal antibody cAbD4 was applied to the Western blot method to identify HBc in HBV-infected cells. The specific experimental steps are as follows:

[0074] This experiment was conducted in a certified biosafety level 2 laboratory. 6HepG2-NTCP cells were plated in 6-well plates coated with type I rat tail collagen (354236, Corning) and cultured for more than 18 hours. 1800 HBV gene copies / cell of HBV virus (purified from HepAD38 supernatant) were added to DMEM medium containing 4% PEG8000 (89510, Sigma), 2.5% DMSO, and 5% FBS, mixed and added to HepG2-NTCP cells. After 20 hours of HBV infection, PBS was diluted 3 times and cultured in DMEM medium containing 2.5% DMSO and 16% FBS. The medium was changed every two days. On the 7th day, HepG2-NTCP cells were washed twice with PBS and 300 μL 1.5×Protein Loading Buffer (DL101-02, Quanshijin) was added to collect the cells. The cell samples treated at 100°C for 10 minutes were transferred to PVDF membranes after SDS-PAGE electrophoresis and blocked with 5% skim milk powder at room temperature for 1 hour. After washing with TBST twice, 0.5 μg / mL cAbD4 was added and incubated at 4°C overnight. After washing with TBST five times, HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) was added at a dilution of 1:5000 and incubated at room temperature for 1 hour. After washing with TBST five times, the membrane was developed. The experimental results are shown in Figure 3 shown.

[0075] Western blot results showed that compared with HBV-uninfected cells, the expression level of β-actin in the reference band of HBV-infected cells was the same, and the total number of cells in both was similar; while the HBc-specific band was only expressed in infected cells and could be identified by the monoclonal antibody cAbD4. The above shows that the monoclonal antibody cAbD4 can specifically bind to HBc in cells infected with HBV.

[0076] Example 5: Verification of the function of monoclonal antibody cAbD4 using immunofluorescence assay (IFA)

[0077] In this example, the monoclonal antibody cAbD4 was applied to IFA detection to identify HBc in HBV-infected cells. The specific experimental steps are as follows:

[0078] The HBV infection procedure was the same as that in Example 4. After the 7-day infected HepG2-NTCP cells were washed twice with PBS, they were fixed with 4% paraformaldehyde solution at room temperature for 30 minutes. After washing once with PBS, PBS solution containing 0.2% Triton X-100 was added to permeabilize the cells for 30 minutes, and QuickBlock TMImmunostaining blocking solution (P0260, Bio-Tech) was used for 1 hour at room temperature. After washing twice with PBS, 10 μg / mL cAbD4 antibody was added and incubated overnight at 4°C. After washing five times with PBS, Alexa Fluor TM 488-Goat anti-Human IgG (H+L) Secondary Antibody (A11013, ThermoFisher) was incubated at room temperature for 1 hour. After washing with PBS three times, DAPI staining solution (C1002, Bio-Tech) was added for 10 minutes. After washing with PBS twice, the fluorescence microscope was used to take pictures. The experimental results are shown in the figure. Figure 4 shown.

[0079] The results showed that strong HBc fluorescence intensity was observed under the field of view, indicating that the monoclonal antibody cAbD4 can clearly recognize HBc in the cytoplasm and nucleus of HBV-infected cells.

[0080] Example 6: Verification of the ability of monoclonal antibody cAbD4 to recognize HBV-infected cells using flow cytometry

[0081] In this example, the monoclonal antibody cAbD4 was applied to flow cytometry to verify its recognition of HBc in HBV-infected cells. The specific experimental steps are as follows:

[0082] The HBV infection steps in this experiment are the same as those in Example 4. After 7 days of infection, the cells were washed twice with PBS and digested with 0.5% trypsin at 37°C for 5 minutes. Then, 10% FBS DMEM medium was added to terminate the culture and the cells were gently pipetted until more than 90% of the cells were in a single cell state. The single cell suspension was pipetted into a 15 mL centrifuge tube and centrifuged at 300 g for 5 minutes. The supernatant was discarded and 3 mL of complete DMEM medium was added. 20 μL of the cell suspension was mixed with an equal volume of AO / PI fuel and the live cells were counted according to 5×10 5 The subsequent staining and data collection steps were the same as in Example 3. The experimental results are shown in Figure 5 shown.

