An intestinal microbiota Bacteroides fragilis toxin-neutralizing nanobody and its application

Abstract

The present invention discloses an intestinal microbiota Bacteroides fragilis toxin-neutralizing nanobody and its application. By deeply studying the enterotoxigenic Bacteroides fragilis and the toxin BFT secreted by it in the bodies of patients with colorectal cancer and breast cancer, nanobodies against BFT are expressed by phage display technology; nanobodies with high binding affinity to the antigen are screened out through biopanning technology; a nanobody with the ability to neutralize the toxicity of BFT, also known as Nb2.43, is obtained. The present invention expresses and optimizes to obtain a BFT nanobody protein with high affinity, stability and uniformity, opening up a new field for the treatment of colorectal cancer and breast cancer with nanobodies targeting BFT, and having good research value and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to an antibody that can neutralize the activity of BFT, specifically to a toxin-neutralizing nanobody of Bacteroides fragilis and its application. Background Technology

[0002] Bacteroides fragilis is ubiquitous in the human gut microbiota, accounting for 0.1%-0.5% of the total gut bacteria. It is the most common anaerobic bacterium isolated from abdominal abscesses and blood infection samples. Bacteroides fragilis can ferment sugars, starches, and fibers into volatile fatty acids, which are absorbed and utilized by host cells to provide nutrition and energy. Bacteroides fragilis is an opportunistic pathogen; when the host's intestinal barrier is compromised, it can spread to areas outside the gastrointestinal tract, causing abscesses and infections in organs and tissues such as the central nervous system, neck, mouth, lungs, and abdomen. Based on toxin production, Bacteroides fragilis can be divided into enterotoxigenic Bacteroides fragilis (ETBF) and non-toxigenic Bacteroides fragilis (NTBF). ETBF can rapidly secrete Bacteroides fragilis toxin (BFT), leading to ulcerative colitis, toxin-mediated acute diarrhea, and bacteremia. Studies have found that persistent co-colonization of ETBF and pks+ Escherichia coli in the colonic mucosa can promote tumor formation in patients with familial adenomatous polyposis (FAP). In a follow-up study of 150 patients who underwent colonoscopy, it was found that about 80% of patients who carried ETBF at the first colonoscopy developed precancerous lesions 12-15 years later. ETBF colonization may be a potential marker for the early occurrence of colorectal cancer. At the same time, ETBF can colonize the breast and intestines, rapidly inducing breast epithelial hyperplasia and promoting the occurrence and metastasis of breast cancer [5]. BFT is the only recognized virulence factor in enterotoxigenic Bacteroides fragilis (ETBF). Therefore, BFT can be used as a biomarker to predict the transformation of colorectal cancer and the occurrence and development of diseases such as breast cancer. New therapies for cancer and related diseases can be developed by studying treatments that target BFT.

[0003] Currently, antibiotics remain the primary treatment for ETBF, with carbapenems and metronidazole being the most effective methods for treating Bacteroides fragilis infections. Surveys have found that the reported rate of metronidazole-resistant Bacteroides fragilis ranges from 0.5% to 7.8%. Bacteroides fragilis exhibits high antibiotic resistance and possesses a wealth of antibiotic resistance mechanisms. Over the past decade, the number of multidrug-resistant isolates of Bacteroides fragilis has increased, and its antimicrobial resistance (AMR) is rising, particularly with carbapenems and metronidazole, two commonly used antibiotics for treating Bacteroides fragilis infections. Therefore, developing new treatment and detection methods is crucial.

