Monoclonal antibody of akkermansia muciniphila specific protein and application thereof

Abstract

The application discloses a monoclonal antibody of a mucinophilic Akkermansia specific protein and application thereof. The monoclonal antibody is any one of monoclonal antibody No. 8, monoclonal antibody No. 17 and monoclonal antibody No. 23, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody No. 8 are respectively shown as SEQ ID NO: 7 and SEQ ID NO: 8, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody No. 17 are respectively shown as SEQ ID NO: 9 and SEQ ID NO: 10, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody No. 23 are respectively shown as SEQ ID NO: 11 and SEQ ID NO: 12. The monoclonal antibody can be used to enrich Akkermansia muciniphila in feces, can be used for strain identification sequencing, and can be used for identification, qualitative and quantitative research of metabolic products of Akkermansia muciniphila.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against a specific protein of Akkermansia myxophilus and its application. Background Technology

[0002] Akkermansia muciniphila (AKK) belongs to the phylum Verrucomicrobia, family Akkermansiaceae, and genus Akkermansia. Akkermansia is the only member of the family Akkermansiaceae and is the dominant group within Verrucomicrobia, accounting for approximately 83%. AKK is an oval-shaped, non-motile, non-spore-forming bacterium that can grow singly or in pairs. Research on AKK is relatively recent; it was isolated, identified, and named in 2004 by researchers at the Microbiology Laboratory of Wageningen University in the Netherlands through anaerobic culture from human feces. It is a Gram-negative, strictly anaerobic intestinal bacterium. In 2007, Collado et al. analyzed the 16S rRNA gene sequence of AKK in fecal samples from healthy subjects of different ages in Europe, demonstrating that AKK is widely present in the human gut, with an average colonization rate of approximately 74.70%.

[0003] In early childhood, AKK colonizes the gut and reaches levels approaching those of healthy adults within a year, while older subjects show lower AKK levels in their feces compared to adults. Numerous studies have shown that the presence of AKK is associated with human health. In most disease states, AKK abundance is significantly lower than in healthy individuals, exhibiting a negative correlation. For example, compared to healthy individuals, AKK abundance is reduced in patients with inflammatory bowel disease (IBD), and the number of AKKs is decreased in patients with acute appendicitis, with its abundance inversely proportional to the severity of appendicitis. Furthermore, AKK abundance is also negatively correlated with diseases such as appendicitis and adolescent autism.

[0004] AKK possesses the ability to degrade mucin, thus it primarily colonizes the intestinal mucus layer. The AKK genome contains numerous genes encoding mucin-degrading enzymes, totaling 78 genes (14% of the total genes), including those encoding glycosidases, sialidases, proteases, and sulfatases. The main component of mucus is mucin, a complex gel composed primarily of water, immunoglobulin A, bioactive peptides, and metabolites. Mucin plays a dual role in the intestine, acting as both a lubricant and a physical barrier, serving as the intestine's first line of defense. Intestinal mucus not only lubricates but also reduces the erosion of the intestinal mucosa by acids and proteases, while also providing a suitable living environment for intestinal microorganisms. Mucin is a nutrient source for AKK. AKK exists in this mucus layer and metabolizes the mucus, producing acetate and propionate. Sugars released by mucin proteases also promote the production of butyrate by other bacteria. Like many anaerobic bacteria, AKK is described as a fermentative bacterium that produces short-chain fatty acids (SCFAs).

[0005] AKK can ferment sugars including galactose, fucose, glucose, Glc-NAc, and GalNAc, but these sugars can only be degraded in the presence of mucin or large amounts of trypsin digests or casein. Due to mucus degradation, AKK produces oligosaccharides and SCFAs, which can stimulate microbial interactions and host responses. Oligosaccharides and acetates can stimulate the growth and metabolic activity of bacteria colonizing near the mucus layer, while simultaneously resisting pathogenic bacteria that must cross the mucus layer to reach intestinal cells.

[0006] Given the various roles of *Ackermania myxophilus*, and the limited research on preparing antibodies against *Ackermania myxophilus*-specific proteins, there is an urgent need to develop a method for preparing monoclonal antibodies based on *Ackermania myxophilus* cell wall surface proteins for the research and analysis of this bacterium. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a monoclonal antibody against a specific protein of *Ackermania myxophilus* and its application. This invention uses the outer membrane proteins Amuc_1100, Porin, and TolC of *Ackermania myxophilus* to immunize mice. Monoclonal antibodies 8, 17, and 23 are prepared using hybridoma technology. These monoclonal antibodies are then conjugated with magnetic beads, and *Ackermania myxophilus* is enriched based on the magnetic bead antibody conjugates. This invention uses the magnetic bead method to enrich *Ackermania myxophilus* in feces, which can be used for bacterial sequencing and identification, as well as for the culture of this bacterium and as a beneficial bacteria addition to regulate bacterial flora imbalance.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0009] This invention provides a monoclonal antibody against a specific protein of *Akermansia myxophilus*, wherein the monoclonal antibody is any one of monoclonal antibody 8, monoclonal antibody 17, and monoclonal antibody 23, wherein...

