Monoclonal antibody tc1 to eubacterium zürichnese specific protein and its use

Abstract

The application discloses a kind of zürich true bacillus genus specific protein monoclonal antibody TC1 and its application.The monoclonal antibody TC1 includes heavy chain variable region and light chain variable region, and the amino acid sequences of heavy chain variable region and light chain variable region are respectively shown as SEQ ID NO:3 and 4.The antibody conjugated magnetic bead of the application is coupled from the monoclonal antibody TC1 and activated carboxyl magnetic bead.The antibody conjugated magnetic bead of the application can specifically enrich zürich true bacillus genus, and the enrichment efficiency is high, and the enrichment operation is simple, can be applied to the analysis of zürich true bacillus genus metabolite, and can be used for its qualitative and quantitative analysis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody TC1 specific to a Zurich Eubacterium genus protein and its applications. Background Technology

[0002] *Turicibacter bilis* is an anaerobic, Gram-positive bacterium belonging to the order Bifidobacteria. Studies have demonstrated that *Turicibacter bilis*, as a beneficial gut microbiota, can regulate glucose and lipid metabolism through the influence of polysaccharides and, as a target group for short-chain fatty acid production, possess butyrate-producing capabilities. For example, in some studies, *Turicibacter bilis* has been found to accumulate in the gut microbiota of mice with colon tumors and can significantly increase the number and size of tumors in germ-free mice. Therefore, *Turicibacter bilis* can serve as a key microbiota for studying human gut health.

[0003] Research on *Eubacterium zurichense* is currently very limited, and the specific mechanisms by which *Eubacterium zurichense* is associated with certain intestinal diseases are still under investigation. Further research into the applications of *Eubacterium zurichense* can be conducted by preparing specific antibodies against its target proteins. Currently, no literature or patents report on the preparation of specific antibodies against *Eubacterium zurichense* target proteins or on how to enrich *Eubacterium zurichense* from feces. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a monoclonal antibody TC1 specific to the genus *Eubacterium zurichense* and its applications. This invention uses the anchoring protein GMA92_05645 of *Eubacterium zurichense* to immunize mice, prepares the monoclonal antibody TC1 using hybridoma technology, and then conjugates the monoclonal antibody TC1 with magnetic beads. *Eubacterium zurichense* is then enriched based on the antibody-conjugated magnetic beads. The antibody-conjugated magnetic beads of this invention capture *Eubacterium zurichense* with high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to the analysis of metabolites of *Eubacterium zurichense*, as well as its qualitative and quantitative analysis.

[0005] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0006] This invention provides a monoclonal antibody for a specific protein of *Eubacterium zurichense*, wherein the monoclonal antibody is monoclonal antibody TC1, and monoclonal antibody TC1 includes a heavy chain variable region and a light chain variable region.

[0007] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody TC1 is shown in SEQ ID NO: 3;

[0008] The amino acid sequence of the variable region of the light chain of the monoclonal antibody TC1 is shown in SEQ ID NO: 4.

[0009] Furthermore, the heavy chain variable region of the monoclonal antibody TC1 includes four heavy chain backbone regions FR-H and three heavy chain complementarity-determining regions CDR-H, namely FR-H1, FR-H2, FR-H3 and FR-H4, and the three heavy chain complementarity-determining regions CDR-H are CDR-H1, CDR-H2 and CDR-H3.

[0010] The amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are shown in SEQ ID NO: 5 to 8, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO: 9 to 11, respectively.

[0011] The monoclonal antibody TC1 light chain variable region includes four light chain backbone regions FR-L and three light chain complementarity-determining regions CDR-L. The four light chain backbone regions FR-L are FR-L1, FR-L2, FR-L3 and FR-L4, and the three light chain complementarity-determining regions CDR-L are CDR-L1, CDR-L2 and CDR-L3.

[0012] The amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 are shown in SEQ ID NO: 12-15, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 16-18, respectively.

[0013] Furthermore, the monoclonal antibody TC1 is prepared from a hybridoma cell line.

[0014] This invention also provides a method for preparing a hybridoma cell line, comprising the following steps:

[0015] (1) The optimized nucleotide sequence of protein GMA92_05645 was transformed into E. coli BL21, expressed, and purified by sonication to obtain the purified protein.

