Protective monoclonal antibody targeting mycobacterium tuberculosis MPT83 as well as preparation method and application of protective monoclonal antibody

Abstract

The invention relates to the technical field of biology, in particular to a protective monoclonal antibody targeting mycobacterium tuberculosis MPT83 as well as a preparation method and application of the protective monoclonal antibody. The antibody or an antigen binding fragment comprises a CDR sequence selected from at least one of the following sequences or a sequence with one amino acid substituted, deleted or increased: a heavy chain variable region CDR sequence: SEQ ID NO: 5-7; cDR sequences of a light chain variable region are as follows: SEQ ID NO: 9-10 and LAS. The monoclonal antibody 9C9 can be specifically combined with MPT83 protein on the surface of Mycobacterium tuberculosis (MTB) and shows efficient antituberculous activity in vivo and in vitro, the activity mainly comprises the aspects of remarkably enhancing the phagocytosis of macrophages on tubercle bacillus, inhibiting tubercle bacillus growth and the like, and in addition, the monoclonal antibody 9C9 is clear in sequence and structure, easy to develop and modify and capable of being used for preparing the anti-tubercle bacillus monoclonal antibody 9C9. The monoclonal antibody 9C9 disclosed by the invention has the advantages that the monoclonal antibody 9C9 can be used for preventing and treating tuberculosis, an immune-mediated treatment mechanism of the monoclonal antibody 9C9 does not easily induce pathogens to generate drug resistance, a new choice is provided for the treatment of tuberculosis when the monoclonal antibody 9C9 is applied to prevention and treatment of tuberculosis, an important technical basis is also provided for response of drug-resistant strains, development of diagnostic reagents, vaccine development and the like, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a protective monoclonal antibody targeting MPT83 of Mycobacterium tuberculosis and a preparation method and application thereof. BACKGROUND

[0002] Tuberculosis (TB) is a serious zoonotic infectious disease that seriously endangers human health caused by Mycobacterium tuberculosis (MTB) through aerosols such as droplets. At present, vaccination is the most economical and effective method for preventing tuberculosis, and the only licensed BCG vaccine can effectively prevent childhood disseminated tuberculosis, while the treatment of MTB mainly relies on antibiotic therapy. Although some progress has been made in the prevention and control of tuberculosis, there are still many problems and challenges, for example, the protective effect of BCG vaccine is limited, especially in adults and immunocompromised populations, and its effect in preventing tuberculosis is not ideal; secondly, with the widespread use of antibiotics in the field of anti-tuberculosis, drug-resistant strains are still increasing, which significantly increases the difficulty and complexity of traditional drug therapy, prolongs the treatment cycle, and easily leads to treatment failure or recurrence.

[0003] With the in-depth study of the immune mechanism of tuberculosis, the specificity and non-resistance of antibodies make them a good choice for the prevention and treatment of tuberculosis. Related studies have shown that the surface protein MPT83 of MTB, as an important secretory protein, has been reported to be able to induce a strong immune response in the host in the early stage of TB infection, and its specific antibodies can be used as a potential marker for the diagnosis of tuberculosis, and can inhibit the growth and spread of Mycobacterium tuberculosis by neutralizing pathogens or enhancing the phagocytosis of immune cells.

[0004] Therefore, it is of great significance to develop a protective monoclonal antibody that can precisely target Mycobacterium tuberculosis MPT83 for the prevention and control of tuberculosis. SUMMARY

[0005] The present application aims to at least partly solve at least one of the technical problems existing in the prior art. To this end, the present application provides a protective monoclonal antibody targeting MPT83 of Mycobacterium tuberculosis and a preparation method and application thereof. The monoclonal antibody 9C9 of the present application can specifically bind to the MPT83 protein on the surface of Mycobacterium tuberculosis (MTB) and exhibits high anti-tuberculosis activity in vivo and in vitro, mainly including significantly enhancing the phagocytosis of macrophages to Mycobacterium tuberculosis, inhibiting the growth of Mycobacterium tuberculosis, etc. In addition, the sequence and structure of the monoclonal antibody 9C9 of the present application are clear and easy to develop and modify, and the immune-mediated treatment mechanism thereof is not easy to induce drug resistance of pathogens. When the monoclonal antibody 9C9 of the present application is applied to the prevention and treatment of tuberculosis, it not only provides a new choice for the treatment of tuberculosis, but also provides an important technical basis for the response to drug-resistant strains, the development of diagnostic reagents and the development of vaccines, etc., and has a wide application prospect.

[0006] In a first aspect of the present application, an antibody or antigen binding fragment is provided. According to embodiments of the present application, the antibody or antigen binding fragment comprises CDR sequences selected from at least one of the following: heavy chain variable region CDR sequences: SEQ ID NO: 5-7; light chain variable region CDR sequences: SEQ ID NO: 9-10 and LAS, or sequences having one amino acid substitution, deletion or addition.

[0007] According to embodiments of the present application, the antibody or antigen binding fragment described above can further have the following additional technical features:

[0008] According to embodiments of the present application, the antibody or antigen binding fragment comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 5, 6 and 7, respectively, or sequences having one amino acid substitution, deletion or addition; and / or, light chain variable region CDRL1, CDRL2, CDRL3 sequences as shown in SEQ ID NO: 9, LAS and SEQ ID NO: 10, respectively, or sequences having one amino acid substitution, deletion or addition.

