Monoclonal antibodies against hepatitis B virus core protein and their preparation and application

Abstract

The present invention discloses a monoclonal antibody against the core protein of hepatitis B virus and its preparation and application. The monoclonal antibody recognizes the core protein HBc of hepatitis B virus HBV. The monoclonal antibody is one of cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, cAbH8 or any combination thereof. The monoclonal antibody obtained by screening in the present invention has a strong ability to bind to HBc. The present invention verifies the application of the monoclonal antibody in various biochemical methods through examples and verifies its ability to detect different HBV genotypes. The monoclonal antibody of the present invention has the characteristics of high efficiency, broad spectrum and versatility, and has broad application prospects in the field of diagnosis of HBV infection.

Description

Technical Field

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

[0002] Hepatitis B virus (HBV) infection remains a major global public health problem. While current antiviral drugs for chronic hepatitis B, such as nucleoside analogs (NAs) and interferon-α (IFN-α), can effectively inhibit HBV DNA replication and disease progression, functional cure rates are low. Therefore, there remains 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 approximately 42 nm. Its inner capsid is composed of the core protein (HBc). In most HBV genotypes, the core protein consists of 183 amino acids (aa), including an N-terminal assembly domain (1-149 aa) for capsid formation and an arginine-rich C-terminal domain (150-183 aa) for nucleic acid binding and nuclear localization. HBc first forms dimers, and typically 120 dimers self-assemble into a capsid with T=4 icosahedral symmetry. The capsid encapsidates the viral RNA and polymerase, providing a closed environment for reverse transcription of the viral RNA and promoting nucleocapsid packaging and virion formation. HBc also regulates capsid transport into the nucleus and is associated with cccDNA formation during infection. In addition to serving as a structural protein and regulating multiple stages of the viral life cycle, HBc also plays a crucial role in the pathogenesis of HBV-related diseases by suppressing host immune responses or activating multiple signaling pathways, including the MAPK and Wnt / β-catenin pathways. These functions of HBc suggest that it could be an important target for HBV detection and intervention.

[0004] HBV-related research primarily assesses HBV infection titers indirectly through the detection of surface antigens, e-antigens, and HBV DNA in cell supernatants. Currently, there are few anti-HBc monoclonal antibodies available for the detection of multiple HBV genotypes and various biochemical assays, such as enzyme-linked immunosorbent assay (ELISA), Western blot, immunofluorescence assay (IFA), flow cytometry, immunospot assay, and immunohistochemistry (IHC). This makes direct detection of HBV-infected cells difficult. Therefore, the development of more sensitive and broad-spectrum anti-HBc monoclonal antibodies is crucial for the advancement of HBV-related research. Summary of the Invention

[0005] To enable the detection of multiple HBV genotypes and various biochemical assays, the present invention screened and obtained eight monoclonal antibodies targeting HBV core protein, which are characterized by high efficiency, broad spectrum, and versatility. The present invention further provides the use of monoclonal antibodies targeting HBc for the detection of HBV infection using various methods. These monoclonal antibodies have strong specific binding ability to HBV core protein and can detect HBV core protein using various biochemical assays. These monoclonal antibodies are expected to become antibodies for detecting live HBV 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 solutions:

[0007] The first aspect of the present invention is to provide a monoclonal antibody 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 monoclonal antibody is one of cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, cAbH8, or any combination thereof; wherein the amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbB8 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 cAbB8 are SEQ ID NO.4, "WSS," and SEQ ID NO.5, respectively;

[0010] The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the cAbD8 are SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17, respectively; the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the cAbD8 are SEQ ID NO. 18, "DAT" and SEQ ID NO. 19, respectively;

[0011] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbE2 are SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 31, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbE2 are SEQ ID NO. 32, "GAT," and SEQ ID NO. 33, respectively;

[0012] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbE5 are SEQ ID NO. 43, SEQ ID NO. 44, and SEQ ID NO. 45, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbE5 are SEQ ID NO. 46, "GKN," and SEQ ID NO. 47, respectively;

[0013] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of cAbE11 are SEQ ID NO. 57, SEQ ID NO. 58, and SEQ ID NO. 59, respectively. The amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of cAbE11 are SEQ ID NO. 60, "DVS," and SEQ ID NO. 61, respectively.

[0014] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbF5 are SEQ ID NO.71, SEQ ID NO.72, and SEQ ID NO.73, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbF5 are SEQ ID NO.74, "KAS," and SEQ ID NO.75, respectively;

[0015] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbF12 are SEQ ID NO.85, SEQ ID NO.86, and SEQ ID NO.87, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbF12 are SEQ ID NO.88, "AAS," and SEQ ID NO.89, respectively;

[0016] The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the cAbH8 are SEQ ID NO.99, SEQ ID NO.100 and SEQ ID NO.101, respectively, and the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the cAbH8 are SEQ ID NO.102, "GAS" and SEQ ID NO.103, respectively.

[0017] Furthermore, the amino acid sequence of the heavy chain variable region VH of the cAbB8 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 cAbB8 is as shown in SEQ ID NO. 12 or an amino acid sequence having at least 80% identity thereto;

[0018] The amino acid sequence of the heavy chain variable region VH of the cAbD8 is as shown in SEQ ID NO. 25 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 cAbD8 is as shown in SEQ ID NO. 26 or an amino acid sequence having at least 80% identity thereto;

[0019] The amino acid sequence of the heavy chain variable region VH of the cAbE2 is as shown in SEQ ID NO. 39 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 cAbE2 is as shown in SEQ ID NO. 40 or an amino acid sequence having at least 80% identity thereto;

[0020] The amino acid sequence of the heavy chain variable region VH of the cAbE5 is as shown in SEQ ID NO. 53 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 cAbE5 is as shown in SEQ ID NO. 54 or an amino acid sequence having at least 80% identity thereto;

[0021] The amino acid sequence of the heavy chain variable region VH of the cAbE11 is shown in SEQ ID NO. 67 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 cAbE11 is shown in SEQ ID NO. 68 or an amino acid sequence having at least 80% identity thereto;

[0022] The amino acid sequence of the heavy chain variable region VH of the cAbF5 is as shown in SEQ ID NO.81 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 cAbF5 is as shown in SEQ ID NO.82 or an amino acid sequence having at least 80% identity thereto;

[0023] The amino acid sequence of the heavy chain variable region VH of the cAbF12 is as shown in SEQ ID NO. 95 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 cAbF12 is as shown in SEQ ID NO. 96 or an amino acid sequence having at least 80% identity thereto;

[0024] The amino acid sequence of the heavy chain variable region VH of the cAbH8 is shown in SEQ ID NO.109 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 cAbH8 is shown in SEQ ID NO.110 or an amino acid sequence having at least 80% identity thereto.

