Protective monoclonal antibody for targeting mycobacterium tuberculosis PspA 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 PspA and a preparation method and application thereof, the protective monoclonal antibody comprises at least one of the following CDR sequences or a sequence with one amino acid substituted, deleted or increased: a heavy chain variable region CDR sequence: SEQ ID NO: 4-6; the light chain variable region CDR sequences are as shown in SEQ ID NO: 8-9 and WAS. The antibody or the antigen binding fragment can specifically bind to PspA protein of mycobacterium tuberculosis (MTB), shows efficient antituberculous activity in vivo and in vitro, specifically, can significantly enhance phagocytosis of macrophages on tubercle bacillus, inhibit tubercle bacillus growth and the like, and is clear in sequence and structure, so that the antibody or the antigen binding fragment is easy to develop and transform, and has broad application prospects. And moreover, an immune-mediated treatment mechanism is not easy to induce pathogens to generate drug resistance, so that when being applied to prevention and treatment of tuberculosis, not only is a new choice provided for treatment of tuberculosis, but also an important basis is 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] This invention relates to the field of biotechnology, specifically to a protective monoclonal antibody targeting Mycobacterium tuberculosis PspA, its preparation method, and its application. Background Technology

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis Tuberculosis (TB) is a serious zoonotic infectious disease caused by infection, resulting in approximately 10 million new cases and 1.3 million deaths annually (WHO, 2024), posing a continuous threat to global public health. The only currently approved vaccine, BCG, has a certain protective effect against TB in children, but its preventive effect against pulmonary TB in adults is limited and it is difficult to curb the spread of TB.

[0003] In the pathogenic mechanism of Mycobacterium tuberculosis, the PhoP-PhoR two-component system serves as a core hub regulating bacterial virulence and host adaptation, directly controlling the expression of multiple key genes. Among them, phage shock protein A (PspA), as a key downstream effector protein, plays an important protective role in the process of MTB responding to host stresses such as acidic environment, membrane damage, and oxidative stress, and thus has become an important potential target for intervention in tuberculosis infection. However, protective monoclonal antibodies against MTB PspA protein have not yet been reported.

[0004] Therefore, developing a protective monoclonal antibody that can precisely target Mycobacterium tuberculosis PspA is of great significance for the prevention and control of tuberculosis. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, this invention provides a protective monoclonal antibody targeting Mycobacterium tuberculosis PspA, its preparation method, and its applications. The antibody or antigen-binding fragment of this invention can specifically bind to the PspA protein of Mycobacterium tuberculosis (MTB), exhibiting highly efficient anti-tuberculosis activity both in vivo and in vitro. Specifically, it significantly enhances the phagocytic activity of macrophages against Mycobacterium tuberculosis and inhibits the growth of Mycobacterium tuberculosis. Furthermore, its sequence and structure are well-defined, making it easy to develop and modify. Moreover, its immune-mediated therapeutic mechanism is less likely to induce drug resistance in pathogens. Its application in the prevention and treatment of tuberculosis not only provides a new treatment option for tuberculosis but also lays an important foundation for dealing with drug-resistant strains, developing diagnostic reagents, and developing vaccines, demonstrating broad application prospects.

[0006] In a first aspect, the present invention provides an antibody or antigen-binding fragment. According to embodiments of the invention, the antibody or antigen-binding fragment comprises a CDR sequence selected from at least one of the following or a sequence having an amino acid substitution, deletion, or addition: heavy chain variable region CDR sequence: SEQ ID NO: 4-6; light chain variable region CDR sequence: SEQ ID NO: 8-9; and WAS.

[0007] According to embodiments of the present invention, the above-mentioned antibody or antigen-binding fragment may further have the following additional technical features: According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4, 5, and 6, or having a sequence with an amino acid substitution, deletion, or addition; and / or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 8, WAS, and SEQ ID NO: 9, or having a sequence with an amino acid substitution, deletion, or addition.

[0008] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: the heavy chain variable region CDR1 sequence as shown in SEQ ID NO: 4, the heavy chain variable region CDR2 sequence as shown in SEQ ID NO: 5, the heavy chain variable region CDR3 sequence as shown in SEQ ID NO: 6, the light chain variable region CDR1 sequence as shown in SEQ ID NO: 8, the light chain variable region CDR2 sequence as shown in WAS, and the light chain variable region CDR3 sequence as shown in SEQ ID NO: 9.

[0009] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region sequence as shown in SEQ ID NO: 7; and / or, a light chain variable region sequence as shown in SEQ ID NO: 10.

[0010] According to embodiments of the present invention, 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.

[0011] According to an embodiment of the present invention, the constant region of the antibody or antigen-binding fragment is derived from IgG1.

[0012] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect.

[0013] According to embodiments of the present invention, the above-mentioned nucleic acid molecule may also have the following additional technical features: According to an embodiment of the present invention, the nucleic acid molecule comprises: a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO: 11; and / or, a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO: 12.

[0014] In a third aspect, the present invention provides an expression vector. According to embodiments of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect.

[0015] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or is capable of expressing the antibody or antigen-binding fragment described in the first aspect.