[0010] Monoclonal antibody 8 includes a heavy chain variable region 8 and a light chain variable region 8, the amino acid sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

[0011] Monoclonal antibody 17 includes a 17 heavy chain variable region and a 17 light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively;

[0012] Monoclonal antibody 23 includes a heavy chain variable region 23 and a light chain variable region 23, the amino acid sequences of which are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0013] Furthermore, in the monoclonal antibody 8, the variable region of the heavy chain 8 includes three complementarity-determining regions (CDR-Hs), namely:

[0014] 8-CDR-H1: ARNVW;

[0015] No. 8-CDR-H2: WAKTPSYMAALQQNHVHV;

[0016] No. 8-CDR-H3: DVCFEQDGPEQ;

[0017] The variable region of light chain 8 includes three complementarity-determining regions (CDR-Ls):

[0018] No. 8-CDR-L1: CANMDLSFAETPR;

[0019] 8-CDR-L2: HNNHPNV;

[0020] No. 8-CDR-L3: YYVKQMVAS;

[0021] In the monoclonal antibody 17, the variable region of the 17 heavy chain includes three complementarity-determining regions (CDR-Hs), namely:

[0022] 17-CDR-H1: WNAVT;

[0023] No. 17-CDR-H2: YACSQSMRFSCMDDIRF;

[0024] 17-CDR-H3: ENFESLFS;

[0025] The variable region of light chain 17 includes three complementary determinant regions (CDR-Ls), namely:

[0026] No. 17-CDR-L1: RPCYYMECEMRYSVQ;

[0027] 17th - CDR - L2: CPCYPDY;

[0028] 17th - CDR - L3: YYYQDEAYC;

[0029] In the monoclonal antibody 23, the variable region of the heavy chain 23 includes three complementarity-determining regions (CDR-Hs), namely:

[0030] 23-CDR-H1: YNVMY;

[0031] 23-CDR-H2: KPKWCCQQQLCMCGEYMRE;

[0032] 23-CDR-H3: FECKAC;

[0033] The variable region of light chain 23 includes three complementarity-determining regions (CDR-Ls):

[0034] 23-CDR-L1: RPNCSMPSQFM;

[0035] 23-CDR-L2: CPCWKCY;

[0036] 23-CDR-L3: CCSTSCAFW.

[0037] Furthermore, the monoclonal antibody is prepared from a hybridoma cell line.

[0038] This invention also provides a method for preparing a hybridoma cell line, comprising the following steps:

[0039] (1) The protein was transformed into Escherichia coli BL21 with a codon-optimized nucleotide sequence, expressed, and purified by sonication to obtain the purified protein; wherein the protein is any one of Amuc_1100, Porin, and TolC.

[0040] (2) The purified protein was mixed with Freund's adjuvant, emulsified and then used to immunize mice. Then, the spleen cells of the immunized mice were fused with myeloma cells SP2 / 0 and the hybridoma cell line was obtained by detection and screening. The hybridoma cell line is any one of hybridoma cell No. 8, hybridoma cell No. 17 and hybridoma cell No. 23.

[0041] Furthermore, when the protein is protein Amuc_1100, the codon-optimized nucleotide sequence of protein Amuc_1100 is shown in SEQ ID NO: 1;

[0042] When the protein is protein Porin, the codon-optimized nucleotide sequence of protein Porin is shown in SEQ ID NO: 3;

[0043] When the protein is protein TolC, the codon-optimized nucleotide sequence of protein TolC is shown in SEQ ID NO: 5.

[0044] Furthermore, the amino acid sequence of the protein Amuc_1100 is shown in SEQ ID NO: 2, the amino acid sequence of the protein Porin is shown in SEQ ID NO: 4, and the amino acid sequence of the protein TolC is shown in SEQ ID NO: 6.

[0045] The present invention also provides the application of the monoclonal antibody in the preparation of magnetic bead antibody conjugates.

[0046] This invention also provides a method for preparing magnetic bead antibody conjugates, comprising the following steps:

[0047] (1) Dilute the monoclonal antibody using MES buffer;

[0048] (2) The magnetic beads were activated by carboxyl groups to obtain activated carboxyl magnetic beads;

[0049] (3) The monoclonal antibody from step (1) is coupled with activated carboxyl magnetic beads to obtain a magnetic bead antibody conjugate. The magnetic bead antibody conjugate is any one of magnetic bead antibody conjugate No. 8, magnetic bead antibody conjugate No. 17 and magnetic bead antibody conjugate No. 23. The diameter of the activated carboxyl magnetic beads is 50-1000 nm and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5-10.

[0050] Furthermore, the diameter of the activated carboxyl magnetic beads is 200 nm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5.

[0051] The present invention also provides the application of a magnetic bead antibody conjugate in the enrichment or isolation of Akkermansia myxophilus, wherein the magnetic bead antibody conjugate is any one or more of magnetic bead antibody conjugate No. 8, magnetic bead antibody conjugate No. 17 and magnetic bead antibody conjugate No. 23.