[0025] 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 antibody's binding recognition and specificity for the antigen, and antibody specificity depends on the interaction between the antibody binding site and the antigenic determinant. The binding sites of the heavy and light chains are mainly composed of residues of three complementary determining regions (CDRs), and the CDRs are connected by framework regions (FRs). Thus, those skilled in the art can easily determine the framework regions after knowing the amino acid sequences of the CDRs. That is, when the CDR sequences of the heavy and light chains remain unchanged, the functions and applications of the monoclonal antibodies of the present invention can be basically achieved. Therefore, the monoclonal antibodies of the present invention, their specific sequences are not limited to the above specific heavy chain sequence variable regions and light chain sequence variable regions, and the variable regions of the above specific sequences are only the monoclonal antibody sequences specifically adopted in one embodiment of the present invention.

[0026] 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.

[0027] The second aspect of the present invention is to provide a biomaterial related to the monoclonal antibody described in the first aspect of the present invention, wherein the biomaterial is selected from one of the following (A) to (B):

[0028] (A) a nucleic acid molecule encoding the monoclonal antibody according to the first aspect of the present invention;

[0029] (B) Expression cassettes, recombinant vectors, and recombinant cell lines containing the nucleic acid molecule described in (A).

[0030] Furthermore, the expression cassette, recombinant vector, and recombinant cell line can be used to express the aforementioned heavy chain sequence, light chain sequence, or monoclonal antibody.

[0031] Furthermore, the nucleic acid sequence encoding CDR1-H of cAbB8 shown in SEQ ID NO.1 is shown in SEQ ID NO.6;

[0032] The nucleic acid sequence encoding CDR2-H of cAbB8 shown in SEQ ID NO. 2 is shown in SEQ ID NO. 7;

[0033] The nucleic acid sequence encoding CDR3-H of cAbB8 shown in SEQ ID NO. 3 is shown in SEQ ID NO. 8;

[0034] The nucleic acid sequence encoding CDR1-L of cAbB8 shown in SEQ ID NO.4 is shown in SEQ ID NO.9;

[0035] The nucleic acid sequence encoding CDR2-L of cAbB8 is "TGGTCATCT";

[0036] The nucleic acid sequence encoding CDR3-L of cAbB8 shown in SEQ ID NO.5 is shown in SEQ ID NO.10;

[0037] The nucleic acid sequence encoding CDR1-H of cAbD8 shown in SEQ ID NO. 15 is shown in SEQ ID NO. 20;

[0038] The nucleic acid sequence encoding CDR2-H of cAbD8 shown in SEQ ID NO. 16 is shown in SEQ ID NO. 21;

[0039] The nucleic acid sequence encoding CDR3-H of cAbD8 shown in SEQ ID NO. 17 is shown in SEQ ID NO. 22;

[0040] The nucleic acid sequence encoding CDR1-L of cAbD8 shown in SEQ ID NO. 18 is shown in SEQ ID NO. 23;

[0041] The nucleic acid sequence encoding CDR2-L of cAbD8 is "GATGCAACC";

[0042] The nucleic acid sequence encoding CDR3-L of cAbD8 shown in SEQ ID NO. 19 is shown in SEQ ID NO. 24;

[0043] The nucleic acid sequence encoding CDR1-H of cAbE2 shown in SEQ ID NO. 29 is shown in SEQ ID NO. 34;

[0044] The nucleic acid sequence encoding CDR2-H of cAbE2 shown in SEQ ID NO. 30 is shown in SEQ ID NO. 35;

[0045] The nucleic acid sequence encoding CDR3-H of cAbE2 shown in SEQ ID NO. 31 is shown in SEQ ID NO. 36;

[0046] The nucleic acid sequence encoding CDR1-L of cAbE2 shown in SEQ ID NO. 32 is shown in SEQ ID NO. 37;

[0047] The nucleic acid sequence encoding CDR2-L of cAbE2 is "GGTGCAACC";

[0048] The nucleic acid sequence encoding CDR3-L of cAbE2 shown in SEQ ID NO. 33 is shown in SEQ ID NO. 38;

[0049] The nucleic acid sequence encoding CDR1-H of cAbE5 shown in SEQ ID NO. 43 is shown in SEQ ID NO. 48;

[0050] The nucleic acid sequence encoding CDR2-H of cAbE5 shown in SEQ ID NO. 44 is shown in SEQ ID NO. 49;

[0051] The nucleic acid sequence encoding CDR3-H of cAbE5 shown in SEQ ID NO. 45 is shown in SEQ ID NO. 50;

[0052] The nucleic acid sequence encoding CDR1-L of cAbE5 shown in SEQ ID NO. 46 is shown in SEQ ID NO. 51;

[0053] The nucleic acid sequence encoding CDR2-L of cAbE5 is "GGTAAAAAC";

[0054] The nucleic acid sequence encoding CDR3-L of cAbE5 shown in SEQ ID NO. 47 is shown in SEQ ID NO. 52;

[0055] The nucleic acid sequence encoding CDR1-H of cAbE11 shown in SEQ ID NO. 57 is shown in SEQ ID NO. 62;

[0056] The nucleic acid sequence encoding CDR2-H of cAbE11 shown in SEQ ID NO. 58 is shown in SEQ ID NO. 63;

[0057] The nucleic acid sequence encoding CDR3-H of cAbE11 shown in SEQ ID NO. 59 is shown in SEQ ID NO. 64;

[0058] The nucleic acid sequence encoding CDR1-L of cAbE11 shown in SEQ ID NO. 60 is shown in SEQ ID NO. 65;

[0059] The nucleic acid sequence encoding CDR2-L of cAbE11 is "GATGTCAGT";

[0060] The nucleic acid sequence encoding CDR3-L of cAbE11 shown in SEQ ID NO. 61 is shown in SEQ ID NO. 66;

[0061] The nucleic acid sequence encoding CDR1-H of cAbF5 shown in SEQ ID NO. 71 is shown in SEQ ID NO. 76;

[0062] The nucleic acid sequence encoding CDR2-H of cAbF5 shown in SEQ ID NO. 72 is shown in SEQ ID NO. 77;

[0063] The nucleic acid sequence encoding CDR3-H of cAbF5 shown in SEQ ID NO. 73 is shown in SEQ ID NO. 78;