[0004] BFT is a Zn 2+ The dependent metalloproteinase is approximately 20 kDa in size. For example... Figure 1As shown, ETBF first synthesizes a BFT precursor protein with a molecular weight of approximately 45 kDa. The precursor protein consists of 397 amino acid residues, including a signal peptide containing 18 amino acid residues, a propeptide region containing 193 amino acid residues, and a catalytically active region containing 186 amino acid residues. The BFT propeptide region inhibits the activity of its catalytic domain through an aspartate transfer mechanism. After processing by the cysteine ​​protease fragipain, the BFT precursor protein secretes the catalytically active domain (active BFT, aBFT) into the culture supernatant. Studies have shown that aBFT can induce extracellular cleavage of epithelial cadherin (E-cadherin) in intestinal epithelial cells, disrupting intercellular junctions and leading to tumor cell invasion and metastasis. E-cadherin is a type I classical cadherin, a transmembrane glycoprotein, and a classic member of the cadherin family, involved in mediating cell signal transduction and cell adhesion. Abnormal E-cadherin function leads to disordered cell adhesion, disruption of tight junctions, and impaired tissue integrity. aBFT, a zinc-dependent metalloproteinase, can specifically cleave the extracellular terminus of E-cadherin within 1 minute, breaking it down into a ~80 kDa soluble extracellular fragment and a ~40 kDa C-terminal fragment. Studies have shown that aBFT promotes inflammatory bowel disease and induces morphological and functional changes in normal breast epithelial cells and breast cancer cells by cleaving E-cadherin, resulting in highly migratory and invasive phenotypes. Cleavage of E-cadherin in intestinal epithelial cells by aBFT leads to impaired cell adhesion, disruption of intercellular junctions, impaired tight junctions, and increased intestinal permeability, activating pro-tumor and pro-inflammatory signaling pathways within intestinal epithelial cells and promoting the occurrence and development of intestinal diseases. Studies by Liam Chung et al. have shown that aBFT can trigger colonic immune cells to produce IL-17, causing mucosal immune responses. IL-17 directly acts on colonic epithelial cells, further promoting the activation of inflammation-related signaling pathways NF-κB and STAT-3, thereby inducing the release of CXC chemokines, which in turn recruits more immune cells to participate in the inflammatory response and promotes colonic tumor development. The intracellular domain of E-cadherin usually binds to α- and β-catenin [8]. When E-cadherin is cleaved by aBFT, it promotes β-catenin to enter the nucleus, binds to the LEF / TCF transcription factor family, and initiates the transcription of the downstream target gene c-myc. ETBF can also colonize in the breast, and by secreting aBFT, it activates the Notch1 and β-catenin signaling pathways in breast cancer and breast cancer epithelial cells, thereby inducing breast hyperplasia and promoting the growth and metastasis of breast cancer cells.

[0005] Nanobodies are single-domain heavy chain antibodies containing only one variable region (VHH) and two CH2 and CH3 regions on the heavy chain. The VHH retains all antigen-binding capacity and is the smallest intact antigen-binding fragment, only 15 kDa. Nanobodies are characterized by small molecular weight, short preparation cycle, high stability, weak immunogenicity, high antigen binding capacity, and good tissue penetration, and are widely used in molecular imaging, tumor diagnosis, immunotherapy, and drug delivery.

[0006] Currently, no neutralizing nanobodies related to intestinal bacterial toxins have been reported. Screening for nanobodies that neutralize BFT toxicity, blocking the binding of aBFT to host cells, and establishing accurate, effective, and specific BFT treatment methods are of significant research value and practical importance for treating diseases caused by ETBF. Therefore, developing BFT neutralizing nanobodies with clinical application potential has great practical significance and application value. Summary of the Invention

[0007] The purpose of this invention is to provide a toxin-neutralizing nanobody of Bacteroides fragilis valvularis and its application.

[0008] To achieve the above objectives, the present invention employs the following technical solution:

[0009] This invention discloses a toxin-neutralizing nanobody of Bacteroides fragilis, a gut microbiome. The heavy chain of this neutralizing nanobody includes three antigen complementarity-determining regions, namely CDR1, CDR2, and CDR3.

[0010] The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0011] Preferably, the heavy chain of the neutralizing nanobody further includes four framework regions, namely FR1, FR2, FR3 and FR4; wherein the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0012] More preferably, the amino acid sequence of the neutralizing nanobody is shown in SEQ ID NO: 8.