[0016] (2) Mix the purified protein with Freund's adjuvant, emulsify it and then immunize the mice;

[0017] (3) Then, spleen cells from immunized mice were fused with myeloma cells SP2 / 0 and the hybridoma cell lines were obtained by screening.

[0018] Furthermore, the optimized nucleotide sequence of the protein GMA92_05645 codon is shown in SEQ ID NO: 2.

[0019] Furthermore, the amino acid sequence of the protein GMA92_05645 is shown in SEQ ID NO: 1.

[0020] The present invention also provides the application of the monoclonal antibody in the preparation of antibody-conjugated magnetic beads, wherein the monoclonal antibody is monoclonal antibody TC1.

[0021] This invention also provides a method for preparing antibody-conjugated magnetic beads, comprising the following steps:

[0022] (1) Dilute monoclonal antibody TC1 to 1.5-2.5 mg / mL using 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) to obtain diluted monoclonal antibody TC1;

[0023] (2) The magnetic beads were activated by carboxyl groups to obtain activated carboxyl magnetic beads;

[0024] (3) The diluted monoclonal antibody TC1 is coupled with activated carboxyl magnetic beads to obtain antibody-coupled magnetic beads, wherein the particle size of the activated carboxyl magnetic beads is 10-30 μm and the molar ratio of monoclonal antibody TC1 to activated carboxyl magnetic beads is 1:5-10.

[0025] Further, the monoclonal antibody TC1 is diluted to 2 mg / mL;

[0026] The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of monoclonal antibody TC1 to activated carboxyl magnetic beads is 1:5.

[0027] The present invention also provides an application of antibody-conjugated magnetic beads prepared by the above preparation method in the enrichment of Zurich Eubacterium bacteria.

[0028] The beneficial effects of this invention are:

[0029] 1. This invention utilizes antibody-conjugated magnetic beads to capture *Eubacterium zurichense*, which has high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to the analysis of metabolites of *Eubacterium zurichense*, as well as its qualitative and quantitative analysis.

[0030] 2. This invention obtains a specific antibody that recognizes the anchoring binding protein GMA92_05645 of the genus *Eubacterium zurichense*. The antibody and magnetic beads are coupled for the enrichment or isolation of *Eubacterium zurichense*. The antibody can bind to specific sites of *Eubacterium zurichense*, forming a synergistic effect, thereby improving the enrichment efficiency by enriching *Eubacterium zurichense* from the antibody-coupled magnetic beads and the specific protein target.

[0031] 3. The method for enriching Zurich Eubacterium spp. of the present invention is simple, does not require the use of expensive instruments such as flow cytometers, reduces costs, and is conducive to widespread application. Attached Figure Description

[0032] Figure 1 Here is an SDS-PAGE electrophoresis image of the purified monoclonal antibody TC1;

[0033] Figure 2 The graph shows the changes in the solution of *Eubacterium zurichense* captured by antibody-conjugated magnetic beads over incubation time. Detailed Implementation

[0034] 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.

[0035] Example 1

[0036] Protein recombinant expression, purity and concentration detection

[0037] The amino acid sequence of the anchoring protein GMA92_05645 of the genus Eubacterium Zurich is shown in SEQ ID NO: 1, and the codon-optimized nucleotide sequence of protein GMA92_05645 is shown in SEQ ID NO: 2.

[0038] The gene GMA92_05645 was transformed into *E. coli* BL21 to express the protein GMA92_05645. After sonication and centrifugation, the supernatant was purified using a nickel column chromatography method to obtain the protein. The protein A280 concentration was measured using a micro spectrophotometer, and the protein purity was analyzed by SDS-PAGE. The results are shown in Table 1. The purity of the purified protein GMA92_05645 reached 92%, which is high.

[0039] Table 1. Protein concentration and purity results after purification.

[0040] protein name Purification Tag A280 concentration SDS-PAGE protein purity GMA92_05645 His 2.4mg / ml 92％

[0041] Example 2

[0042] Mouse immune and antiserum titers and hybridoma cell fusion

[0043] 1. Mouse immunization

[0044] (1) After mixing protein GMA92_05645 with Freund's adjuvant, emulsify the mixture in a mixer. The initial immunization dose was 50 μg protein / mouse, with Freund's complete adjuvant, and the interval between the first and second immunizations was 3 weeks. The doses for the second to fourth immunizations were 50 μg protein / mouse, with Freund's incomplete adjuvant, and the interval between the second and second immunizations was 2 weeks.