[0009] According to embodiments of the present application, the antibody or antigen binding fragment comprises: a heavy chain variable region CDR1 sequence as shown in SEQ ID NO: 5, a heavy chain variable region CDR2 sequence as shown in SEQ ID NO: 6, a heavy chain variable region CDR3 sequence as shown in SEQ ID NO: 7, a light chain variable region CDR1 sequence as shown in SEQ ID NO: 9, a light chain variable region CDR2 sequence as shown in LAS, and a light chain variable region CDR3 sequence as shown in SEQ ID NO: 10.

[0010] According to embodiments of the present application, the antibody or antigen binding fragment comprises: a heavy chain variable region sequence as set forth in SEQ ID NO: 4; and / or, a light chain variable region sequence as set forth in SEQ ID NO: 8.

[0011] According to embodiments of the present application, the constant region of the antibody or antigen binding fragment is derived from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0012] According to embodiments of the present application, the constant region of the antibody or antigen binding fragment is derived from IgG1. In a second aspect, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the antibody or antigen binding fragment of the first aspect.

[0013] According to embodiments of the present application, the nucleic acid molecule can further have the following additional technical features:

[0014] According to embodiments of the present application, the nucleic acid molecule comprises: a heavy chain variable region encoding nucleic acid sequence as set forth in SEQ ID NO: 11; and / or, a light chain variable region encoding nucleic acid sequence as set forth in SEQ ID NO: 12.

[0015] In a third aspect, the present application provides an expression vector. According to embodiments of the present application, the expression vector comprises the nucleic acid molecule of the second aspect.

[0016] In a fourth aspect, the present application provides a recombinant virus. According to embodiments of the present application, the recombinant virus comprises the expression vector of the third aspect, and the recombinant virus is capable of infecting immune cells.

[0017] In a fifth aspect, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell carries the nucleic acid molecule of the second aspect or the expression vector of the third aspect, or is capable of expressing the antibody or antigen binding fragment of the first aspect.

[0018] In a sixth aspect, the present application provides a composition. According to embodiments of the present application, the composition contains one or more of: the antibody or antigen binding fragment of the first aspect; the nucleic acid molecule of the second aspect; the expression vector of the third aspect; the recombinant cell of the fifth aspect.

[0019] In a seventh aspect, the present application provides a medicament. According to embodiments of the present application, the medicament contains one or more of: the antibody or antigen binding fragment of the first aspect; the nucleic acid molecule of the second aspect; the expression vector of the third aspect; the recombinant cell of the fifth aspect; the composition of the sixth aspect.

[0020] In an eighth aspect, the present application provides a kit. According to embodiments of the present application, the kit contains one or more of the following: the antibody or antigen binding fragment of the first aspect; the nucleic acid molecule of the second aspect; the expression vector of the third aspect; the recombinant virus of the fourth aspect; the recombinant cell of the fifth aspect.

[0021] In a ninth aspect, the present application provides use of the antibody or antigen binding fragment of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect in the manufacture of a medicament for preventing, alleviating, adjuvant treating or treating a disease caused by Mycobacterium tuberculosis infection.

[0022] According to embodiments of the present application, the above-mentioned use can further have the following additional technical features:

[0023] According to embodiments of the present application, the Mycobacterium tuberculosis includes one or more of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium caprae.

[0024] According to embodiments of the present application, the disease caused by Mycobacterium tuberculosis infection includes one or more of pulmonary tuberculosis, extrapulmonary tuberculosis, drug-resistant tuberculosis and latent tuberculosis infection.

[0025] Additional aspects and advantages of the present application will be given, in part, in the following description, become apparent from the following description, or be understood through practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0026] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0027] Figure 1 Preparation flow chart of monoclonal antibody 9C9 targeting Mycobacterium tuberculosis surface protein MPT83 in Example 1 of the present application, wherein A is the preparation flow chart of MPT83 antigen protein, and B is the preparation flow chart of MPT83 antigen protein immunization and monoclonal antibody 9C9;

[0028] Figure 2Figure for preparation and identification of purified MPT83 protein in embodiment 1 of the present application, wherein A is a detection result map of amplified mpt83 gene fragment, B is a result map of PCR identification of mpt83 gene fragment, C is a detection result map of MPT83 protein induced expression, D is a detection result map of MPT83 protein Ni + column purification detection result map, E is a detection result map of MPT83 purified protein SDS-PAGE electrophoresis, and F is a detection result map of MPT83 purified protein Western Blot identification;

[0029] Figure 3 Figure for related detection results of monoclonal antibody 9C9 in embodiment 1 of the present application, wherein A is a detection result map of serum titer of MPT83 antigen protein immunized mice, B is a detection result map of macrophage phagocytosis experiment, C is a detection result map of purification effect of monoclonal antibody 9C9, and D is a detection result map of subtype identification of monoclonal antibody 9C9;

[0030] Figure 4 Figure for anti-tuberculosis function characterization experiment results of monoclonal antibody 9C9 in embodiment 2 of the present application, wherein A is a detection result map of titer of monoclonal antibody 9C9 recognizing whole bacteria, B is a detection result map of macrophage phagocytosis experiment of monoclonal antibody 9C9, C is an experimental result map of monoclonal antibody 9C9 inhibiting BCG growth, and D is an experimental result map of monoclonal antibody 9C9 inhibiting M.bovis growth;

[0031] Figure 5 Figure for determination results of in vivo intervention experiment of monoclonal antibody 9C9 on M.bovis infected body in embodiment 2 of the present application, wherein A is a flow chart of in vivo intervention experiment of monoclonal antibody 9C9, B is a detection result map of lung organ index of each group of mice, C is a detection result map of organ index of spleen of each group of mice, and D is a detection result map of bacterial load in lung of each group of mice;

[0032] Figure 6 Figure for structure prediction of variable region of monoclonal antibody 9C9 and MPT83 protein and prediction results of epitope recognized by monoclonal antibody 9C9 in embodiment 3 of the present application, wherein A is a structure prediction result map of variable region of monoclonal antibody 9C9, B is a structure prediction result map of MPT83 antigen protein, C is a combination position (red square represents structure of monoclonal antibody 9C9) of top 4 antigen-antibody (MPT83 antigen protein-monoclonal antibody 9C9) combinations, and D is a situation map of amino acid of antigen-antibody (MPT83 antigen protein-monoclonal antibody 9C9) combination interface. DETAILED DESCRIPTION

[0033] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only, and are not to be understood as limiting the present application.