[0052] The principle of this invention:

[0053] The reasons for selecting the outer membrane proteins Amuc_1100, Porin, and TolC in this invention are as follows:

[0054] 1. Outer membrane proteins (Omps) are located on the surface of bacteria, accounting for approximately 50% of the outer membrane structure. Amuc_1100 is a 28 kDa protein rich in specific type IV fimbriae gene clusters within the outer membrane protein component, and is one of the most abundant membrane proteins. Amuc_1100 participates in the interaction between Akk and the host through TLR2 signaling. Amuc_1100 can directly interact with TLR2 to promote the expression of tryptophan hydroxylase 1 (Tph1), the rate-limiting enzyme in the synthesis of 5-HT, a key signaling molecule regulating gastrointestinal function and other organs, in RIN-14B cells, and reduce the expression of serotonin reuptake transporter (SERT) in Caco-2 cells (human colon cancer cell line), thereby improving 5-HT biosynthesis and extracellular utilization. Amuc_1100 protein also exhibits significant antidepressant effects, the mechanism of which may be related to improving gut microbiota dysbiosis, upregulating brain-derived neurotrophic factor levels, and inhibiting neuroinflammatory responses. These effects suggest that Amuc_1100 protein has the potential to be used as a novel protein drug to treat human metabolic disorders and gut-immune-related diseases.

[0055] 2. Porin proteins are important outer membrane channel proteins located on the outer membrane of Gram-negative bacteria. Composed of 200 amino acids, they are crucial outer membrane proteins for Gram-negative bacteria, primarily responsible for the transport and diffusion of hydrophilic nutrients. They can also induce a strong protective immune response, making them an important candidate antigen for subunit vaccines. Studies have shown that outer membrane phosphoporin pore protein (PhoE) is a major pathway for antibiotics, and changes in its expression are closely related to antibiotic resistance.

[0056] 3. The TolC protein contains 426 amino acids, has an isoelectric point of 4.865, a half-life greater than 8 hours, and an instability index of 35, classifying it as a stable protein. SOPMA software secondary structure prediction shows that the α-helix accounts for 61.1% of the total sequence, random coils account for 27.4%, and extended chains account for 11.5%. SWISS-MODEL software tertiary structure prediction results show that the TolC protein has a porous three-dimensional structure. The random coils in the TolC protein's secondary structure account for 27.4%, indicating a tendency for this structure to twist and coil, potentially exposing the outer layer of the TolC protein and thus becoming a dominant cellular antigenic epitope. This provides a basis for antigenic epitope prediction.

[0057] The beneficial effects of this invention are:

[0058] 1. This invention selects the outer membrane proteins Amuc_1100, Porin, and TolC, which have good immunogenicity on the surface of Akkermansia myxophilus, and uses magnetic bead sorting with magnetic bead antibody conjugates to capture Akkermansia myxophilus, thereby achieving the goal of rapidly isolating Akkermansia myxophilus from fecal samples. The separation is carried out using a magnetic rack, which is convenient and inexpensive. The isolated Akkermansia myxophilus can be used for culture, sequencing, scientific research, etc.

[0059] 2. This invention utilizes magnetic bead antibody conjugates to capture Akkermansia myxophilus, which has high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to the analysis of Akkermansia myxophilus metabolites, as well as its qualitative and quantitative analysis.

[0060] 3. This invention detected the immunogenicity of proteins Amuc_1100, Porin, and TolC. The results showed that the outer membrane proteins of Akkermansia myxophilus have good immunogenicity, laying the foundation for the isolation and identification of Akkermansia myxophilus. Akkermansia myxophilus may also become a very promising probiotic with great potential in the prevention and treatment of diseases such as diabetes, obesity, and metabolic disorders, which is of great significance for future research and development. Attached Figure Description

[0061] Figure 1 This is a plasmid map of the PET32a vector.

[0062] Figure 2 Here are SDS-PAGE images of the three proteins after purification;

[0063] Figure 3 The graph shows the titer test results of three monoclonal antibodies.

[0064] Figure 4 SDS-PAGE images of the purified monoclonal antibodies from the three strains;

[0065] Figure 5 A graph showing the results of Western blotting to identify the specificity of monoclonal antibodies;

[0066] Figure A shows the specificity results of Western blot identification of monoclonal antibody 17, Figure B shows the specificity results of Western blot identification of monoclonal antibody 8, and Figure C shows the specificity results of Western blot identification of monoclonal antibody 23.

[0067] Figure 6 This is a graph showing the sequencing results. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0069] Example 1

[0070] Protein recombinant expression purity and concentration detection

[0071] The amino acid sequence of the N-terminus 34–285 amino acids of protein Amuc_1100 (NCBI accession number WP_197738471.1) is shown in SEQ ID NO: 2, and the nucleotide sequence after codon optimization is shown in SEQ ID NO: 1;

[0072] The amino acid sequence of the N-terminus 22–200 amino acids of protein Porin (NCBI accession number WP_157759309.1) is shown in SEQ ID NO: 4, and the nucleotide sequence after codon optimization is shown in SEQ ID NO: 3;

[0073] The amino acid sequence of the N-terminus 19–426aa of protein TolC (NCBI accession number WP_102742890.1) is shown in SEQ ID NO: 6, and the nucleotide sequence after codon optimization is shown in SEQ ID NO: 5.

