Monoclonal antibody of specific protein of bifidobacterium longum and application of monoclonal antibody

Abstract

The invention discloses a monoclonal antibody of specific protein of bifidobacterium longum and application of the monoclonal antibody, and belongs to the technical field of biology. According to the invention, MetQ and LPXTG proteins on the surface of bifidobacterium longum are used as immunogens, a monoclonal antibody with strong specificity and high binding force with antigens is prepared through a hybridoma technology, and after the monoclonal antibody is coupled with magnetic beads, the obtained magnetic bead antibody conjugate can realize efficient enrichment and / or separation of bifidobacterium longum in a to-be-treated sample; the advantages of convenience and low cost are realized; in addition, the separated bifidobacterium longum can be used for culture, qualitative and quantitative research on metabolites of the bifidobacterium longum, relation of interaction with other microorganisms and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody against a specific protein of Bifidobacterium longum and its application. Background Technology

[0002] Bifidobacterium longum is a Gram-positive, non-spore-forming, rod-shaped anaerobic bacterium. Most commonly found singly, a few occur in pairs or short chains. It belongs to the phylum Firmicutes, order Bifidobacteriales, family Bifidobacteriaceae, and genus Bifidobacterium. It is an important component of the human gut microbiota and is widely distributed in the intestines of humans and animals, especially in infants. It is dominant in the gut of breastfed infants, but its numbers may gradually decrease with age. During its metabolism, Bifidobacterium longum produces beneficial metabolites, such as short-chain fatty acids (e.g., acetic acid, propionic acid, butyric acid), vitamins (e.g., B vitamins), and antimicrobial substances (e.g., bifidobacteriacin). These metabolites play important roles in gut health, such as regulating intestinal pH, inhibiting the growth of harmful bacteria, promoting intestinal motility, maintaining gut microbiota balance, and preventing and treating intestinal infections.

[0003] Because Bifidobacterium longum plays an important role in the gut microbiota, its abundance in the gut is related to the health of the organism. Currently, the enrichment and isolation of Bifidobacterium longum in fecal samples is often performed to clarify its impact on the health of the organism. Among these methods, the immunomagnetic bead method for isolating Bifidobacterium longum from feces has been widely used. The main principle of immunomagnetic bead isolation is to selectively capture Bifidobacterium longum using magnetic beads coated with antibodies that specifically bind to surface proteins of Bifidobacterium longum, and then use magnetic force to achieve mechanical movement, thereby isolating Bifidobacterium longum.

[0004] However, existing antibodies that specifically bind to surface proteins of Bifidobacterium longum have problems such as poor specificity and weak binding affinity to antigens. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against a specific protein of Bifidobacterium longum and its application. This invention uses MetQ and LPXTG proteins on the surface of Bifidobacterium longum as immunogens to prepare a monoclonal antibody with high specificity and high binding affinity to the antigen. After conjugating the monoclonal antibody with magnetic beads, the resulting magnetic bead antibody conjugate can achieve efficient enrichment and / or separation of Bifidobacterium longum.

[0006] In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof of a Bifidobacterium longum-specific protein, comprising a heavy chain variable region and a light chain variable region as shown in A1) or A2) below; A1) the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO: 13-15, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO: 17-19, respectively; A2) the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO: 21-23, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO: 25-27, respectively.

[0007] The monoclonal antibody or antigen-binding fragment of the Bifidobacterium longum specific protein provided by the present invention includes at least one of Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide bond-stabilized Fv (dsFv), single-chain antibody (ScFv), and single-domain antibody (sdAb).

[0008] In some implementations, the monoclonal antibody or its antigen-binding fragment in A1) targets and binds to the MetQ protein on the surface of Bifidobacterium longum, and the monoclonal antibody or its antigen-binding fragment in A2) targets and binds to the LPXTG protein on the surface of Bifidobacterium longum.

[0009] In some embodiments, the amino acid sequence of the MetQ protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the MetQ protein after codon optimization is shown in SEQ ID NO: 2; the amino acid sequence of the LPTG protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the LPTG protein after codon optimization is shown in SEQ ID NO: 6.

[0010] The MetQ and LPXTG proteins provided by this invention can be natural, recombinant, or synthetic active polypeptides. These active polypeptides can be naturally purified products, chemically synthesized products, or products produced from prokaryotic hosts (e.g., Escherichia coli) or eukaryotic hosts (e.g., yeast, higher plants) using recombinant technology.

[0011] In some implementations, the MetQ and LPXTG proteins are obtained by introducing a recombinant vector containing their encoding genes into an expression host (e.g., Escherichia coli BL21(DE3)) to obtain a recombinant genetically engineered strain, then culturing the recombinant genetically engineered strain and inducing expression to obtain the MetQ and LPXTG proteins.