[0016] In a fifth aspect, the invention provides for the use of the PspA protein in the preparation of vaccines or medicaments for the prevention and / or treatment of Mycobacterium tuberculosis infection. According to embodiments of the invention, the amino acid sequence of the PspA protein is as shown in SEQ ID NO: 3.

[0017] In a sixth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a medicament, characterized in that the medicament is used for the prevention, relief, adjunctive treatment, or treatment of Mycobacterium tuberculosis (MBTB). Mycobacterium tuberculosis Diseases caused by infection.

[0018] According to embodiments of the present invention, the above-described uses may also have the following additional technical features: According to an embodiment of the present invention, the Mycobacterium tuberculosis includes Mycobacterium humanis (Mycobacterium tuberculosis). Mycobacterium tuberculosis ), Mycobacterium bovis ( Mycobacterium bovis ), Mycobacterium africanum ( Mycobacterium africanum ) and Mycobacterium caprineis ( Mycobacterium caprae One or more of the following.

[0019] According to embodiments of the present invention, the diseases caused by Mycobacterium tuberculosis infection include one or more of pulmonary tuberculosis, extrapulmonary tuberculosis, drug-resistant tuberculosis, and latent tuberculosis infection.

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The images shown are the identification results of BCG outer membrane protein samples in Example 1 of this invention. In the images, A is the detection result of BCG outer membrane protein extraction, B is the result of identifying BCG outer membrane protein samples using GroeL2 polyclonal antibody serum, C is the result of identifying BCG outer membrane protein samples using MctB polyclonal antibody serum, D is the result of identifying BCG outer membrane protein samples using OmpA polyclonal antibody serum, and E is the result of identifying BCG outer membrane protein samples using FtsH polyclonal antibody serum. Figure 2 The following are the relevant detection results of monoclonal antibody 9C5 in Example 2 of the present invention: A is the result of serum titer determination of mouse immune BCG outer membrane protein sample; B is the result of flow cytometry analysis of the effect of serum immune BCG outer membrane protein sample on macrophage phagocytosis; C is the statistical result of flow cytometry analysis of the effect of serum immune BCG outer membrane protein sample on macrophage phagocytosis; D is the purification result of monoclonal antibody 9C5; and E is the subtype analysis result of monoclonal antibody 9C5. Figure 3 The diagram shows the identification results of the target antigen of monoclonal antibody 9C5 in Example 3 of the present invention. In this diagram, A is the result of Co-IP identification of the antigen recognized by monoclonal antibody 9C5, B is the purification result of the target protein PspA, C is the purification and identification result of the target protein PspA, and D is the identification result of monoclonal antibody 9C5 recognizing antigen PspA. Figure 4 This is a graph showing the functional characterization results of monoclonal antibody 9C5 in Example 4 of the present invention, where A represents the effect of monoclonal antibody 9C5 on BCG and M.bovis The results of the whole-cell identification assay are shown in Figure B. Figure B shows the statistical flow cytometry analysis of the effect of monoclonal antibody 9C5 on macrophage phagocytosis. Figure C shows the results of the monoclonal antibody 9C5 inhibiting BCG growth. Figure D shows the results of the monoclonal antibody 9C5 inhibiting... M.bovis Figure showing the results of the growth experiment; Figure 5 The following is a graph showing the in vivo protective effect of monoclonal antibody 9C5 in Example 4 of this invention. In the graph, A is the experimental design flowchart of the in vivo protective effect of monoclonal antibody 9C5, B is the statistical graph of the effect of monoclonal antibody 9C5 on the organ index of lungs in mice in each group, C is the statistical graph of the effect of monoclonal antibody 9C5 on the organ index of spleen in mice in each group, and D is the statistical graph of the effect of monoclonal antibody 9C5 on the bacterial load in the lungs of mice in each group. Figure 6The diagram shows the predicted structure of the variable region of monoclonal antibody 9C5 and PspA protein in Example 5 of this invention. In this diagram, A is the result of homology modeling of the structure of monoclonal antibody 9C5, B is the result of the predicted structure of antigen PspA, C~G are the conformation results of the antibody (monoclonal antibody 9C5)-antigen (PspA protein) complex with the top five molecular docking scores, and H is the result of detailed docking conformation based on the highest ranking of the RDOCK scoring system. Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0026] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0027] Terms and Definitions In this document, the term "PMSF" refers to benzyl sulfonyl fluoride, an irreversible serine protease inhibitor, which, in the embodiments of the present invention, is used to effectively inhibit protein degradation during protein extraction, thereby ensuring the integrity and yield of the target protein.

[0028] In this document, the term "β-D-glucopyranoside" refers to a nonionic detergent used in the embodiments of the present invention to solubilize cell membrane components, thereby effectively dissolving and releasing outer membrane proteins integrated into the membrane structure into the solution, and is one of the key reagents for extracting membrane proteins.

[0029] In this paper, the term "TBS" refers to Tris buffer solution, whose main components are Tris-HCl and sodium chloride. It is a commonly used physiological condition buffer solution used in immunological experiments (such as immunoprecipitation) to maintain the pH and ionic strength of the reaction system and reduce non-specific binding.