[0074] The proteins Amuc_1100, Porin, and TolC were expressed using *E. coli*. The fusion proteins were purified using a His tag, and prokaryotic expression vectors PET32a-Amuc_1100, PET32a-Porin, and PET32a-TolC were constructed. The plasmid map of vector PET32a is shown below. Figure 1 As shown, prokaryotic expression vectors PET32a-Amuc_1100, PET32a-Porin, and PET32a-TolC were transformed into *E. coli* BL21 competent cells. After sonication and centrifugation, the supernatant was purified by nickel column chromatography. The protein A280 concentration was measured using an ultra-micro spectrophotometer, and the protein purity was detected by SDS-PAGE. The results are shown in Table 1 and... Figure 2 As shown, the proteins Amuc_1100, Porin, and TolC purified in this embodiment have high purity, all reaching 91% or higher.

[0075] Table 1 Protein Purity

[0076]

[0077] Example 2

[0078] Mouse immune and antiserum titers and hybridoma cell fusion

[0079] 1. Mouse immunization

[0080] The purified proteins Amuc_1100, Porin, and TolC were mixed with Freund's complete adjuvant and emulsified in a mixer. The first immunization was performed via intraperitoneal and subcutaneous injection at a dose of 100 μg of protein. The second to fourth immunizations were performed via subcutaneous injection at a dose of 50 μg of protein. Immunization was performed every two weeks. After four immunizations, tail vein blood was collected from mice to detect serum antibody titers. Mice with the highest titers were selected for a shock immunization, with 50 μg of protein injected intraperitoneally. Cell fusion was performed three days later.

[0081] 2. Hybridoma cell fusion

[0082] (1) After immunization, prepare a spleen cell suspension and wash it with PBS. Then mix it with SP2 / 0 cells at a ratio of spleen cells:SP2 / 0 = 7:1. Centrifuge at 1500 rpm for 5 min, then drain the mixed cells and gently tap to loosen the cell clumps. Add 1 mL of PEG1450 preheated at 37℃. After adding the PEG1450, react in a 37℃ water bath for 1 min. Then slowly add 40 mL of RPMI-1640 stop solution along the tube wall.

[0083] (2) After the cell fusion was terminated, the cells were centrifuged at 800 rpm for 5 min and the residual liquid was aspirated. The cells were resuspended in 100 mL of HAT medium containing 20% ​​FBS and seeded into 96-well cell culture plates containing feeder cells. The cells were then cultured in a carbon dioxide incubator at 37°C and 5% CO2.

[0084] (3) Observe the cell status 8 to 10 days after fusion, and use the indirect ELISA method to determine the cell supernatant titer. Select positive cell lines with high titer and good specificity for subcloning.

[0085] (4) Using HT medium, the selected cells were diluted to 1 cell / well using the limiting dilution method. The cells were then seeded into 96-well cell culture plates. Once the monoclonal cells had grown to a medium size and reached a density of approximately 10, the cells were allowed to mature. 4 Titer detection was performed on cells of 100 or more. Positive cells were then collected again for a second subcloning screening. Once all cell supernatants in the microwells were found to be positive, hybridoma cell lines were obtained through three subcloning processes. The cell lines were numbered as follows: Amuc_1100 recognition protein 8, Porin recognition protein 17, and TolC recognition protein 23. The titer results for the three monoclonal antibody lines are as follows: Figure 3 As shown, the antibody titers all reached 1:320000 or higher.

[0086] The antibody subtype identification kit was used to identify the subtypes of three monoclonal antibodies. The results showed that the light chain type of all three monoclonal antibodies was kappa and the heavy chain type was IgG1.

[0087] Example 3

[0088] Serum-free culture of hybridoma cells and purification of monoclonal antibodies

[0089] 1. Resuscitate hybridoma cells No. 8, No. 17 and No. 23 under serum-containing conditions. During several consecutive cell passages, gradually reduce the proportion of serum (e.g., from 20% serum to 15% serum to 10% serum, and then to serum-free), and gradually increase the amount of culture medium used as a substitute, so that the hybridoma cells gradually adapt to the serum-free culture environment.

[0090] 2. In the preparation of monoclonal antibodies, hybridoma cells No. 8, No. 17, and No. 23 were revived with serum-free culture medium. When the cells were in good condition, the amount of culture medium and the container for culturing the cells were increased. When a large number of cells died, the liquid was collected, and the supernatant was collected by centrifugation, filtered, and the sample to be purified was loaded onto a Protein A-agarose affinity chromatography column at a flow rate of 0.5 mL / min to allow the antibody to bind to Protein A. Finally, the antibody was eluted with elution buffer to obtain the purified antibody. The purity was identified by SDS-PAGE.

[0091] The results are as follows Figure 4 As shown, this embodiment successfully obtained purified monoclonal antibody No. 8, monoclonal antibody No. 17 and monoclonal antibody No. 23.

[0092] Example 4

[0093] Western blot detection of recombinant protein and antibody specificity

[0094] 1. Prepare separating and stacking gels of appropriate concentrations according to the sizes of proteins Amuc_1100, Porin, and TolC. The pre-stained marker volume is 5 μL, and the protein sample volume is 20 μL. The initial voltage is 80V. After the bromophenol blue in the protein sample enters the separating gel, the voltage is adjusted to 120V until the bromophenol blue reaches the bottom of the separating gel. The electrophoresis is then stopped. The entire electrophoresis process takes 2 hours.

