Monoclonal antibody for detecting or blocking FomA protein with fusobacterium nucleatum as well as application and product of monoclonal antibody

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a monoclonal antibody for detecting or blocking FomA protein with fusobacterium nucleatum as well as application and a product of the monoclonal antibody. The monoclonal antibodies obtained through screening or antigen binding fragments FAMD6 and FAME67 of the monoclonal antibodies can be specifically combined with different sites of Fn-FomA protein, an immunodetection or diagnosis kit constructed by the monoclonal antibodies or the antigen binding fragments FAMD6 and FAME67 is high in sensitivity and specificity and can rapidly and accurately detect Fn infection, the detection sensitivity can reach 3.9 ng / mL, the highest dilution of Fn culture supernatant is 1: 1600, the sensitivity is high, and the Fn infection can be rapidly and accurately detected. Good application prospects are realized. Besides, in a mouse model, the monoclonal antibody or the antigen binding fragments FAMD6 and FAME67 thereof have higher protectiveness on pregnant mice, and the bad pregnancy rate can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to monoclonal antibodies for detecting or blocking the FomA protein of Fusobacterium nucleatum, their applications and products. Background Technology

[0002] Fusobacterium nucleatum (Fn) is a slender, fusiform, Gram-negative anaerobic bacillus that does not spore, and its nucleic acid staining reveals a nucleus-like structure. Fn's elongated shape helps form a unique structural support in oral biofilms, making it a resident member of the oral microbiota, widely present on the tooth surface and gingival sulcus of healthy individuals. It acts as a "bridge bacterium," connecting primary colonizing bacteria and secondary anaerobes by co-aggregating with various microorganisms (such as Streptococcus and Porphyromonas gingivalis) to form dental plaque biofilms. Fn's metabolic pathways include amino acid fermentation and glycolysis, and it is significantly influenced by other microorganisms. It promotes biofilm formation and metabolic regulation by expressing various adhesins (such as RadD, FadA, and Fap2) to mediate interactions with host cells and microorganisms.

[0003] 1. Fn is closely related to a variety of oral and systemic diseases: Porphyromonas gingivalis (Fn) is one of the main pathogens of periodontitis, and it works synergistically with Porphyromonas gingivalis (Pg) to enhance inflammatory responses and tissue destruction. It can induce the expression of antimicrobial peptides (such as β-defensin 2) and pro-inflammatory factors (such as IL-6 and IL-8) in oral epithelial cells, promoting the progression of periodontitis. Fn may promote disease progression in oral tumor models by activating the NF-κB pathway and the expression of pro-inflammatory factors (such as TNF-α); recent studies have found that Fn is significantly enriched in colorectal cancer tissues and is associated with tumorigenesis, proliferation, metastasis, and chemotherapy resistance. Its surface adhesion protein Fap2 can recognize Gal-GalNAc overexpressed on tumor cells, prompting Fn to migrate to the colorectal tumor microenvironment via the bloodstream; FadA protein promotes tumor cell proliferation by binding to E-cadherin and activating the β-catenin and Wnt signaling pathways; Fn is abundant in the intestinal tissue and feces of IBD patients, and may induce inflammatory responses through proteins such as FomA, exacerbating the pathological processes of Crohn's disease and ulcerative colitis; Fn is a common bacterium in the amniotic fluid of preterm pregnant women, and can invade umbilical cord endothelial cells, leading to placental inflammation and increasing the risk of preterm birth, miscarriage, and neonatal sepsis; Fn targets the Rab5a-YB-1 axis through FadA-containing outer membrane vesicles (OMVs), delivering them to joints, inducing local inflammation, and exacerbating rheumatoid arthritis symptoms.

[0004] 2. The FomA protein of Fn has attracted much attention due to its immunogenicity and potential vaccine target value: FomA is the major outer membrane pore protein of Fusobacterium nucleatum, with a molecular weight of approximately 41 kDa. It is located on the outer membrane of Fn and is responsible for substance transport and bacterial-host cell interactions. As a receptor protein, FomA participates in the co-aggregation of Fn with other oral pathogens (such as P. gingivalis), promoting dental plaque biofilm formation, which is a key step in the development of periodontitis. FomA can induce host cells (such as macrophages and epithelial cells) to secrete pro-inflammatory factors (such as IL-1β and IL-6), exacerbating the inflammatory response of periodontitis and IBD. FomA enhances the adhesion and invasion ability of Fn to host tissues, especially in the oral and intestinal environments.

[0005] FomA is highly immunogenic and can induce the body to produce specific antibodies (such as IgG and SIgA). Studies have shown that subcutaneous injection or oral administration of recombinant bacteria expressing FomA (such as Lactobacillus acidophilus and Lactobacillus plantarum) can induce high-titer antibodies in mouse serum and intestinal fluid, with antibody titers reaching their peak two weeks after secondary immunization. Oral administration of recombinant Lactobacillus plantarum expressing FomA can induce the production of SIgA and IgG in mouse intestines and serum, stimulate B cells, T cells, and dendritic cells, and significantly alleviate Fn-induced IBD symptoms (such as colonic atrophy and inflammatory cell infiltration).

[0006] 3. The significance of preparing monoclonal antibodies against Fn-FomA protein: Given that FomA is a major pore protein of the Fn outer membrane, it plays a crucial role not only in bacterial adhesion, biofilm formation, and inflammation induction, but also serves as an ideal antigen target due to its high immunogenicity. Therefore, the development of monoclonal antibodies against the Fn-FomA protein has significant scientific and clinical implications.