[0012] In this invention, monoclonal antibodies or antigen-binding fragments of Bifidobacterium longum specific proteins can target and bind to MetQ and LPXTG proteins on the surface of Bifidobacterium longum, and have the advantages of high specificity and high affinity.

[0013] In some embodiments, in A1), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 16, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 16; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 20, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 20; in A2), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 24, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 24; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 28, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 28.

[0014] As used herein, the term “sequence identity” can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). Molecules are identical at that position when positions in the compared sequences are occupied by the same bases or amino acids. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. “Sequence identity” of a polynucleotide or amino acid sequence with another sequence having a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0015] In a second aspect, the present invention provides a biological material selected from any of the following: B1) a nucleic acid molecule encoding any of the above-mentioned monoclonal antibodies or their antigen-binding fragments; B2) a recombinant vector containing the nucleic acid molecule B1); B3) a recombinant cell containing the nucleic acid molecule B1 or the recombinant vector B2.

[0016] The nucleic acid molecules provided by this invention can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as mRNA or hnRNA; and these nucleic acid molecules can usually be obtained by PCR amplification or artificial synthesis.

[0017] The recombinant vector provided by this invention includes a cloning vector and an expression vector. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be a pET28a(+) vector.

[0018] In a third aspect, the present invention provides a magnetic bead antibody conjugate comprising a magnetic bead and any of the above-described monoclonal antibodies or antigen-binding fragments thereof conjugated to the magnetic bead.

[0019] The magnetic bead antibody conjugate provided by this invention can achieve efficient enrichment and / or separation of Bifidobacterium longum.

[0020] In a fourth aspect, the present invention provides a method for preparing a magnetic bead antibody conjugate, comprising the following steps: diluting any of the above-mentioned monoclonal antibodies or their antigen-binding fragments to obtain a diluent of the monoclonal antibody or its antigen-binding fragment; performing carboxyl activation treatment on magnetic beads to obtain activated carboxyl magnetic beads; and conjugating the diluent of the monoclonal antibody or its antigen-binding fragments with the activated carboxyl magnetic beads to obtain a magnetic bead antibody conjugate.

[0021] In some embodiments, in the step of diluting any of the above monoclonal antibodies or their antigen-binding fragments, a buffer solution is used for dilution, and the buffer solution is preferably MES buffer with a concentration of 10-20 mM, preferably 15 mM.

[0022] In some embodiments, in the step of carboxyl activation treatment of the magnetic beads, EDC solution and NHS solution are used for activation, wherein the concentration of EDC solution is 15-25 mg / mL, preferably 20 mg / mL; the concentration of NHS solution is 20-30 mg / mL, preferably 24 mg / mL; the activation treatment includes: activation at a temperature of 20-30°C (preferably 25°C) for 25-35 min, preferably 30 min.

[0023] In some embodiments, in the step of coupling the monoclonal antibody or its antigen-binding fragment dilution with the activated carboxyl magnetic beads, the molar ratio of the monoclonal antibody or its antigen-binding fragment dilution to the activated carboxyl magnetic beads is 1:(5-10), and the diameter of the activated carboxyl magnetic beads is 50-1000 nm.

[0024] In some implementations, the diameter of the activated carboxyl magnetic beads is 200 nm.

[0025] In this invention, by controlling the parameters in the preparation process of magnetic bead antibody conjugates within a specific range, the performance of the magnetic bead antibody conjugates can be improved, making it easier to enrich and / or isolate Bifidobacterium longum more efficiently.

[0026] In a fifth aspect, the present invention provides the use of any of the above-described monoclonal antibodies or their antigen-binding fragments, the above-described biological materials, the above-described magnetic bead antibody conjugates, and the magnetic bead antibody conjugates prepared by any of the above-described preparation methods in the enrichment and / or isolation of Bifidobacterium longum.

[0027] In a sixth aspect, the present invention provides a method for enriching and / or separating Bifidobacterium longum using the magnetic bead antibody conjugate or the magnetic bead antibody conjugate prepared by any of the above preparation methods, comprising the following steps: providing a sample to be treated; adding the magnetic bead antibody conjugate to the sample to be treated, and after incubation, separating magnetic beads bound to Bifidobacterium longum; suspending the magnetic beads bound to Bifidobacterium longum in a solution, adding a protease and incubating, and separating to obtain a suspension of Bifidobacterium longum.

[0028] In some implementations, the samples to be processed include fecal samples.