[0095] 2. Cut the PDVF membrane to an appropriate size according to the number of wells for the pre-stained marker and protein samples. Immerse the PDVF membrane in methanol for 5 min, and stack them in a sandwich shape in the following order: anode plate - filter paper - SDS - PAGE gel - PDVF membrane - filter paper - cathode plate. Perform electrotransfer using a wet transfer apparatus at a constant current of 300 mA for 1 h. After the transfer is complete, remove the PDVF membrane. The pre-stained marker will be visibly imprinted on the PDVF membrane. Wash the membrane three times with TBST for 5 min each time.

[0096] 3. Block the PDVF membrane overnight at 4°C in TBST containing 5% BSA. After blocking, wash the PDVF membrane three times with TBST for 10 min each time. Immerse the PDVF membrane in primary antibody diluted with 1% BSA (primary antibody is monoclonal antibody No. 8, monoclonal antibody No. 17, or monoclonal antibody No. 23; the dilution factor is determined according to the preliminary experiment, generally 500-2000 times) and incubate at 37°C for 1 h.

[0097] 4. Wash the PDVF membrane three times with TBST for 10 minutes each time. Immerse the PDVF membrane in secondary antibody diluted with 1% BSA (the secondary antibody is goat anti-mouse-HRP labeled, and the appropriate dilution ratio is determined according to the preliminary experiment, generally diluted 5000 to 50000 times), and incubate at 37°C for 1 hour.

[0098] 5. Wash the PDVF membrane three times with PBST for 10 minutes each time, develop the color with ECL, and take pictures and save them using an electrochemiluminescence analyzer.

[0099] The results are as follows Figure 5 As shown, monoclonal antibody 8 specifically reacts with protein Amuc_1100, monoclonal antibody 17 specifically reacts with protein Porin, and monoclonal antibody 23 specifically reacts with protein TolC, all exhibiting a distinct single band. These results indicate that proteins Amuc_1100, Porin, and TolC possess good immunogenicity, and monoclonal antibodies with good specificity were prepared.

[0100] Example 5

[0101] Cell line sequencing

[0102] 1. Hybridoma cells No. 8, No. 17, and No. 23 were cultured separately, lysed, and total RNA was extracted. mRNA was reverse transcribed to synthesize cDNA using random hexamer primers (5'-Pd(NNNNNN)-3'N=G, A, T, or C). Then, nested PCR was performed in two rounds: the first-strand cDNA was used as a template for amplification, with the forward primer being a sequence complementary to the corresponding heavy and light chain leader sequences, and the reverse primer being a sequence within the constant region of the heavy and light chains.

[0103] Heavy chain forward primer: CGGCCCAGCCGGCC;

[0104] Heavy chain reverse primer: TGAACCGCCTCCACC;

[0105] Light chain forward primer: GGTTCCACTGGT;

[0106] Light chain reverse primer: GTGCAGCATCAGC

[0107] The PCR amplification program was as follows: denaturation at 94℃ for 2 min; denaturation at 94℃ for 20 s, annealing at 58℃ for 20 s, extension at 72℃ for 60 s, for 40 PCR extension cycles; and termination extension at 72℃ for 5 min.

[0108] The second round of amplification yielded gene products with restriction enzyme sites (EcoRI and XhoI), which were ligated into the pTIG cloning vector. Sequencing and analysis then yielded the heavy and light chain variable region sequences of monoclonal antibodies 8, 17, and 23.

[0109] 2. The amino acid sequence of the heavy chain variable region of monoclonal antibody 8 is shown in SEQ ID NO: 7:

[0110] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-H) of the monoclonal antibody heavy chain, while the underlined regions indicate the backbone region (FR-H) of the monoclonal antibody heavy chain.

[0111] MGWIWIFLFLLSGTAGVHS EVQLQQSGPELVKTGASVKISCKASGYSFTARNVWWVKQRPGKSLEWIGW AKTPSYMAALQQNHVHVKATFTVDTSSSTAYMQFNSLTSEDSAVYYCGRDVCFEQDGPEQWGQGTSVTVSS

[0112] No. 8-FR-H1: EVQLQQSGPELVKTGASVKISCKASGYSFT;

[0113] 8-CDR-H1: ARNVW;

[0114] No. 8-FR-H2: WVKQRPGKSLEWIG;

[0115] No. 8-CDR-H2: WAKTPSYMAALQQNHVHV;

[0116] No. 8-FR-H3:KATFTVDTSSSTAYMQFNSLTSEDSAVYYCGR;

[0117] No. 8-CDR-H3: DVCFEQDGPEQ;

[0118] 8-FR-H4: WGQGTSVTVSS.

[0119] The amino acid sequence of the variable region of the 8th light chain of the monoclonal antibody is shown in SEQ ID NO: 8.

[0120] Note: The bold and underlined areas indicate the complementarity-determining region (CDR-L) of the monoclonal antibody's 8th light chain, while the underlined areas indicate the backbone region (FR-L) of the monoclonal antibody's 8th light chain.