[0045] (2) Take blood from the tail vein of mice and detect the serum antibody titer. Mice with a titer of 1:10000 or higher are generally selected for fusion.

[0046] (3) Three days before hybridoma cell fusion, mice were immunized with 100 μg protein per mouse without adjuvant. The immunization method is shown in Table 2.

[0047] Table 2. Mouse Immunization Schedule

[0048] Number of immunizations Immune sites adjuvant Immunization dose 1 subcutaneous Freund's complete adjuvant 50μg 2 abdominal cavity Freund's incomplete adjuvant 50μg 3 abdominal cavity Freund's incomplete adjuvant 50μg 4 abdominal cavity Freund's incomplete adjuvant 50μg 5 abdominal cavity No adjuvants 100μg

[0049] 2. Hybridoma cell fusion

[0050] (1) After blood was collected from the eyeballs of mice after immunization, spleen cell suspension was prepared from mice with good immunization effect and washed with PBS solution. SP2 / 0 cells were then mixed in at a ratio of spleen cells:SP2 / 0 = 10:1. After centrifugation at 1000 rpm for 5 min, the mixed cells were drained and the cell clumps were loosened by tapping.

[0051] (2) Add 1 mL of PEG 1450 to the fused cells in a 37°C water bath and react for 2 min in a 37°C water bath. Then slowly add 20 mL of RPMI-1640 stop solution along the tube wall.

[0052] (3) 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 DMEM complete medium containing HAT and pipetted into 96-well cell culture plates using a multichannel pipette.

[0053] (4) Cell colonies can generally be observed 3 days after fusion, and the medium should be changed and tested after 7 days.

[0054] (5) Based on the growth of the fused cells, when the colony size reaches approximately 1 / 4 of the bottom area of ​​the well, it is considered for detection. Take 100 μL of supernatant and perform detection using the indirect ELISA method. Select positive wells with high OD values ​​and good colony status for subcloning.

[0055] (6) Using HT medium, the selected positive cell colonies 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 grow. 4 The titer can be detected with a number of cells or more; then, the positive cell wells are taken again, and the subcloning screening is repeated. After all the cell supernatants in all the microwells are positive, the same subcloning is performed again until all the results are positive again, which confirms that a positive hybridoma cell line has been screened. The cell line is numbered TC1. This example successfully obtained a monoclonal antibody, which is named monoclonal antibody TC1.

[0056] 3. Antiserum titer detection

[0057] The titer of monoclonal antibody TC1 detected by indirect ELISA is shown in Table 3. The isotype of monoclonal antibody TC1 was identified using an antibody isotype identification kit, and the results are shown in Table 4. The isotype of monoclonal antibody TC1 is IgG2b.

[0058] Table 3. Titer determination of one monoclonal antibody strain

[0059]

[0060] Table 4. Isotype determination of monoclonal antibodies

[0061] Cell line number Subtype TC1 IgG2b

[0062] Example 3

[0063] Preparation of ascites fluid and monoclonal antibody TC1

[0064] 1. One week before fusing hybridoma cells, BALB / c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant per mouse, followed by inoculation with 5 × 10⁶ cells per mouse. 6 A number of positive hybridoma cells were collected, and ascites fluid was collected 7–12 days later to determine the monoclonal antibody titer.

[0065] 2. Centrifuge the collected ascites fluid at 10,000 rpm for 10 min to remove cellular components and other precipitates. Collect the supernatant and test the monoclonal antibody titer. Aliquot and store at -80℃ for later use.

[0066] 3. Load the monoclonal antibody sample to be purified onto a Protein A-agarose affinity chromatography column at a flow rate of 0.5 mL / min to allow the monoclonal antibody to bind to Protein A. Finally, elute with elution buffer to obtain monoclonal antibody TC1. Identify its purity using SDS-PAGE. Figure 1 As shown, the monoclonal antibody TC1 has high purity.

