Monoclonal antibody of fecal coccus and application

Abstract

The invention discloses a monoclonal antibody of fecal coccus and application of the monoclonal antibody. The monoclonal antibody is a monoclonal antibody CE1 or a monoclonal antibody CE2; the method comprises the following steps: selecting two proteins expressing an S-layer protein and an anchoring protein binding region of coprococcus faecalis, preparing two specific antibodies CE1 and CE2 for identifying the coprococcus faecalis by using the two proteins, respectively coupling the two antibodies to magnetic beads, and enriching the coprococcus faecalis by using the two magnetic bead-antibody conjugates to obtain the coprococcus faecalis magnetic bead-antibody conjugate. The principle that CE1 and CE2 antibodies are combined with specific antigens on the surfaces of bacteria is utilized, so that the regular faecalis coccus can be specifically enriched.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a monoclonal antibody of Coprococcus eutactus and application thereof. BACKGROUND

[0002] Coprococcus eutactus is the type strain of the genus Coprococcus, which is an obligate anaerobe, morphologically spherical, usually in pairs. β-glucan is the main growth substrate of human intestinal bacteria associated with Coprococcus eutactus, which metabolizes to produce butyrate and formate or lactic acid. Coprococcus eutactus can produce a wide range of carbohydrate substrates, showing good growth on glucose, and metabolizing to produce butyrate and propionate. Coprococcus eutactus is a gram-positive bacterium, with spherical cells, 0.8-1.5 μm in diameter, sometimes oval, in pairs or short chains, gram-positive, non-motile, strictly anaerobic. The optimum growth temperature is 37℃, most species are isolated from human intestine and feces, and rarely from human clinical specimens, indicating that the bacteria mainly colonize the intestinal tract of healthy people. Studies have shown that the decrease in the abundance of Coprococcus eutactus in the intestine is closely related to various diseases such as inflammatory bowel disease, type II diabetes, breast cancer and Crohn's disease, and taking the antibiotic ciprofloxacin can also cause a decrease in Coprococcus eutactus in the intestinal flora. These findings show that Coprococcus eutactus has the potential to be a microbial biomarker for assessing human gastrointestinal health.

[0003] At present, there is an urgent need for a product that can enrich Coprococcus eutactus in feces, so as to further provide technical support for the study of the metabolism, positioning, qualitative and quantitative analysis of Coprococcus eutactus. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art, and provides a monoclonal antibody of Coprococcus eutactus and application thereof. The specific antibody of the target protein of Coprococcus eutactus prepared by the present application can be used for enriching Coprococcus eutactus in feces, and can also be used for identifying the metabolic products of Coprococcus eutactus, and for qualitative and quantitative research of Coprococcus eutactus.

[0005] To achieve the above-mentioned purpose, the technical scheme designed by the present application is as follows:

[0006] The present application provides a monoclonal antibody of Coprococcus eutactus, which is a monoclonal antibody CE1 or a monoclonal antibody CE2.

[0007] The monoclonal antibody CE1 includes a CE1 heavy chain and a CE1 light chain, wherein,

[0008] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody CE1 is:

[0009] QIQLVQSGPELKKPGETVTISCKTSGFTFTGNYIM WVRQPP GKGFKWMG NTWITDEGTPAYEDKFG RFAFSLETSASTAYLQIS NLKNEDMASYFCAK DEDGSIAYD WGQGTSVTVSS;

[0010] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody CE1 is:

[0011] DVLMTQTPLSLPVSLGDQASISC SQSSSHVIGNNTNLEY WY LQKPGQSPRLLIY RPWCHMW GVPDRFSGSGSGTDFTLKISRVEA EDLGVYYC PTACRMLLC FGSGTKLEIK;

[0012] The monoclonal antibody CE2 comprises a CE2 heavy chain and a CE2 light chain, wherein,

[0013] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody CE2 is:

[0014] QVTLKESGPGILQPSQTLSLTCSFSGFSLS CWAFPLT WIRQPS GKGLEWLA RPCNEEERHNPNLSSK RLTISKDSSNKQVFLKFTSV DTADTATYYCAH FAYYNYDGAVDS WGQGTSVTVSS;

[0015] The amino acid sequence of the variable region of the light chain of the monoclonal antibody CE2 is:

[0016] DIQLTQSPAILSVSPGERVSFSC KKGQTCPACYVR WYQQRT NGSPRLLIK NSQDWPN GLPSRFSGSGSGTDFTLTISSVESEDIADY YC SSQNWSFCAL FGGGTKLEI.