[0030] In this article, the term "PspA protein" refers to phage shock protein A, which in Mycobacterium tuberculosis is produced by... pspA The gene encodes a stress response protein, which is a key downstream effector regulated by the PhoP-PhoR two-component system. It plays a core protective role in bacteria's response to host environmental stresses (such as acidic pH and membrane damage) and is the target antigen specifically recognized by the monoclonal antibody 9C5 (antibody or antigen-binding fragment) described in this invention.

[0031] Antibody or antigen-binding fragment This invention proposes an antibody or antigen-binding fragment. According to embodiments of the invention, the antibody or antigen-binding fragment comprises a CDR sequence selected from at least one of the following or a sequence having an amino acid substitution, deletion, or addition: heavy chain variable region CDR sequence: SEQ ID NO: 4-6; light chain variable region CDR sequence: SEQ ID NO: 8-9; and WAS. The antibody or antigen-binding fragment according to embodiments of the invention, by comprising specific CDR sequences (SEQ ID NO: 4-6 and 8-9 and WAS) or variants thereof (having an amino acid substitution, deletion, or addition), achieves precise recognition and specific binding to the Mycobacterium tuberculosis (MTB) PspA protein. This specific binding endows the antibody or antigen-binding fragment of the invention with highly efficient anti-tuberculosis activity and low drug resistance in vitro and in vivo, providing a foundation for subsequent drug development, diagnostic reagent development, etc.

[0032] In this paper, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen. It consists of two lighter light chains and two heavier heavy chains, which are linked by disulfide bonds to form a tetrapeptide molecule. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is called the variable region (V-terminus), while the carboxyl terminus (C-terminus) is relatively stable and varies little and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are called VL and VH, respectively. Both the variable regions of the heavy chain and the variable regions of the light chain have three CDR regions.

[0033] In this document, the term "chimeric antigen receptor (CAR)" is a molecule that binds an antibody-based specificity against a desired antigen (e.g., a tumor antigen) to a T-cell receptor-activated intracellular domain to produce a chimeric protein exhibiting specific anti-tumor cellular immune activity; in this invention, the antibody or antigen-binding fragment of the invention specifically recognizes the Mycobacterium tuberculosis (MTB) PspA protein.

[0034] It should be noted that the sequence information such as the heavy chain variable region sequence and the light chain variable region sequence in the embodiments of the present invention are shown from the N end to the C end.

[0035] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4, 5, and 6, or having a sequence with an amino acid substitution, deletion, or addition; and / or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 8, WAS, and SEQ ID NO: 9, or having a sequence with an amino acid substitution, deletion, or addition.

[0036] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: the heavy chain variable region CDR1 sequence as shown in SEQ ID NO: 4, the heavy chain variable region CDR2 sequence as shown in SEQ ID NO: 5, the heavy chain variable region CDR3 sequence as shown in SEQ ID NO: 6, the light chain variable region CDR1 sequence as shown in SEQ ID NO: 8, the light chain variable region CDR2 sequence as shown in WAS, and the light chain variable region CDR3 sequence as shown in SEQ ID NO: 9.

[0037] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region sequence as shown in SEQ ID NO: 7; and / or, a light chain variable region sequence as shown in SEQ ID NO: 10.

[0038] According to embodiments of the present invention, 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.

[0039] According to an embodiment of the present invention, the constant region of the antibody or antigen-binding fragment is derived from IgG1.

[0040] It should be noted that the antibody or antigen-binding fragment described in this invention comprises a variable region and a constant region, the specificity of which is determined by the sequence of the variable region. The "constant region" refers to a relatively conserved region in the antibody molecule that does not directly participate in antigen recognition. Since this part of the sequence varies very little among different antibodies, and the constant region sequence is known in the art and can be conventionally selected, the core of this invention lies in the specific recognition function of the antibody or antigen-binding fragment (achieved by the variable region). Therefore, the specific sequence of the constant region of the antibody or antigen-binding fragment is not shown in detail. Furthermore, those skilled in the art can, according to actual needs (such as regulating effector function, half-life, etc.), replace and combine the constant regions of different subtypes or species origins already existing in the prior art without affecting the binding specificity of the antibody or antigen-binding fragment of this invention. Such cases are all within the scope of protection of this invention.

[0041] Nucleic acid molecules This invention proposes a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment. The nucleic acid molecule according to embodiments of the invention enables the mass production of the antibody or antigen-binding fragment of the invention using genetic engineering techniques, and lays the foundation for subsequent construction of expression vectors, recombinant viruses, and recombinant cells, thereby achieving efficient expression and application of the antibody or antigen-binding fragment of the invention.

[0042] According to an embodiment of the present invention, the nucleic acid molecule comprises: a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO: 11; and / or, a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO: 12.

[0043] It should be noted that those skilled in the art should understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands; although in most cases only one strand is given, the other complementary strand is actually disclosed as well; in addition, the nucleic acid molecule sequences in this invention include DNA or RNA forms, and disclosing one of them means that the other is also disclosed.