[0013] The present invention also discloses a nucleic acid encoding a toxin-neutralizing nanobody of Bacteroides fragilis fragilis with the above-mentioned amino acid sequence as shown in SEQ ID NO: 8, the nucleotide sequence of which is shown in SEQ ID NO: 9.

[0014] The present invention also discloses a prokaryotic expression vector containing the nucleic acid of the above-mentioned Bacteroides fragilis toxin neutralizing nanobody.

[0015] The present invention also discloses a prokaryotic host cell containing the above-mentioned prokaryotic expression vector.

[0016] This invention also discloses the application of the above-mentioned intestinal microorganism Bacteroides fragilis toxin neutralizing nanobodies, nucleic acids, prokaryotic expression vectors or prokaryotic host cells in the preparation of formulations for treating tumors.

[0017] Preferably, the tumor is colorectal cancer.

[0018] Preferably, the tumor is breast cancer.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention, through in-depth research on Bacteroides fragilis toxin, employs phage display technology to express BFT nanobodies. Using bioscreening technology, nanobodies with high binding affinity to the antigen are selected, specifically those containing the catalytically active domain (active B. fragilis toxin, hereinafter referred to as aBFT), also known as Nb2.43. This invention expresses and optimizes to obtain aBFT-neutralizing nanobody protein with high affinity and stable homogeneity. ITC testing of antibody binding activity showed that nanobody Nb2.43 exhibits the highest binding affinity (KD) at 5.59 nM, making it the first specific antibody capable of neutralizing aBFT. It can be used to identify aBFT blockade in patients and, as one of the treatment methods for colorectal cancer and breast cancer, has significant scientific and clinical application value. This invention opens up a new field for testing the therapeutic potential of Bacteroides fragilis and their toxins produced by human intestinal bacteria, demonstrating excellent research value and application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the BFT metalloproteinase protein.

[0022] Figure 2 Image showing the purification of recombinant BFT1-sFL antigen protein;

[0023] Figure 3 The ELISA method was used to evaluate the immunogenicity.

[0024] Figure 4 The graph shows the results of calculating the insertion rate using PCR by randomly selecting 20 colonies.

[0025] Figure 5To verify the experimental results of nanobody size and integrity based on SDS-PAGE Coomassie Brilliant Blue staining results;

[0026] Figure 6 This is a gel image after purification using aBFT molecular sieves.

[0027] Figure 7 Figure 1 shows the results of aBFT bioactivity validation; (a) represents the control group; (b) represents the group treated with aBFT; scale bar: 50 μm.

[0028] Figure 8 Affinity detection of Nb2.43 with aBFT.

[0029] Figure 9 Nb2.43 has a neutralizing effect on all three subtypes of aBFT.

[0030] Figure 10 The effect of Nb2.43 on the expression of E-cadherin in NCM460 and HT29 cells by aBFT was investigated; (a) represents the expression level of E-cadherin protein in NCM460 cells; (b) represents the expression level of E-cadherin protein in HT29 cells; GAPDH was used as an internal control protein.

[0031] Figure 11 To verify the efficacy of the neutralizing antibody in mice in vivo, HE staining was performed on mouse colon tissue. (Image of the results). Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0033] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0034] The present invention will now be described in further detail with reference to the accompanying drawings:

[0035] This invention utilizes phage display technology to screen nanobody clones that specifically bind to the target recombinant protein BFT mature somatic protein (aBFT) from single-domain heavy chain antibodies used in alpaca immunization. Alpacas were immunized with universal BFT recombinant protein, and leukocytes were isolated from the blood. A phage display library was constructed using phage display technology. Phages binding to the BFT protein were obtained through three consecutive biopanning processes. After sequencing and bioalignment, high-affinity nanobodies against aBFT were screened using enzyme-linked immunosorbent assay (ELISA).