[0017] Further, in the monoclonal antibody CE1, the amino acid sequence of the variable region of the heavy chain of CE1 comprises three complementarity determining regions (CDRs), which are respectively:

[0018] CE1-CDR-H1: GNYIM,

[0019] CE1-CDR-H2: NTWITDEGTPAYEDKFG,

[0020] CE1-CDR-H3: DEDGSIAYD;

[0021] The amino acid sequence of the variable region of the light chain of CE1 includes three complementarity determining regions (CDRs), which are respectively:

[0022] CE1-CDR-L1: SQSSSHVIGNNTNLEY,

[0023] CE1-CDR-L2: RPWCHMW,

[0024] CE1-CDR-L3: PTACRMLLC.

[0025] In the monoclonal antibody CE2, the amino acid sequence of the variable region of the heavy chain of CE2 includes three complementarity determining regions (CDRs), which are respectively:

[0026] CE2-CDR-H1: CWAFPLT,

[0027] CE2-CDR-H2: RPCNEEERHNPNLSSK,

[0028] CE2-CDR-H3: FAYYNYDGAVDS;

[0029] The amino acid sequence of the variable region of the light chain of CE2 includes three complementarity determining regions (CDRs), which are respectively:

[0030] CE2-CDR-L1: KKGQTCPACYVR,

[0031] CE2-CDR-L2: NSQDWPN,

[0032] CE2-CDR-L3: SSQNWSFCAL.

[0033] Further, the monoclonal antibody is prepared by a hybridoma cell strain.

[0034] The application further provides a preparation method of a hybridoma cell strain, including the following steps:

[0035] 1) transforming the protein-optimized nucleotide sequence into Escherichia coli BL21 to express and ultrasonically purify to obtain a purified protein; wherein the protein is any one of COPEUT_01511 and COPEUT_01706;

[0036] 2) mixing the purified protein with Freund's adjuvant, immunizing a mouse after emulsification, then fusing the spleen cells of the immunized mouse with myeloma cells SP2 / 0 to obtain a hybridoma cell strain through detection and screening, wherein the hybridoma cell strain is any one of the hybridoma cell strain CE1 and the hybridoma cell strain CE2.

[0037] Further, when the protein is COPEUT_01511, the optimized nucleotide sequence of COPEUT_01511 is shown as SEQ ID NO: 2,

[0038] When the protein is COPEUT_01706, the optimized nucleotide sequence of COPEUT_01706 is shown as SEQ ID NO: 4.

[0039] Further, the amino acid sequence of COPEUT_01511 is shown as SEQ ID NO: 1, and the amino acid sequence of COPEUT_01706 is shown as SEQ ID NO: 3.

[0040] The application further provides an application of the monoclonal antibody in preparation of a magnetic bead-antibody conjugate.

[0041] The application further provides a preparation method of the magnetic bead-antibody conjugate, comprising the following steps:

[0042] 1) diluting the monoclonal antibody with a MES buffer,

[0043] 2) performing carboxyl activation on the magnetic beads to obtain activated carboxyl magnetic beads, wherein the particle size of the activated carboxyl magnetic beads is 10-30 μm;

[0044] 3) coupling the monoclonal antibody in step 1) with the activated carboxyl magnetic beads to obtain a magnetic bead-antibody conjugate, wherein the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5-10; and the magnetic bead-antibody conjugate is any one of magnetic bead-antibody conjugate CE1 and magnetic bead-antibody conjugate CE2.

[0045] Further, the particle size of the activated carboxyl magnetic beads is 10 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5.

[0046] The application further provides an application of the antibody conjugate in enriching or separating Faecalibacterium prausnitzii, wherein the antibody conjugate is any one or both of the magnetic bead-antibody conjugate CE1 and the magnetic bead-antibody conjugate CE2.