[0067] Example 4

[0068] Cell line sequencing

[0069] 1. Culture hybridoma cells TC1, lyse them, and extract total RNA and mRNA from the lysate. Use random hexamer primers (5'-Pd(NNNNNN)-3'N=G,A,T or C) to reverse transcribe mRNA to synthesize cDNA. Then, perform two rounds of nested PCR: amplify with the first-strand cDNA as a template, the forward primer is a sequence complementary to the corresponding heavy and light chain leader sequences of the monoclonal antibody TC1, and the reverse primer is a sequence in the constant region of the heavy and light chains.

[0070] Heavy chain forward primer: CGGCCCAGCCGGCC;

[0071] Heavy chain reverse primer: TGAACCGCCTCCACC;

[0072] Light chain forward primer: GGTTCCACTGGT;

[0073] Light chain reverse primer: GTGCAGCATCAGC.

[0074] 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.

[0075] 2. The second round of amplification yielded the gene product with restriction enzyme sites (EcoRI and HindIII), which was ligated into the pMD19-T cloning vector. Then, through sequencing and analysis, the light chain and heavy chain variable region sequences of the monoclonal antibody TC1 were obtained.

[0076] Heavy chain forward primer: TGAATTCCGGCCCAGCCGGCC;

[0077] Heavy chain reverse primer: TAAGCTTTGAACCGCCTCCACC;

[0078] Light chain forward primer: TGATTCGGTTCCACTGGT;

[0079] Light chain reverse primer: TAAGCTTGTGCAGCATCAGC.

[0080] 3. The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody TC1 is shown in SEQ ID NO: 3:

[0081]

[0082] Note: The bold and underlined regions indicate the heavy chain complementarity-determining regions (CDR-H), while the bolded regions only indicate the heavy chain backbone regions (FR-H); the amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 are shown in SEQ ID NO: 5, 6, 7, and 8, respectively; the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO: 9, 10, and 11, respectively.

[0083] FR-H1:EVQLQQSGPELVKPGASVKMSCKASGYTFT;

[0084] CDR-H1: WNPFR;

[0085] FR-H2: WVKQKPGQGLEWIG;

[0086] CDR-H2: SACFCCEACRNCDRPHA;

[0087] FR-H3:KATLTSDKSSSTAYMELSSLTSEDSAVYYCAR;

[0088] CDR-H3: ACNHCEPKEPEN;

[0089] FR-H4: WGQGTTLTVSS.

[0090] The amino acid sequence of the variable region of the light chain of the monoclonal antibody TC1 is shown in SEQ ID NO: 4.

[0091]

[0092] Note: The bold and underlined regions indicate the light chain complementarity-determining regions (CDR-L), while the bolded regions only indicate the light chain backbone regions (FR-L); the amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 are shown in SEQ ID NO: 12, 13, 14, and 15, respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 16, 17, and 18, respectively.

[0093] FR-L1:DVLVTQTPLSLPVSLGDQASISC;

[0094] CDR-L1: KQQSWLPRWCACWNAP;

[0095] FR-L2: WYLQKPGQSPKLLIY;

[0096] CDR-L2: KPWCKPT;

[0097] FR-L3: GVPDRFSGSGSGTDFTLKITRVEAEDLGVYYC;

[0098] CDR-L3: PSAWRPFSC;

[0099] FR-L4: FGGGTKLEIK.

[0100] Example 5

[0101] Preparation of antibody-conjugated magnetic beads

[0102] 1. Dilute monoclonal antibody TC1

[0103] Replace the monoclonal antibody TC1 buffer with 15mM MES buffer (pH 6.0), and dilute the monoclonal antibody TC1 to 2mg / mL with MES buffer to obtain the diluted monoclonal antibody TC1.

[0104] 2. Activation of carboxyl groups on the surface of magnetic beads

[0105] (1) After mixing the magnetic beads, take 100 μL of Mag COOH magnetic beads (70113-5, Suzhou Beaver Biotechnology) into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, wash twice with 200 μL of MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20), and then remove the supernatant.

[0106] (2) Quickly add 100 μL of freshly prepared EDC solution (10 mg / mL, using the above MEST solution as a dispersant) and 100 μL of NHS solution (10 mg / mL, using the above MEST solution as a dispersant) to the centrifuge tube containing the magnetic beads, vortex to mix and fully suspend the magnetic beads, activate at 25°C for 30 min, during which time keep the magnetic beads in suspension (a vertical mixer can be used for inverted mixing).