[0064] The nucleic acid sequence encoding CDR1-L of cAbF5 shown in SEQ ID NO. 74 is shown in SEQ ID NO. 79;

[0065] The nucleic acid sequence encoding CDR2-L of cAbF5 is "AAGGCGTCG";

[0066] The nucleic acid sequence encoding CDR3-L of cAbF5 shown in SEQ ID NO. 75 is shown in SEQ ID NO. 80;

[0067] The nucleic acid sequence encoding CDR1-H of cAbF12 shown in SEQ ID NO. 85 is shown in SEQ ID NO. 90;

[0068] The nucleic acid sequence encoding CDR2-H of cAbF12 shown in SEQ ID NO. 86 is shown in SEQ ID NO. 91;

[0069] The nucleic acid sequence encoding CDR3-H of cAbF12 shown in SEQ ID NO. 87 is shown in SEQ ID NO. 92;

[0070] The nucleic acid sequence encoding CDR1-L of cAbF12 shown in SEQ ID NO. 88 is shown in SEQ ID NO. 93;

[0071] The nucleic acid sequence encoding CDR2-L of cAbF12 is "GCTGCGTCC";

[0072] The nucleic acid sequence encoding CDR3-L of cAbF12 shown in SEQ ID NO. 89 is shown in SEQ ID NO. 94;

[0073] The nucleic acid sequence encoding CDR1-H of cAbH8 shown in SEQ ID NO. 99 is shown in SEQ ID NO. 104;

[0074] The nucleic acid sequence encoding CDR2-H of cAbH8 shown in SEQ ID NO. 100 is shown in SEQ ID NO. 105;

[0075] The nucleic acid sequence encoding CDR3-H of cAbH8 shown in SEQ ID NO. 101 is shown in SEQ ID NO. 106;

[0076] The nucleic acid sequence encoding CDR1-L of cAbH8 shown in SEQ ID NO. 102 is shown in SEQ ID NO. 107;

[0077] The nucleic acid sequence encoding CDR2-L of cAbH8 is "GGTGCGTCC";

[0078] The nucleic acid sequence encoding CDR3-L of cAbH8 shown in SEQ ID NO. 103 is shown in SEQ ID NO. 108.

[0079] 5. The biomaterial according to claim 3, characterized in that

[0080] The nucleic acid sequence encoding the VH of cAbB8 shown in SEQ ID NO.11 is shown in SEQ ID NO.13;

[0081] The nucleic acid sequence encoding the VL of cAbB8 shown in SEQ ID NO. 12 is shown in SEQ ID NO. 14;

[0082] The nucleic acid sequence encoding the VH of cAbD8 shown in SEQ ID NO. 25 is shown in SEQ ID NO. 27;

[0083] The nucleic acid sequence encoding the VL of cAbD8 shown in SEQ ID NO. 26 is shown in SEQ ID NO. 28;

[0084] The nucleic acid sequence encoding the VH of cAbE2 shown in SEQ ID NO. 39 is shown in SEQ ID NO. 41;

[0085] The nucleic acid sequence encoding the VL of cAbE2 shown in SEQ ID NO.40 is shown in SEQ ID NO.42;

[0086] The nucleic acid sequence encoding the VH of cAbE5 shown in SEQ ID NO. 53 is shown in SEQ ID NO. 55;

[0087] The nucleic acid sequence encoding the VL of cAbE5 shown in SEQ ID NO. 54 is shown in SEQ ID NO. 56;

[0088] The nucleic acid sequence encoding VH of cAbE11 shown in SEQ ID NO. 67 is shown in SEQ ID NO. 69;

[0089] The nucleic acid sequence encoding the VL of cAbE11 shown in SEQ ID NO. 68 is shown in SEQ ID NO. 70;

[0090] The nucleic acid sequence encoding the VH of cAbF5 shown in SEQ ID NO.81 is shown in SEQ ID NO.83;

[0091] The nucleic acid sequence encoding the VL of cAbF5 shown in SEQ ID NO.82 is shown in SEQ ID NO.84;

[0092] The nucleic acid sequence encoding VH of cAbF12 shown in SEQ ID NO. 95 is shown in SEQ ID NO. 97;

[0093] The nucleic acid sequence encoding the VL of cAbF12 shown in SEQ ID NO.96 is shown in SEQ ID NO.98;

[0094] The nucleic acid sequence encoding the VH of cAbH8 shown in SEQ ID NO. 109 is shown in SEQ ID NO. 111;

[0095] The nucleic acid sequence encoding VL of cAbH8 shown in SEQ ID NO.110 is shown in SEQ ID NO.112.

[0096] 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 the codons, while ensuring that the encoded amino acid sequence remains unchanged, in addition to the nucleic acid sequences 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, including nucleotide substitutions, deletions and additions) are all within the scope of protection of the present invention.

[0097] 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. 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.

[0098] Among the aforementioned biological materials, a vector is a nucleic acid delivery vehicle that can insert a polynucleotide encoding a protein and enable protein expression. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within them. Vectors can be plasmids, cosmids, bacteriophages, or viruses.

[0099] In the aforementioned biological materials, host cells refer to cells into which the vector is introduced, including prokaryotic cells, fungal cells, insect cells, animal cells, and the like, such as Escherichia coli, yeast cells, S2 Drosophila cells, BHK cells, CHO cells, and HEK293 cells. The aforementioned expression cassettes, recombinant vectors, and recombinant cell lines can all be prepared using conventional methods in the art.

[0100] The third aspect of the present invention is to provide a method for preparing the monoclonal antibody 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; culturing the transfected host cells and collecting the supernatant, and purifying to obtain the monoclonal antibody.

[0101] Furthermore, the recombinant vector is a pCDNA3.4 plasmid, and the host cell is a 293F cell.

[0102] The fourth aspect of the present invention is to provide the use of the monoclonal antibody described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the monoclonal antibody prepared by the preparation method described in the third aspect of the present invention in the preparation of products for detecting or diagnosing HBV.

[0103] The fifth aspect of the present invention is to provide a product for detecting or diagnosing HBV, which comprises the monoclonal antibody described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the monoclonal antibody prepared by the preparation method described in the third aspect of the present invention.

[0104] Furthermore, the above-mentioned product can be a diagnostic reagent (diagnostic kit), a detection reagent (detection kit), etc., which may also include other reaction reagents. The above-mentioned product can be used for ELISA, flow cytometry, and immunofluorescence methods to detect HBV core protein HBc.