[0044] expression carrier This invention proposes an expression vector. According to embodiments of the invention, the expression vector comprises the aforementioned nucleic acid molecule. The expression vector according to embodiments of the invention enables the nucleic acid molecule to be efficiently expressed in host cells and provides a key tool for the subsequent construction of recombinant viruses and recombinant cells.

[0045] It should be noted that the expression vector mentioned in this article refers to a nucleic acid molecule that can be inserted into a suitable host and self-replicate, transferring the inserted nucleic acid molecule to host cells and / or between host cells; the expression vector may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for transcription and / or translation of DNA or RNA; the expression vector may be a polynucleotide that can be transcribed and translated when introduced into a suitable host cell; by culturing a suitable host cell containing the vector, the expression vector produces the aforementioned nucleic acid molecule, and the expression vector includes viral vectors, plasmids, bacteriophages, etc.

[0046] Recombinant cells This invention proposes a recombinant cell. According to embodiments of the invention, 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. The recombinant cell according to embodiments of the invention can be introduced into the body via cell transplantation or other methods to exert an anti-tuberculosis effect, providing more options for the treatment of tuberculosis.

[0047] Use of PspA protein in the preparation of vaccines or drugs for the prevention and / or treatment of Mycobacterium tuberculosis infection This invention proposes the use of PspA protein in the preparation of vaccines or drugs for the prevention and / or treatment of Mycobacterium tuberculosis infection. According to embodiments of the invention, the amino acid sequence of the PspA protein is shown in SEQ ID NO: 3. According to the use according to embodiments of the invention, specific antibodies against the PspA protein (such as monoclonal antibody 9C5 in the embodiments of the invention) can mediate a highly effective anti-tuberculosis protective effect, demonstrating that this protein can serve as an effective vaccine target. Vaccines or drugs designed based on this target can effectively stimulate the body to produce a specific protective immune response against Mycobacterium tuberculosis, thereby achieving the prevention, relief, adjunctive treatment, or treatment of diseases caused by Mycobacterium tuberculosis infection.

[0048] Uses in drug preparation This invention proposes the use of the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in the preparation of a drug, characterized in that the drug is used for the prevention, relief, adjunctive treatment, or treatment of Mycobacterium tuberculosis (MBTB). Mycobacterium tuberculosis Diseases caused by infection.

[0049] According to an embodiment of the present invention, the Mycobacterium tuberculosis includes Mycobacterium humanis (Mycobacterium tuberculosis). Mycobacterium tuberculosis ), Bovine tuberculosis mycobacterium ( Mycobacterium bovis African tuberculosis mycobacterium (Mycobacterium africanum ) and Mycobacterium tuberculosis of caprines ( Mycobacterium caprae One or more of the following.

[0050] According to embodiments of the present invention, the diseases caused by Mycobacterium tuberculosis infection include one or more of pulmonary tuberculosis, extrapulmonary tuberculosis, drug-resistant tuberculosis, and latent tuberculosis infection.

[0051] It should be noted that the antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, or recombinant cells described in this invention are not limited to the specific Mycobacterium tuberculosis species or disease types mentioned above. They also have potential application value in the prevention, relief, adjuvant treatment, or treatment of other diseases or infections caused by Mycobacterium tuberculosis, and these applications should all be within the scope of protection of this invention.

[0052] The specific sequences involved in this invention are detailed in Table 1.

[0053] Table 1

[0054] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0055] Example 1: Preparation and Identification of BCG Outer Membrane Proteins 1. BCG culture After resuscitation and activation, the BCG strain (BCGPasteur1173P2 strain) was washed three times with PBS, transferred into fresh Soton medium, and cultured at 37°C and 100 rpm until the logarithmic growth phase to obtain BCG bacterial culture.

[0056] The formula of the Sutong medium is as follows: Weigh 4 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of ferric ammonium citrate, 2 g of citric acid, and dissolve 60 mL of glycerol in 700 mL of ddH2O. Adjust the pH to 7.4 using 5M NaOH, and bring the volume to 900 mL. Then sterilize at 121℃ under high temperature and high pressure for 20 min. After the temperature drops to room temperature, add 100 mL of 4% asparagine solution to obtain the Sutong medium.

[0057] 2. BCG outer membrane protein extraction The BCG bacterial culture obtained in step 1 (300 mL, OD) 600nm =0.8) Add 100 µg / mL ciprofloxacin, shake at 37 ℃ and 100 rpm for 1 h to inactivate the bacteria, then resuspend the bacteria in 10 mL of outer membrane protein extraction buffer, add 1 mM MPMSF to the final concentration, add 1 / 3 volume of 0.1 mm glass beads, and sonicate in a tissue homogenizer (program set: run for 30 s, stand on ice for 1 min; cycle 8 times); then add outer membrane protein extraction buffer to make up the bacterial culture volume to 30 mL, add 1 mM MPMSF to the final concentration, and sonicate (program set: 300 w, sonicate for 2 s, pause for 4 s, total time 20 min); centrifuge at 2300 g for 5 min at 4 ℃ to obtain supernatant I, add 1% β-D-glucopyranoside to supernatant I to the final concentration, stand at room temperature for 2 h, then centrifuge at 27000 g for 1 h to obtain supernatant II, add 1 mM PMSF was transferred to a dialysis bag and dialyzed overnight. The samples were then aliquoted to obtain BCG outer membrane protein samples, which were stored at -80 ℃ for later use.