[0107] After the above steps, the carboxyl groups on the surface of the magnetic beads are activated, resulting in an activated carboxyl magnetic bead solution, which can be covalently coupled with biological ligands containing primary amino groups. (The activated state should not be stored for a long time; immediate coupling is recommended.)

[0108] 3. Covalent coupling of magnetic beads and monoclonal antibody TC1

[0109] (1) Take 200 μg of diluted monoclonal antibody TC1 and mix it with 100 μL of activated carboxyl magnetic bead solution (10 μm in diameter). After coupling at 25 °C for 1 h, place it at 4 °C overnight. Keep the magnetic beads in suspension during coupling (you can use a vertical mixer to invert and mix them).

[0110] (2) Magnetic separation: aspirate the supernatant and simultaneously detect the remaining antibody content in the supernatant. Calculate the amount and concentration of the magnetic bead-coupled antibody. Wash the magnetic beads 2 to 3 times with physiological saline and resuspend them with physiological saline to obtain the antibody-coupled magnetic bead solution, i.e., TC1 magnetic bead solution.

[0111] Example 6

[0112] Zurich Eubacterium enrichment effect verification

[0113] 1. Determination of antigen capture time by TC1 magnetic beads

[0114] (1) 4×10 4 CFU / mL Zurich Eubacterium solution was added to the TC1 magnetic bead solution prepared in Example 5 and incubated at room temperature on a horizontal shaker at 100 rpm / min.

[0115] (2) Magnetic separation was performed every 10 minutes, and then the number of bacteria in the supernatant was detected by plate counting.

[0116] like Figure 2As shown, the TC1 magnetic bead constructed in this invention is used in conjunction with 4×10 4 The capture limit was reached after incubation with CFU / mL Zurich Eubacterium solution for 30 minutes.

[0117] 2. Sensitivity verification analysis of TC1 magnetic beads for enriching Zurich Eubacterium spp.

[0118] The sensitivity was verified by enriching the above Zurich Eubacterium solution with TC1 magnetic beads at 10-fold serial dilutions. The specific steps are as follows:

[0119] (1) Add the TC1 magnetic bead solution prepared in Example 5 to 50 mL of Zurich Eubacterium solution after a 10-fold serial dilution, mix and incubate at 37°C for 30 min.

[0120] (2) TC1 magnetic beads were separated using a magnetic rack. Microorganisms and supernatant that were not bound to TC1 magnetic beads were removed. Then, the TC1 magnetic beads (i.e., labeled magnetic beads) bound to Zurich Eubacterium were resuspended in physiological saline. The labeled magnetic beads were then mixed with an antibody label removal reagent, i.e., 0.05% papain, and incubated at 37°C for 30 min to cut the Fc and Fab of the mouse monoclonal antibody, thereby separating the TC1 magnetic beads from Zurich Eubacterium.

[0121] (3) Then, the TC1 magnetic beads were collected using a magnetic rack, and the supernatant was the Zurich eubacterium suspension. After diluting the Zurich eubacterium, it was added to a hemocytometer and counted under a microscope. Three sets of replicate experiments were performed, and the average value was taken. The sensitivity was calculated as follows: sensitivity % = yield / number of Zurich eubacterium before enrichment * 100. The results are shown in Table 5. The TC1 magnetic beads of the present invention have high sensitivity to Zurich eubacterium.

[0122] Table 5. Sensitivity of TC1 magnetic beads for enriching Zurich eubacterium.

[0123] Serial Number Combination type Yield (CFU / mL) Sensitivity 1 TC1 <![CDATA[3.82*10 3 ]]> 95.5％

[0124] 3. Specificity analysis of TC1 magnetic bead enrichment for *Eubacterium* genus in Zurich

[0125] Enrichment specificity tests were performed on bacterial solutions of several intestinal probiotics, including *Eubacterium zurichense*, *Bifidobacterium bisporum*, *Lactobacillus*, *Faecalibacterium faecium*, and *Butyric acid-producing cocci*, to verify the specificity of the TC1 magnetic beads of the present invention for enriching *Eubacterium zurichense*. The specific steps are as follows:

[0126] (1) Take 50 mL of each of the 4×10⁻⁶ mL samples. 3 The solutions of the above-mentioned strains (Bifidobacterium, Lactobacillus, Coccidia foetida, and Butyric acid-producing cocci) at CFU / mL were each mixed with the TC1 magnetic bead solution prepared in Example 5 and incubated at 37°C for 30 min.