[0034] It should be noted that the terms "first", "second" are used only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0035] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be approximations which can vary depending on the intended application. For numerical ranges, the endpoints are provided as a separate value from but the endpoints are included in the described range or the individual points are included in the described range or the individual points are included in the described range.

[0036] In this document, the terms "comprising" or "including" are open-ended, that is, the inclusion of the indicated elements does not preclude the addition of further elements.

[0037] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0038] Antibody or antigen binding fragment

[0039] The present application provides an antibody or antigen binding fragment. According to an embodiment of the present application, the antibody or antigen binding fragment comprises CDR sequences selected from at least one of the following: heavy chain variable region CDR sequences: SEQ ID NO: 5-7; light chain variable region CDR sequences: SEQ ID NO: 9-10 and LAS, or sequences with one amino acid substitution, deletion or addition. The antibody or antigen binding fragment according to the embodiment of the present application, by comprising specific CDR sequences (SEQ ID NO: 5-7 and 9-10 and LAS) or variants thereof (sequences with one amino acid substitution, deletion or addition), realizes precise recognition and specific binding to the MPT83 protein on the surface of Mycobacterium tuberculosis (MTB), and such specific binding endows the antibody or antigen binding fragment of the present application with high-efficiency anti-tuberculosis activity and low drug resistance in vitro and in vivo, providing a basis for subsequent drug development, diagnostic reagent development, etc.

[0040] In the present text, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen, which includes two light chains with relatively light molecular weight and two heavy chains with relatively heavy molecular weight, the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form a four-chain peptide chain molecule; wherein the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable and varies little, which is called the constant region (C region); the V regions of L chain and H chain are called VL and VH respectively, and there are three CDR regions on the heavy chain variable region and the light chain variable region.

[0041] In the present text, the term "chimeric antigen receptor (CAR)" is a molecule that combines the specificity of an antibody-based antigen (such as a tumor antigen) with a T cell receptor-activated intracellular domain to produce a chimeric protein that exhibits specific anti-tumor cell immune activity; in the present application, the antibody or antigen binding fragment of the present application specifically recognizes the MPT83 protein on the surface of Mycobacterium tuberculosis (MTB).

[0042] It should be noted that the sequence information of the heavy chain variable region sequence, the light chain variable region sequence, etc. of the embodiments of the present application is shown from the N-terminal to the C-terminal.

[0043] According to an embodiment of the present application, the antibody or antigen binding fragment comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 5, 6 and 7 respectively, or sequences with one amino acid substitution, deletion or addition; and / or, light chain variable region CDRL1, CDRL2, CDRL3 sequences as shown in SEQ ID NO: 9, LAS and SEQ ID NO: 10 respectively, or sequences with one amino acid substitution, deletion or addition.

[0044] According to an embodiment of the present application, the antibody or antigen binding fragment comprises: a heavy chain variable region CDR1 sequence as shown in SEQ ID NO: 5, a heavy chain variable region CDR2 sequence as shown in SEQ ID NO: 6, a heavy chain variable region CDR3 sequence as shown in SEQ ID NO: 7, a light chain variable region CDR1 sequence as shown in SEQ ID NO: 9, a light chain variable region CDR2 sequence as shown in LAS, and a light chain variable region CDR3 sequence as shown in SEQ ID NO: 10.

[0045] According to an embodiment of the present application, the antibody or antigen binding fragment comprises: a heavy chain variable region sequence as shown in SEQ ID NO: 4; and / or, a light chain variable region sequence as shown in SEQ ID NO: 8.

[0046] According to an embodiment of the present application, the constant region of the antibody or antigen binding fragment is derived from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0047] According to an embodiment of the present application, the constant region of the antibody or antigen binding fragment is derived from IgG1.

[0048] Nucleic acid molecule

[0049] The present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the aforementioned antibody or antigen binding fragment. According to an embodiment of the present application, the nucleic acid molecule makes it possible to mass-produce the antibody or antigen binding fragment of the present application by genetic engineering technology, and lays a foundation for subsequent construction of expression vectors, recombinant viruses and recombinant cells, and further realizes efficient expression and application of the antibody or antigen binding fragment of the present application.

[0050] According to an embodiment of the present application, the nucleic acid molecule comprises: a heavy chain variable region encoding nucleic acid sequence as shown in SEQ ID NO: 11; and / or, a light chain variable region encoding nucleic acid sequence as shown in SEQ ID NO: 12.

[0051] It should be noted that for the nucleic acid molecule mentioned herein, those skilled in the art should understand that any one or both of the complementary double strands are actually included; although only one strand is given in most cases, the other complementary strand is also disclosed; in addition, the nucleic acid molecule sequence in the present application includes DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0052] Expression vector

[0053] The present application provides an expression vector. According to an embodiment of the present application, the expression vector comprises the aforementioned nucleic acid molecule. According to the expression vector of an embodiment of the present application, the nucleic acid molecule can be efficiently expressed in a host cell, and provides a key tool for subsequent construction of recombinant viruses and recombinant cells.