[0083] The results showed that the monoclonal antibody cAbD4 could specifically recognize HBc in HBV-infected cells and clearly distinguish between HBV-infected and non-infected cells.

[0084] Example 7: Validation of monoclonal antibody cAbD4 for detecting HBV infection using live virus immunospot assay

[0085] In this example, the monoclonal antibody cAbD4 was used in the immunospot assay to detect HBc in HBV-infected cells. The specific experimental steps are as follows:

[0086] This experiment was conducted in a certified biosafety level 2 laboratory. 5 HepG2-NTCP cells were plated in a 96-well plate coated with type I rat tail collagen (354236, Corning) and cultured for more than 18 hours. 1800, 600, 300, and 0 gene copies / cell of HBV virus were added to the DMEM medium containing 4% PEG8000 (89510, Sigma), 2.5% DMSO, and 5% FBS, respectively, and the mixture was added to the HepG2-NTCP cells. After 20 hours of HBV infection, PBS was diluted three times and cultured with DMEM medium containing 2.5% DMSO and 16% FBS. The medium was changed every two days. On the 7th day, HepG2-NTCP cells were washed twice with PBS and fixed with 4% paraformaldehyde solution at room temperature for 30 minutes. After washing once with PBS, a PBS solution containing 0.2% Triton X-100 was added to permeabilize the cells for 30 minutes. QuickBlock was then added TM Immunostaining blocking solution (P0260, BioTek) was used for 1 hour at room temperature. After washing twice with PBS, 10 μg / mL cAbD4 antibody was added and incubated overnight at 4°C. After washing 5 times with PBS, 1:5000 diluted HRP-sheep anti-human IgG (ZB-2304, Zhongshan Jinqiao) was added and incubated at room temperature for 1 hour. After washing 5 times with PBS, KPL TrueBlue peroxidase substrate (Seracare Life Sciences) was used for color development. The cell imaging multifunctional microplate detection instrument Cytation 7 (BioTek) was used to take pictures and count the number of HBV-infected cells. The experimental results are shown in the figure. Figure 6 shown.

[0087] The results showed that cAbD4 could clearly identify HBV-infected cells and the number of spots decreased with the decrease of HBV infection virus amount, indicating that cAbD4 can be used for the detection of HBV core protein HBc by immunospot assay and has virus dose dependence.

[0088] Example 8: Validation of the ability of monoclonal antibody cAbD4 to recognize HBV-infected liver tissue using immunohistochemistry

[0089] In this example, the monoclonal antibody cAbD4 was applied to IHC detection to verify its ability to recognize HBc in HBV-infected liver tissue. The specific experimental steps are as follows:

[0090] Liver tissues of two clinical chronic HBV patients, Donor 1 and Donor 2, were fixed in formalin, embedded in paraffin, and sliced, then dewaxed with xylene and hydrated with different concentrations of ethanol. After rinsing with tap water for 1 minute, 100 μL of endogenous peroxidase blocker (Kit-0014, Maixin Biotechnology Development Co., Ltd.) was added to the tissue and incubated at room temperature for 10 minutes. After washing with PBS three times, 100 μL of antibody diluent (ABD-0030, Maixin Biotechnology Development Co., Ltd.) was added and blocked at room temperature for 1 hour. After washing with PBS three times, 100 μL of 20 μg / mL HRP-coupled cAbD4 antibody (cAbD4-HRP) was added and incubated at room temperature for 2 hours. After washing with PBS five times, DAB reagent (Kit-0014, Maixin Biotechnology Development Co., Ltd.) was used for color development at room temperature for 5-10 minutes. After rinsing with tap water, hematoxylin staining was performed for 20 seconds, and PBS returned to blue after rinsing with tap water. After dehydration, transparency and sealing, the slides were photographed using Motic VM1000 (Motic Electric Group Co., Ltd). Figure 7 shown.