[0029] It is understood that the samples to be processed can be conventionally selected according to actual needs, as long as they contain Bifidobacterium longum to be enriched and / or isolated. For example, in this invention, the samples to be processed preferably include fecal samples.

[0030] In some implementations, the step of adding magnetic bead antibody conjugates to the sample to be treated and then incubating specifically includes incubating at a temperature of 30-45°C (preferably 37°C) for 1-3 hours, preferably 2 hours.

[0031] In some embodiments, in the step of suspending magnetic beads conjugated with Bifidobacterium longum in a solution, adding a protease and then incubating, the amount of protease added is 0.05%, and the protease includes papain; the incubation specifically includes incubation at a temperature of 30-45°C (preferably 37°C) for 2-4 hours, preferably 3 hours.

[0032] It is understood that the type and amount of protease added can be routinely adjusted according to actual usage needs, as long as it has efficient activity in cleaving Fc and Fab fragments. For example, in this invention, the amount of protease added is preferably 0.05%, and the protease preferably includes papain.

[0033] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention uses MetQ and LPXTG proteins on the surface of Bifidobacterium longum as immunogens, and prepares monoclonal antibodies with high specificity and high antigen binding affinity through hybridoma technology. After conjugating this monoclonal antibody with magnetic beads, the resulting magnetic bead antibody conjugate can achieve efficient enrichment and / or separation of Bifidobacterium longum in the sample to be treated, which has the advantages of convenience and low cost. In addition, the isolated Bifidobacterium longum can be used for culture, qualitative and quantitative research on its metabolites, and its interaction with other microorganisms, which has good application prospects. Attached Figure Description

[0034] Figure 1 The results of SDS-PAGE analysis of the purified MetQ and LPXTG proteins expressed in Example 1 of this invention are shown. Figure 2 The results of the titer test for monoclonal antibody 1A5 in Example 2 of this invention; Figure 3 This refers to the titer test results of monoclonal antibody 4D6 in Example 2 of this invention; Figure 4 The results of SDS-PAGE analysis of the purified monoclonal antibodies 1A5 and 4D6 in Example 2 of this invention; Figure 5 This is the WB detection result of monoclonal antibody 1A5 specifically recognizing MetQ protein in Example 2 of the present invention; Figure 6 This is the WB detection result of the monoclonal antibody 4D6 specifically recognizing the LPXTG protein in Example 2 of the present invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0037] Example 1: Preparation of MetQ and LPXTG proteins on the surface of Bifidobacterium longum 1.1 Construction of MetQ and LPXTG recombinant expression vectors Bioinformatics analysis of the MetQ protein (NCBI, protein_id: WP_011068221.1) identified its extracellular region as consisting of 51-326 amino acids (aa), and its expression was performed in vitro in prokaryotes. The amino acid sequence of the MetQ protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the synthesized MetQ gene after codon optimization is shown in SEQ ID NO: 2.

[0038] Similarly, bioinformatics analysis of the LPXTG protein (NCBI, protein_id: WP_434280316.1) clarified that the LPXTG protein consists of a transmembrane structure, an intracellular region, and an extracellular region. An extracellular region of 35-678 amino acids (aa) was selected for in vitro prokaryotic expression. The amino acid sequence of the LPXTG protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the synthesized LPXTG gene after codon optimization is shown in SEQ ID NO: 6.

[0039] Primers for amplifying the MetQ and LPTSG genes were designed and synthesized, with the specific sequences as follows: MetQ-F:5'-TTTTGAATTCGGTGCCAACGAATCCTCTGCGA-3' (SEQ ID NO: 3); MetQ-R:5'-TTTTAAGCTTTCACTGCTGTGCTTTCGCGTCCT-3' (SEQ ID NO: 4); LPXTG-F:5'-TTTTGAATTCGGTGGTGGCGGTTGGCCCCT-3' (SEQ ID NO: 7); LPXTG-R:5'-TTTTAAGCTTGTCACCACCAACCTTATCAGA-3' (SEQ ID NO: 8); The primers mentioned above contain EcoRI and HindIII restriction sites.

[0040] Gene amplification is performed according to the following steps: a) Using the primers designed above, the MetQ and LPXTG genes were amplified respectively using PCR technology; b) After amplification, the PCR products are obtained by gel extraction to remove excess impurities; c) The PCR product and vector pET28a(+) were double-digested with EcoRI and HindIII respectively to generate sticky ends that could be complementary and linked together. d) The enzyme digestion product is purified to obtain the target fragment and linearized vector.