[0058] The formulation of the outer membrane protein extraction buffer (OMP buffer) is as follows: Tris base 1.2114 g, NaCl 8.766 g, ddH2O 1000 mL, adjust pH to 7.4.

[0059] 3. Identification of BCG outer membrane proteins The BCG outer membrane protein samples obtained in step 2 were identified by Western blotting using polyclonal antibodies against outer membrane proteins (OmpA, MctB), inner membrane proteins (FtsH), and cytoplasmic proteins (GroeL2).

[0060] The identification results of the BCG outer membrane protein sample are shown below. Figure 1 .

[0061] The results showed that specific bands of outer membrane proteins OmpA and MctB could be detected in the BCG outer membrane protein sample obtained in step 2, indicating that the outer membrane protein extraction was successful. Although a small amount of inner membrane proteins FtsH and GroeL2 were detected in the total extract, the sample was sufficient for immunization to screen for antibodies targeting outer membrane proteins.

[0062] Example 2: Preparation and screening of monoclonal antibody 9C5 targeting BCG outer membrane protein 1. Mouse immunization and serum titer determination The treatment methods for the experimental group mice are as follows: The BCG outer membrane protein sample prepared and identified in Example 1 was mixed with an emulsion adjuvant (purchased from Thermofisher, catalog number 77161) and used to immunize BALB / c mice (purchased from Beijing Spaford Biotechnology Co., Ltd.) twice to obtain immunized BALB / c mice; and its serum titer was detected by ELISA.

[0063] The negative control group mice were treated as follows: Without any treatment, negative control mice (NC) were obtained.

[0064] 2. Functional verification of immunized mouse serum After complement was inactivated, serum from immunized BALB / c mice was added to a system of BCG-infected macrophages J774a.1 (MOI=10:1). Serum from the negative control group mice obtained in step 1 was used as a negative control. The phagocytic effect of macrophages J774a.1 on BCG was analyzed by flow cytometry.

[0065] 3. Cell fusion and hybridoma screening The experimental group of mice with the highest serum titers (BALB / c mice after immunization) were selected. After booster immunization with BCG outer membrane protein samples without water emulsion adjuvant, the spleens of the mice were harvested. Myeloma cell line SP2 / 0 cells and spleen cells were fused using PEG1450. Then, a hybridoma cell line that could stably secrete specific antibodies was obtained by screening using HAT / HT medium (purchased from Sigma-Aldrich, catalog numbers H0262 and H0137) and named 9C5 (monoclonal cell line 9C5).

[0066] 4. Purification and identification of monoclonal antibody 9C5 Ascites fluid was prepared from paraffin-immunized mice and purified using Protein G resin to obtain high-purity monoclonal antibody 9C5, which was stored at -80℃ for later use.

[0067] Antibody 9C5 was prepared from mouse ascites fluid and purified by Protein G resin affinity chromatography.

[0068] 5. Identification of monoclonal antibody 9C5 subtypes The specific subtype of the high-purity monoclonal antibody 9C5 obtained in step 4 was determined using an antibody subtype identification kit (purchased from Frdbio).

[0069] The relevant detection results for monoclonal antibody 9C5 are shown below. Figure 2 .

[0070] The results showed that after two immunizations of mice with the extracted BCG outer membrane protein sample combined with an aqueous emulsion adjuvant, the mouse serum titer reached 1.024 × 10⁻⁶.6 (See details) Figure 2 (A) When complement-inactivated serum from immunized mice was added to a BCG-infected macrophage system, it was found that, compared to serum from unimmunized mice (negative control), serum from immunized mice significantly promoted macrophage phagocytosis. FcR blocking inhibited this phagocytic effect, demonstrating that the antibody mediated the phagocytic response via FcR (see [reference needed] for details). Figure 2 (B and C in the text); Ascites fluid was prepared from paraffin-immunized mice, purified with Protein G resin, and analyzed by Coomassie staining and gel electrophoresis. The obtained monoclonal antibody 9C5 showed two distinct bands, one at 55 kDa for the heavy chain and the other at 25 kDa for the light chain (see details in B and C). Figure 2 (D in the text), and identified the monoclonal antibody 9C5 subtype as IgG1 (see details). Figure 2 (E in the text).

[0071] Example 3: Identification of the target antigen of monoclonal antibody 9C5 1. Identification of target antigens by immunoprecipitation combined with mass spectrometry analysis Using an antibody crosslinking immunoprecipitation kit (purchased from Beyotime Biotechnology, catalog number P2180S), 5 µg of the high-purity monoclonal antibody 9C5 obtained in Example 2 was incubated with Protein G magnetic beads for 1 h. After washing with TBS, 50 µg of the BCG outer membrane protein sample prepared in Example 1 and identified was added, and the mixture was incubated at room temperature for 2 h. After washing with TBS, 100 µL of acidic elution buffer was added, and the mixture was incubated at room temperature for 5 min. Then, 10 µL of neutralization buffer was added to the supernatant, and the eluted sample was analyzed by Western blot and mass spectrometry.