[0105] 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: mixing the product described in the fifth aspect of the present invention with a sample to be tested to detect the core protein HBc of HBV.

[0106] 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.

[0107] Furthermore, detection methods include: ELISA, flow cytometry and immunofluorescence methods.

[0108] It is understandable that in actual applications, cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8 can be used individually, or a combination of multiple antibodies can be used to detect HBV core protein HBc.

[0109] Compared with the prior art, the present invention adopts the above technical solution to achieve the following beneficial effects:

[0110] The monoclonal antibodies against the hepatitis B virus core protein obtained by screening in the present invention have strong binding ability to HBc. The present invention demonstrates the application of monoclonal antibodies in various biochemical methods and their ability to detect different HBV genotypes through examples. Monoclonal antibodies are highly effective, broad-spectrum, and versatile, and have broad application prospects in the diagnosis of HBV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] 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:

[0112] Figure 1 This is a schematic diagram of the ELISA results of the monoclonal antibody binding activity to genotype C HBV core protein in Example 2 of the present invention;

[0113] Figure 2 This is a schematic diagram of the results of using the monoclonal antibody in Example 3 of the present invention to detect HBc expressed in cells by flow cytometry;

[0114] Figure 3A and Figure 3B This is a schematic diagram of the results of using the monoclonal antibody in Example 4 of the present invention to identify HBc in HBV-infected cells using the IFA method;

[0115] Figure 4A 、 Figure 4B and Figure 4C This is a schematic diagram of the results of identifying HBc of different HBV genotypes using the monoclonal antibodies in Example 5 of the present invention. DETAILED DESCRIPTION

[0116] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. It should be understood that the described embodiments are only some of the embodiments of the present invention, and are not intended to be exhaustive. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national standards. Experimental materials in the following examples where the sources are not specified are all commercially available. The equipment used in each step in the following examples is conventional equipment. Where there are no corresponding national standards, the procedures are carried out in accordance with generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise specified, all parts are by weight, and all percentages are by mass. Unless otherwise defined or indicated, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention.

[0117] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0118] Example 1: Screening, expression and purification of antibodies

[0119] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (approval number: 2021-030). All participants provided written informed consent for subsequent sample processing and analysis. The required peripheral blood mononuclear cell (PBMC) samples and liver tissue sections were obtained from patients with chronic HBV infection and stored in liquid nitrogen.

[0120] This example uses two PBMC samples, and the monoclonal antibody screening and purification steps are as follows:

[0121] (1) Monoclonal antibody screening

[0122] 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 viable cells. The number of cells is 1×10 6 Cells were aliquoted into 50 μL of staining buffer (PBS + 2% fetal bovine serum) containing Live / Dead dye (L34964, Invitrogen) and resuspended in peripheral blood mononuclear cells. The cells were stained at 4°C for 30 minutes. After washing twice with PBS, 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) were added to 50 μL of staining buffer and the peripheral blood mononuclear cells were resuspended and stained at 4°C for 30 minutes. After washing twice with PBS, peripheral blood mononuclear cells were resuspended in 50 μL of staining buffer containing genotype C HBc (P2301, Shenzhen Innocell Biotechnology Co., Ltd.) labeled with DyLight 550 (84531, Invitrogen) and DyLight 650 (84536, Invitrogen) 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.

[0123] Single B cells were sorted into 96-well PCR plates containing lysis buffer. RT-PCR and nested PCR were then performed to amplify the variable regions of the heavy and light chains, respectively, according to the methods described in 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.). The PCR amplification products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the resulting antibody heavy and light chain variable region sequences were synthesized by GenScript Biotech Co., Ltd. and constructed into the full-length IgG1 heavy and light chain expression vectors pCDNA3.4 (GenScript Biotech Co., Ltd.), respectively, to ultimately produce large quantities of antibody heavy and light chain plasmids.

[0124] (2) Antibody expression and purification

[0125] The monoclonal antibody in this example was expressed and purified from 293F cells. Taking 400 mL of 293F cells as an example, the specific steps are as follows:

[0126] 1. Prepare expression cells: 400 mL, 1.2 × 10 6 293F cells at 100 μg / mL were cultured in a shaker in an incubator at 8% CO2 and 37°C for 2 hours;

[0127] 2. Prepare transfection plasmid A: Take 200 μg each of antibody heavy chain plasmid and antibody light chain plasmid and dilute them into 10 mL of Opti-MEM (31985070, Gibco);

[0128] 3. Prepare transfection reagent B: Add 2 mL of 1 mg / mL PEI transfection reagent (24885-2, Polysciences) to 10 mL of opti-MEM and let it stand for 5 minutes;

[0129] 4. Prepare transfection mixture AB: add solution B to solution A, mix, and let stand for 20 minutes;

[0130] 5. Transfection: Add 22 mL of AB mixture dropwise to 293F cells, shaking well while adding;

[0131] 6. Collect cell supernatant: After culturing 293F cells for 5-6 days, centrifuge at 3500g for 30 minutes, collect the supernatant, and filter through a 0.45μm filter membrane;

[0132] 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, wash the gravity column with 3 column volumes of PBS solution, and finally elute with 5 volumes of 0.1 M glycine-hydrochloric acid solution (pH = 3.0); place the eluate in a 30KD ultrafiltration concentrator tube and centrifuge at 3500g at 4°C until the liquid in the concentrator tube is less than 1 mL; discard the waste liquid in the collection tube and fill the concentrator tube with PBS solution, centrifuge under the same conditions until the liquid in the concentrator tube is less than 1 mL, and measure the antibody concentration.

[0133] Example 2: Enzyme-linked immunosorbent assay (ELISA) to verify the binding ability of monoclonal antibodies

[0134] From the antibodies obtained in Example 1, 8 antibodies were selected for monoclonal antibody expression and related function verification, and were named cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8, respectively.

[0135] Among them, the amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of cAbB8 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 cAbB8 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, CDR2-L is "WSS", 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 "TGGTCATCT". The amino acid sequence of the heavy chain variable region VH of cAbB8 is shown in SEQ ID NO.11, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.11 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region VL of cAbB8 is shown in SEQ ID NO.12, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.12 is shown in SEQ ID NO.14.