[0007] Monoclonal antibodies targeting FomA can be used to develop highly sensitive diagnostic tools that aid in the early diagnosis of periodontitis, colorectal cancer, or inflammatory bowel disease by detecting the abundance of fibroblasts (Fn) in the patient's oral cavity, intestines, or tumor tissue. For example, FomA-based ELISA or immunofluorescence assays can accurately identify Fn infection, particularly in colorectal cancer patients. FomA antibodies can help assess the enrichment of Fn in the tumor microenvironment by recognizing Fn surface antigens, providing a basis for disease staging and prognosis. Furthermore, FomA antibodies can be combined with imaging techniques for non-invasive monitoring of the dynamic changes in Fn-related infections.

[0008] FomA monoclonal antibodies possess potential therapeutic value. By blocking FomA-mediated adhesion of fibroblasts (Fn) to host cells or symbiotic bacteria and inhibiting biofilm formation, they can alleviate pathological damage associated with periodontitis or intestinal inflammation. Secondly, they can neutralize the pathogenic factors of Fn, reducing the expression of pro-inflammatory factors (such as IL-1β and IL-6) and alleviating inflammatory responses. Furthermore, through antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), FomA antibodies may possess potential killing efficacy against Fn, reducing their abundance in lesions. In the treatment of colorectal cancer, FomA antibodies may be used in combination with chemotherapy or immune checkpoint inhibitors to target Fn, thereby disrupting the pro-tumor microenvironment and improving treatment efficacy. Summary of the Invention

[0009] To address the aforementioned technical problems in existing technologies, this invention develops a monoclonal antibody for detecting the FomA protein in Fusobacterium nucleatum, which can specifically bind to the Fn-FormA protein. It also provides related applications and products, such as immunoassay or diagnostic kits, which are highly sensitive and specific, enabling rapid and accurate detection of Fusobacterium nucleatum abundance.

[0010] The objectives of this invention include: The first aspect of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0011] A second aspect of the present invention is to provide a recombinant protein.

[0012] A third aspect of the present invention aims to provide biological materials related to the monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention or the recombinant protein of the second aspect of the present invention.

[0013] A fourth aspect of the present invention is to provide a coupling agent.

[0014] The fifth aspect of this invention aims to provide the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fourth aspect in the preparation of products.

[0015] The sixth aspect of this invention aims to provide a product.

[0016] The seventh aspect of this invention is to provide a medicine.

[0017] The object of the eighth aspect of the present invention is to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0018] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof targeting the FomA protein of Fusobacterium nucleatum, said monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain.

[0019] In some embodiments of the present invention, the monoclonal antibody includes one or both of A) or B); A) FAMD6 The heavy chain includes: Heavy chain variable region, which includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; The light chain comprises: The light chain variable region, which includes CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region, The light chain variable region has the amino acid sequence shown in SEQ ID NO: 9; B) FAMD67 The heavy chain includes: Heavy chain variable region, which includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 17; The light chain comprises: Light chain variable region, which includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region; The light chain variable region has the amino acid sequence shown in SEQ ID NO: 25.

[0020] In some embodiments of the present invention, when IMGT is used as the definition scheme: A) FAMD6 The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. B) FAMD67 The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively. The amino acid sequences of the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0021] In some embodiments of the present invention, those skilled in the art may adopt other definition schemes recognized in the art, such as Kabat, Chothia, and Contact. Other CDR sequences obtained based on the heavy chain and light chain variable regions of the present invention are still within the protection scope of the present invention.

[0022] In some embodiments of the present invention: A) FAMD6 The amino acid sequence of the heavy chain variable region is SEQ ID NO: 1; The amino acid sequence of the light chain variable region is SEQ ID NO: 9; B) FAMD67 The amino acid sequence of the heavy chain variable region is SEQ ID NO: 17; The amino acid sequence of the variable region of the light chain is SEQ ID NO: 25.

[0023] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment further includes a constant region: A) FAMD6 The heavy chain includes four framework regions FR, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. The light chain comprises four framework regions FR, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. B) FAMD67 The heavy chain includes four framework regions FR, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively. The light chain includes four frame regions FR, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively.

[0024] In some embodiments of the present invention, based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the antibody light chain can be classified as a lambda (λ) chain or a kappa (κ) chain. The antibodies disclosed in this invention can be any of the above types or subtypes.

[0025] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment may be an isotype selected from IgG, IgA, IgM, IgE and IgD.

[0026] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment FAMD6 subclass is IgG3, and the monoclonal antibody FAME67 subclass is IgG2b. In some embodiments of the invention, the antigen-binding fragment comprises (i) a Fab fragment, i.e., a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) an F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments connected by disulfide bonds at the hinge region; (iii) a Fab' fragment, which is essentially a Fab fragment having a portion of the hinge region (see Fundamental Immunology (edited by Paul, Supplement 3, 1993); (iv) an Fd fragment consisting of a VH domain and a CH1 domain; (v) an Fd' fragment having a VH domain and a CH1 domain and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL domain and the VH domain of an antibody arm; (vii) a dAb fragment (Ward et al. (1989), Nature, Vol. 341: 544). (546 pages) It consists of (viii) a separate complementarity-determining region (CDR); and (ix) a nanobody, i.e., a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, these domains can still be linked via synthetic linkers using recombination methods, allowing these domains to be fabricated into a single protein chain where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988), Science, Vol. 242: p. 423). 426 pages; and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, Vol. 85: 5879 (Page 5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibodies. Furthermore, antigen-binding fragments also include "linear antibodies," which contain a pair of tandem Fd segments (VH...). CH1 VH CH1) and any of the aforementioned segments in modified form, this pair of tandem Fd segments together with the complementary light chain polypeptide form an antigen-binding region, and the modified forms of these segments retain antigen-binding activity.