[0054] It should be noted that the expression vector mentioned herein refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule to the host cell and / or between host cells; the expression vector can include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for transcription and / or translation of expression of DNA or RNA; the expression vector can be a polynucleotide capable of being transcribed and translated when introduced into a suitable host cell; the aforementioned nucleic acid molecule is produced by culturing a suitable host cell comprising the vector, and the expression vector includes viral vectors, plasmids, bacteriophages and the like.

[0055] Recombinant virus

[0056] The present application provides a recombinant virus. According to an embodiment of the present application, the recombinant virus comprises the aforementioned expression vector, and the recombinant virus is capable of infecting immune cells. According to the recombinant virus of the embodiment of the present application, the gene of the antibody or antigen-binding fragment can be efficiently introduced into the target cells by using the infection ability of the recombinant virus, and then applied to in vivo gene therapy or vaccine development and the like.

[0057] Recombinant cell

[0058] The present application provides a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the aforementioned nucleic acid molecule or the aforementioned expression vector or is capable of expressing the aforementioned antibody or antigen-binding fragment. According to the recombinant cell of the embodiment of the present application, the recombinant cell expressing the antibody or antigen-binding fragment of the present application can be introduced into the body by cell transplantation and the like, and then play an anti-tuberculosis role, providing more choices for the treatment of tuberculosis.

[0059] Composition

[0060] The present application provides a composition. According to an embodiment of the present application, the composition contains one or more of the following: the aforementioned antibody or antigen-binding fragment; the aforementioned nucleic acid molecule; the aforementioned expression vector; and the aforementioned recombinant cell. According to the composition of the embodiment of the present application, it can be applied to various application scenarios, including drug development, diagnostic reagent development and vaccine development and the like; in addition, the composition can be designed according to the specific needs to improve its effect and adaptability in different applications, and provide diversified technical means for the prevention and control of tuberculosis.

[0061] Medicine

[0062] The present application provides a medicine. According to an embodiment of the present application, the medicine contains one or more of the following: the aforementioned antibody or antigen-binding fragment; the aforementioned nucleic acid molecule; the aforementioned expression vector; the aforementioned recombinant cell; and the aforementioned composition. According to the medicine of the embodiment of the present application, it can be used for preventing, relieving, assisting treatment or treating diseases caused by Mycobacterium tuberculosis infection, providing a new choice for the treatment of tuberculosis; further, compared with traditional medicines, the medicine based on the antibody or antigen-binding fragment of the present application has higher specificity and lower drug resistance risk, which can effectively cope with the challenge of drug-resistant strains, and then provide a new choice for the prevention and control of tuberculosis.

[0063] Kit

[0064] The present application provides a kit. According to an embodiment of the present application, the kit contains one or more of the following: the aforementioned antibody or antigen-binding fragment; the aforementioned nucleic acid molecule; the aforementioned expression vector; the aforementioned recombinant virus; and the aforementioned recombinant cell. The kit according to the embodiment of the present application can be used for diagnosing, detecting or researching the infection of Mycobacterium tuberculosis, and exemplarily, early diagnosis of tuberculosis can be achieved by detecting the binding ability of the antibody or antigen-binding fragment to the MPT83 protein, thereby providing important technical support for the prevention and control of tuberculosis.

[0065] Use

[0066] The present application provides the use of the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell or the aforementioned composition in the preparation of a medicament, characterized in that the medicament is used for preventing, alleviating, adjuvant therapy or treating diseases caused by Mycobacterium tuberculosis infection.

[0067] According to an embodiment of the present application, the Mycobacterium tuberculosis includes one or more of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium caprae.

[0068] According to an embodiment of the present application, the diseases caused by Mycobacterium tuberculosis infection include one or more of pulmonary tuberculosis, extrapulmonary tuberculosis, drug-resistant tuberculosis and latent tuberculosis infection.

[0069] It should be noted that the antibody or antigen-binding fragment, nucleic acid molecule, expression vector, recombinant cell or composition described in the present application are not limited to the above-mentioned specific Mycobacterium tuberculosis species or disease types, and also have potential application value in preventing, alleviating, adjuvant therapy or treating other diseases or infections caused by Mycobacterium tuberculosis, and these applications shall be within the protection scope of the present application.

[0070] It can be understood by those skilled in the art that the features and advantages described above for the nucleic acid molecule, expression vector, recombinant cell or the aforementioned composition also apply to the use, which will not be repeated here.

[0071] The amino acid sequence involved in the present application is shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075]

[0076] The schemes of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be commercially available.

[0077] Example 1: Preparation of monoclonal antibody 9C9

[0078] 1. Preparation of MPT83 protein

[0079] (1) Primer design

[0080] The upstream primer MPT83-F and the downstream primer MPT83-R were designed according to the mpt83 gene sequence of Mycobacterium tuberculosis BCG (Gene ID: 4699266) in GenBank:

[0081] MPT83-F:

[0082] 5'-ATGGGTCGCGGATCCGAATTCATGATCAACGTTCAGGCCAAA-3' (SEQ ID NO: 1)

[0083] MPT83-R:

[0084] 5'-CTCGAGTGCGGCCGCAAGCTTCTGTGCCGGGGGCATCAG-3' (SEQ ID NO: 2)

[0085] (2) Gene amplification and construction of recombinant plasmid

[0086] First, the mpt83 gene fragment was amplified by PCR, then the vector pET28a(+) was double digested with EcoRI and HindIII, then the recombinant plasmid was constructed by homologous recombination method, and then the recombinant plasmid with correct sequence was transformed into BL21 competent cells, and IPTG was used for induction of expression, then the obtained MPT83 protein was purified by Ni2+ column affinity chromatography, the purified MPT83 protein was obtained, and the obtained purified MPT83 protein was identified by SDS-PAGE and Western Blot.