[0091] The results showed that cAbD4 could clearly identify HBc in HBV-infected hepatocytes in the liver tissues of Donor 1 and Donor 2; HBc was distributed differently in the hepatocytes of the two, existing in both the cytoplasm and nucleus of Donor 1, and mainly in the cytoplasm of Donor 2. This suggests that cAbD4 can be used for clinical immunohistochemical detection of the core protein HBc in the liver tissues of HBV patients.

[0092] It can be seen from the above examples that the monoclonal antibody cAbD4 obtained by screening in the present invention has a strong binding ability with the core protein HBc of the hepatitis B virus, and can be used in a variety of detection methods to diagnose HBc of the hepatitis B virus, showing good broad spectrum and sensitivity.

[0093] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody cAbD4 against the core protein of hepatitis B virus, It is characterized in that The monoclonal antibody recognizes the core protein HBc of the hepatitis B virus HBV; the amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the monoclonal antibody are SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the monoclonal antibody are SEQ ID NO.4, "LGS" and SEQID NO.5, respectively.

2. The monoclonal antibody cAbD4 according to claim 1, It is characterized in that The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is as shown in SEQ ID NO.11 or an amino acid sequence having at least 80% identity thereto, and the amino acid sequence of the light chain variable region VL of the monoclonal antibody is as shown in SEQ ID NO.12 or an amino acid sequence having at least 80% identity thereto.

3. A biomaterial related to the monoclonal antibody cAbD4 according to any one of claims 1 to 2, It is characterized in that The biological material is selected from one of the following (A) to (B): (A) a nucleic acid molecule encoding the monoclonal antibody cAbD4 according to any one of claims 1 to 2; (B) An expression cassette, a recombinant vector, and a recombinant cell line containing the nucleic acid molecule described in (A).

4. The biomaterial according to claim 3, It is characterized in that The nucleic acid sequence encoding CDR1-H shown in SEQ ID NO.1 is shown in SEQ ID NO.6; The nucleic acid sequence encoding CDR2-H shown in SEQ ID NO.2 is shown in SEQ ID NO.7; The nucleic acid sequence encoding CDR3-H shown in SEQ ID NO.3 is shown in SEQ ID NO.8; The nucleic acid sequence encoding CDR1-L shown in SEQ ID NO.4 is shown in SEQ ID NO.9; The nucleic acid sequence encoding CDR2-L is "TTGGGTTCT"; The nucleic acid sequence encoding CDR3-L shown in SEQ ID NO.5 is shown in SEQ ID NO.

10.

5. The biomaterial according to claim 3, It is characterized in that The nucleic acid sequence encoding the VH shown in SEQ ID NO.11 is shown in SEQ ID NO.13; The nucleic acid sequence encoding the VL shown in SEQ ID NO.12 is shown in SEQ ID NO.

14.

6. A method for preparing the monoclonal antibody cAbD4 according to any one of claims 1 to 2, It is characterized in that include: The recombinant vector containing the nucleic acid molecule encoding the monoclonal antibody cAbD4 is transfected into a host cell; the transfected host cell is cultured and the supernatant is collected and purified to obtain the monoclonal antibody cAbD4.

7. The preparation method according to claim 6, It is characterized in that The recombinant vector is pCDNA3.4 plasmid, and the host cell is 293F cell.

8. Use of the monoclonal antibody cAbD4 according to any one of claims 1 to 2, the biomaterial according to any one of claims 3 to 5, or the monoclonal antibody cAbD4 prepared by the preparation method according to any one of claims 6 to 7 in the preparation of products for detecting or diagnosing HBV.

9. A product for detecting or diagnosing HBV, It is characterized in that The product comprises the monoclonal antibody cAbD4 according to any one of claims 1 to 2, the biological material according to any one of claims 3 to 5, or the monoclonal antibody cAbD4 prepared by the preparation method according to any one of claims 6 to 7.

10. A method for detecting HBV or HBc for non-diagnostic purposes, It is characterized in that The method comprises: using the product of claim 9 and mixing it with a sample to be tested to detect the core protein HBc of HBV.

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