[0072] 2. Preparation of recombinant PspA protein Further expression analysis was performed on BCG_2760c (PspA), the protein ranked highest in Step 1. Based on the expression of Mycobacterium tuberculosis BCG in GenBank... pspA Primers were designed based on the gene sequence (SEQ ID NO: 1~2), and the recombinant expression plasmid pET28a-pspA was constructed. After transformation into BL21(DE3) competent cells, expression was induced, and the plasmid was expressed via Ni... 2+ Recombinant PspA protein was prepared by column-induced expression and affinity chromatography purification, and the prepared recombinant PspA protein samples were identified by SDS-PAGE and Western blotting.

[0073] The upstream primer PspA-F and the downstream primer PspA-R are shown in SEQ ID NO: 1~2 (lowercase letters are vector homologous arms), and the amino acid sequence of the PspA protein is shown in SEQ ID NO: 3.

[0074] PspA-F: atgggtcgcggatccgaattcATGGCCAATCCGTTCGTTAA (SEQ ID NO: 1) PspA-R: ctcgagtgcggccgcaagcttCTGACCGTAGGGCTGCTCG (SEQ ID NO: 2) PspA protein: MANPFVKAWKYLMALFSSKIDEHADPKVQIQQAIEEAQRTHQALTQQAAQVIGNQRQLEMRLNRQLADIEKLQVNVRQALTLADQATAAGDAAKATEYNNAAEAFAAQLVTAEQSVEDLKTLHDQALSAAAQAKKAV ERNAMVLQQKIAERTKLLSQLEQAKMQEQVSASLRSMSELAAPGNTPSLDEVRDKIERRYANAIGSAELAESSVQGRMLEVEQAGIQMAGHSRLEQIRASMRGEALPAGGTTATPRPATETSGGAIAEQPYGQ (SEQ ID NO: 3) 3. Verification of the specific binding of monoclonal antibody 9C5 to PspA protein Using the recombinant PspA protein obtained in step 2 as the antigen, and the high-purity monoclonal antibody 9C5 prepared in Example 2 as the primary antibody, Western blotting analysis was performed.

[0075] The identification results of the target antigen of monoclonal antibody 9C5 are shown in [the table]. Figure 3 .

[0076] The results showed that the target protein could be extracted from the BCG outer membrane protein sample using immunoprecipitation (see details). Figure 3 (A in the original text). Then, mass spectrometry analysis was performed, and further expression analysis was conducted on the top-ranked protein BCG_2760c (PspA). 2+ The target protein PspA was successfully prepared by column affinity purification (see details). Figure 3 (B in the text); then Western blot analysis revealed that the anti-His antibody, used as the primary antibody, successfully developed the target band, and the Mycobacterium tuberculosis protein PspA was successfully obtained (see details). Figure 3(C in the text); then, using monoclonal antibody 9C5 as the primary antibody to incubate the protein PspA, analysis revealed that monoclonal antibody 9C5 specifically recognizes the Mycobacterium tuberculosis antigen PspA (see details). Figure 3 (D in the middle).

[0077] Example 4: Functional characterization of monoclonal antibody 9C5 1. Whole-cell recognition capability Using the ELISA method, firstly... M.bovis The bacterial suspension and BCG suspension were inactivated at 80°C for 20 min to obtain inactivated [the desired product]. M.bovis Bacterial solution, inactivated BCG bacterial solution; then the inactivated... M.bovis Bacterial suspension and inactivated BCG bacterial suspension were respectively administered at 1×10 7 CFU / well was coated into 96-well ELISA plates and dried in a 60°C oven. Then, the high-purity monoclonal antibody 9C5 obtained in Example 2 was used as the primary antibody, initially diluted at 500 µg / ml, and serially diluted to 100 µL / well. The plates were then co-incubated with bacteria at 37°C for 1 h. 100 µL of horseradish peroxidase (HRP)-labeled secondary antibody (purchased from Kangwei Century Company, catalog number CW0102) (1:10000) was added, and the plates were incubated at 37°C for 1 h. After TMB color development, OD was measured. 450 absorbance value in nm.

[0078] 2. Macrophage phagocytosis The high-purity monoclonal antibody 9C5 obtained in Example 2 was co-incubated with BCG bacterial culture (the concentration of monoclonal antibody 9C5 used was 50 μg / mL), and then macrophages J774a.1 were treated with an infection multiple of 10:1 at an MOI of 10:1. The phagocytic ratio of BCG by macrophages J774a.1 was analyzed by flow cytometry.