[0136] The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of cAbD8 are shown in SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, respectively, and the nucleic acid sequences encoding SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17 are shown in SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbD8 are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively, CDR2-L is "DAT", the nucleic acid sequences encoding SEQ ID NO.18 and SEQ ID NO.19 are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively, and the nucleic acid sequence encoding CDR2-L is "GATGCAACC". The amino acid sequence of the cAbD8 heavy chain variable region VH is shown in SEQ ID NO.25, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.25 is shown in SEQ ID NO.27; the amino acid sequence of the cAbD8 light chain variable region VL is shown in SEQ ID NO.26, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.26 is shown in SEQ ID NO.28.

[0137] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of cAbE2 are shown in SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31, respectively, and the nucleic acid sequences encoding SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31 are shown in SEQ ID NO.34, SEQ ID NO.35, and SEQ ID NO.36, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbE2 are shown in SEQ ID NO.32 and SEQ ID NO.33, respectively, and CDR2-L is "GAT." The nucleic acid sequences encoding SEQ ID NO.32 and SEQ ID NO.33 are shown in SEQ ID NO.37 and SEQ ID NO.38, respectively, and the nucleic acid sequence encoding CDR2-L is "GGTGCAACC." The amino acid sequence of the heavy chain variable region VH of cAbE2 is shown in SEQ ID NO.39, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.39 is shown in SEQ ID NO.41; the amino acid sequence of the light chain variable region VL of cAbE2 is shown in SEQ ID NO.40, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.40 is shown in SEQ ID NO.42.

[0138] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of cAbE5 are shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, respectively, and the nucleic acid sequences encoding SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45 are shown in SEQ ID NO.48, SEQ ID NO.49, and SEQ ID NO.50, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbE5 are shown in SEQ ID NO.46 and SEQ ID NO.47, respectively, and CDR2-L is "GKN." The nucleic acid sequences encoding SEQ ID NO.46 and SEQ ID NO.47 are shown in SEQ ID NO.51 and SEQ ID NO.52, respectively, and the nucleic acid sequence encoding CDR2-L is "GGTAAAAAC." The amino acid sequence of the heavy chain variable region VH of cAbE5 is shown in SEQ ID NO.53, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.53 is shown in SEQ ID NO.55; the amino acid sequence of the light chain variable region VL of cAbE5 is shown in SEQ ID NO.54, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.54 is shown in SEQ ID NO.56.

[0139] The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of cAbE11 are shown in SEQ ID NOs. 57, 58, and 59, respectively, and the nucleic acid sequences encoding SEQ ID NOs. 57, 58, and 59 are shown in SEQ ID NOs. 62, 63, and 64, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbE11 are shown in SEQ ID NOs. 60 and 61, respectively, and CDR2-L is "DVS." The nucleic acid sequences encoding SEQ ID NOs. 60 and 61 are shown in SEQ ID NOs. 65 and 66, respectively, and the nucleic acid sequence encoding CDR2-L is "GATGTCAGT." The amino acid sequence of the heavy chain variable region VH of cAbE11 is shown in SEQ ID NO.67, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.67 is shown in SEQ ID NO.69; the amino acid sequence of the light chain variable region VL of cAbE11 is shown in SEQ ID NO.68, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.68 is shown in SEQ ID NO.70.

[0140] The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of cAbF5 are shown in SEQ ID NO.71, SEQ ID NO.72 and SEQ ID NO.73, respectively, and the nucleic acid sequences encoding SEQ ID NO.71, SEQ ID NO.72 and SEQ ID NO.73 are shown in SEQ ID NO.76, SEQ ID NO.77 and SEQ ID NO.78, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbF5 are shown in SEQ ID NO.74 and SEQ ID NO.75, respectively, CDR2-L is "KAS", the nucleic acid sequences encoding SEQ ID NO.74 and SEQ ID NO.75 are shown in SEQ ID NO.79 and SEQ ID NO.80, respectively, and the nucleic acid sequence encoding CDR2-L is "AAGGCGTCG". The amino acid sequence of the cAbF5 heavy chain variable region VH is shown in SEQ ID NO.81, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.81 is shown in SEQ ID NO.83; the amino acid sequence of the cAbF5 light chain variable region VL is shown in SEQ ID NO.82, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.82 is shown in SEQ ID NO.84.

[0141] The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of cAbF12 are shown in SEQ ID NO.85, SEQ ID NO.86 and SEQ ID NO.87, respectively, and the nucleic acid sequences encoding SEQ ID NO.85, SEQ ID NO.86 and SEQ ID NO.87 are shown in SEQ ID NO.90, SEQ ID NO.91 and SEQ ID NO.92, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbF12 are shown in SEQ ID NO.88 and SEQ ID NO.89, respectively, CDR2-L is "AAS", the nucleic acid sequences encoding SEQ ID NO.88 and SEQ ID NO.89 are shown in SEQ ID NO.93 and SEQ ID NO.94, respectively, and the nucleic acid sequence encoding CDR2-L is "GCTGCGTCC". The amino acid sequence of the heavy chain variable region VH of cAbF12 is shown in SEQ ID NO.95, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.95 is shown in SEQ ID NO.97; the amino acid sequence of the light chain variable region VL of cAbF12 is shown in SEQ ID NO.96, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.96 is shown in SEQ ID NO.98.

[0142] The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of cAbH8 are shown in SEQ ID NO.99, SEQ ID NO.100 and SEQ ID NO.101, respectively, and the nucleic acid sequences encoding SEQ ID NO.99, SEQ ID NO.100 and SEQ ID NO.101 are shown in SEQ ID NO.104, SEQ ID NO.105 and SEQ ID NO.106, respectively; the amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbH8 are shown in SEQ ID NO.102 and SEQ ID NO.103, respectively, CDR2-L is "GAS", the nucleic acid sequences encoding SEQ ID NO.102 and SEQ ID NO.103 are shown in SEQ ID NO.107 and SEQ ID NO.108, respectively, and the nucleic acid sequence encoding CDR2-L is "GGTGCGTCC". The amino acid sequence of the heavy chain variable region VH of cAbH8 is shown in SEQ ID NO.109, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.109 is shown in SEQ ID NO.111; the amino acid sequence of the light chain variable region VL of cAbH8 is shown in SEQ ID NO.110, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.110 is shown in SEQ ID NO.112.