[0121] MDFQMQIISLLLISVTVIVSNG EIVLTQSPTTMAASPGEKITITCCANMDLSFAETPRWYQQKPGFSPK LLIYHNNHPNVGVPARFSGGGSGTSYSLTIGTMEAEDVATYYCYYVKQMVASLRGGESSWNIN

[0122] No. 8-FR-L1: EIVLTQSPTTMAASPGEKITITC;

[0123] No. 8-CDR-L1: CANMDLSFAETPR;

[0124] No. 8-FR-L2:WYQQKPPGFSPKLLIY;

[0125] 8-CDR-L2: HNNHPNV;

[0126] No. 8-FR-L3: GVPARFSGGGSGTSYSLTIGTMEAEDVATYYC;

[0127] No. 8-CDR-L3: YYVKQMVAS;

[0128] 8-FR-L4: LRGG.

[0129] 3. The amino acid sequence of the variable region of the heavy chain 17 of the monoclonal antibody is shown in SEQ ID NO: 9:

[0130] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-H) of the monoclonal antibody heavy chain 17, and the underlined regions indicate the backbone region (FR-H) of the monoclonal antibody heavy chain 17.

[0131] QVQLQQSGPELKKPGETVKISCKASGYTFTWNAVTWVKQAPGKGLKWMGYACSQSMRFSCMDDIRFRFA FSLETSASTAYLQINNLKNEDTGTYFCARENFESLFSWGQGTLVTVSA

[0132] No. 17-FR-H1: QVQLQQSGPELKKPGETVKISCKASGYTFT;

[0133] 17-CDR-H1: WNAVT;

[0134] No. 17-FR-H2: WVKQAPGKGLKWMG;

[0135] No. 17-CDR-H2: YACSQSMRFSCMDDIRF;

[0136] No. 17-FR-H3: RFAFSLETSASTAYLQINNLKNEDTGTYFCAR;

[0137] 17-CDR-H3: ENFESLFS;

[0138] 17-FR-H4: WGQGTLVTVSA.

[0139] The amino acid sequence of the variable region of the 17th light chain of the monoclonal antibody is shown in SEQ ID NO: 10:

[0140] Note: The bold and underlined areas indicate the complementarity-determining region (CDR-L) of the 17th light chain of the monoclonal antibody, and the underlined areas indicate the backbone region (FR-L) of the 17th light chain of the monoclonal antibody.

[0141] EIVLTQSPASLAVSLGQRATISCRPCYYMECEMRYSVQWYQQKPGQPPKLLIYCPCYPDYGIPARFSGS GSGTDFTLNIHPVEEEDAATYYCYYYQDEAYCFGGGTKLEIK

[0142] No. 17-FR-L1:EIVLTQSPASLAVSLGQRATISC;

[0143] No. 17-CDR-L1: RPCYYMECEMRYSVQ;

[0144] No. 17-FR-L2: WYQQKPGQPPKLLIY;

[0145] 17th - CDR - L2: CPCYPDY;

[0146] No. 17-FR-L3:GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC;

[0147] 17th - CDR - L3: YYYQDEAYC;

[0148] 17-FR-L4: FGGGTKLEIK.

[0149] 4. The amino acid sequence of the variable region of the heavy chain 23 of the monoclonal antibody is shown in SEQ ID NO: 11:

[0150] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-H) of the heavy chain 23 of the monoclonal antibody, and the underlined regions indicate the backbone region (FR-H) of the heavy chain 23 of the monoclonal antibody.

[0151] EVQLQESGGGLVQPKGSLKLSCTASGFTFNYNVMYWVRQAPGKGLEWVAKPKWCCQQQLCMCGEYMRER FTISRDDSQSMLYLQMNNLRTEDTAMYYCVRFECKACWGQGTTLTVSS

[0152] No. 23-FR-H1: EVQLQESGGGLVQPKGSLKLSCTASGFTFN;

[0153] 23-CDR-H1: YNVMY;

[0154] No. 23-FR-H2: WVRQAPGKGLEWVA;

[0155] 23-CDR-H2: KPKWCCQQQLCMCGEYMRE;

[0156] No. 23-FR-H3: RFTISRDDSQSMLYLQMNNLRTEDTAMYYCVR;

[0157] 23-CDR-H3: FECKAC;

[0158] 23-FR-H4: WGQGTTLTVSS.

[0159] The amino acid sequence of the variable region of the light chain 23 of the monoclonal antibody is shown in SEQ ID NO: 12:

[0160] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-L) of the light chain 23 of the monoclonal antibody, and the underlined regions indicate the backbone region (FR-L) of the light chain 23 of the monoclonal antibody.

[0161] DIVMTQSQKFMSTSVGDRVSVTCRPNCSMPSQFMWYQQKPGQFPKALIYCPCWKCYGVPDRFKGSGSGT DFTLTINNVQSEDLAEYSCCCSTSCAFWFGGGTKLEIK R

[0162] No. 23-FR-L1: DIVMTQSQKFMSTSVGDRVSVTC;

[0163] 23-CDR-L1: RPNCSMPSQFM;

[0164] No. 23-FR-L2: WYQQKPGQFPKALIY;

[0165] 23-CDR-L2: CPCWKCY;

[0166] No. 23-FR-L3:GVPDRFKGSGSGTDFTLTINNVQSEDLAEYSC;

[0167] 23-CDR-L3: CCSTSCAFW;

[0168] 23-FR-L4:FGGGTKLEIK.