[0027] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the use of the fragments can be screened in the same manner as for intact antibodies.

[0028] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv or scFv.

[0029] A second aspect of the invention provides a recombinant protein comprising: a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

[0030] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag, HA tag, GST tag, SUMO tag.

[0031] A third aspect of the invention provides biological materials relating to the monoclonal antibody or its antigen-binding fragment of the first aspect of the invention or the recombinant protein of the second aspect; said biological material comprising at least one of 1) to 12): 1) A nucleic acid molecule encoding a monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention, or a recombinant protein of the second aspect of the present invention; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A carrier containing the nucleic acid molecule described in 1); 4) A carrier containing the expression box described in 2); 5) Transgenic cell lines containing the nucleic acid molecules described in 1); 6) A transgenic cell line containing the expression cassette described in 2); 7) Transgenic cell lines containing the vector described in 3); 8) A transgenic cell line containing the vector described in 4); 9) Microorganisms containing the nucleic acid molecules described in 1); 10) Microorganisms containing the expression cassette described in 2); 11) Microorganisms containing the carrier described in 3); 12) Microorganisms containing the carrier described in 4).

[0032] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0033] In some embodiments of the present invention, the vector may be any suitable recombinant expression vector, including plasmids for amplifying nucleic acid molecules, or viral vectors for transfecting cells, including but not limited to retroviral vectors, DNA vectors, murine leukemia virus vectors, SFG vectors, plasmids, RNA vectors, adenovirus vectors, baculovirus vectors, Epstein-Barr virus vectors, papillomavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, adenovirus-associated vectors, lentiviral vectors, or any combination thereof. In some embodiments of the present invention, the transgenic cell line is a host cell that produces the monoclonal antibody or antigen-binding fragment of the present invention, including but not limited to prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.

[0034] In some embodiments, the transgenic cell line includes human cells, such as CHO cells (including but not limited to CHOS cells, CHO-K1 cells) and HEK293 cells (including but not limited to HEK293A, HEK293T and HEK293FS).

[0035] A fourth aspect of the present invention provides a conjugate comprising at least one of a monoclonal antibody or an antigen-binding fragment thereof from the first aspect of the present invention or a recombinant protein from the second aspect; and a conjugation portion comprising a detectable marker.

[0036] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, enzymes, fluorescent compounds, or any combination thereof; In some embodiments of the present invention, the fluorescent compounds include fluorescein, fluorescein isothiocyanate, rhodamine, and 5... dimethylamine l Naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), etc.

[0037] In some embodiments of the present invention, the enzymes include horseradish peroxidase, β-carotene... Galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When an antibody or antigen-binding fragment conjugates with a detectable enzyme, detection can be achieved by adding additional reagents. The enzyme uses these additional reagents to produce a distinguishable reaction product. For example, when horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product.

[0038] In some embodiments of the present invention, the radioactive labeling includes, but is not limited to, the following radioactive isotopes or radioactive nucleotides: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0039] In some embodiments of the present invention, monoclonal antibodies or antigen-binding fragments may also be conjugated with biotin and detected by indirect measurement of binding to avidin or streptavidin.

[0040] A fifth aspect of the invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fourth aspect in the preparation of a product.

[0041] In some embodiments of the present invention, the product includes one of d1) to d4): The product includes one of d1) to d4): d1) Products tested for Clostridium nucleatum; d2) Products containing the FomA protein from Fusobacterium nucleatum; d3) Drugs that inhibit Clostridium nucleatum infection; d4) Medications for the prevention and / or treatment of diseases associated with Fusobacterium nucleatum.

[0042] In some embodiments of the present invention, the diseases associated with Fusobacterium nucleatum include, but are not limited to: periodontitis, odontogenic infection, halitosis, colorectal cancer, Crohn's disease, ulcerative colitis, appendicitis, bacteremia, organ abscess, bacterial vaginosis, and adverse pregnancy.

[0043] In some embodiments of the present invention, the adverse pregnancy includes, but is not limited to, infertility, premature birth, premature rupture of membranes, and chorioamnionitis.

[0044] A sixth aspect of the present invention provides a product comprising a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the invention, a recombinant protein of the second aspect, and / or a conjugate of the fourth aspect.

[0045] In some embodiments of the present invention, the product includes at least one of reagents, detection plates, reagent kits, and detection chips.

[0046] In some embodiments of the present invention, the product includes supporting auxiliary reagents and consumables for testing.

[0047] A seventh aspect of the present invention provides a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the invention, a recombinant protein of the second aspect and / or a conjugate of the fourth aspect, and pharmaceutically acceptable excipients.

[0048] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: propellants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0049] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0050] In some embodiments of the present invention, the dosage form of the product includes one of the following: powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0051] In some embodiments of the present invention, the product is administered via the gastrointestinal tract or non-gastrointestinal route.

[0052] In some embodiments of the present invention, the gastrointestinal administration includes one of oral administration, sublingual administration, and rectal administration.

[0053] In some embodiments of the present invention, the non-gastrointestinal administration includes one of intravenous injection, subcutaneous injection, and mucosal administration.

[0054] In some embodiments of the present invention, the drug is one of d3) to d4): d3) Drugs that inhibit Clostridium nucleatum infection; d4) Medications for the prevention and / or treatment of diseases associated with Fusobacterium nucleatum.

[0055] The eighth aspect of the present invention provides a method for preparing a monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention, obtained by culturing the transgenic cell line or microorganism of the third aspect of the present invention.