[0127] (2) Use a magnetic rack to separate TC1 magnetic beads, remove microorganisms and supernatant that have not bound to TC1 magnetic beads, and follow the same steps as in step 2 of this embodiment. Perform three sets of repeated experiments, take the average value, and calculate the specificity. Specificity% = yield / number of colonies in the bacterial solution before enrichment * 100.

[0128] The results are shown in Table 6. The TC1 magnetic beads in this invention showed significant specificity only for Eubacterium zurichense, with a specificity of 95.9%, while no significant specificity was found for Bifidobacterium, Lactobacillus, Faecalibacterium, and Butyrica proliferators.

[0129] Table 6. Specificity of TC1 magnetic beads in enriching different bacterial genera.

[0130] Serial Number strains Yield (CFU / mL) Specificity 1 Zurich Eubacterium <![CDATA[3.79*10 3 ]]> 95.9％ 2 Bifidobacteria 9 0.23％ 3 Lactobacilli 8 0.20％ 4 fecal bacteria 10 0.25％ 5 butyric acid cocci 8 0.20％

[0131] 4. Since *Zurichella zetaeniorhynchus* is generally found in the intestines and can be detected in feces, fecal samples were used to further test the enrichment effect of *Zurichella zetaeniorhynchus*. The specific steps are as follows:

[0132] (1) Add 5g of feces to physiological saline at a mass-volume ratio of 1:5 (for example, add 25mL of physiological saline to 5g of feces), filter through gauze, and collect the pre-treated fecal bacteria liquid.

[0133] (2) Take the pre-treated fecal microbial solution, add 0.1 mg of TC1 magnetic beads, mix and incubate at 37°C for 0.5 h, separate the TC1 magnetic beads using a magnetic rack, and remove the microorganisms and supernatant that have not bound to the TC1 magnetic beads.

[0134] (3) Then, the TC1 magnetic beads (i.e. labeled magnetic beads) bound to Eubacterium zurich were resuspended with physiological saline. The labeled magnetic beads were then mixed with an antibody label removal reagent, i.e., 0.05% papain, and incubated at 37°C for 0.5 h to cut the Fc and Fab of the mouse monoclonal antibody, thereby separating the TC1 magnetic beads from Eubacterium zurich.

[0135] (4) Then, the TC1 magnetic beads were collected using a magnetic rack, and the supernatant was the *Eubacterium zurichense* suspension. The *Eubacterium zurichense* was diluted and added to a hemocytometer, and counted under a microscope. Each experiment was performed in triplicate. The results are shown in Table 7. The yield of *Eubacterium zurichense* enriched by the TC1 magnetic beads of this invention was 1.45 * 10-1. 5 CFU / mL, the TC1 magnetic beads of the present invention can efficiently enrich Zurich Eubacterium in fecal samples.

[0136] Table 7. Production of fecal bacteria enriched by TC1 magnetic beads.

[0137] Serial Number Combination type Yield (CFU / mL) 1 TC1 <![CDATA[1.45*10 5 ]]>

[0138] 5. Culture and sequencing of *Eubacterium* species in Zurich

[0139] (1) Dilute the *Bacillus zurichensis* isolated from the feces to 10 μL. 3 -10 4 The culture conditions were as follows: cells / mL, cultured on tryptone-soybean agar (TSA) medium, which consisted of 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g sodium chloride, 13.0 g agar, 1.0 L distilled water, pH adjusted to 7.3 ± 0.2, and cultured for 72 h.

[0140] (2) Observe the colony growth. Observe the colony characteristics: round, transparent teardrop-shaped colonies grow, with a diameter of about 0.5 mm.