[0087] The amino acid sequence of the MPT83 protein is shown in SEQ ID NO: 3.

[0088] MINVQAKPAAAASLAAIAIAFLAGCSSTKPVSQDTSPKPATSPAAPVTTAAMADPAADLIGRGCAQYAAQNPTGPGSVAGMAQDPVATAASNNPMLSTLTSALSGKLNPDVNLVDTLNGGEYTVFAPTNAAFDKLPAATIDQLKTDAKLLSSILTYHVIAGQASPSRIDGTHQTLQGADLTVIGARDDLMVNNAGLVCGGVHTANATVYMIDTVLMPPAQ (SEQ ID NO: 3)

[0089] Preparation of monoclonal antibody 9C9 targeting Mycobacterium tuberculosis surface protein MPT83 is shown in Figure 1 Preparation of purified MPT83 protein and identification results are shown in Figure 2 .

[0090] The results show that after IPTG induction, MPT83 protein is expressed in large amounts. In addition, the results of SDS-PAGE and Western Blot identification show that the successfully prepared protein is purified MPT83 protein.

[0091] 2. Preparation of monoclonal antibody 9C9

[0092] (1) Pretreatment of mice in each group

[0093] After 4 female BALB / c mice of 7 weeks old were adaptively cultured for one week, they were numbered as BALB / c-1 ~ BALB / c-3, NC. The treatment method of mice numbered as BALB / c-1 ~ BALB / c-3 is as follows:

[0094] After 100 μg (1 mg / mL) of purified MPT83 protein prepared in step 1 was uniformly mixed with aluminum adjuvant (purchased from Thermo fisher company, item number 77161) (v / v = 3:1), MPT83 protein injection was obtained, and MPT83 protein injection (133 μL per mouse) was injected subcutaneously through the neck of the BALB / c mouse. Immunization injection was performed once every two weeks, and a total of 3 times, to obtain immunization treated mice (BALB / c-1 ~ BALB / c-3).

[0095] The treatment method of mice numbered as NC is as follows:

[0096] It is not treated, and the control group mice (NC) are obtained.

[0097] (2) Antibody titer detection

[0098] The immune-treated mice and control mice were subjected to intravenous plexus blood collection, and the serum was collected after centrifugation at 3000 rpm for 10 min at room temperature. The serum titer of each mouse was determined by ELISA.

[0099] (3) Macrophage phagocytosis experiment

[0100] After inactivating the complement of the serum of the immune-treated mice obtained in step (1), the serum was added to the system of BCG-infected macrophage J774a.1 (MOI = 10:1), and the serum of the control mice obtained in step (1) was used as a negative control. The phagocytosis of BCG by macrophage J774a.1 was analyzed by flow cytometry.

[0101] (4) Monoclonal antibody 9C9 cell strain

[0102] The immune-treated mouse (BALB / c-3) with the highest antibody titer was selected, and 100 μg (1 mg / ml) of the purified MPT83 protein obtained in step 1 was injected intraperitoneally. Three days later, the spleen of the mouse was removed, and the myeloma cell line SP2 / 0 cells and the spleen cells were fused using PEG1450. Then, the specific antibody cell line against the MPT83 antigen was screened using HAT / HT medium (purchased from Sigma-Aldrich, H0262 and H0137), and the screened cell line was cloned to obtain the monoclonal antibody 9C9 cell strain. Thus, the antibody capable of specifically recognizing the MPT83 protein was obtained and named monoclonal antibody 9C9.

[0103] (5) Purification of monoclonal antibody 9C9

[0104] Eight-week-old BALB / c mice were selected, and each mouse was sensitized by intraperitoneal injection of 500 μL of liquid paraffin. Fourteen days later, each mouse was intraperitoneally injected with 500 μL of 1 × 10 5 The monoclonal antibody 9C9 cell suspension (from the monoclonal antibody 9C9 cell strain) was diluted 1:1 with the balanced buffer (Na2HPO4 2.8338 g, NaCl 8.766 g, ddH2O to 1000 mL), and the antibody was purified by protein G resin affinity chromatography after filtration with a 0.45 μm filter. Then, the antibody was eluted with 15 mL of elution buffer (7.507 g Glycine, ddH2O to 1000 mL, pH = 2.5), and 1 / 10 volume of neutralization buffer (121.14 g Tris, ddH2O to 1000 mL, pH = 8.5) was quickly added. After mixing, the purified antibody was identified for purity by SDS-PAGE electrophoresis, filtered with a 0.22 μm filter to remove bacteria, and aliquoted to obtain high-purity monoclonal antibody 9C9, which was stored at -80°C for later use.

[0105] (6) Identification of monoclonal antibody 9C9 subtypes

[0106] The specific subtype of the high-purity monoclonal antibody 9C9 obtained in step 2-(5) was determined using an antibody subtype identification kit (purchased from Frdbio).

[0107] The relevant detection results for monoclonal antibody 9C9 are shown below. Figure 3 .