[0041] The following system was used: MetQ or LPTSG fragment: 1 μL; vector fragment: 3 μL; T4 DNA ligase: 1 μL; 10× buffer: 1 μL; water: 4 μL; total volume: 10 μL. After thoroughly mixing all components, the mixture was placed at 16°C and ligated overnight to ensure complete ligation of the MetQ fragment and the vector fragment.

[0042] Add 5 μL of the ligation product to 100 μL of competent cells, gently mix, and incubate on ice for 30 minutes to ensure full contact between the ligation product and the competent cells. Then, heat shock the cells at 42°C for 45 seconds to induce the ligation product to enter the competent cells. After heat shock, immediately incubate on ice for 3 minutes. Next, add 500 μL of LB medium and incubate at 37°C for 1 hour to allow the transformed cells to return to normal growth. Finally, plate the bacterial culture onto LB agar plates containing kanamycin (50 μg / mL) and incubate at 37°C for 12-16 hours to allow successfully transformed cells to form single colonies.

[0043] Three single colonies were randomly selected from LB agar plates and inoculated into 2 mL of LB liquid medium containing 50 μg / mL kanamycin. The cultures were incubated overnight at 37°C and 220 rpm. After incubation, plasmids were extracted from each culture and double-digested with EcoRI and HindIII, followed by sequencing for verification. Based on the digestion and sequencing results, the correct prokaryotic expression vectors pET28a(+)-MetQ and pET28a(+)-LPXTG were selected and constructed for subsequent experimental studies.

[0044] 1.2 Expression and purification of MetQ and LPXTG proteins The expression and purification of MetQ and LPTG proteins were performed according to the following steps: Transformation culture: The pET28a(+)-MetQ and pET28a(+)-LPXTG recombinant plasmids verified by sequencing in step 1.1 were electrotransformed into E.coli BL21(DE3) competent cells, and seeded into LB solid medium containing 50 μg / mL kanamycin and cultured upside down at 37°C for 12-16 hours.

[0045] Seed culture expansion: Select a single colony and inoculate it into 2 mL of LB liquid medium (containing 50 μg / mL kanamycin), and incubate overnight at 37°C with shaking at 220 rpm; transfer to 200 mL of LB medium (same antibiotic concentration) at a ratio of 1:200, and incubate at 37°C with shaking at 220 rpm until OD reaches 1 / 2. 600 =0.6-0.8.

[0046] Protein induction: IPTG was added to a final concentration of 1 mM, and expression was induced at 37°C and 220 rpm for 16 hours; the bacterial culture was collected and centrifuged at 4°C and 12,000 × g for 1 minute, the supernatant was discarded and the bacterial pellet was collected.

[0047] Cell disruption: Resuspend the bacterial pellet in PBS and sonicate (5 seconds working, 5 seconds interval, total 10 minutes) until the suspension is clear; collect the supernatant by centrifugation at 12,000×g for 10 minutes at 4°C and filter through a 0.22μm filter membrane.

[0048] Nickel column chromatography: Column packing: After uniformly suspending the Ni-NTA agarose gel packing material (GE Healthcare), pack it into the chromatography column and wash with deionized water (5-10 column volumes) at a flow rate of 1-2 mL / min.

[0049] Equilibration: Equilibrate with equilibration buffer (20mM Na2HPO4, 0.5M NaCl, pH 7.4) until the conductivity of the effluent is stable.

[0050] Sample loading: Load the filtrate onto the nickel column at a flow rate of 0.5 mL / min to ensure sufficient binding of the His-tag protein.

[0051] Washing: Wash off contaminating proteins with washing buffer (equilibration buffer of 20 mM imidazole) and monitor absorbance at 280 nm until baseline stability.

[0052] Elution: Elute the target protein stepwise with elution buffer (equilibration buffer of 250mM imidazole) and collect the elution peaks.

[0053] Post-processing: The eluent was dialyzed (PBS, 4°C overnight) to remove imidazole, concentrated by ultrafiltration (Amicon Ultra-15, 10kDa cutoff), and the purity was determined by SDS-PAGE.

[0054] SDS-PAGE test results are as follows: Figure 1 As shown.

[0055] from Figure 1 As can be seen, the MetQ and LPXTG proteins are consistent with the expected molecular weight. Among them, the purity of MetQ protein reached 88%, with a concentration of 1.8 mg / mL; the purity of LPXTG protein reached 91%, with a concentration of 1.0 mg / mL.

[0056] Example 2: Preparation of monoclonal antibodies targeting and binding to MetQ and LPXTG proteins on the surface of Bifidobacterium longum 2.1 Mouse Immunization The MetQ and LPXTG proteins prepared in Example 1 were mixed with Freund's complete adjuvant and emulsified in a mixer. The first immunization was performed by intraperitoneal and subcutaneous injection, with an immunization dose of 100 μg protein. The second to fourth immunizations were performed by subcutaneous injection, with an immunization dose of 50 μg 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 subsequent preparation of hybridoma cells.