[0047] The application has the following beneficial effects:

[0048] 1. The application selects two proteins expressing the S-layer protein (Surface-layer) and the anchor protein binding region (LPxTG structure) of Eubacterium rectum, and uses the two proteins to prepare two specific antibodies CE1 and CE2 for recognizing Eubacterium rectum, and then couples the two antibodies to magnetic beads, respectively, and uses the two magnetic bead-antibody conjugates to enrich Eubacterium rectum, and uses the principle that the antibodies CE1 and CE2 bind to specific antigens on the surface of bacteria to specifically enrich Eubacterium rectum.

[0049] 2. The magnetic bead sorting method using a magnetic stand according to the application has low cost compared with a flow sorting method, and can retain the activity of target bacteria to a greater extent compared with other affinity column purification methods. The finally enriched E. rectale can be applied to analysis of metabolites of E. rectale, and can also be applied to qualitative or quantitative analysis of E. rectale. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 FIG. 2 shows electrophoretograms of two purified antibodies. DETAILED DESCRIPTION

[0051] The application will be further described in detail below with reference to specific examples, so as to be understood by those skilled in the art.

[0052] Example 1: Detection of protein recombinant expression purity and concentration

[0053] The codon-optimized nucleotide sequences SEQ2, SEQ4 of COPEUT_01511 and COPEUT_01706 were expressed in E. coli, and transformed into E. coli BL21. After ultrasonic disruption and centrifugation, the supernatant was purified by a nickel column to obtain the protein. The protein A280 concentration was measured by an ultramicro spectrophotometer, and the protein purity was analyzed by SDS-PAGE (see Table 1). In the table,

[0054] The amino acid sequence of COPEUT_01511 is shown as SEQ ID NO: 1:

[0055] MVLCEKIKASGSCLRRRRSEKVMKKVMTHMIRRIFSRKRLAVYAMTMVMLLGFGITAMANESPDRDFGTDSSHILLSEPDTSIEDIDDLVMGYDPAGVLGDSTLKGFSTLAVFSVTAVDDYDFSNLEITKIRGAVITSDMTVQVRFLPTDGAWEKLSFEVVDGAVNVKLPSEGQLAIFVKDEVDIKDDDGDGNSMTKPTTESGTDGNVDPGKAPQTGDRGGGGSGGGGSGGGGSKKIRK;

[0056] The nucleotide sequence of COPEUT_01511 is shown as SEQ ID NO: 2:

[0057] ATGGTCCTGTGCGAAAAAATTAAAGCTTCTGGCTCCTGTCTGCGCCGTCGTCGTAGCGAGAAAGTGATGAAAAAAGTTATGACCCACATGATCCGCCGTATCTTCTCCCGTAAACGTCTGGCTGTGTATGCTATGACCATGGTTATGCTGCTGGGTTTTGGTATCACCGCAATGGCCAACGAATCTCCGGATCGTGACTTCGGCACCGACAGCTCTCATATTCTGCTGTCTGAACCGGATACCTCCATCGAAGACATTGACGACCTGGTTATGGGTTATGATCCGGCTGGCGTGCTGGGTGACAGCACCCTGAAAGGCTTCTCTACGCTGGCTGTGTTCTCCGTTACTGCGGTTGACGATTACGACTTCTCCAACCTGGAAATCACTAAGATCCGTGGTGCAGTCATCACCTCCGATATGACCGTTCAGGTGCGTTTCCTGCCTACCGACGGCGCCTGGGAAAAACTGAGCTTCGAAGTGGTCGATGGTGCAGTAAATGTAAAACTGCCATCCGAAGGCCAGCTGGCGATCTTCGTCAAAGATGAGGTTGATATCAAGGACGATGACGGCGATGGCAACTCCATGACCAAGCCGACCACTGAATCTGGCACCGATGGTAACGTCGATCCGGGTAAAGCACCGCAGACTGGCGATCGTGGTGGTGGTGGTAGCGGCGGTGGTGGTAGCGGTGGCGGCGGTTCCAAAAAAATCCGTAAG;

[0058] COPEUT_01706 amino acid sequence is shown in SEQ ID NO: 3:

[0059] MRAILYVGCSIEGMINMMKSKLTIVKKYAVLMMTVIVCLCMQPFAMTVQASQPKVMVVDCKIVQKEIIAGQTFDLAVTIKNTGSRYVDNLKISVTSDTGDIVPAEGAGNGFLEELPNGEESTFTFRLRAADGLAEKSYKLSVVNEYDDIWGNPYSATDVIYLPVKLEQRASITDLYVDDDAVLGDSVEVIGSVNNMGAGMLYNVRVRVESNYLEENDTFIGNIDTGKSGSIDILTNASATTSETGELSNVVVTYEDKEGNETELTGKFKVTVNSPVYDNVEKIKDTTTKINKNGGGGSGGGGSGGGGSRRNKRKKRILEEF;

[0060] The nucleotide sequence of the COPEUT_01706 protein is shown in SEQ ID No. 4:

[0061] ATGCGCGCTATCCTGTACGTCGGCTGCTCCATCGAAGGCATGATCAACATGATGAAATCCAAGCTGACTATCGTTAAAAAATACGCGGTTCTGATGATGACCGTAATTGTTTGCCTGTGCATGCAGCCATTCGCCATGACTGTGCAGGCTTCCCAACCGAAAGTCATGGTAGTAGACTGCAAGATCGTGCAGAAAGAAATTATTGCGGGCCAGACTTTTGACCTGGCTGTGACCATTAAAAACACCGGTTCTCGTTACGTGGACAATCTGAAAATCAGCGTTACGTCTGACACGGGCGACATCGTTCCGGCCGAAGGTGCAGGTAACGGTTTTCTGGAAGAACTGCCAAACGGCGAAGAATCTACTTTCACTTTCCGCCTGCGTGCAGCGGATGGTCTGGCAGAAAAAAGCTACAAACTGTCTGTCGTCAACGAGTACGACGACATCTGGGGTAACCCGTACTCTGCTACTGATGTCATCTATCTGCCTGTTAAACTGGAACAACGCGCGTCCATCACTGACCTGTACGTAGATGATGACGCGGTTCTGGGTGACTCTGTTGAAGTGATTGGCAGCGTTAACAACATGGGTGCAGGCATGCTGTATAACGTCCGTGTGCGCGTCGAGTCTAACTATCTGGAAGAGAACGACACCTTCATTGGTAACATCGACACCGGTAAATCTGGCAGCATCGATATTCTGACGAATGCATCCGCTACTACCAGCGAAACCGGCGAACTGAGCAACGTAGTTGTTACCTATGAAGACAAAGAGGGCAACGAGACGGAACTGACTGGTAAATTCAAAGTCACTGTGAACTCCCCGGTATACGATAACGTGGAGAAGATCAAAGACACTACCACCAAGATCAACAAAAACGGCGGTGGCGGTTCTGGTGGCGGTGGTAGCGGCGGTGGCGGCTCCCGTCGCAACAAACGTAAAAAACGTATTCTGGAGGAATTC。

[0062] Table 1. Concentration and purity results of the two proteins after purification.

[0063] Protein name Purification tag A280 concentration SDS-PAGE protein purity COPEUT_01511 His 1.4 mg / ml 93％ COPEUT_01706 His 1.3 mg / ml 94％

[0064] Example 2: Detection of mouse immune and antiserum titers and hybridoma cell fusion

[0065] COPEUT_01706 and COPEUT_01511 proteins were mixed with Freund's adjuvant and emulsified using a homogenizer. The initial immunization dose was 50 μg protein / mouse, using Freund's complete adjuvant, with a 3-week interval between the first and second immunizations. The second to fourth immunizations used 50 μg protein / mouse, using Freund's incomplete adjuvant, with a 2-week interval between the first and second immunizations. Blood was collected from the tail vein of mice to detect serum antibody titers. Mice with a titer of 1:10,000 or higher were generally selected for fusion. The immunization methods are shown in Table 2.