[0169] Example 6

[0170] Preparation of magnetic bead antibody conjugates

[0171] 1. Monoclonal antibody dilution

[0172] The buffer solutions for monoclonal antibodies 8, 17, and 23 were replaced with 15 mL MES buffer at pH 6.0. The antibodies were then diluted to 2 mg / mL with MES buffer to obtain antibody dilution solutions 8, 17, and 23, respectively.

[0173] 2. Activation of carboxyl magnetic beads

[0174] (1) Take 1 mg of carboxyl magnetic beads, wash them 3 times with MES buffer, and resuspend them in 0.1 mL of MES buffer to 10 mg / mL;

[0175] (2) Weigh EDC and NHS and dissolve them in MES buffer. The concentration of EDC solution is 20 mg / mL and the concentration of NHS solution is 24 mg / mL. Take 50 μL of EDC solution and 50 μL of NHS solution respectively and add them to the magnetic beads treated in step (1). Activate at 25℃ for 30 min to obtain activated carboxyl magnetic beads.

[0176] 3. Covalent coupling of magnetic beads and antibodies

[0177] Antibody dilution buffer was mixed with 1 mg of activated carboxyl magnetic beads (50 nm, 200 nm, and 1000 nm in diameter) at an antibody-magnetic bead molar ratio of 1:10 or 1:5. The mixture was reacted at 25 °C for 16 h, followed by magnetic separation. The supernatant was collected, and the remaining antibody content was simultaneously measured. The amount and concentration of the magnetic bead-conjugated antibody were calculated. The magnetic beads were washed two to three times with physiological saline and resuspended in physiological saline to obtain magnetic bead antibody conjugates No. 8①, ②, ③, and ④, respectively. The following are examples of magnetic bead antibody conjugates: Magnetic Bead Antibody Conjugate No. 8⑤, Magnetic Bead Antibody Conjugate No. 8⑥, Magnetic Bead Antibody Conjugate No. 17①, Magnetic Bead Antibody Conjugate No. 17②, Magnetic Bead Antibody Conjugate No. 17③, Magnetic Bead Antibody Conjugate No. 17④, Magnetic Bead Antibody Conjugate No. 7⑤, Magnetic Bead Antibody Conjugate No. 7⑥, Magnetic Bead Antibody Conjugate No. 23①, Magnetic Bead Antibody Conjugate No. 23②, Magnetic Bead Antibody Conjugate No. 23③, Magnetic Bead Antibody Conjugate No. 23④, Magnetic Bead Antibody Conjugate No. 23⑤, and Magnetic Bead Antibody Conjugate No. 23⑥. The conditions for antibody conjugation with magnetic beads are shown in Table 2.

[0178] Table 2 Conditions for Antibody-Conjugated Magnetic Beads

[0179]

[0180]

[0181] Example 7

[0182] Enrichment and sequencing verification of Akkermansia myxophilus

[0183] 1. Take 5 mL of fecal suspension (5 g of feces suspended in 5 mL of physiological saline), add 1 mg of the magnetic bead antibody conjugate prepared in Example 6, mix and incubate at 37 °C for 2 h, separate the magnetic beads using a magnetic rack, and remove the unbound microorganisms and supernatant.

[0184] 2. Then, the magnetic beads (labeled magnetic beads) conjugated with Akkermansia myxophilus were resuspended in physiological saline. The labeled magnetic beads were then mixed with an antibody label removal reagent, namely 0.05% papain (S10011, Yuanye Biotechnology), and incubated at 37°C for 3 hours to cleave the Fc and Fab of the mouse monoclonal antibody, thereby separating the magnetic beads from Akkermansia myxophilus.

[0185] 3. Then, the magnetic beads are collected using a magnetic rack. The supernatant is the Akkermansia myxophilus suspension. After diluting the Akkermansia myxophilus suspension, it is added to a hemocytometer and counted under a microscope. The yield of Akkermansia myxophilus enrichment is shown in Table 2. Based on the yield calculation, Akkermansia myxophilus enrichment is subsequently carried out under the conditions of 200 nm and an antibody to magnetic bead molar ratio of 1:5. Furthermore, the magnetic bead antibody conjugate of the present invention is used to enrich Akkermansia myxophilus, resulting in high enrichment yield and strong specificity.

[0186] 4. Dilute the enriched and isolated Akkermansia mycotoxin-loving bacteria to 10. 5 PCR amplification of 16S rDNA was performed using bacterial culture as a template at a concentration of [number] cells / ml. The amplification products were verified by sequencing, and the sequencing results are as follows: Figure 6 As shown, the sequencing results were compared with the NCBI database using BLAST. The results showed that the 16S rDNA gene sequence of this bacterium had 100% homology with hundreds of Akkermansia myxophilus strains. Therefore, the isolate was identified as Akkermansia myxophilus.

[0187] This invention selects the outer membrane proteins Amuc_1100, Porin, and TolC, which have good immunogenicity on the surface of *Ackermania obliterans*, to prepare monoclonal antibodies. Monoclonal antibodies No. 8, No. 17, and No. 23 prepared can specifically bind to the outer membrane proteins of *Ackermania obliterans*, laying the foundation for capturing *Ackermania obliterans*. Furthermore, this invention employs a magnetic bead sorting method, utilizing magnetic bead antibody conjugates to capture *Ackermania obliterans*, achieving the goal of rapidly isolating *Ackermania obliterans* from fecal samples. The separation is convenient and inexpensive, utilizing a magnetic rack. The isolated *Ackermania obliterans* can be used for culture, sequencing, and scientific research.