[0057] 2.2 Preparation of hybridoma cells 1) Take mice with the highest antibody titer after immunization, prepare spleen cell suspension, wash with PBS, and mix 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 PEG 1450 preheated at 37℃, and react in a 37℃ water bath for 1 min. Slowly add 40 mL of RPMI-1640 stop solution along the tube wall.

[0058] 2) After cell fusion is terminated, centrifuge at 800 rpm for 5 min and aspirate any residual liquid. Resuspend the cells in 100 mL of HAT medium containing 20% ​​FBS, plate them into 96-well cell culture plates containing feeder cells, and incubate at 37°C in a 5% CO2 incubator.

[0059] 3) Observe the cell status 8-10 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.

[0060] 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 reached a medium size and a density of approximately 102, the cells were allowed to mature. 4 Titer can be detected with a number of cells or more. Then, positive cells are collected again for a second subcloning screening. Once all cell supernatants in the microwells are positive, three subcloning processes are performed to obtain hybridoma cell lines. The cell line targeting MetQ protein is named hybridoma cell line 1A5, and the cell line targeting LPXTG protein is named hybridoma cell line 4D6. The antibodies secreted by these cells are named monoclonal antibodies 1A5 and 4D6, respectively. The titers of all the above monoclonal antibodies reached above 1:320000. The detection results are shown in Table 1 and below. Figure 2-3 As shown.

[0061] Table 1. Results of monoclonal antibody 1A5 and 4D6 titers

[0062] 2.3 Purification of Monoclonal Antibodies Hybridoma cell lines 1A5 and 4D6 obtained in step 2.2 were revived under serum-containing conditions. During several consecutive cell passages, the proportion of serum was gradually reduced (e.g., from 20% serum to 15% serum to 10% serum, and then to serum-free), while the amount of culture medium was gradually increased to allow the hybridoma cells to gradually adapt to the serum-free culture environment.

[0063] In preparing monoclonal antibodies, hybridoma cell lines 1A5 and 4D6 obtained in step 2.2 were revived using serum-free medium. When the cells were in good condition, the amount of medium and the culture vessel were increased. When a large number of cells died, the culture medium was collected, and the supernatant was centrifuged, 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, elution was performed with elution buffer to obtain the purified antibody. SDS-PAGE was used to identify its purity, and the results are as follows: Figure 4 As shown.

[0064] from Figure 4 As can be seen, the sizes of monoclonal antibodies 1A5 and 4D6 are consistent with expectations, and they have high purity.

[0065] 2.4 Specificity analysis of monoclonal antibodies The specificity of the monoclonal antibody was validated using Western blotting. Specifically, the process included the following steps: 1) Polyacrylamide gel electrophoresis (SDS-PAGE) Gel preparation: A vertical electrophoresis system consisting of 10% separating gel and 5% stacking gel was used.

[0066] Loading parameters: The pre-stained protein marker (5 μL) and the test sample (20 μL) were loaded separately, and the empty BL21(DE3) lysis buffer was used as a negative control (NC).

[0067] Electrophoresis procedure: Start at a constant voltage of 80V, and increase the voltage to 120V after the bromophenol blue indicator enters the separating gel. The entire process takes about 2 hours.

[0068] 2) Transfer operation Membrane treatment: Cut a PVDF membrane to match the size of the gel and activate it with methanol for 5 minutes.

[0069] Sandwich assembly: Assemble in the following order: anode plate → filter paper → gel → PVDF membrane → filter paper → cathode plate, ensuring no air bubbles.

[0070] Electrotransfer conditions: Wet transfer at a constant current of 300mA for 1 hour, followed by washing with TBST buffer 3 times for 5 minutes each time.

[0071] 3) Immunoblotting detection Blocking: Block overnight at 4°C with 5% BSA-TBST solution.

[0072] Primary antibody incubation: Incubate 1A5 and 4D6 monoclonal antibodies diluted with 1% BSA-TBST (1:1000) at 37°C for 1 hour.

[0073] Secondary antibody incubation: Goat anti-mouse HRP-labeled antibody (1:20000 dilution), reacted at 37°C for 1 hour.

[0074] Washing procedure: 3 washes with TBST buffer, 10 minutes each time; 3 washes with PBST buffer, 10 minutes each time.

[0075] 4) Color imaging ECL chemiluminescence method for color development, and electrochemiluminescence imaging system for recording experimental results.