[0066] Table 2. Mouse Immunization Schedule

[0067]

[0068]

[0069] Three days prior to fusion, mice were immunized with 50 μg protein per mouse without adjuvant. Blood was collected from the eyeballs of mice. Spleen cell suspensions were prepared from mice with good immunization response, washed thoroughly with PBS, and then mixed with SP2 / 0 cells at a ratio of 10:1 (spleen cells:SP2 / 0). The mixture was centrifuged at 1000 rpm for 5 minutes, and the cells were drained and the cell clumps were gently tapped to loosen them. 1 ml of PEG1450 was added to a 37°C water bath, and the mixture was incubated at 37°C for 2 minutes. Then, 20 ml of RPMI-1640 stop solution was slowly added along the tube wall. After fusion termination, the cells were centrifuged at 800 rpm for 5 minutes, and any remaining liquid was aspirated. The cells were resuspended in DMEM complete medium containing HAT and pipetteed into 96-well cell culture plates. Cell colonies can generally be observed 3 days after fusion, and the medium is changed and analyzed on day 7.

[0070] Based on cell growth, colonies that have grown to approximately one-quarter of the well bottom area are considered for detection. 100 μL of supernatant is collected and analyzed using an indirect ELISA method. Subcloning is performed on positive wells with high OD values ​​and good colony condition.

[0071] The selected positive cell colonies were diluted to 1 cell / well using the limiting dilution method on HT medium. The cells were then seeded into 96-well cell culture plates. Once the monoclonal cells reached a medium size and a density of approximately 10⁻⁶ cells / well, they were allowed to grow. 4Antibody titer can be detected with a number of cells or more. Then, positive cell wells are taken again for subcloning screening. Once all cell supernatants in the wells are positive, the same subcloning process is repeated until a completely positive result is obtained again, confirming the selection of positive hybridoma cell lines. Cell lines are designated CE1 and CE2. Antibody titers detected by indirect ELISA are shown in Table 3. Antibody subtype identification kits were used to identify the subtypes of the two monoclonal antibodies. The subtype results are shown in Table 4.

[0072] Table 3. Titer determination of two monoclonal antibodies

[0073]

[0074]

[0075] Table 4. Isotype determination of two monoclonal antibodies

[0076] Cell line number Subtype CE1 IgGl CE2 IgG2a

[0077] Example 3: Serum-free culture of hybridoma cells and purification of antibodies

[0078] Hybridoma cells were resuscitated in the presence of serum. During several successive 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 substitute additive was gradually increased to allow the hybridoma cells to adapt and continue growing. In the preparation of monoclonal antibodies, hybridoma cells were resuscitated in serum-free medium. When the cells were in good condition, the volume of medium and the culture vessel were increased. When a large number of cells died, the medium was collected, centrifuged, and the supernatant was filtered and collected. 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 antibody. SDS-PAGE was used to determine its purity. Figure 1 As shown.

[0079] Example 4: Cell line sequencing

[0080] CE1 and CE2 hybridoma cells were cultured and lysed. Total RNA and mRNA were extracted from the lysates. 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.

[0081] Heavy chain forward primer: CGGCCCAGCCGGCC

[0082] Heavy chain reverse primer: TGAACCGCCTCCACC;

[0083] Light chain forward primer: GGTTCCACTGGT,

[0084] Light chain reverse primer: GTGCAGCATCAGC;

[0085] 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.

[0086] The second round of amplification produced gene products with restriction enzyme sites (EcoRI and HindIII), which were then ligated into the pMD19-T cloning vector. Sequencing and analysis were then used to obtain the antibody light chain and heavy chain variable region sequences of CE1 and CE2.

[0087] Heavy chain forward primer: TGAATTCCGGCCCAGCCGGCC,

[0088] Heavy chain reverse primer: TAAGCTTTGAACCGCCTCCACC;

[0089] Light chain forward primer: TGATTCGGTTCCACTGGT,

[0090] Light chain reverse primer: TAAGCTTGTGCAGCATCAGC.