[0108] The results showed that the serum antibody titer was highest in immunized mice (BALB / c-3), reaching 1.024 × 10⁻⁶. 6 (See details) Figure 3 In addition, serum from immunized mice significantly promoted macrophage phagocytosis of BCG (see A for details). Figure 3 (B) ; Testing revealed that the high-purity monoclonal antibody 9C9 obtained was IgG1 (see details...). Figure 3 D)

[0109] Example 2: Characterization of the anti-tuberculosis function of monoclonal antibody 9C9

[0110] 1. Detection of the recognition ability of monoclonal antibody 9C9 against Mycobacterium tuberculosis

[0111] To determine whether monoclonal antibody 9C9 has a recognition effect on MPT83 protein, Mycobacterium bovis (M. bovis), and Bacille Calmette-Guérin (BCG), a bacterial ELISA method was used for the assay. The specific steps are as follows:

[0112] (1) Detection of the ability of monoclonal antibody 9C9 to recognize MPT83 protein

[0113] The purified MPT83 protein was diluted to 1 μg / mL with carbonate-buffered saline (PBS) and coated into 96-well ELISA plates (100 μL / well). The high-purity monoclonal antibody 9C9 prepared in Example 1 was then used as the primary antibody, serially diluted to an initial concentration of 500 μg / mL, and incubated with bacteria at 37°C for 1 h at 100 μL / well. Then, 100 μL of horseradish peroxidase (HRP)-labeled secondary antibody (1:10000) was added and incubated at 37°C for 1 h. After 3,3',5,5'-tetramethylbenzidine (TMB) color development, the OD was measured. 450 nm Absorbance value.

[0114] (2) Detection of the ability of monoclonal antibody 9C9 to recognize M. bovis and BCG

[0115] M.bovis and BCG bacterial solution (with / without Tween-80) were inactivated at 80℃ for 20 min to obtain BCG-T80 bacterial solution (experimental group I, BCG-T80), M.bovis-T80 bacterial solution (experimental group II, M.bovis-T80), M.bovis bacterial solution (experimental group III, M.bovis) and BCG bacterial solution (experimental group IV, BCG), and the inactivated bacterial solution was coated in 96-well ELISA plates at 1×10 7 CFU / well, dried in an oven at 60℃, and then incubated with the high-purity monoclonal antibody 9C9 prepared in Example 1 as the primary antibody at an initial concentration of 500 μg / mL for 1 h at 37℃, followed by the addition of 100 μL of horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000) for 1 h at 37℃, and then the OD value was measured after color development with 3,3',5,5'-tetramethylbenzidine (TMB). 450 nm Absorbance value.

[0116] 2. Macrophage phagocytosis experiment

[0117] The BCG strain was labeled with fluorescein isothiocyanate (FITC) to obtain a FITC-labeled BCG strain, and then the FITC-labeled BCG strain was incubated with PBS (blank control group, PBS), the high-purity monoclonal antibody 9C9 prepared in Example 1 at concentrations of 1 μg / mL (experimental group I, 9C9-1 μg / mL), 5 μg / mL (experimental group II, 9C9-5 μg / mL), 50 μg / mL (experimental group III, 9C9-50 μg / mL) and 250 μg / mL (experimental group IV, 9C9-250 μg / mL) at 37℃ for 1 h, and then the incubated FITC-labeled BCG strain was co-incubated with the macrophage cell line J774a.1 for 3 h (multiplicity of infection (MOI) = 10), and then the fluorescence intensity of each group of cells was detected by flow cytometry, and the antibody phagocytosis index was calculated.

[0118] 3. Mycobacterium tuberculosis growth inhibition experiment

[0119] (1) Monoclonal antibody 9C9 inhibits M.bovis growth experiment

[0120] In the peripheral blood (PBMC) of BALB / c mice, M.bovis bacterial solution (MOI = 10:1 of M.bovis bacterial solution and cell infection) was incubated with PBS (blank control group, PBS), mGO53 antibody with a concentration of 50 μg / mL (negative control group, mGO53), high-purity monoclonal antibody 9C9 prepared in Example 1 with concentrations of 5 μg / mL (experimental group I, 9C9-5 μg / mL), 50 μg / mL (experimental group II, 9C9-50 μg / mL), and 250 μg / mL (experimental group III, 9C9-250 μg / mL) respectively at 37℃ for 4 days, and then the number of surviving M.bovis strains was counted.

[0121] (2) Monoclonal antibody 9C9 inhibits BCG growth experiment

[0122] In the peripheral blood (PBMC) of BALB / c mice, BCG bacterial solution (MOI = 10:1 of BCG bacterial solution and cell infection) was incubated with PBS (blank control group, PBS), mGO53 antibody with a concentration of 50 μg / mL (negative control group, mGO53), high-purity monoclonal antibody 9C9 prepared in Example 1 with concentrations of 5 μg / mL (experimental group I, 9C9-5 μg / mL), 50 μg / mL (experimental group II, 9C9-50 μg / mL), and 250 μg / mL (experimental group III, 9C9-250 μg / mL) respectively at 37℃ for 4 days, and then the number of surviving BCG strains was counted.

[0123] 4. Monoclonal antibody 9C9 against tuberculosis infection verification

[0124] Ten C57BL / 6J mice (purchased from Beijing Sbiopharm Co., Ltd.) were randomly divided into a blank control group (PBS) and an experimental group (9C9). After one week of adaptive culture, the mice in the experimental group were injected intraperitoneally with 500 μg of high-purity monoclonal antibody 9C9 prepared in Example 1, and the mice in the blank control group were injected intraperitoneally with an equal amount of PBS. Five hours later, the mice in both groups were infected with 500 CFU of M.bovis by nasal instillation. Fourteen days later, the mice in both groups were sacrificed, and the lungs and spleens of the mice in both groups were taken for determination of lung organ index and spleen organ index. The lungs of the mice were ground and plated for counting of lung bacterial load.