[0076] The results are as follows Figure 5 and 6 As shown.

[0077] from Figure 5 and 6 As can be seen, monoclonal antibody 1A5 specifically recognizes MetQ protein, and monoclonal antibody 4D6 specifically recognizes LPTG protein, both showing a single immune response band, confirming the high specificity of monoclonal antibodies.

[0078] 2.5 Sequencing of monoclonal antibodies Hybridoma cell lines 1A5 and 4D6 obtained in step 2.2 were cultured separately, lysed, and total RNA was extracted. cDNA was synthesized from the mRNA using random hexamer primers (5'-Pd(NNNNNN)-3'; where N = G, A, T, or C). Then, two rounds of nested PCR were performed: amplification was performed using the first-strand cDNA as a template, with the forward primer containing sequences complementary to the corresponding heavy and light chain leader sequences, and the reverse primer containing sequences within the constant regions of the heavy and light chains. The specific primer sequences are as follows: Heavy chain forward primer: 5'-CGGCCCAGCCGGCC-3' (SEQ ID NO: 9); Heavy chain reverse primer: 5'-TGAACCGCCTCCACC-3' (SEQ ID NO: 10); Light chain forward primer: 5'-GGTTCCACTGGT-3' (SEQ ID NO: 11); Light chain reverse primer: 5'-GTGCAGCATCAGC-3' (SEQ ID NO: 12).

[0079] 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 cycles; final extension at 72℃ for 5 min.

[0080] The second round of amplification yielded gene products containing 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 1A5 and 4D6, as follows: The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 1A5 is shown below: EVQLQQSGAELVKPGASVKLSCTASGFNIKETYMLWVKQRPEQGLEWIGKIHVAGTCTRHVPKRAGKATITADTSSNTAYLQLSSLTSEDTAVYYCGRKGLSSWGQGTLVTVSA (SEQ ID NO: 16).

[0081] The complementarity-determining region of the heavy chain variable region of monoclonal antibody 1A5 is shown below: CDR-H1: ETYML (SEQ ID NO: 13); CDR-H2: KIHVAGTCTRHVPKRAG (SEQ ID NO: 14); CDR-H3: KGLSS (SEQ ID NO: 15).

[0082] The amino acid sequence of the variable region of the light chain of monoclonal antibody 1A5 is shown below: DIVMSSQSPSSLAVSVGELVTMSSCRTTWTLSTTCTSRSTILWYQQKPGQSPKLLITSGASTKMLGVPDRFTGSGSGTDFTLISSVKAEDLVYYCCCCSSSTVTFGGGTKEIK (SEQ ID NO: 20).

[0083] The complementarity-determining region of the light chain variable region of monoclonal antibody 1A5 is shown below: CDR-L1: RTTWTLSTTCTSRSTIL (SEQ ID NO: 17); CDR-L2: SGASTKML (SEQ ID NO: 18); CDR-L3: CCSSSTVT (SEQ ID NO: 19).

[0084] The amino acid sequence of the variable region of the 4D6 heavy chain of the monoclonal antibody is shown below: DVQLVESGGDLVQPGGSLKFSCAASGFSFSRYGMLWVRQTPDKRLELVAQIQSRQAGSSSPERIKGRFTISRDDNAKNTLYLQMSSLKSEDTAMYYCARAATTESSYELIWGAGTTVIVSS (SEQ ID NO: 24).

[0085] The complementarity-determining region of the 4D6 heavy chain variable region of the monoclonal antibody is shown below: CDR-H1: RYGML (SEQ ID NO: 21); CDR-H2: QIQSRQAGSSSPERIKG (SEQ ID NO: 22); CDR-H3: AATTESSYELI (SEQ ID NO: 23).

[0086] The amino acid sequence of the variable region of the light chain of monoclonal antibody 4D6 is shown below: DVVMTQTPLTLSVTIGQPASISCRTTWIIIEAAEAHRSCQWLFQMPGQSPKRLLYIVRKRCEGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWCASTLHRVFGGGTKLEIK (SEQ ID NO: 28).

[0087] The complementarity-determining region of the variable region of the 4D6 light chain of the monoclonal antibody is shown below: CDR-L1: RTTWIIIEAAEAHRSCQ (SEQ ID NO: 25); CDR-L2: IVRKRCE (SEQ ID NO: 26); CDR-L3: WCASTLHRV (SEQ ID NO: 27).