[0056] The beneficial effects of this invention are: The monoclonal antibodies or their antigen-binding fragments FAMD6 and FAME67 obtained through screening in this invention can capture and detect Fn-FomA in a double-sandwich manner. Immunoassay or diagnostic kits constructed using the monoclonal antibodies FAMD6 and FAME67 can accurately detect recombinant Fn-FomA protein with a detection sensitivity of up to 3.9 ng / mL. The highest dilution for detecting Fn culture supernatant is 1:1600, demonstrating high sensitivity and promising application prospects. Furthermore, in mouse models, the monoclonal antibodies or their antigen-binding fragments FAMD6 and FAME67 exhibit higher protective efficacy in pregnant mice, effectively improving the rate of adverse pregnancy outcomes. Attached Figure Description

[0057] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The titer identification results are for the antibody prepared in Example 1.

[0058] Figure 2 The affinity assay results of the antibody prepared in Example 1 of this invention were determined using a Gator molecular interaction analyzer.

[0059] Figure 3 The results show the sensitivity determination of the detection kit prepared with antibodies in Example 2 of this invention.

[0060] Figure 4 The results show the protective effect of FomA monoclonal antibody in a pregnant mouse model of Fn gavage. Detailed Implementation

[0061] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0062] Example 1: Preparation and Characterization of Fn-FomA Monoclonal Antibody 1. Preparation of immunoantigens Natural FomA protein: The supernatant from Fn culture was concentrated and precipitated with 40% ammonium sulfate to obtain crude protein. The protein was dissolved in 20 mM PBS buffer (pH 7.4) and further purified using a Bio-Rad Enrich™ SEC 650 gel filtration (medium pressure) column. The molecular weight of the target FomA protein was approximately 42 kDa. Recombinant FomA protein: The gene corresponding to Fn-fomA 51–269aa (ATCC NO: 25586) was cloned and constructed into the PET28b expression plasmid. This plasmid was transformed into *E. coli* BL21(DE3) competent cells, and bacterial amplification and protein expression were induced according to standard procedures. The protein was further purified using a Bio-Rad IMAC (Immobilized Metal Affinity Chromatography) column. The molecular weight of the target protein was approximately 37 kDa. The purified recombinant protein was quantified using a standard Coomassie protein assay kit (PIERCE, Cat, No. ED62976). The obtained natural Fn-FomA protein can be used as an immunogenic antigen, while the recombinant FomA protein can be used for subsequent antibody screening and identification.

[0063] 2. Immunizing mice Three 4-6 week old female BALB / c mice were used for the first immunization. Freund's complete adjuvant was mixed with an equal volume of natural FomA protein and emulsified (final antigen concentration of 500 μg / mL). 50 μg of the protein was administered subcutaneously to each mouse at multiple sites. Subsequently, every 10 days, Freund's incomplete adjuvant was mixed with an equal volume of natural FomA protein and emulsified (dose as before). After a total of 4 immunizations, 100 μg of natural FomA protein was injected intraperitoneally into each mouse 3 days before fusion for a booster immunization.

[0064] 3. Determination of antibody titer in immune serum An indirect ELISA method for determining antibody titers in immune serum was established, and the specific procedure is as follows: Polystyrene microplates were coated with 50 mM pH 9.6 carbonate buffer containing 0.5 μg / mL natural Fn-FomA protein at 100 μL / well at 4°C overnight. The next day, the plates were blocked with 300 μL / well of blocking buffer containing 0.25% casein (Sigma) at 4°C overnight. The blocked buffer was discarded, dried, and stored at 4°C for use in the determination of antibody titers in mouse immune serum.

[0065] Blood was collected from the tail vein of mice 10 days after the first immunization. The mouse immune serum was diluted with PBS containing 0.1% BSA 10mM at 10°C. 3 ~10 6 Dilute the solution 1:1000 and add it to the above 96-well plate. Incubate at 37°C for 30 minutes. Wash the plate five times with 10mM PBS containing 0.1% Tween-20. Add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Sigma, INC.) diluted 1:1000 and incubate at 37°C for 30 minutes. Wash the plate (washing procedure as before). Add 100 μL of TMB chromogenic solution and incubate at room temperature in the dark for 10 minutes. Stop the reaction by adding 100 μL of 1M H2SO4 per well. Measure the absorbance at 450 nm. Use pre-immunization mouse serum as a negative control. A positive result is defined as a ratio of ≥2.1 between the measured value and the control value.

[0066] 4. Hybridoma preparation and screening Select serum antibody titers of 1×10⁻⁶ 6 Three days prior to fusion, mice were intraperitoneally injected with 100 μg of natural Fn-FomA protein. On the day of fusion, the spleens of the mice were aseptically removed, and a spleen cell suspension was prepared and mixed with the logarithmically growing mouse myeloma cell line NS-1 (commercial) at a ratio of 10:1. Fusion was then performed using 45% polyethylene glycol (PEG, MW4000, Sigma). The PEG solution was added to the cells as follows: 1.0 mL of PEG was slowly added to the cell-containing culture medium at 37°C, gently shaking while adding. The fusion was terminated with serum-free RPMI-1640 medium. The cells were centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were gently resuspended in 60 mL of RPMI-1640 medium containing 15% fetal bovine serum. This cell suspension was added to six 96-well culture plates and placed in a CO2 incubator (37°C, 5% CO2). The following day, 100 μL of selection medium containing hypoxanthine, aminopterin, and thymidine deoxyribonucleoside (HAT, Sigma) was added to each well. The culture was then changed with this selection medium every 3 days until cell clones formed.