[0036] 1. Construction and protein expression of the BFT prokaryotic expression system

[0037] Full-length prokaryotic expression plasmids of bft1-sFL without the signal peptide were constructed. Fpn was constructed on the pET28a vector using NcoI and EcoRI restriction endonuclease sites, with a 6*HIS tag added to the N-terminus of the protein. Fpn was also constructed on the pET28a vector using NcoI and EcoRI restriction endonuclease sites, with a 6*HIS tag added to the C-terminus of the protein. Expression of the recombinant BFT protein was induced by IPTG. When the OD600 of the bacterial culture medium was approximately 0.6, 0.4 M IPTG was added, and induction was performed overnight at 18°C. BFT1-sFL exhibited good solubility. The size exclusion chromatography results of the BFT1-sFL recombinant protein are shown below. Figure 2 As shown.

[0038] 2. Construction of a nanobody library using BFT1-sFL as antigen

[0039] 1) Antibody detection in alpaca immune serum

[0040] A healthy 5-year-old female alpaca was subcutaneously injected six times weekly with 100 μg of purified BFT1-sFL protein and an immune adjuvant. Peripheral blood was collected from the jugular vein before the first injection and on day 7 after the last injection. Serum was separated, and antibody titers were compared using ELISA. 100 μg / mL of BFT1-sFL protein was coated onto 96-well plates, with PBS used as a negative control. After washing and blocking, serially diluted pre- and post-immunization sera were added. The reaction was performed using HRP-conjugated goat anti-Llama antibody as a secondary antibody and ABTS reagent. Absorbance was measured at 405 nm using a microplate reader. Results are shown below. Figure 3 ,from Figure 3The results show that the level of antibodies targeting BFT1-sFL in alpaca serum was significantly higher after immunization than before immunization, proving that the method of subcutaneous injection of BFT recombinant protein mixed with immune adjuvant can successfully induce humoral immune response in alpacas. The immunization of experimental animals achieved the expected purpose, and subsequent work such as library construction can be carried out.

[0041] 2) Construction of phage libraries

[0042] Seven days after the last immunization, 100 mL of peripheral blood was collected from the jugular vein of alpacas. Peripheral blood mononuclear cells were isolated using Sepmate tubes and Lymphoprep. Total RNA was extracted from PBMCs using Trizol reagent, and cDNA was synthesized using Random primers and reverse transcriptase.

[0043] Nested PCR amplification of the VHH gene was performed using the following primers:

[0044] CALL001(5'-GTCCTGGCTGTTCTCTCTCCAAGG-3')

[0045] CALL002(5'-GGTACTGCTGTTTGAACTGTCC-3')

[0046] Gene fragments (700 bp) encoding heavy chain antibodies were extracted by rapid gel electrophoresis using 1% agarose gel electrophoresis.

[0047] Then, the following primers were used as templates for the second PCR:

[0048] VHH-for(5'-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3')

[0049] VHH-Back(5'-GATGTGCA GCAGGA GTCT GGRGGAG-3')

[0050] These primers were designed for frame 1 and frame 4 regions of VHH and contain PstI and Eco91I restriction sites. The second-round PCR products were recovered by electrophoresis, and the phage vector pMES4 was digested with PstI, XbaI, and Eco91I restriction endonucleases. The second-round PCR products were then digested with PstI and Eco91I. The digested pMES4 and PCR products were ligated using T4 DNA ligase. The recombinant vector was transfected into *E. coli* TG1 competent cells and cultured on LB agar plates containing ampicillin.

[0051] Randomly select 20 colonies and perform colony PCR using the following vector primers. Calculate the insertion rate:

[0052] GIII(5'-CCACAGACCCCTCATAG-3')

[0053] MP57(5'-TTATGCTTCCGGCTCGTATG-3').

[0054] Transformed TG1 cells were infected with M13K07 helper phage to display the VHH fragment. Infected bacteria were cultured overnight in a medium containing ampicillin and kanamycin. After centrifugation at medium temperature, the supernatant was mixed with PEG6000 / NaCl to separate the phages, and the phage particles were then resuspended in 1 mL of ice-cold PBS. The VHH library reached a size of 1.93 × 10⁻⁶. 7 / mL. PCR screening of 20 randomly selected colonies showed that most clones had inserted the VHH gene, with an insertion rate of 95%. See [link to results]. Figure 4 , Figure 4 For agarose gel electrophoresis to detect the size of PCR products from colonies (1-20 colonies), a band with a molecular weight of 700 bp was considered a clone with the inserted VHH fragment, and a band with a molecular weight of approximately 400 bp was considered to have been transformed into an empty plasmid. M: DL2000 nucleic acid marker. 1-20 refers to 20 randomly selected colonies.