[0088] Example 3: Preparation of magnetic bead antibody conjugates and enrichment and isolation of Bifidobacterium longum 3.1 Preparation of magnetic bead antibody conjugates First, the magnetic bead antibody conjugate is prepared, specifically including the following steps: 3.1.1 Monoclonal antibody dilution The monoclonal antibodies 1A5 and 4D6 obtained in Example 2 were diluted with MES buffer (concentration of 15mM, pH 6.0) to an antibody concentration of 2mg / mL to obtain monoclonal antibody 1A5 dilution and monoclonal antibody 4D6 dilution, respectively.

[0089] 3.12 Activation of Carboxyl Magnetic Beads 1) Take 1 mg of carboxyl magnetic beads, wash 3 times with MES buffer, and resuspend them in 0.1 mL of MES buffer to a concentration of 10 mg / mL; 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 °C for 30 min to obtain activated carboxyl magnetic beads.

[0090] 3.1.3 Covalent coupling of magnetic beads and antibodies The antibody dilution obtained in step 3.1 was mixed with 1 mg of activated carboxyl magnetic beads (diameters of 50 nm, 200 nm, and 1000 nm, respectively) 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 aspirated, and the remaining antibody content in the supernatant was simultaneously detected. 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 the magnetic bead antibody. The following are listed: 1A5①, 1A5②, 1A5③, 1A5④, 1A5⑤, 1A5⑥, 4D6①, 4D6②, 4D6③, 4D6④, 4D6⑤, and 4D6⑥. The conditions for conjugating the magnetic beads are shown in Table 2.

[0091] 3.2 Enrichment and Isolation of Bifidobacterium longum Then, the magnetic bead antibody conjugate prepared in section 3.1 was used to enrich and isolate Bifidobacterium longum, specifically including the following steps: 1) Preparation of fecal microbial suspension Take 10g of fecal sample and add physiological saline at a ratio of 1:5 (i.e., 5mL of physiological saline per 1g of feces). Stir thoroughly to ensure the feces and saline are evenly mixed. First, filter the mixture through a 10-mesh sieve to remove larger fecal residues. Then, further filter the bacterial suspension through 30-mesh and 60-mesh sieves.

[0092] Place the filtered bacterial suspension in a centrifuge and centrifuge at 8000g for 5 minutes. After centrifugation, discard the supernatant, add 40mL of physiological saline to the precipitate, and mix thoroughly to redisperse the bacterial precipitate in the physiological saline.

[0093] Repeat the above steps of centrifugation, discarding supernatant, and resuspending three times to finally obtain a relatively clean fecal microbiota suspension.

[0094] 2) Incubation of magnetic bead antibody conjugate with fecal suspension Take 5 mL of the prepared fecal suspension and add 1 mg of the magnetic bead antibody conjugate prepared in step 3.1. Place the mixture in a 37°C environment, mix well, and incubate for 2 hours to allow the magnetic bead antibody conjugate to fully bind to Bifidobacterium longum bacteria. After incubation, use a magnetic rack to separate the magnetic beads, and remove the unbound microorganisms and supernatant.

[0095] 3) Isolation of magnetic beads and Bifidobacterium longum bacteria The magnetic beads contaminated with Bifidobacterium longum (labeled magnetic beads) were resuspended in physiological saline. An antibody label removal reagent, namely 0.05% papain (product number S10011, Yuanye Biotechnology), was mixed with the labeled magnetic beads and incubated at 37°C for 3 hours. Papain cleaves the Fc and Fab fragments of the mouse monoclonal antibody, thereby separating the magnetic beads from the Bifidobacterium longum bacteria.

[0096] 4) Counting and determination of conditions for Bifidobacterium longum bacteria The magnetic beads were collected again using a magnetic rack, and the resulting supernatant was the Bifidobacterium longum bacterial suspension. This suspension was diluted and added to a hemocytometer for counting under a microscope. The yield of enriched Bifidobacterium longum bacteria is shown in Table 2.

[0097] Table 2. Antibody-conjugated magnetic bead conditions and yield results

[0098] As shown in Table 2, the magnetic bead antibody conjugate containing monoclonal antibodies 1A5 and 4D6 of this invention exhibits good enrichment efficiency for *Bifidobacterium longum*; and the best enrichment efficiency is achieved when the magnetic bead antibody conjugate contains both monoclonal antibodies 1A5 and 4D6. Furthermore, the enrichment of *Bifidobacterium longum* using magnetic beads with a diameter of 200 nm and an antibody-to-magnetic bead molar ratio of 1:5 demonstrates good efficiency. The magnetic bead antibody conjugate of this invention offers advantages such as high separation and enrichment yield and strong specificity for the enrichment of *Bifidobacterium longum*.