[0067] To detect the presence of antibody-producing clones, the indirect ELISA method described above was used to detect the cell culture supernatant. Strongly positive hybridoma cells were selected for cloning, and the cloning was repeated 2-3 times using limiting dilution to obtain two stable antibody-secreting hybridoma cell lines, named FAMD6 and FAME67. Cells with 100% cloning positivity were expanded and cultured before being cryopreserved in liquid nitrogen.

[0068] 5. Preparation of ascites fluid and purification of anti-Fn-FomA protein monoclonal antibody The monoclonal antibodies FAMD6 and FAME67 were prepared by inoculating mice with corresponding hybridoma cells using an in vivo induction method.

[0069] The specific preparation method is as follows: Each mouse was injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant (Sigma). Within 1–2 weeks, 2 × 10⁻⁶ mg / mL of the adjuvant was administered. 6 One hybridoma cell line, either FAMD6 or FAME67, was suspended in 0.2 mL of serum-free RPMI 1640 medium and injected into the peritoneal cavity of mice. After 1–2 weeks, ascites fluid was drained using a No. 9 needle; this process could be repeated several times. The ascites fluid was then centrifuged, clarified, and stored at 4°C for later use.

[0070] The ascites antibody was purified using the caprylic acid-ammonium sulfate precipitation method. The ascites was diluted 2-fold with 60 mM, pH 5.0 acetate buffer, and the pH was adjusted to 4.8 with 0.1N hydrochloric acid. The liquid changed from clear to turbid. Caprylic acid was slowly added dropwise over 30 minutes at room temperature while stirring, with 33 μL of caprylic acid added per milliliter of pre-diluted ascites. A large amount of precipitate appeared. The solution was incubated at 4°C for 2 hours, centrifuged at 10,000 g at 4°C for 30 minutes, and the supernatant was collected. 1 / 10 volume of 100 mM phosphate buffer (pH 7.4) was added, and the pH was adjusted to 7.4 with 0.1N sodium hydroxide. Ammonium sulfate was slowly added with stirring in an ice bath, with 0.277 g of ammonium sulfate added per milliliter of liquid to achieve 45% saturation. The solution was incubated at 4°C overnight, centrifuged at 10,000 g at 4°C for 30 minutes, and the supernatant was discarded. The precipitate was dissolved in an appropriate amount of 10 mM phosphate buffer, and dialyzed overnight at 4°C using the same solution. The solution was changed three times. Quantification was performed using Coomassie Brilliant Blue Protein Assay Reagent (PIERCE, Cat, No. ED62976). After concentration determination, the antibody was added to a final concentration of 50% glycerol and stored at -80°C.

[0071] 6. Identification of subclasses of anti-Fn-FomA protein monoclonal antibodies Indirect ELISA was used to detect positive clones and determine their antibody subclasses. The specific procedure was as follows: 96-well plates were coated with natural Fn-FomA protein, blocked, and incubated with hybridoma cell culture supernatant. Then, the plates were incubated separately with 1:1000 diluted anti-mouse subclass-specific immunoglobulins (Suzhou Bio-Long Technology Co., Ltd., catalog number BF16001). HRP-labeled anti-mouse antibodies of various types and subclasses were added for reaction. Finally, the reaction was performed using a TMB substrate system and terminated with dilute sulfuric acid. The absorbance was measured using a microplate reader to determine the class or subclass of the tested monoclonal antibody.

[0072] The test results showed that the monoclonal antibody FAMD6 subclass was IgG3 and the monoclonal antibody FAME67 subclass was IgG2b.

[0073] 7. Identification of the binding ability of Fn-FomA monoclonal antibody Microplates were coated with natural Fn-FomA protein and identified using an indirect ELISA method. After blocking, serially diluted monoclonal antibodies FAMD6 or FAME67 (1000 ng / mL to 0.9 ng / mL) were added and incubated at 37°C for 1 h. Horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, Inc.) diluted 1:1000 was added at 100 μL / well and incubated at 37°C for 30 min. TMB chromogenic buffer was added and the reaction was developed at room temperature in the dark for 10 min. The reaction was terminated by adding 1 M H2SO4, and the absorbance at 450 nm was measured. A 450 ).

[0074] The results are as follows Figure 1 As shown, both the monoclonal antibodies FAMD6 and FAME67 have good binding ability to the natural Fn-FomA protein.

[0075] 8. Affinity identification of Fn-FomA monoclonal antibody Experiments were performed using a Gator™ molecular interaction instrument (Gator Bio, Suzhou, China) (all probes and buffers used were products of this company). First, the anti-mouse IgG Fc biosensor probe was pre-equilibrated in Q buffer until baseline stability, then incubated in 10 mg / mL mAbs solution for 240 seconds to achieve antibody immobilization. After capture, the sensor was again equilibrated in Q buffer for 60 seconds, then incubated with serially diluted native Fn-FomA protein (or Q buffer as a blank control) in the same buffer system for 180 seconds, and finally incubated in Q buffer for 300 seconds to complete the dissociation process. Experimental data were processed using the Gator™ system's accompanying analysis software, and the binding rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) were calculated through kinetic curve fitting. This method offers advantages such as label-free operation, real-time monitoring, and high-throughput analysis, and can accurately characterize the interaction features between mAbs and target proteins.

[0076] The results are as follows Figure 2 As shown, the calculated affinity KD values ​​for monoclonal antibodies FAMD6 and FAME67 are 0.514 nM and 1.38 nM, respectively, both meeting the affinity requirements for diagnostic antibodies.