[0055] 3. Nanobody Screening

[0056] 1) Biological screening

[0057] Three rounds of biopanning were performed on 96-well plates coated with 100 μL of BFT1-sFL protein to enrich phages specifically binding to BFT1-sFL. After blocking, the plates were washed five times. The phage library was added to the antigen and negative wells, incubated at room temperature for 2 h, washed 10-15 times, and eluted with protease. 10 μL of phage eluted from the antigen and negative wells were serially diluted and used to infect logarithmic-phase *E. coli* TG1, and streaked onto LB agar plates containing ampicillin. The enrichment of phages containing BFT1-sFL specifically binding to VHHs was evaluated by comparing the titers of phages in the antigen and negative wells, as shown in Table 1. The remaining phages were used to infect TG1 and cultured overnight. M13K07 helper phage was added to the bacterial culture, and the precipitation process in the previous section was repeated to amplify the phage sublibrary for the next round of biopanning. After three rounds of screening, the enrichment rate reached 4 × 10⁻⁶. 4 As shown in Table 1 below, the titer of the antigen wells after the third round was more than 1000 times higher than that of the negative wells, indicating that the portion of the phage library that specifically binds to BFT1-sFL has been sufficiently enriched, meeting the conditions for screening positive clones.

[0058] Table 1. Enrichment level of bacteriophages

[0059]

[0060]

[0061] CFU: Colony forming unit.

[0062] 2) Bacterial cytoplasmic extract ELISA

[0063] E. coli TG1 cells infected with second and third rounds of phage sub-libraries were cultured on LB agar plates containing ampicillin. Ninety-four colonies from each of the second and third rounds of sub-libraries were randomly selected and cultured in TB medium containing ampicillin. Induction was performed overnight at 28°C with 1M IPTG. Cytoplasmic proteins were extracted using TES solution. Mouse anti-HIS antibody was used as the primary antibody, and goat anti-mouse antibody conjugated with HRP was used as the secondary antibody. TMB reagent was used for colorimetric analysis. Anti-BFT antibody and goat anti-rabbit antibody conjugated with HRP were used as positive controls. Absorbance values ​​were measured using a microplate reader. Clones with an OD450 value more than twice that of the negative wells were considered positive. Plasmids were extracted from positive clones for sequencing and classification according to the CDR3 region. See Table 2:

[0064] Table 2 Screening of phage libraries

[0065]

[0066] 4. Expression, purification, and identification of nanobiomes

[0067] 1) Expression purification

[0068] The specific nanobody sequence was inserted into the pHEN6c plasmid and transfected into *E. coli* WK6 cells. The HIS-labeled recombinant nanobody was induced and extracted in 1 L TB medium with 1 mM IPTG, then purified using a Ni-NTA column and immobilized metal affinity chromatography. The nanobody was dialyzed from imidazole to PBS. The nanobody expression level was good at 10.8 mg / L.

[0069] 2) SDS-PAGE analysis

[0070] Add 40 μL of purified nanobody to 10 μL of 5× loading buffer and incubate at 100℃ for 5 min. Load 5 μL of 4%-15% SDS-PAGE gel and perform electrophoresis, staining with Coomassie Brilliant Blue for 2 h. See results below. Figure 5 , Figure 5 It is clearly shown that there is only one 15kDa band. Specifically, the Nb2.43 nanobody bands are all located around 15kDa, consistent with the sequencing results.