[0143] The specific binding ability of the monoclonal antibody to the HBV core protein HBc was detected by ELISA. 2μg / mL, 100μL / well of genotype C HBc (P2301, Shenzhen Innocell Biotechnology Co., Ltd.) was coated in a 96-well enzyme-labeled plate and coated overnight at 4°C. A PBS solution of 0.05% Tween-20 (PBST) was prepared and the 96-well plate was washed 5 times. A blocking solution of 5% skim milk + 2% BSA (prepared in PBS) was prepared, 200μL / well, and blocked at room temperature for 1 hour. After washing 3 times, the monoclonal antibody was diluted with blocking solution to a maximum concentration of 10μg / mL and gradually diluted 5-fold for 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 five times with PBST and HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) was diluted 1:5000 in blocking buffer at 100 μL / well. The plate was incubated at 37°C for 1 hour. The plate was washed five times with PBST and a 1:1 mixture of colorimetric solution A and solution B (E661007, Sangon) was added at 100 μL / well. The plate was developed for 20 minutes at room temperature in the dark. The reaction was terminated by adding 50 μL of 2 mol / L H2SO4. The optical density was measured at 450 nm (OD) using a Varioskan LUX multimode microplate reader (Thermo Scientific).

[0144] The binding activity of monoclonal antibodies to HBV core protein HBc was analyzed by ELISA. Figure 1 As shown in the figure, monoclonal antibodies cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8 can specifically bind to HBc of HBV genotype C with strong binding ability, and the half effective concentration EC 50 It reached the nanogram level, 1.52-4.92ng / mL.

[0145] Example 3: Verification of the function of monoclonal antibodies using flow cytometry

[0146] In this example, a monoclonal antibody was applied to flow cytometry to verify its recognition of intracellular HBc. The experimental steps are as follows:

[0147] 293T cells were transfected with HBV genotype C HBc protein expression plasmid and empty vector respectively. After 48 hours, the cells were digested with 0.5% trypsin and terminated by adding complete DMEM medium containing 10% FBS. The cells were gently blown with a pipette and observed under a microscope until more than 90% of the cells were in a single cell state. The single cell suspension was aspirated into a 15mL centrifuge tube and centrifuged at 400g for 5 minutes. The supernatant was discarded and 3mL of complete DMEM was added for culture. Take 20μL of cell suspension, add 20μL of AO / PI dye (RE010212, Countstar), count the number of live cells, and the number of cells was 5×10 5 cells / tube. The cells were fixed and permeabilized using a cell fixation / permeabilization kit (554714, BD Biosciences), centrifuged at 400g for 5 minutes, and the supernatant was discarded. 5μg / mL monoclonal antibody was then added to each flow cytometry tube and incubated at 4°C for 30 minutes; after washing, the cells were stained with AlexaFluor 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). Figure 2 shown.

[0148] The results showed that monoclonal antibodies cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8 could specifically recognize HBc in cells and clearly distinguish between HBc-expressing and non-expressing cells.

[0149] Example 4: Verification of the function of monoclonal antibodies using immunofluorescence assay (IFA)

[0150] In this example, monoclonal antibodies were used in IFA to identify HBc in HBV-infected cells. The experimental steps were as follows:

[0151] This experiment was conducted in a certified biosafety level 2 laboratory. 6 HepG2-NTCP cells were plated in 6-well plates coated with type I rat tail collagen (354236, Corning) and cultured for more than 18 hours. HBV virus (purified from HepAD38 supernatant) with 1800 HBV gene copies / cell was added to DMEM culture medium containing 4% PEG8000 (89510, Sigma), 2.5% DMSO, and 5% FBS, mixed well, and added to HepG2-NTCP cells. After 20 hours of HBV infection, PBS was diluted three times and cultured with DMEM culture 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, the cells were permeabilized with a PBS solution containing 0.2% Triton X-100 for 30 minutes, and QuickBlock was added. TM Immunostaining blocking solution (P0260, Beyotime) was added at room temperature for 1 hour. After washing with PBS twice, 10 μg / mL monoclonal antibodies were added and incubated at 4°C overnight. After washing with PBS five times, Alexa Fluor was added at a ratio of 1:1000. 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, Biyuntian) was added for 10 minutes. After washing with PBS twice, fluorescence microscopy was used to take pictures. The experimental results are shown in Figure 2. Figure 3A and Figure 3B shown.

[0152] The results showed that strong HBc fluorescence intensity was observed under the field of view, indicating that monoclonal antibodies cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8 were able to clearly recognize HBc in the cytoplasm and nucleus of HBV-infected cells.

[0153] Example 5: Verification of the broad-spectrum recognition ability of monoclonal antibodies using flow cytometry

[0154] This example uses flow cytometry to verify that monoclonal antibodies recognize HBc expressed in cells of different HBV genotypes. The specific experimental steps are as follows:

[0155] 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. The cells were gently blown with a pipette and observed under a microscope until more than 90% of the cells were in a single cell state. The single cell suspension was aspirated into a 15mL centrifuge tube and centrifuged at 400g for 5 minutes. The supernatant was discarded and 3mL of complete DMEM was added for culture. Take 20μL of cell suspension, add 20μL of AO / PI dye (RE010212, Countstar), count the number of live cells, and the number of cells was 5×10 5 cells / tube. The cells were fixed and permeabilized using a cell fixation / permeabilization kit (554714, BD Biosciences), centrifuged at 400g for 5 minutes, and the supernatant was discarded. 5μg / mL monoclonal antibody was then added to each flow cytometry tube and incubated at 4°C for 30 minutes; after washing, the cells were stained with AlexaFluor 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). Figure 4A 、 Figure 4B and Figure 4C shown.

[0156] The results showed that monoclonal antibodies cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8 could clearly detect HBc expressed in cells. Among them, cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, and cAbF12 could widely identify HBc derived from different HBV genotypes A, B, C, D, E, F, G, H, I, and J, and cAbH8 could widely identify HBc derived from different HBV genotypes C, D, E, and G.

[0157] As can be seen from the above examples, the monoclonal antibodies cAbB8, cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8 obtained by screening in the present invention all have strong binding ability to the core protein HBc of hepatitis B virus, and can be used in a variety of detection methods to diagnose HBc of hepatitis B virus, showing good broad spectrum and sensitivity.

[0158] 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 against hepatitis B virus core protein, characterized in that: The monoclonal antibody recognizes the core protein HBc of the hepatitis B virus HBV; the monoclonal antibody is cAbB8, the amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the cAbB8 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 cAbB8 are SEQ ID NO.4, WSS and SEQ ID NO.5, respectively.