[0077] 9. Sequence identification of monoclonal antibodies According to testing by a commercial testing company, the heavy chain variable region sequence of the monoclonal antibody FAMD6 is as follows: EVQLQQSGPELVKPGASVKMSCKTSGYSFTDFYMKWMKQSHGKSLEWIGHINPNNGDTFYNQKFQVKATLTVDKSSSTAYMQLNSLTSEDSAVYFCAIITTVPPMDCWGQGTSVTVSS (SEQ ID NO: 1).

[0078] It contains three CDRs: heavy chain CDR1 (sequence: GYSFTDFY, SEQ ID NO: 2), heavy chain CDR2 (sequence: INPNGDT, SEQ ID NO: 3), and heavy chain CDR3 (sequence: AIITTVPPMDC, SEQ ID NO: 4); and four frame regions FR: heavy chain FR1 (sequence: EVQLQQSGPELVKPGASVKMSCKTS, SEQ ID NO: 5), heavy chain FR2 (sequence: MKWMKQSHGKSLEWIGH, SEQ ID NO: 6), heavy chain FR3 (sequence: FYNQKFQVKATLTVDKSSSTAYMQLNSLTSEDSAVYFC, SEQ ID NO: 7), and heavy chain FR4 (sequence: WGQGTSVTVSS, SEQ ID NO: 8).

[0079] The light chain variable region sequence of the monoclonal antibody FAMD6 is as follows: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEVEDLGVYYCSQGSHVPYTFGGGTKLEIK (SEQ ID NO: 9).

[0080] It contains three CDRs: light chain CDR1 (sequence: QSIVHSNGNTY, SEQ ID NO: 10), light chain CDR2 (sequence: KVS, SEQ ID NO: 11), and light chain CDR3 (sequence: SQGSHVPYT, SEQ ID NO: 12); and four frame regions FR: light chain FR1 (sequence: DVLMTQTPLSLPVSLGDQASISCRSS, SEQ ID NO: 13), light chain FR2 (sequence: LEWYLQKPGQPPKLLIY, SEQ ID NO: 14), light chain FR3 (sequence: NRFSGVPDRFSGSGSGTDFTLKISRVEVEDLGVYYC, SEQ ID NO: 15), and light chain FR4 (sequence: FGGGTKLEIK, SEQ ID NO: 16).

[0081] The heavy chain variable region sequence of the monoclonal antibody FAME67 is as follows: QVQLQQSGAELVRPGTSVKLSCKALGYKFIDFEMYWVKQTPVHGLEWIGAIHPGSGGTAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCRPLLLLEEPMDYWGQGTSVTVSS (SEQ ID NO: 17).

[0082] It contains three CDRs: heavy chain CDR1 (sequence: GYKFIDFE, SEQ ID NO: 18), heavy chain CDR2 (sequence: IHPGSGGT, SEQ ID NO: 19), and heavy chain CDR3 (sequence: RPLLLLEEPMDY, SEQ ID NO: 20); and four frame regions FR: heavy chain FR1 (sequence: QVQLQQSGAELVRPGTSVKLSCKAL, SEQ ID NO: 21), heavy chain FR2 (sequence: MYWVKQTPVHGLEWIGA, SEQ ID NO: 22), heavy chain FR3 (sequence: AYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYC, SEQ ID NO: 23), and heavy chain FR4 (sequence: WGQGTSVTVSS, SEQ ID NO: 24).

[0083] The light chain variable region sequence of the monoclonal antibody FAME67 is as follows: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYVNWFLQRPGQSPKRLISLVSKLDSGVPDRFAGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTNLEIK (SEQ ID NO: 25).

[0084] It contains three CDRs: light chain CDR1 (sequence: QSLLDSDGKTY, SEQ ID NO: 26), light chain CDR2 (sequence: LVS, SEQ ID NO: 27), and light chain CDR3 (sequence: WQGTHFPQT, SEQ ID NO: 28); and four frame regions FR: light chain FR1 (sequence: DVVMTQTPLTLSVTIGQPASISCKSS, SEQ ID NO: 29), light chain FR2 (sequence: VNWFLQRPGQSPKRLIS, SEQ ID NO: 30), light chain FR3 (sequence: KLDSGVPDRFAGSGSGTDFTLKISRVEAEDLGVYYC, SEQ ID NO: 31), and light chain FR4 (sequence: FGGGTNLEIK, SEQ ID NO: 32).

[0085] Example 2: Preparation of FomA protein immunoassay or diagnostic kit This embodiment describes the development of an immunoassay or diagnostic kit for Fusobacterium nucleatum FomA protein (i.e., Fn-FomA protein) based on the monoclonal antibodies FAMD6 and FAME67 obtained in Example 1.

[0086] 1. Reagent composition 1) Microplate coated with monoclonal antibody FAMD6.

[0087] The specific preparation process is as follows: The monoclonal antibody FAMD6 was diluted to 10 μg / mL with 10 mM phosphate buffer (pH 7.6), and 150 μL / well was used to coat polystyrene microplates. The plate was incubated overnight at 4°C. After drying, 300 μL / well of 0.25% casein (Sigma) blocking buffer was added to each well, and the plate was incubated overnight at 4°C to block non-specific binding sites. The strips were then spin-dried, vacuum-dried for 12–24 h, and vacuum-packed in aluminum foil bags for storage at 4°C for later use.

[0088] 2) Sample processing solution: composed of 0.1% BSA and 0.1% Tween-20 PBS.

[0089] 3) Working solution of horseradish peroxidase-labeled monoclonal antibody FAME67.