[0188] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against a specific protein of Akkermansia myxophilus, characterized in that: The monoclonal antibody is any one of monoclonal antibody 8, monoclonal antibody 17, and monoclonal antibody 23, wherein... Monoclonal antibody 8 includes a heavy chain variable region 8 and a light chain variable region 8, the amino acid sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; Monoclonal antibody 17 includes a 17 heavy chain variable region and a 17 light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; Monoclonal antibody 23 includes a heavy chain variable region 23 and a light chain variable region 23, the amino acid sequences of which are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

2. The monoclonal antibody according to claim 1, characterized in that: In the monoclonal antibody 8, the variable region of the heavy chain includes three complementarity-determining regions (CDRs-H), namely: 8-CDR-H1: ARNVW; No. 8-CDR-H2: WAKTPSYMAALQQNHVHV; No. 8-CDR-H3: DVCFEQDGPEQ; The variable region of light chain 8 includes three complementarity-determining regions (CDR-Ls): No. 8-CDR-L1: CANMDLSFAETPR; 8-CDR-L2: HNNHPNV; No. 8-CDR-L3: YYVKQMVAS; In the monoclonal antibody 17, the variable region of the 17 heavy chain includes three complementarity-determining regions (CDRs-H), namely: 17-CDR-H1: WNAVT; No. 17-CDR-H2: YACSQSMRFSCMDDIRF; 17-CDR-H3: ENFESLFS; The variable region of light chain 17 includes three complementary determinant regions (CDR-Ls), namely: No. 17-CDR-L1: RPCYYMECEMRYSVQ; 17th - CDR - L2: CPCYPDY; 17th - CDR - L3: YYYQDEAYC; In the monoclonal antibody 23, the variable region of the heavy chain 23 includes three complementarity-determining regions (CDR-Hs), namely: 23-CDR-H1: YNVMY; 23-CDR-H2: KPKWCCQQQLCMCGEYMRE; 23-CDR-H3: FECKAC; The variable region of light chain 23 includes three complementarity-determining regions (CDR-Ls): 23-CDR-L1: RPNCSMPSQFM; 23-CDR-L2: CPCWKCY; 23-CDR-L3: CCSTSCAFW.

3. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody was prepared from a hybridoma cell line.

4. A method for preparing a hybridoma cell line, characterized in that: Includes the following steps: (1) The protein was transformed into Escherichia coli BL21 with a codon-optimized nucleotide sequence, expressed, and purified by sonication to obtain the purified protein; wherein the protein is any one of Amuc_1100, Porin, and TolC. (2) The purified protein was mixed with Freund's adjuvant, emulsified and then used to immunize mice. Then, the spleen cells of the immunized mice were fused with myeloma cells SP2 / 0 and the hybridoma cell line was obtained by detection and screening. The hybridoma cell line is any one of hybridoma cell No. 8, hybridoma cell No. 17 and hybridoma cell No.

23.

5. The preparation method according to claim 4, characterized in that: When the protein is protein Amuc_1100, the codon-optimized nucleotide sequence of protein Amuc_1100 is shown in SEQ ID NO: 1; When the protein is protein Porin, the codon-optimized nucleotide sequence of protein Porin is shown in SEQ ID NO: 3; When the protein is protein TolC, the codon-optimized nucleotide sequence of protein TolC is shown in SEQ ID NO:

5.

6. The preparation method according to claim 4, characterized in that: The amino acid sequence of the protein Amuc_1100 is shown in SEQ ID NO: 2, the amino acid sequence of the protein Porin is shown in SEQ ID NO: 4, and the amino acid sequence of the protein TolC is shown in SEQ ID NO:

6.

7. The use of the monoclonal antibody of claim 1 in the preparation of magnetic bead antibody conjugates.

8. A method for preparing a magnetic bead antibody conjugate, characterized in that: Includes the following steps: (1) Dilute the monoclonal antibody of claim 1 using MES buffer; (2) The magnetic beads were activated by carboxyl groups to obtain activated carboxyl magnetic beads; (3) The monoclonal antibody from step (1) is coupled with activated carboxyl magnetic beads to obtain a magnetic bead antibody conjugate. The magnetic bead antibody conjugate is any one of magnetic bead antibody conjugate No. 8, magnetic bead antibody conjugate No. 17 and magnetic bead antibody conjugate No.

23. The diameter of the activated carboxyl magnetic beads is 50-1000 nm and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5-10.

9. The preparation method according to claim 8, characterized in that: The activated carboxyl magnetic beads have a diameter of 200 nm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:

5.

10. The application of a magnetic bead antibody conjugate in the enrichment or isolation of Akkermansia myxophilia, characterized in that: The magnetic bead antibody conjugate is any one or more of magnetic bead antibody conjugate No. 8, magnetic bead antibody conjugate No. 17, and magnetic bead antibody conjugate No. 23.

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