[0071] 5. Obtain mature BFT protein (aBFT)

[0072] 30 mg of BFT-sFL (N-terminal 6*His tag) protein was digested with 6 mg Fpn (C-terminal 6*His tag). BFT-sFL and Fpn were mixed and reacted at room temperature for 30 min. The protein reaction mixture was purified using a Ni-NTA affinity chromatography column, and the elution was collected simultaneously with sample loading. After sample loading, elution was performed with Buffer C, and the elution was collected until the UV280 absorbance was equal to that of Buffer C. All elutions were concentrated and further purified using a Superdex 75PG (GE Heathcare) molecular sieve. 1 mL was collected from each tube, and the protein concentration was determined using a NanoDrop 2000. The collected protein samples were analyzed by 12% SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue to verify the protein separation and purification effect, yielding high-purity aBFT protein with consistent molecular weight. The results are shown in [link to results]. Figure 6 In the figure, A1, A5, B1, B4, B5, C1, C4, D1, and D3 represent protein samples collected from different centrifuge tubes using molecular sieves. The protein size is approximately 20 kDa, consistent with the expected molecular weight.

[0073] 6. Validation of aBFT bioactivity

[0074] After purification, the bioactivity of aBFT needs to be verified. HT29 cells are relatively sensitive to BFT and can undergo significant morphological changes. HT29 cells were cultured at 1.5 × 10⁻⁶ cells / year. 6 Cells were seeded at a density of 1 cells / well in 12-well plates and treated when cell confluence was approximately 70%. After treatment with 3 μg / mL aBFT at 37°C for 20 min, significant morphological changes were observed, including increased intercellular spacing and rounded cells, indicating that purified aBFT possessed biological activity. (See results below) Figure 7 )

[0075] 7. Nb2.43 and aBFT Affinity Detection

[0076] Isothermal titration calorimetry (ITC) can accurately determine the binding constant (Kd), reaction stoichiometry (N), enthalpy (ΔH), and entropy (ΔS) by measuring heat transfer during the binding process. It provides complete thermodynamic information about molecular interactions; ITC can not only determine binding affinity but also elucidate the mechanisms of potential molecular interactions. ITC was performed using a Microcal ITC200 calorimeter at 20°C. For both Nb2.43 and aBFT protein samples, the buffer was replaced with buffer A (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% Glycerol). Protein samples were quantified using a Nanodrop spectrophotometer; the Nb2.43 protein concentration was approximately 200 μM, and the aBFT protein concentration was approximately 20 μM. Experimental data were processed using the Origin software included with the Microcal ITC 200 calorimeter.

[0077] like Figure 8 As shown, the ITC results for Nb2.43 and aBFT show that their dissociation constant (Kd) is 5.59 μM, ΔH is 16 kcal / mol, ΔG is -7.17 kcal / mol, -TΔS is -23.2 kcal / mol, and the stoichiometric coefficient N for the binding reaction is 1, indicating that one Nb2.43 molecule binds to one aBFT molecule.

[0078] Nb2.43 effectively blocked the hydrolysis of E-cadherin protein in cells by aBFT-1. The three aBFT isoforms showed high homology in amino acid sequences. Next, the ability of Nb2.43 to neutralize the activity of aBFT-2 and aBFT-3 proteins was tested. First, Nb2.43 was pre-incubated with aBFT-1, aBFT-2, and aBFT-3 to form complexes, respectively. Then, recombinant E-cadherin extracellular terminal protein was added. The reacted samples were analyzed using 12.5% ​​SDS-PAGE gel and stained with Coomassie brilliant blue. The results showed that compared with the aBFT-only treatment group, Nb2.43 significantly blocked the hydrolytic activity of the three aBFT isoforms on the recombinant E-cadherin extracellular terminal protein, and Nb2.43 had a neutralizing effect on all three aBFT isoforms. Figure 9 ).

[0079] 8. BFT neutralizing antibody-cell assay

[0080] BFT can induce extracellular cleavage of E-cadherin in intestinal epithelial cells, disrupting intercellular junctions and leading to tumor cell invasion and metastasis. Cleavage of E-cadherin can also increase intestinal permeability and facilitate the translocation of intestinal microbiota metabolites. Furthermore, E-cadherin cleavage can activate the β-catenin signaling pathway, thereby exacerbating intestinal inflammatory responses.