[0090] The specific preparation process is as follows: Dissolve 5 mg of horseradish peroxidase in 1 mL of distilled water, add 0.2 mL of freshly prepared 0.1 M sodium periodate and stir for 30 min in the dark. Dialyze overnight at 4°C in 1 mM pH 4.4 sodium acetate buffer. The next day, adjust the pH of 10 mg of monoclonal antibody FAME67 to 9.5 with 0.2 M pH 9.5 carbonate buffer, add the dialyzed horseradish peroxidase, and adjust the pH to 9.5 again with 0.2 M pH 9.5 carbonate buffer. Stir gently at room temperature in the dark for 2–3 h, then add 0.1 mL of freshly prepared 4 mg / mL sodium borohydride and incubate overnight at 4°C in the dark. The next day, add an equal volume of saturated ammonium sulfate dropwise with stirring on an ice bath in the dark (adjust the pH of the ammonium sulfate to 7.2 with ammonia before use). Let stand at 4°C for 6 h, then centrifuge at 12000 rpm for 30 min at 4°C. Discard the supernatant and use an appropriate amount of 10 mM pH 7.2 buffer. The precipitate was resuspended in PBS buffer and dialyzed overnight at 4°C, with the buffer changed three times. The conjugate was collected and a protectant containing 1% BSA and 50% glycerol was added. Finally, it was diluted 1000 times with phosphate buffer to obtain the working solution of horseradish peroxidase-labeled monoclonal antibody FAME67.

[0091] 4) Concentrated washing solution: 20×PBS containing 2% Tween-20, i.e., 1 L of solution contains 4.56 g NaH2PO4, 58.02 g Na2HPO4•12H2O, and 175.3 g NaCl. After autoclaving at 15 psi for 20 min, add 20 mL of Tween-20 and stir well. Dilute 20 times before use.

[0092] 5) Positive control solution: recombinant Fn-FomA protein expressed by Escherichia coli at a concentration of 1 µg / mL.

[0093] 6) Negative control solution: 10mM pH7.4 PBS containing 0.1% Tween-20, i.e., 1 L of solution contains 4.56g NaH2PO4, 58.02g Na2HPO4•12H2O, 175.3g NaCl, autoclaved at 15 psi for 20 min, diluted 20 times and then 0.1% Tween-20 is added.

[0094] 7) Developing solution: Composed of developing solutions A and B. Use equal amounts of both and mix thoroughly. The components of developing solutions A and B are as follows: Colorimetric solution A: Dissolve 0.89 g citric acid and 0.16 g disodium EDTA in 1000 mL of water, autoclave at 115 °C for 30 min, cool to 90 °C, add 0.25 g TMB, shake well, and store at 4 °C in the dark. Colorimetric solution B: Dissolve 9.33g citric acid and 14.6g disodium EDTA in 1000 mL of water, pressurize at 115℃ for 30 min, then reduce the temperature to 90℃ and add 12.8 mL of 0.75% hydrogen peroxide urea. Shake well and store at 4℃ in the dark.

[0095] 8) Termination solution: 1M H2SO4.

[0096] 2. Instructions for using the kit (double antibody sandwich ELISA detection method) 1) Sample testing Take 100 μL of the sample to be tested and add it to a microplate coated with monoclonal antibody FAMD6. Set up negative and positive controls at the same time. Incubate at 37℃ for 1 h. Wash the strip with concentrated washing buffer diluted 20 times. After washing the plate five times, add 100 μL of HRP-labeled monoclonal antibody FAME67 diluted 1:500 per well. Incubate at room temperature for 30 min. Wash the plate eight times as above. Add 100 μL of colorimetric reagent (colorimetric reagent A and B are mixed in equal volumes and prepared fresh for use). Incubate at room temperature in the dark for 10 min. Add 100 μL of stop solution per well to stop the reaction.

[0097] 2) Result Interpretation: Zero the instrument using the blank aperture and measure the absorbance at a wavelength of 450 nm. A (Value). The experiment is valid if the average value of the positive control is ≥0.50 and the average value of the negative control is ≤0.10. Sample A Value ≥ negative control A If the average value is multiplied by 2.1, it is considered positive; otherwise, it is considered negative.

[0098] Example 3: Efficacy test of the FomA protein immunoassay or diagnostic kit This example demonstrates the sensitivity determination of the immunoassay or diagnostic kit from Example 2.

[0099] 1. Sensitivity determination of recombinant Fn-FomA protein Recombinant FomA protein was serially diluted 11 times starting at 1000 ng / mL and detected using the diagnostic kit from Example 2. A negative control solution was used as a negative control. A 450 The lowest concentration of recombinant FomA protein corresponding to a value greater than or equal to 2.1 times the average value of the negative control is used as the sensitivity of this method for detecting the recombinant antigen.

[0100] The results are as follows Figure 3 As shown in the left figure, when the recombinant FomA protein was diluted to 3.9 ng / mL, A 450 =0.259, which is higher than the cutoff value (0.25). Therefore, according to the above judgment criteria, the sensitivity of the diagnostic kit in Example 2 for detecting recombinant FomA protein is approximately 3.9 ng / mL.

[0101] 2. Sensitivity determination of natural Fn-FomA protein The Fn culture supernatant was serially diluted eight times, starting at 1:100, and detected using the diagnostic kit from Example 2. Normal culture medium was used as a negative control. A 450 The lowest dilution concentration corresponding to a value greater than or equal to 2.1 times the average value of the negative control is used as the sensitivity of this method for detecting natural antigens.

[0102] The results are as follows Figure 3 As shown in the right figure, when the supernatant of the Fn culture was diluted to 1:1600, A 450 =0.289, which is higher than the cutoff value (0.25). Therefore, according to the above judgment criteria, the sensitivity of the diagnostic kit in Example 2 for detecting natural FomA protein is approximately equivalent to 1:1600 diluted Fn culture supernatant.