[0091] Monoclonal antibody CE1 consists of a CE1 heavy chain and a CE1 light chain, wherein,

[0092] The amino acid sequence of the variable region of the CE1 heavy chain is shown in SEQ ID NO:5:

[0093] QIQLVQSGPELKKPGETVTISCKTSGFTFT GNYIM WVRQPP GKGFKWMG NTWITDEGTPAYEDKFG RFAFSLETSASTAYLQIS NLKNEDMASYFCAK DEDGSIAYD WGQGTSVTVSS;

[0094] Note: The bold and underlined areas indicate the complementarity determination region (CDR);

[0095] FR-H1: QIQLVQSGPELKKPGETVTISCKTSGFTFT,

[0096] CDR-H1: GNYIM

[0097] FR-H2: WVRQPPGKGFKWMG

[0098] CDR-H2: NTWITDEGTPAYEDKFG

[0099] FR-H3:RFAFSLETSASTAYLQISNLKNEDMASYFCAK,

[0100] CDR-H3: DEDGSIAYD

[0101] FR-H4: WGQGTSVTVSS;

[0102] The amino acid sequence of the variable region of the CE1 light chain is shown in SEQ ID NO:6:

[0103] DVLMTQTPLSLPVSLGDQASISC SQSSSHVIGNNTNLEY WY LQKPGQSPRLLIY RPWCHMW GVPDRFSGSGSGTDFTLKISRVEA EDLGVYYC PTACRMLLC FGSGTKLEIK;

[0104] Note: The bold and underlined areas indicate the complementarity determination region (CDR).

[0105] FR-L1: DVLMTQTPLSLPVSLGDQASISC,

[0106] CDR-L1: SQSSSHVIGNNTNLEY,

[0107] FR-L2: WYLQKPGQSPRLLIY

[0108] CDR-L2: RPWCHMW,

[0109] FR-L3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC,

[0110] CDR-L3: PTACRMLLC,

[0111] FR-L4: FGSGTKLEIK;

[0112] Monoclonal antibody CE2 consists of a CE2 heavy chain and a CE2 light chain, wherein,

[0113] The amino acid sequence of the variable region of the CE2 heavy chain is shown in SEQ ID NO:7:

[0114] QVTLKESGPGILQPSQTLSLTCSFSGFSLS CWAFPLT WIRQPS GKGLEWLA RPCNEEERHNPNLSSKRLTISKDSSNKQVFLKFTSVDTADTATYYCAH FAYYNYDGAVDS WGQGTSVTVSS;

[0115] Note: The bold and underlined areas indicate the complementarity determination region (CDR).

[0116] FR-H1: QVTLKESGPGILQPSQTLSLTCSFSGFSLS,

[0117] CDR-H1: CWAFPLT,

[0118] FR-H2: WIRQPSGKGLEWLA

[0119] CDR-H2: RPCEEERHNPNLSSK,

[0120] FR-H3:RLTISKDSSNKQVFLKFTSVDTADTATYYCAH,

[0121] CDR-H3: FAYYNYDGAVDS

[0122] FR-H4: WGQGTSVTVSS;

[0123] The amino acid sequence of the variable region of the CE2 light chain is shown in SEQ ID NO:8:

[0124] DIQLTQSPAILSVSPGERVSFSC KKGQTCPACYVR WYQQRT NGSPRLLIK NSQDWPN GLPSRFSGSGSGTDFTLTISSVESEDIADY YC SSQNWSFCAL FGGGTKLEI;

[0125] Note: The bold and underlined areas indicate the complementarity determination region (CDR).

[0126] FR-L1:DIQLTQSPAILSVSPGERVSSFSC,

[0127] CDR-L1: KKGQTCPACYVR

[0128] FR-L2: WYQQRTNGSPRLLIK,

[0129] CDR-L2: NSQDWPN,

[0130] FR-L3: GLPSRFSGSGSGTDFTLTISSVESEDIADYYC,

[0131] CDR-L3: SSQNWSFCAL

[0132] FR-L4: FGGGTKLEI.

[0133] Example 5: Magnetic bead sorting and enrichment of regular fecal cocci.

[0134] 1. Activation of carboxyl groups on the surface of magnetic beads

[0135] 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, and wash twice with 200 μL MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20), then remove the supernatant; quickly add 100 μL of freshly prepared LEDC solution (10 mg / mL, using the above MEST solution as a dispersant) and 100 μL of NHS (10 mg / mL, using the above MEST solution as a dispersant) to the centrifuge tube containing the magnetic beads, vortex to fully suspend the magnetic beads, and activate at 25 °C for 30 min, keeping the magnetic beads in suspension during this period (vertical mixing can be used for inverting). After the above steps, the carboxyl groups on the surface of the magnetic beads have been activated and can be covalently coupled with biological ligands containing primary amino groups (the activated state should not be stored for a long time, and coupling is recommended immediately).