[0081] To investigate the neutralizing effect of Nb2.43 at the cellular level, nanobody (0.1 mg / mL) and aBFT (3 μg / mL) were mixed and incubated at 4 °C for 30 min to form a complex. The BFT-nanobody mixture was then used to treat cells at 37 °C for 10 min, and total cellular protein was extracted. E-cadherin protein expression was detected using Western blotting.

[0082] Human normal colonic epithelial cells NCM460 and human colon cancer cells HT29 were treated, and compared with the control group, aBFT significantly reduced the expression level of E-cadherin protein in NCM460 and HT29 cells. Nb2.43 can form a complex with aBFT, which can block the cleavage of E-cadherin protein by aBFT in NCM460 and HT29 cells and upregulate the expression level of E-cadherin (results are shown in Figure 1). Figure 10 (As shown).

[0083] 9. BFT neutralizing antibody—mouse experiment

[0084] Seven-week-old, SPF-grade female C57BL / 6 mice were divided into four groups: ETBF, NTBF, ETBF+Nb2.43 (BFT neutralizing antibody), and ETBF+Nb119 (peer control nanobody). One week prior to infection with *Bacteroides fragilis*, the mice were treated with 100 mg / L clindamycin in their drinking water. One week later, ETBF and NTBF were converted into 1×10⁻⁶ nanoparticles. 10 CFU / ml bacterial suspension was administered to each mouse via gavage at a dose of 100 μL for two consecutive days. Twenty-four hours after the first gavage, Nb2.43 nanobody intervention was initiated. Mice in the nanobody intervention group received a 4 mg tail vein injection of the nanobody, while the remaining groups received an equal volume of PBS via tail vein injection. The medication was administered four times a week for a total of one week. Colonic tissue from the mice was collected for HE staining, and the results are as follows. Figure 11As shown, the upper layer of the colonic mucosa in the NTBF group mice remained intact, with no obvious inflammatory cell infiltration. The colonic tissue of the ETBF-induced colitis group mice showed obvious inflammatory cell infiltration. Compared with the Nb2.43 treatment group, the colonic tissue of the Nb119 (peer control) treatment group mice showed obvious inflammatory cell infiltration. Compared with the ETBF treatment group, the colonic tissue of the Nb2.43 treatment group mice showed no obvious inflammatory cell infiltration.

[0085] In summary, this invention began with the immunization of alpacas, an experimental animal, and constructed a large-capacity phage display library of nanobodies. After three rounds of screening using a bio-panning method, high-affinity BFT nanobodies were obtained through large-scale expression and purification. ITC testing of antibody binding activity showed that nanobodies Nb2.43 exhibited the highest binding affinity (KD) of 5.59 nM, making it the first specific antibody capable of neutralizing aBFT. This can be used to identify the blocking of aBFT in patients and, as one of the treatment methods for colorectal cancer and breast cancer, has significant scientific and clinical application value.

[0086] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A toxin-neutralizing nanobody for Bacteroides fragilis, characterized in that, The heavy chain of this neutralizing nanobody includes three antigen complementarity-determining regions, namely CDR1, CDR2 and CDR3. The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

2. The enteric microorganism Bacteroides fragilis toxin neutralizing nanobody according to claim 1, characterized in that, The heavy chain of this neutralizing nanobody also includes four framework regions, namely FR1, FR2, FR3 and FR4; wherein the amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

3. The enteric microorganism Bacteroides fragilis toxin neutralizing nanobody according to claim 1, characterized in that, The amino acid sequence of the neutralizing nanobody is shown in SEQ ID NO:

8.

4. A prokaryotic expression vector, characterized in that, The present invention contains nucleic acid encoding a toxin-neutralizing nanobody of Bacteroides fragilis as described in any one of claims 1-3.

5. A prokaryotic host cell, characterized in that, It contains the prokaryotic expression vector as described in claim 4.

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