[0141] (3) Twenty single colonies were selected and used as templates for PCR amplification. The upstream primer sequence was 5'-AGAGTTTGATCCTGGCTCAGPCR-3', and the downstream primer sequence was 5'-GGTTACCTTGTTACGACTT-3'. PCR reaction system: DNA template (10 ng / μL) 1 μL, upstream and downstream primers (10 μmol / L) 2 μL each, 10×PCR Buffer 5 μL, dNTPs (2.5 mmol / L) 4 μL, Taq enzyme (5 U / μL) 0.5 μL, ddH2O 35.5 μL. PCR reaction conditions: 94℃ for 10 min; 94℃ for 1 min, 56℃ for 1 min, 72℃ for 25 s, 72℃ for 10 min, 30 cycles.

[0142] (4) After the reaction, the results were identified by agarose gel electrophoresis. The target band was recovered, purified and sequenced by gel electrophoresis. The sequencing results were compared with BLAST in the NCBI database. The results showed that the 16S rRNA gene sequence of the 20 colonies had 97% homology with Turicibacter bilis PIG517 (GenBank: GCA_024499545.1). Therefore, the isolate was identified as Turicibacter bilis. The above results further confirm that TC1 magnetic beads can efficiently enrich Turicibacter bilis.

[0143] 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 TC1 specific to a *Eubacterium zurichense* protein, characterized in that: The monoclonal antibody TC1 includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody TC1 is shown in SEQ ID NO: 3; The amino acid sequence of the variable region of the light chain of the monoclonal antibody TC1 is shown in SEQ ID NO:

4.

2. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody TC1 heavy chain variable region includes four heavy chain backbone regions FR-H and three heavy chain complementarity-determining regions CDR-H. The four heavy chain backbone regions FR-H are FR-H1, FR-H2, FR-H3 and FR-H4, and the three heavy chain complementarity-determining regions CDR-H are CDR-H1, CDR-H2 and CDR-H3. The amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are shown in SEQ ID NO: 5 to 8, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO: 9 to 11, respectively. The monoclonal antibody TC1 light chain variable region includes four light chain backbone regions FR-L and three light chain complementarity-determining regions CDR-L. The four light chain backbone regions FR-L are FR-L1, FR-L2, FR-L3 and FR-L4, and the three light chain complementarity-determining regions CDR-L are CDR-L1, CDR-L2 and CDR-L3. The amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 are shown in SEQ ID NO: 12-15, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 16-18, respectively.

3. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody TC1 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 optimized nucleotide sequence of protein GMA92_05645 was transformed into E. coli BL21, expressed, and purified by sonication to obtain the purified protein. (2) Mix the purified protein with Freund's adjuvant, emulsify it and then immunize the mice; (3) Then, spleen cells from immunized mice were fused with myeloma cells SP2 / 0 and the hybridoma cell lines were obtained by screening.

5. The preparation method according to claim 4, characterized in that: The optimized nucleotide sequence of the protein GMA92_05645 codon is shown in SEQ ID NO:

2.

6. The preparation method according to claim 4, characterized in that: The amino acid sequence of the protein GMA92_05645 is shown in SEQ ID NO:

1.

7. The application of the monoclonal antibody as described in claim 1 in the preparation of antibody-conjugated magnetic beads, characterized in that: The monoclonal antibody is monoclonal antibody TC1.

8. A method for preparing antibody-conjugated magnetic beads, characterized in that: Includes the following steps: (1) Dilute monoclonal antibody TC1 to 1.5-2.5 mg / mL with 2-(N-morpholino)ethanesulfonic acid buffer to obtain diluted monoclonal antibody TC1; (2) The magnetic beads were activated by carboxyl groups to obtain activated carboxyl magnetic beads; (3) The diluted monoclonal antibody TC1 is coupled with activated carboxyl magnetic beads to obtain antibody-coupled magnetic beads, wherein the particle size of the activated carboxyl magnetic beads is 10-30 μm and the molar ratio of monoclonal antibody TC1 to activated carboxyl magnetic beads is 1:5-10.

9. The preparation method according to claim 8, characterized in that: The monoclonal antibody TC1 was diluted to 2 mg / mL; The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of monoclonal antibody TC1 to activated carboxyl magnetic beads is 1:

5.

10. The application of antibody-conjugated magnetic beads prepared by the method described in claim 8 in the enrichment of Zurich Eubacterium bacteria.

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