[0136] 2. Covalent coupling of magnetic beads and antibodies

[0137] Monoclonal antibody CE1 and monoclonal antibody CE2 were respectively replaced with 15mM MES buffer at pH 6.0. The antibodies were diluted to 2mg / mL with MES buffer. 200ug of the antibody was mixed with 100μL of the above activated carboxyl magnetic beads (10μm in diameter) and reacted at 25℃ for 2h, or coupled at 25℃ for 1h and then incubated at 4℃ overnight. During the coupling, the magnetic beads were kept in suspension (they could be mixed by inverting using a vertical mixer). The supernatant was magnetically separated, and the remaining antibody content in the supernatant was detected at the same time. The amount and concentration of the antibody coupled to the magnetic beads were calculated. The magnetic beads were washed 2 to 3 times and resuspended in physiological saline to obtain magnetic bead-antibody conjugate CE1 and magnetic bead-antibody conjugate CE2.

[0138] 3. Enrichment

[0139] a. Take the above two types of magnetic bead-antibody conjugates (magnetic bead-antibody conjugate CE1 magnetic beads and magnetic bead-antibody conjugate CE2 magnetic beads);

[0140] b. Add 5g of feces to physiological saline at a ratio of 1:5 (e.g., 5g of feces to 25mL of physiological saline), filter through gauze, and collect the pre-treated fecal microbial solution. Take the pre-treated fecal microbial solution and add 0.1mg of magnetic bead-antibody conjugate CE1, magnetic bead-antibody conjugate CE2, or a mixture of the two magnetic bead-antibody conjugates (mixed in a 1:1 mass ratio), mix and incubate at 37℃ for 0.5h, separate the magnetic beads using a magnetic rack, and remove the unbound microorganisms and supernatant;

[0141] c. Then, the magnetic beads bound with regular fecal cocci were resuspended in physiological saline. The labeled magnetic beads were then mixed with an antibody labeling removal reagent, namely 0.05% papain (S10011, Yuanye Biotechnology), and incubated at 37°C for 0.5 h to cleave the Fc and Fab of the mouse monoclonal antibody, thereby separating the magnetic beads from the regular fecal cocci. The magnetic beads were then collected using a magnetic rack, and the supernatant was the regular fecal cocci suspension.

[0142] d. Dilute regular fecal cocci and add them to a hemocytometer, counting them under a microscope. Calculate the yield.

[0143] The results are shown in Table 5, demonstrating that the two magnetic bead antibody conjugates are more effective than one.

[0144] Table 5. Types and yields of antibody-conjugated magnetic beads combinations

[0145]

[0146]

[0147] Example 6: Regular fecal cocci culture and sequencing

[0148] The isolated regular fecal cocci were diluted to 10. 3 ~10 4 Cells / mL were plated onto tryptone soybean broth containing 5% sterile defibrinated sheep blood and anaerobically cultured at 37°C and pH 6 for 24 h. Colony characteristics were observed: cells were spherical, with weak α-hemolysis, 0.8–1.5 μm in diameter, sometimes elliptical, occurring in pairs or short chains, and non-motile. Twenty single colonies were picked and used as templates for PCR amplification of 16S rRNA. The amplified products were separated by agarose gel electrophoresis, revealing a specific band of approximately 1500 bp, consistent with the expected band size. The target band was purified by gel extraction and nucleotide sequencing. The sequencing results were compared with the NCBI database using BLAST. The results showed that the 16S rDNA gene sequence of the 20 colonies had 97% homology with *Clostridium falciparum* (GenBank: NZ_CP102278.1), thus identifying the isolate as *Clostridium falciparum*.