2. A monoclonal antibody composition against hepatitis B virus core protein, characterized in that: The monoclonal antibody composition is any combination of cAbB8 according to claim 1 and monoclonal antibodies cAbD8, cAbE2, cAbE5, cAbE11, cAbF5, cAbF12, and cAbH8; The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the cAbD8 are SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, respectively; the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the cAbD8 are SEQ ID NO.18, DAT and SEQ ID NO.19, respectively; The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbE2 are SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 31, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbE2 are SEQ ID NO. 32, GAT, and SEQ ID NO. 33, respectively; The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbE5 are SEQ ID NO. 43, SEQ ID NO. 44, and SEQ ID NO. 45, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbE5 are SEQ ID NO. 46, GKN, and SEQ ID NO. 47, respectively; The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of cAbE11 are SEQ ID NO. 57, SEQ ID NO. 58, and SEQ ID NO. 59, respectively. The amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of cAbE11 are SEQ ID NO. 60, DVS, and SEQ ID NO. 61, respectively. The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbF5 are SEQ ID NO.71, SEQ ID NO.72, and SEQ ID NO.73, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbF5 are SEQ ID NO.74, KAS, and SEQ ID NO.75, respectively; The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of the cAbF12 are SEQ ID NO.85, SEQ ID NO.86, and SEQ ID NO.87, respectively; the amino acid sequences of CDR1-L, CDR2-L, and CDR3-L of the light chain variable region VL of the cAbF12 are SEQ ID NO.88, AAS, and SEQ ID NO.89, respectively; The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the cAbH8 are SEQ ID NO.99, SEQ ID NO.100 and SEQ ID NO.101, respectively, and the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the cAbH8 are SEQ ID NO.102, GAS and SEQ ID NO.103, respectively.

3. The monoclonal antibody composition against hepatitis B virus core protein according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region VH of the cAbB8 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 cAbB8 is as shown in SEQ ID NO. 12 or an amino acid sequence having at least 80% identity thereto; The amino acid sequence of the heavy chain variable region VH of the cAbD8 is as shown in SEQ ID NO. 25 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 cAbD8 is as shown in SEQ ID NO. 26 or an amino acid sequence having at least 80% identity thereto; The amino acid sequence of the heavy chain variable region VH of the cAbE2 is as shown in SEQ ID NO. 39 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 cAbE2 is as shown in SEQ ID NO. 40 or an amino acid sequence having at least 80% identity thereto; The amino acid sequence of the heavy chain variable region VH of the cAbE5 is as shown in SEQ ID NO. 53 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 cAbE5 is as shown in SEQ ID NO. 54 or an amino acid sequence having at least 80% identity thereto; The amino acid sequence of the heavy chain variable region VH of the cAbE11 is as shown in SEQ ID NO. 67 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 cAbE11 is as shown in SEQ ID NO. 68 or an amino acid sequence having at least 80% identity thereto; The amino acid sequence of the heavy chain variable region VH of the cAbF5 is as shown in SEQ ID NO.81 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 cAbF5 is as shown in SEQ ID NO.82 or an amino acid sequence having at least 80% identity thereto; The amino acid sequence of the heavy chain variable region VH of the cAbF12 is as shown in SEQ ID NO. 95 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 cAbF12 is as shown in SEQ ID NO. 96 or an amino acid sequence having at least 80% identity thereto; The amino acid sequence of the heavy chain variable region VH of the cAbH8 is shown in SEQ ID NO.109 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 cAbH8 is shown in SEQ ID NO.110 or an amino acid sequence having at least 80% identity thereto.

4. A biomaterial, 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 against hepatitis B virus core protein according to claim 1 or the monoclonal antibody composition against hepatitis B virus core protein according to claim 2; (B) Expression cassettes, recombinant vectors, and recombinant cell lines containing the nucleic acid molecule described in (A).