[0125] The results of the characterization experiment of the anti-tuberculosis function of monoclonal antibody 9C9 are shown in Figure 4 , and the determination results of the in vivo intervention experiment of M.bovis infection by monoclonal antibody 9C9 are shown in Figure 5 .

[0126] The results show that:

[0127] (1) Monoclonal antibody 9C9 can effectively recognize whole bacteria BCG and M. bovis, and the ability of monoclonal antibody 9C9 to bind M. bovis is significantly greater than that of BCG, in addition, the ability of monoclonal antibody 9C9 to bind the two strains is significantly reduced when Tween-80 is present in the culture medium (see A) in Figure 4 ; in addition, monoclonal antibody 9C9 promotes the phagocytosis of macrophages to BCG in a concentration-dependent manner (see B) in Figure 4 ; further, monoclonal antibody 9C9 exhibits a significant intracellular growth inhibition effect on both BCG and M. bovis strains in a concentration-dependent manner (see C and D) in Figure 4 ;

[0128] (2) Monoclonal antibody 9C9 can effectively reduce the colonization of M. bovis in the lungs of C57BL / 6J mice (see Figure 5 ).

[0129] Example 3: Sequencing and structure prediction of monoclonal antibody 9C9

[0130] 1. Sequencing of the variable region amino acid sequence of monoclonal antibody 9C9

[0131] After the monoclonal antibody cell strain 9C9 was cultured to the logarithmic growth phase, the cells were suspended by blowing with DMEM medium, and were sent to Jinweizhi Biotechnology Co., Ltd. for sequencing; wherein, the total RNA of the hybridoma cells was extracted using HiPure RNA Mini Columns (Magen) according to the instructions, after analyzing the RNA concentration and integrity using NanoDrop and gel electrophoresis, the cDNA was reverse transcribed using SMART Scribe Reverse Transcriptase (Vazyme), the forward primer anchored in Template switch oligo (TSO) and the reverse primer against the constant region of the antibody were designed, the variable regions of the heavy chain and light chain of the antibody were amplified, and the nucleic acid sequence of the antibody variable region was sequenced using Illumina MiSeq PE300.

[0132] Among them, the sequencing result information of monoclonal antibody 9C9 is as follows:

[0133] (1) The heavy chain variable region of monoclonal antibody 9C9

[0134] Monoclonal antibody 9C9-VH:

[0135] DVKLVESGGGLVRLGGSLKLSCAASGFTFSSYYMSWVRQTPEKRLEWVAAINTNGV NTYYPDTVRGRFTISRDNAKNTLYLQMSSLKSEDTALYYCARHELGRAWFAYWGQGTLV TVSA (SEQ ID NO:4)

[0136] Monoclonal antibody 9C9 heavy chain variable region CDR1 (CDRH1):

[0137] GFTFSSYY (SEQ ID NO:5)

[0138] Monoclonal antibody 9C9 heavy chain variable region CDR2 (CDRH2):

[0139] INTNGVNT (SEQ ID NO:6)

[0140] Monoclonal antibody 9C9 heavy chain variable region CDR3 (CDRH3):

[0141] ARHELGRAWFAY (SEQ ID NO:7)

[0142] (2) Light chain variable region of monoclonal antibody 9C9

[0143] Monoclonal antibody 9C9-VL:

[0144] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNL ESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHNRELPYTFGGGTKLEIK (SEQ ID NO:8)

[0145] Monoclonal antibody 9C9 light chain variable region CDR1 (CDRL1):

[0146] KSVSTSGYSY (SEQ ID NO:9)

[0147] Monoclonal antibody 9C9 light chain variable region CDR2 (CDRL2):

[0148] LAS (SEQ ID NO: 13)

[0149] Monoclonal antibody 9C9 light chain variable region CDR3 (CDRL3):

[0150] QHNRELPYT (SEQ ID NO: 10)

[0151] (3) Heavy chain variable region encoding nucleic acid sequence of monoclonal antibody 9C9

[0152] GACGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGAGGCTTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATTACATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCAGCCATTAATACTAATGGTGTTAACACCTACTATCCAGACACTGTGAGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCTTGTATTACTGTGCAAGACATGAACTGGGAAGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 11)

[0153] (4) Light chain variable region encoding nucleic acid sequence of monoclonal antibody 9C9

[0154] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGGCAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAATAGGGAGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 12)

[0155] 2. Construction of 3D model of variable region of monoclonal antibody 9C9

[0156] Firstly, the framework template with high homology to monoclonal antibody 9C9 was retrieved in IMGT database, and the homology to 5TLJ CD reached 98.3%. Then the variable region structure of antibody 9C9 was constructed using homology modeling software Modeller with 5TLJ CD as the template, and the positions of complementarity determining regions (CDRs) were labeled. The rationality of the model was evaluated by the verification tool Procheck to ensure that its stereochemistry and energetics meet the physical and chemical properties of biological macromolecules. Finally, the variable region structure of antibody 9C9 was displayed using molecular visualization software PyMOL, thus obtaining a high-quality 3D structure model of the variable region of monoclonal antibody 9C9.

[0157] 3. 3D model construction of antigen protein (MPT83 protein)

[0158] Firstly, the amino acid sequence of MPT83 protein was submitted to Alphafold 3 protein structure prediction platform, and the 3D structure model of MPT83 protein was obtained by structure prediction through Alphafold 3. Then the predicted structure was optimized by energy minimization and molecular dynamics simulation GROMACS to ensure the stability and reliability of the model. Finally, the quality of the 3D model was evaluated using the verification tool ProCheck, and a high-quality 3D structure model of MPT83 protein (antigen protein) was obtained.