[0079] 3. Intracellular bactericidal activity Will M.bovis Alternatively, BCG was used to prepare a single-cell suspension, which was added to peripheral blood mononuclear cells (PBMCs) containing different concentrations of the high-purity monoclonal antibody 9C5 (1 µg / ml, 5 µg / ml, 50 µg / ml and 100 µg / ml) prepared in Example 2 or mGO53 antibody at a concentration of 50 μg / mL (negative control group, mGO53 as the control group) (MOI=10:1). The cells were cultured at 37°C for 4 days, centrifuged at 8000 rpm for 5 min to collect the cells, and after complete lysis, the surviving bacteria in the system were plated on 7H10 plates (purchased from BDBiosciences, catalog number 262710).

[0080] 4. In vivo protective effect C57BL / 6J mice (purchased from Beijing SPAF Biotechnology Co., Ltd.) were randomly divided into a blank control group (PBS) and an experimental group (9C9). After one week of adaptive culture, the experimental group mice were intraperitoneally injected with 500 µg of the high-purity monoclonal antibody 9C5 prepared in Example 2, while the blank control group mice were intraperitoneally injected with an equal volume of PBS. M.bovis (500 CFU) After 14 days, the mice in both groups were sacrificed, and the lungs and spleens of the mice in both groups were taken to measure the organ index of the lungs and the organ index of the spleen. The lungs of the mice were ground and smeared on plates to count the bacterial load in the lungs.

[0081] The functional characterization results of monoclonal antibody 9C5 are shown in the figure. Figure 4 The in vivo protective efficacy of monoclonal antibody 9C5 can be found in the following results. Figure 5 .

[0082] The results showed that the monoclonal antibody 9C5 could effectively recognize whole bacterial BCG and... M.bovis (See details) Figure 4 (A in the text); In addition, the monoclonal antibody 9C5 can significantly promote the phagocytosis of BCG by the macrophage line J774A.1 (see details in A). Figure 4 (B in the text); Meanwhile, monoclonal antibody 9C5 showed concentration-dependent susceptibility to BCG and... M.bovis All showed significant intracellular growth inhibition effects (see details). Figure 4 (C and D in the text). Then, to further determine the effect of the monoclonal antibody 9C5 on mice... M.bovis Protective effect against infection, establishing a mouse model for preventing infection (see details). Figure 5 Analysis of antibody prevention revealed that monoclonal antibody 9C5 could reduce infection. M.bovis Colonization in mouse lungs (see details) Figure 5 (B in the middle).

[0083] Example 5: Sequencing and structural simulation of monoclonal antibody 9C5 1. Amino acid sequence sequencing of the 9C5 variable region of the monoclonal antibody. After the monoclonal cell line 9C5 obtained in step 3 of Example 2 was cultured to the logarithmic growth phase, the cells were suspended in DMEM medium (purchased from Thermo Fisher Scientific, catalog number C11995500BT) and sent to Genewiz Biotechnology Co., Ltd. for sequencing. Total RNA was extracted from the hybridoma cells using HiPure RNA Mini Columns (Magen) according to the manufacturer's instructions. RNA concentration and integrity were analyzed by NanoDrop and gel electrophoresis. cDNA was reverse transcribed using SMART Scribe Reverse Transcriptase (Vazyme). Forward primers anchored to the Template switch oligo (TSO) and reverse primers for the antibody constant region were designed to amplify the variable regions of the antibody heavy and light chains. The nucleic acid sequence of the antibody variable region was sequenced using an Illumina MiSeq PE300.

[0084] The sequencing results for monoclonal antibody 9C5 are as follows: (1) Heavy chain variable region of monoclonal antibody 9C5 Monoclonal antibody 9C5 heavy chain variable region CDR1 (CDRH1): GYTFTDYS (SEQ ID NO: 4) Monoclonal antibody 9C5 heavy chain variable region CDR2 (CDRH2): INTETGEP (SEQ ID NO: 5) Monoclonal antibody 9C5 heavy chain variable region CDR3 (CDRH3): ARSGGYYVFDY (SEQ ID NO: 6) Monoclonal antibody 9C5-VH: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSIQWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSASTAFLQINNLKNEDTATYFCARSGGYYVFDYWGQGTTLTVSS (SEQ ID NO: 7) (2) Light chain variable region of monoclonal antibody 9C5 Monoclonal antibody 9C5 light chain variable region CDR1 (CDRL1): QSLLNSRTKNS (SEQ ID NO: 8) Monoclonal antibody 9C5 light chain variable region CDR2 (CDRL2): WAS (SEQ ID NO: 13) Monoclonal antibody 9C5 light chain variable region CDR3 (CDRL3): KHSYNLYT (SEQ ID NO: 9) Monoclonal antibody 9C5-VL: DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNSLAWYQQKPGQSPKLLIYWASTRVSGVPDRFTGSGSGTDFTLTISSVQGEDLAVYYCKHSYNLYTFGGGTKLEIK (SEQ ID NO: 10) (3) The heavy chain variant region of monoclonal antibody 9C5 encodes the nucleic acid sequence. CAGATCCAGTTGGTGCAGTCTGGACCTGAGTTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTTATAACCTTCACAGACTATTCAATACAGTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATA TGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTTTTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGATCGGGGGGTTACTACGTTTTTGACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 11) (3) The light chain variant region of monoclonal antibody 9C5 encodes the nucleic acid sequence. GACATTGTGATGTCCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTCCTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGC ATCCACTAGGGTATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGGTGAAGACCTGGCAGTTTTACTGCAAGCACTCTTATAATCTTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 12) 2. Construction of a 3D model of the 9C5 variable region of a monoclonal antibody First, a framework template with high homology to the monoclonal antibody 9C5 was searched in the IMGT database, and it was found that the homology with 1NCA reached 96.5%. Then, the variable region structure of the monoclonal antibody 9C5 was constructed using the homology modeling software Modeller as a template, and the position of the complementarity-determining region (CDR) was marked. The rationality of the model was evaluated by the validation tool Procheck to ensure that its stereochemistry and energy conform to the physicochemical properties of biomolecules. Finally, the structure of the variable region of the monoclonal antibody 9C5 was displayed using the molecular visualization software PyMOL, thus obtaining a high-quality 3D structural model of the variable region of the monoclonal antibody 9C5.