[0149] 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 *Factococcus faecium*, characterized in that: The monoclonal antibody is either monoclonal antibody CE1 or monoclonal antibody CE2. Monoclonal antibody CE1 consists of a CE1 heavy chain and a CE1 light chain, wherein, The amino acid sequence of the variable region of the heavy chain of monoclonal antibody CE1 is as follows: QIQLVQSGPELKKPGETVTISCKTSGFTFTGNYIMWVRQPP GKGFKWMGNTWTDEGTPAYEDKFGRFAFSLETSASTAYLQISN LKNEDMASYFCAKDEDGSIAYDWGQGTSVTVSS; The amino acid sequence of the variable region of the light chain of monoclonal antibody CE1 is as follows: DVLMTQTPLSLPVSLGDQASISCSQSSSHVIGNNTNLEYWYLQKPGQSPRLLIYRPWCHMWGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCPTACRMLLCFGSGTKLEIK Monoclonal antibody CE2 consists of a CE2 heavy chain and a CE2 light chain, wherein, The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody CE2 is as follows: QVTLKESGPGILQPSQTLSLTCSFSGFSLSCWAFPLTWIRQPS GKGLEWLARPCNEEERHNPNLSSKRLTISKDSSNKQVFLKFTSV DTADTATYYCAHFAYYNYDGAVDSWGQGTSVTVSS; The amino acid sequence of the variable region of the light chain of monoclonal antibody CE2 is as follows: DIQLTQSPAILSVSPGERVSFSCKKGQTCPACYVRWYQQRT NGSPRLLIKNSQDWPNGLPSRFSGSGSGTDFTLTISSVESEDIADY YCSSQNWSFCALFGGGTKLEI.

2. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody CE1 contains three complementarity-determining regions (CDRs) in its heavy chain variable region amino acid sequence: CE1-CDR-H1: GNYIM CE1-CDR-H2:NTWITDEGTPAYEDKFG, CE1-CDR-H3: DEDGSIAYD; The CE1 light chain variable region amino acid sequence includes three complementarity-determining regions (CDRs), namely: CE1-CDR-L1: SQSSSHVIGNNTNLEY, CE1-CDR-L2: RPWCHMW CE1-CDR-L3: PTACRMLLC; In the monoclonal antibody CE2, the amino acid sequence of the CE2 heavy chain variable region includes three complementarity-determining regions (CDRs), namely: CE2-CDR-H1: CWAFPLT, CE2-CDR-H2: RPCNEEERHNPNLSSK, CE2-CDR-H3: FAYYNYDGAVDS; The amino acid sequence of the CE2 light chain variable region includes three complementarity-determining regions (CDRs), namely: CE2-CDR-L1: KKGQTCPACYVR CE2-CDR-L2: NSQDWPN, CE2-CDR-L3:SSQNWSFCAL.

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 optimized nucleotide sequence of the protein was transformed into E. coli BL21, expressed, and purified by sonication to obtain the purified protein; wherein the protein is either COPEUT_01511 or COPEUT_01706. 2) Mix the purified protein with Freund's adjuvant, emulsify it and then immunize mice; then take the spleen cells of the immunized mice and fuse them with myeloma cells SP2 / 0, and screen to obtain hybridoma cell lines, wherein the hybridoma cell line is either hybridoma cell line CE1 or hybridoma cell line CE2.

5. The preparation method according to claim 4, characterized in that: When the protein is COPEUT_01511, the optimized nucleotide sequence of COPEUT_01511 is shown in SEQ ID NO:

2. When the protein is COPEUT_01706, the optimized nucleotide sequence of COPEUT_01706 is shown in SEQ ID NO:

4.

6. The preparation method according to claim 4, characterized in that: The amino acid sequence of COPEUT_01511 is shown in SEQ ID NO:1, and the amino acid sequence of COPEUT_01706 is shown in SEQ ID NO:

3.

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) Activate the carboxyl groups of the magnetic beads 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, wherein the magnetic bead-antibody conjugate is either magnetic bead-antibody conjugate CE1 or magnetic bead-antibody conjugate CE2. The activated carboxyl magnetic beads have a particle size of 10–30 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5–10.

9. The method for preparing the magnetic bead-antibody conjugate according to claim 8, characterized in that: The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:

5.

10. The application of an antibody conjugate in the enrichment or isolation of Clostridium pluvialis, characterized in that: The antibody conjugate is any one or both of magnetic bead-antibody conjugate CE1 and magnetic bead-antibody conjugate CE2.

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