5. The biomaterial according to claim 4, characterized in that The nucleic acid sequence encoding CDR1-H of cAbB8 shown in SEQ ID NO.1 is shown in SEQ ID NO.6; The nucleic acid sequence encoding CDR2-H of cAbB8 shown in SEQ ID NO. 2 is shown in SEQ ID NO. 7; The nucleic acid sequence encoding CDR3-H of cAbB8 shown in SEQ ID NO. 3 is shown in SEQ ID NO. 8; The nucleic acid sequence encoding CDR1-L of cAbB8 shown in SEQ ID NO.4 is shown in SEQ ID NO.9; The nucleic acid sequence encoding CDR2-L of cAbB8 is TGGTCATCT; The nucleic acid sequence encoding CDR3-L of cAbB8 shown in SEQ ID NO.5 is shown in SEQ ID NO.10; The nucleic acid sequence encoding CDR1-H of cAbD8 shown in SEQ ID NO. 15 is shown in SEQ ID NO. 20; The nucleic acid sequence encoding CDR2-H of cAbD8 shown in SEQ ID NO. 16 is shown in SEQ ID NO. 21; The nucleic acid sequence encoding CDR3-H of cAbD8 shown in SEQ ID NO. 17 is shown in SEQ ID NO. 22; The nucleic acid sequence encoding CDR1-L of cAbD8 shown in SEQ ID NO. 18 is shown in SEQ ID NO. 23; The nucleic acid sequence encoding CDR2-L of cAbD8 is GATGCAACC; The nucleic acid sequence encoding CDR3-L of cAbD8 shown in SEQ ID NO. 19 is shown in SEQ ID NO. 24; The nucleic acid sequence encoding CDR1-H of cAbE2 shown in SEQ ID NO. 29 is shown in SEQ ID NO. 34; The nucleic acid sequence encoding CDR2-H of cAbE2 shown in SEQ ID NO. 30 is shown in SEQ ID NO. 35; The nucleic acid sequence encoding CDR3-H of cAbE2 shown in SEQ ID NO. 31 is shown in SEQ ID NO. 36; The nucleic acid sequence encoding CDR1-L of cAbE2 shown in SEQ ID NO. 32 is shown in SEQ ID NO. 37; The nucleic acid sequence encoding CDR2-L of cAbE2 is GGTGCAACC; The nucleic acid sequence encoding CDR3-L of cAbE2 shown in SEQ ID NO. 33 is shown in SEQ ID NO. 38; The nucleic acid sequence encoding CDR1-H of cAbE5 shown in SEQ ID NO. 43 is shown in SEQ ID NO. 48; The nucleic acid sequence encoding CDR2-H of cAbE5 shown in SEQ ID NO. 44 is shown in SEQ ID NO. 49; The nucleic acid sequence encoding CDR3-H of cAbE5 shown in SEQ ID NO. 45 is shown in SEQ ID NO. 50; The nucleic acid sequence encoding CDR1-L of cAbE5 shown in SEQ ID NO. 46 is shown in SEQ ID NO. 51; The nucleic acid sequence encoding CDR2-L of cAbE5 is GGTAAAAAC; The nucleic acid sequence encoding CDR3-L of cAbE5 shown in SEQ ID NO. 47 is shown in SEQ ID NO. 52; The nucleic acid sequence encoding CDR1-H of cAbE11 shown in SEQ ID NO. 57 is shown in SEQ ID NO. 62; The nucleic acid sequence encoding CDR2-H of cAbE11 shown in SEQ ID NO. 58 is shown in SEQ ID NO. 63; The nucleic acid sequence encoding CDR3-H of cAbE11 shown in SEQ ID NO. 59 is shown in SEQ ID NO. 64; The nucleic acid sequence encoding CDR1-L of cAbE11 shown in SEQ ID NO. 60 is shown in SEQ ID NO. 65; The nucleic acid sequence encoding CDR2-L of cAbE11 is GATGTCAGT; The nucleic acid sequence encoding CDR3-L of cAbE11 shown in SEQ ID NO. 61 is shown in SEQ ID NO. 66; The nucleic acid sequence encoding CDR1-H of cAbF5 shown in SEQ ID NO. 71 is shown in SEQ ID NO. 76; The nucleic acid sequence encoding CDR2-H of cAbF5 shown in SEQ ID NO. 72 is shown in SEQ ID NO. 77; The nucleic acid sequence encoding CDR3-H of cAbF5 shown in SEQ ID NO. 73 is shown in SEQ ID NO. 78; The nucleic acid sequence encoding CDR1-L of cAbF5 shown in SEQ ID NO. 74 is shown in SEQ ID NO. 79; The nucleic acid sequence encoding CDR2-L of cAbF5 is AAGGCGTCG; The nucleic acid sequence encoding CDR3-L of cAbF5 shown in SEQ ID NO. 75 is shown in SEQ ID NO. 80; The nucleic acid sequence encoding CDR1-H of cAbF12 shown in SEQ ID NO. 85 is shown in SEQ ID NO. 90; The nucleic acid sequence encoding CDR2-H of cAbF12 shown in SEQ ID NO. 86 is shown in SEQ ID NO. 91; The nucleic acid sequence encoding CDR3-H of cAbF12 shown in SEQ ID NO. 87 is shown in SEQ ID NO. 92; The nucleic acid sequence encoding CDR1-L of cAbF12 shown in SEQ ID NO. 88 is shown in SEQ ID NO. 93; The nucleic acid sequence encoding CDR2-L of cAbF12 is GCTGCGTCC; The nucleic acid sequence encoding CDR3-L of cAbF12 shown in SEQ ID NO. 89 is shown in SEQ ID NO. 94; The nucleic acid sequence encoding CDR1-H of cAbH8 shown in SEQ ID NO. 99 is shown in SEQ ID NO. 104; The nucleic acid sequence encoding CDR2-H of cAbH8 shown in SEQ ID NO. 100 is shown in SEQ ID NO. 105; The nucleic acid sequence encoding CDR3-H of cAbH8 shown in SEQ ID NO. 101 is shown in SEQ ID NO. 106; The nucleic acid sequence encoding CDR1-L of cAbH8 shown in SEQ ID NO. 102 is shown in SEQ ID NO. 107; The nucleic acid sequence encoding CDR2-L of cAbH8 is GGTGCGTCC; The nucleic acid sequence encoding CDR3-L of cAbH8 shown in SEQ ID NO. 103 is shown in SEQ ID NO.

108.

6. The biomaterial according to claim 4, characterized in that The nucleic acid sequence encoding the VH of cAbB8 shown in SEQ ID NO.11 is shown in SEQ ID NO.13; The nucleic acid sequence encoding the VL of cAbB8 shown in SEQ ID NO. 12 is shown in SEQ ID NO. 14; The nucleic acid sequence encoding the VH of cAbD8 shown in SEQ ID NO. 25 is shown in SEQ ID NO. 27; The nucleic acid sequence encoding the VL of cAbD8 shown in SEQ ID NO. 26 is shown in SEQ ID NO. 28; The nucleic acid sequence encoding the VH of cAbE2 shown in SEQ ID NO. 39 is shown in SEQ ID NO. 41; The nucleic acid sequence encoding the VL of cAbE2 shown in SEQ ID NO.40 is shown in SEQ ID NO.42; The nucleic acid sequence encoding the VH of cAbE5 shown in SEQ ID NO. 53 is shown in SEQ ID NO. 55; The nucleic acid sequence encoding the VL of cAbE5 shown in SEQ ID NO. 54 is shown in SEQ ID NO. 56; The nucleic acid sequence encoding VH of cAbE11 shown in SEQ ID NO. 67 is shown in SEQ ID NO. 69; The nucleic acid sequence encoding the VL of cAbE11 shown in SEQ ID NO. 68 is shown in SEQ ID NO. 70; The nucleic acid sequence encoding the VH of cAbF5 shown in SEQ ID NO.81 is shown in SEQ ID NO.83; The nucleic acid sequence encoding the VL of cAbF5 shown in SEQ ID NO.82 is shown in SEQ ID NO.84; The nucleic acid sequence encoding VH of cAbF12 shown in SEQ ID NO. 95 is shown in SEQ ID NO. 97; The nucleic acid sequence encoding the VL of cAbF12 shown in SEQ ID NO. 96 is shown in SEQ ID NO. 98; The nucleic acid sequence encoding the VH of cAbH8 shown in SEQ ID NO. 109 is shown in SEQ ID NO. 111; The nucleic acid sequence encoding VL of cAbH8 shown in SEQ ID NO.110 is shown in SEQ ID NO.

112.

7. The method for preparing a monoclonal antibody against hepatitis B virus core protein according to claim 1, characterized in that: include: transfecting a host cell with a recombinant vector containing a nucleic acid molecule encoding the monoclonal antibody; The transfected host cells are cultured and the supernatant is collected and purified to obtain the monoclonal antibody.

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

9. Use of the monoclonal antibody against hepatitis B virus core protein according to claim 1, or the monoclonal antibody composition against hepatitis B virus core protein according to any one of claims 2 to 3, or the biomaterial according to any one of claims 4 to 6 in the preparation of a product for detecting HBV.

10. A product for detecting HBV, characterized in that: The product comprises the monoclonal antibody against the hepatitis B virus core protein according to claim 1, or the monoclonal antibody composition against the hepatitis B virus core protein according to any one of claims 2 to 3, or the biomaterial according to any one of claims 4 to 6.

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

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