[0085] 3. Construction of a 3D model of the antigen protein (PspA protein) First, the amino acid sequence of the PspA protein was submitted to the Alphafold 3 protein structure prediction platform. The structure was predicted using Alphafold 3 to obtain a three-dimensional structural model of the PspA protein. 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 3D model quality was evaluated using the validation tool ProCheck to obtain a high-quality 3D structural model of the PspA protein (antigen protein).

[0086] Structural prediction results of the 9C5 variable region of monoclonal antibody and PspA protein Figure 6 .

[0087] The results showed that the homologous framework template of the monoclonal antibody antibody 9C5 was retrieved from the IMGT database to model the variable region structure of the antibody (see details). Figure 6The A in the figure was used to predict the amino acid residues on the antigen-antibody contact surface; and the structure of the PspA antigen was predicted using Alphafold 3 (see details). Figure 6 (B) From this result, it can be seen that the PspA protein exhibits a protein structure with a long tail, which is anchored to the N-terminus of the bacterial outer membrane, while most of the C-terminal structure is exposed on the bacterial surface, which will facilitate antibody recognition; the top five antibody-antigen complex conformations in molecular docking score (numbered 10 / 3 / 9 / 15 / 2), the antibody structure is shown in the red box (see details). Figure 6 (C~G in the text), and provides a detailed demonstration of the docking conformation with the highest ranking based on the RDOCK scoring system (see details). Figure 6 (H in the text).

[0088] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0089] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen binding fragment, characterized in that, comprises CDR sequences selected from at least one of: heavy chain variable region CDR sequences: SEQ ID NOs: 4~6; light chain variable region CDR sequences: SEQ ID NOs: 8~9 and WAS.

2. The antibody or antigen-binding fragment of claim 1, wherein, comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively, or sequences with one amino acid substitution, deletion or addition; and / or, light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs: 8, WAS, and 9, respectively, or sequences with one amino acid substitution, deletion or addition; optionally, the antibody or antigen-binding fragment comprises: a heavy chain variable region CDR1 sequence as set forth in SEQ ID NO: 4, a heavy chain variable region CDR2 sequence as set forth in SEQ ID NO: 5, a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO: 6, a light chain variable region CDR1 sequence as set forth in SEQ ID NO: 8, a light chain variable region CDR2 sequence as set forth in WAS, and a light chain variable region CDR3 sequence as set forth in SEQ ID NO:

9.

3. The antibody or antigen-binding fragment of claim 1, wherein comprises: a heavy chain variable region sequence as set forth in SEQ ID NO: 7; and / or, a light chain variable region sequence as set forth in SEQ ID NO:

10.

4. 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.

5. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the antibody or antigen-binding fragment of any one of claims 1~4.

6. An expression vector, characterized by, the expression vector comprises the nucleic acid molecule of claim 5.

7. A recombinant cell, wherein, the recombinant cell harbors the nucleic acid molecule of claim 5 or the expression vector of claim 6, or is capable of expressing the antibody or antigen-binding fragment of any one of claims 1~4.

8. Use of a PspA protein for the preparation of a vaccine or a medicament for the prevention and / or treatment of Mycobacterium tuberculosis infection, characterized in that, the amino acid sequence of the PspA protein is set forth in SEQ ID NO:

3.

9. Use of the antibody or antigen binding fragment of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the expression vector of claim 6 or the recombinant cell of claim 7 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. Mycobacterium tuberculosis ) 10. Use according to claim 9, characterized in that, The Mycobacterium tuberculosis includes one or more of Mycobacterium tuberculosis (M. tuberculosis) Mycobacterium tuberculosis ), Mycobacterium bovis (M. bovis) Mycobacterium bovis ), Mycobacterium africanum (M. africanum) Mycobacterium africanum ), and Mycobacterium caprae (M. caprae) Mycobacterium caprae ). optionally, the disease caused by the Mycobacterium tuberculosis infection comprises one or more of pulmonary tuberculosis, extrapulmonary tuberculosis, drug-resistant tuberculosis, and latent tuberculosis infection.

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