[0103] Example 4: Validation of the therapeutic function of Fn-FomA monoclonal antibody This embodiment demonstrates the protective function of the antibody prepared in Example 1 in animals.

[0104] 1. Experimental Methods The experimental procedure was as follows: Thirty-two 6-8 week old female C57BL / 6 mice were randomly divided into four groups (n=8 per group, 4 mice per cage, for a total of 8 cages). All mice were administered a quadruple antibiotic ABX (vancomycin 100 mg / kg, neomycin, metronidazole, and ampicillin 200 mg / kg each) by gavage for 5 consecutive days to clear intestinal bacteria. On the evening of the seventh day after antibiotic treatment, eight 8-week-old male mice of similar weight were randomly assigned to the eight cages in the four groups for co-hospitalization. On the morning of the eighth day, the female mice were examined, and any mating plugs were immediately removed. Simultaneously, male mice were exchanged between cages, continuing this process for 2-3 days until all female mice were pregnant. During this period, the protective effect of the antibodies on the mice was observed by continuous Fn / PBS gavage and tail vein injection. The experiment was repeated three times.

[0105] The specific experimental plans for the four groups are as follows: 1) PBS Control Group: Mice that had completed ABX treatment were administered 200 μL / mouse of PBS by gavage once a day for 25 consecutive days; 2) Fn treatment group (Fn): Fn was counted by colony count at a ratio of 10-1. 8 Mice that had completed ABX treatment were administered CFU / 200 μL / mouse via gavage once a day for 25 consecutive days. 3) Fn combined with FAME67 monoclonal antibody treatment group (Fn+FAME67): After 5 days of ABX treatment, mice were first injected with FAME67 monoclonal antibody (100ug / mouse) via tail vein, followed by 10 8 Mice were administered CFU at a dose of 200 μL / mouse once a day for 25 consecutive days. 4) Fn combined with FAMD6 monoclonal antibody treatment group (Fn+FAMD6): After 5 days of ABX treatment, mice were first injected with FAMD6 monoclonal antibody (100ug / mouse) via tail vein, followed by 10 8 Mice were administered CFU / 200μL / mouse via gavage once a day for 25 consecutive days.

[0106] 2. Experimental Results The results are as follows Figure 4 As shown, compared with the Fn treatment group alone, the injection of monoclonal antibodies FAME67 and FAMD6 improved the pregnancy rate of female mice. Among them, FAMD6 had higher protective effect and the difference was statistically significant (P = 0.001). The pregnancy rate of mice reached 75%, which was not significantly different from the PBS control group (P>0.05).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof targeting a Fusobacterium nucleatum FomA protein, characterized in that: the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; the monoclonal antibody comprises one or both of A) or B); A) FAMD6 the heavy chain comprises: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region; the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 1; the light chain comprises: a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region, the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 9; B) FAMD67 the heavy chain comprises: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region; the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 17; the light chain comprises: a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region; the light chain variable region has the amino acid sequence set forth in SEQ ID NO:

25.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, characterized in that: when the CDRs are defined by the IMGT definition scheme: A) FAMD6 the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, respectively; B) FAMD67 the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are set forth in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, respectively.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1, characterized in that: A) FAMD6 the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1; the amino acid sequence of the light chain variable region is SEQ ID NO: 9; B) FAMD67 the amino acid sequence of the heavy chain variable region is SEQ ID NO: 17; the amino acid sequence of the light chain variable region is SEQ ID NO:

25.

4. The monoclonal antibody or antigen-binding fragment thereof of claim 1, characterized in that: The monoclonal antibody or the antigen-binding fragment thereof comprises at least one of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fv, or a scFv.

5. A recombinant protein comprising: the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, and a tag sequence.

6. A biological material associated with the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, or the recombinant protein according to claim 5, the biological material comprising at least one of 1) to 12): 1) a nucleic acid molecule encoding the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, or the recombinant protein according to claim 5; 2) an expression cassette comprising the nucleic acid molecule of 1); 3) a vector comprising the nucleic acid molecule of 1); 4) a vector comprising the expression cassette of 2); 5) a transgenic cell line comprising the nucleic acid molecule of 1); 6) a transgenic cell line comprising the expression cassette of 2); 7) a transgenic cell line comprising the vector of 3); 8) a transgenic cell line comprising the vector of 4); 9) a microorganism comprising the nucleic acid molecule of 1); 10) a microorganism comprising the expression cassette of 2); 11) a microorganism comprising the vector of 3); and 12) a microorganism comprising the vector of 4).

7. A conjugate comprising: at least one of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, and the recombinant protein according to claim 5; and a conjugation moiety comprising a detectable label.

8. Use of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, the recombinant protein according to claim 5, the biological material according to claim 6, or the conjugate according to claim 7, in the manufacture of a product; the product comprising one of d1) to d4): d1) a product for detecting Fusobacterium nucleatum; d2) a product for detecting the FomA protein of Fusobacterium nucleatum; d3) a drug for inhibiting the infection of Fusobacterium nucleatum; and d4) a drug for preventing and / or treating a disease associated with Fusobacterium nucleatum.

9. A product for detecting Fusobacterium nucleatum, comprising at least one of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, the recombinant protein according to claim 5, or the conjugate according to claim 7; the product comprising at least one of a reagent, a detection plate, a kit, and a detection chip.

10. A pharmaceutical composition comprising at least one of the monoclonal antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, the recombinant protein according to claim 5, or the conjugate according to claim 7, and a pharmaceutically acceptable excipient. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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