[0099] Example 4 Identification of Bifidobacterium longum The *Bifidobacterium longum* bacteria isolated and collected in Example 3 were diluted 1000 and 10000 times respectively, and spread on the prepared solid culture medium (tryptone: 10.0g, beef extract powder: 8.0g, yeast extract: 4.0g, glucose: 20.0g, triammonium citrate: 2.0g, dipotassium hydrogen phosphate: 2.0g, sodium acetate: 5.0g, MnSO4·4H2O: 0.05g, MgSO4·7H2O: 0.05g). 2g, Tween-80: 1.0mL, cysteine ​​hydrochloride: 0.5g, agar powder: 15.0g (for solid culture, omit for liquid), distilled water: 1000mL, pH adjustment: adjust to 6.2-6.4 with NaOH before sterilization, sterilize at 121℃ for 15 minutes) and culture in an anaerobic incubator for more than 3 days. Pick 10 single colonies, culture them in vitro in an anaerobic environment, extract DNA, and perform 16S rRNA gene sequencing using bacterial DNA as a template. The sequencing results were compared with BLAST in the NCBI database. The 16S rRNA gene sequence of this bacterium showed more than 99% homology with Bifidobacterium longum, and the 16S RNA gene sequences of the 10 single colonies in the sequencing results were all Bifidobacterium longum. Therefore, the isolated bacteria were identified as Bifidobacterium longum.

[0100] In summary, this invention uses MetQ and LPXTG proteins on the surface of Bifidobacterium longum as immunogens, and prepares monoclonal antibodies with high specificity and high binding affinity to antigens through hybridoma technology. After conjugating the monoclonal antibody with magnetic beads, the resulting magnetic bead antibody conjugate can achieve efficient enrichment and / or separation of Bifidobacterium longum in the sample to be treated.

[0101] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof to a specific protein of Bifidobacterium longum, characterized in that, comprises a heavy chain variable region and a light chain variable region shown in A1) or A2) below; A1) the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences shown in SEQ ID NOs: 13-15, respectively, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences shown in SEQ ID NOs: 17-19, respectively; A2) the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences shown in SEQ ID NOs: 21-23, respectively, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 having the amino acid sequences shown in SEQ ID NOs: 25-27, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, In A1), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO: 16; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO:

20. In A2), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO: 24; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 28, or an amino acid sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO:

28.

3. A biomaterial, characterized by, The biomaterial is selected from any one of the following: B1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2; B2) a recombinant vector comprising the nucleic acid molecule of B1); B3) a recombinant cell comprising the nucleic acid molecule of B1) or the recombinant vector of B2).

4. A magnetic bead antibody conjugate, characterized in that, The magnetic beads and the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2 coupled to the magnetic beads.

5. A method for preparing a magnetic bead antibody conjugate, characterized by, The method comprises the following steps: diluting the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2 to obtain a monoclonal antibody or antigen-binding fragment thereof diluent; carrying out carboxyl activation treatment on the magnetic beads to obtain activated carboxyl magnetic beads; coupling the monoclonal antibody or antigen-binding fragment thereof diluent to the activated carboxyl magnetic beads to obtain a magnetic bead-antibody conjugate.

6. The production method according to claim 5, wherein In the step of coupling the monoclonal antibody or antigen-binding fragment thereof diluent to the activated carboxyl magnetic beads, the molar ratio of the monoclonal antibody or antigen-binding fragment thereof diluent to the activated carboxyl magnetic beads is 1: (5-10), and the diameter of the activated carboxyl magnetic beads is 50-1000 nm.

7. The production method according to claim 6, wherein The diameter of the activated carboxyl magnetic beads is 200 nm.

8. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-2, the biomaterial according to claim 3, the magnetic bead antibody conjugate according to claim 4, or the magnetic bead antibody conjugate prepared by the method according to any one of claims 5-7 for use in enrichment and / or isolation of Bifidobacterium longum.

9. A method for enriching and / or isolating Bifidobacterium longum by using the magnetic bead-antibody conjugate according to claim 4 or the magnetic bead-antibody conjugate prepared by the preparation method according to any one of claims 5 to 7, characterized in that, The method comprises the following steps: providing a sample to be treated; adding the magnetic bead antibody conjugate to the sample to be treated, and after incubation, isolating magnetic beads combined with Bifidobacterium longum; suspending the magnetic beads combined with Bifidobacterium longum in a solution, adding a protease, and after incubation, isolating a Bifidobacterium longum suspension.

10. The method of claim 9, wherein, In the step of suspending the magnetic beads combined with Bifidobacterium longum in a solution, adding a protease, and after incubation, isolating a Bifidobacterium longum suspension, the protease is added in an amount of 0.05%, and the protease comprises papain.

Images