[0159] The results of structure prediction of monoclonal antibody 9C9 variable region and MPT83 protein and prediction of the epitope recognized by monoclonal antibody 9C9 are shown in Figure 6 .

[0160] The results show that the 3D model of monoclonal antibody 9C9 variable region is successfully constructed, and the complementarity determining regions (CDRs) of H and L chains are labeled after homology modeling, which are used to predict the antigen-antibody contact surface amino acid residues (see Figure 6 A and B for details); in addition, the 3D model of MPT83 protein as an antigen is successfully constructed, and MPT83 protein presents a protein structure with a long tail, which is anchored at the N-terminal of the outer membrane of bacteria, while most of the structure of the C-terminal is exposed on the surface of bacteria, which will be beneficial to the recognition of monoclonal antibody 9C9 (see Figure 6 C for details); molecular docking analysis was performed using the modeled antibody structure and predicted antigen structure, and 2000 binding positions were obtained. The binding position was limited to the structure of CDR3, which was optimized and limited. The antigen-antibody binding position was recalculated for interaction energy, and the results after docking were sorted. The interfacial amino acid sequences of the top 4 binding positions were analyzed and displayed in detail (see Figure 6 D for details).

[0161] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0162] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. An antibody or antigen binding fragment, characterized in that, This includes CDR sequences selected from at least one of the following or sequences having one amino acid substitution, deletion, or addition: Heavy chain variable region CDR sequences: SEQ ID NO: 5~7; Light chain variable region CDR sequence: SEQ ID NO:9~10 and LAS.

2. The antibody or antigen-binding fragment of claim 1, wherein, include: The heavy chain variable region CDR1, CDR2, and CDR3 sequences, respectively, as shown in SEQ ID NO:5, 6, and 7 or having a single amino acid substitution, deletion, or addition; And / or, The light chain variable regions CDRL1, CDRL2, and CDRL3 sequences, respectively, as shown in SEQ ID NO:9, LAS, and SEQ ID NO:10, or having a single amino acid substitution, deletion, or addition.

3. The antibody or antigen-binding fragment of claim 2, wherein, include: The heavy chain variable region CDR1 sequence shown in SEQ ID NO:5, the heavy chain variable region CDR2 sequence shown in SEQ ID NO:6, the heavy chain variable region CDR3 sequence shown in SEQ ID NO:7, the light chain variable region CDR1 sequence shown in SEQ ID NO:9, the light chain variable region CDR2 sequence shown in LAS, and the light chain variable region CDR3 sequence shown in SEQ ID NO:

10.

4. The antibody or antigen-binding fragment of claim 1, wherein, include: As shown in SEQ ID NO:4, the heavy chain variable region sequence; And / or, The light chain variable region sequence shown in SEQ ID NO:

8.

5. The antibody or antigen-binding fragment of claim 1, wherein The constant region of the antibody or antigen-binding fragment is derived from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Optionally, the constant region of the antibody or antigen-binding fragment is derived from IgG1.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment as described in any one of claims 1 to 5.

7. The nucleic acid molecule of claim 6, wherein, include: The heavy chain variable region encoding a nucleic acid sequence as shown in SEQ ID NO:11; And / or, The light chain variable region shown in SEQ ID NO:12 encodes a nucleic acid sequence.

8. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule as described in claim 6 or 7.

9. A recombinant virus, wherein, The recombinant virus comprises the expression vector of claim 8, and the recombinant virus is capable of infecting immune cells.

10. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 6 or 7, or the expression vector of claim 8, or an antibody or antigen-binding fragment capable of expressing any one of claims 1 to 5.

11. A composition characterized in that, The composition contains one or more of the following: The antibody or antigen-binding fragment according to any one of claims 1 to 5; The nucleic acid molecule as described in claim 6 or 7; The expression vector as described in claim 8; The recombinant cell according to claim 10.

12. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The drug contains one or more of the following: The antibody or antigen-binding fragment according to any one of claims 1 to 5; The nucleic acid molecule as described in claim 6 or 7; The expression vector as described in claim 8; The recombinant cells of claim 10; The composition according to claim 11.

13. A reagent kit, characterized in that the reagent kit contains one or more of the following: The antibody or antigen-binding fragment according to any one of claims 1 to 5; The nucleic acid molecule of claim 6 or 7; The expression vector of claim 8; The recombinant virus of claim 9; The recombinant cell of claim 10.

14. Use of the antibody or antigen binding fragment of any one of claims 1 to 5, the nucleic acid molecule of claim 6 or 7, the expression vector of claim 8, the recombinant cell of claim 10 or the composition of claim 11 for the manufacture of a medicament, characterized in that, The medicament is used for preventing, alleviating, adjuvant treating or treating diseases caused by Mycobacterium tuberculosis infection.

15. Use according to claim 14, characterized in that, The Mycobacterium tuberculosis includes one or more of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium caprae; Optionally, the diseases caused by Mycobacterium tuberculosis infection include one or more of pulmonary tuberculosis, extrapulmonary tuberculosis, drug-resistant tuberculosis and latent tuberculosis infection. The nucleic acid molecule of claim 6 or 7; The expression vector of claim 8; The recombinant virus of claim 9; The recombinant cell of claim 10. The medicament is used for preventing, alleviating, adjuvant treating or treating diseases caused by Mycobacterium tuberculosis infection. The Mycobacterium tuberculosis includes one or more of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium caprae; Optionally, the diseases caused by Mycobacterium tuberculosis infection include one or more of pulmonary tuberculosis, extrapulmonary tuberculosis, drug-resistant tuberculosis and latent tuberculosis infection.

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