Antibodies to rumenococcus bacteria-specific proteins and uses thereof

Abstract

The application discloses an antibody of a specific protein of a bacterium of the genus Ruminococcus and application thereof. The monoclonal antibody of the application is any one of monoclonal antibody 3F, monoclonal antibody 6D, monoclonal antibody 10E and monoclonal antibody 2C, and the monoclonal antibody 3F, the monoclonal antibody 6D, the monoclonal antibody 10E and the monoclonal antibody 2C recognize proteins glgA, glgB, GH18 and pulA respectively. The magnetic bead antibody conjugate is obtained by coupling the monoclonal antibody with activated carboxyl magnetic beads. The magnetic bead antibody conjugate of the application can specifically enrich the bacterium of the genus Ruminococcus, has high enrichment efficiency, is simple in enrichment operation and can be applied to identification of metabolic products of the bacterium of the genus Ruminococcus and also can be used for qualitative and quantitative analysis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antibody against a rumenococcal bacterial-specific protein and its application. Background Technology

[0002] Ruminococcus is a group of Gram-positive, obligate anaerobic bacteria that do not produce spores, are non-motile, and are widely distributed in the intestines of humans and animals, as well as the rumen of ruminants. Their unique abilities include digesting resistant starch and breaking down cellulose, making them closely involved in food digestion and metabolism and crucial to the host's digestive process. They support intestinal barrier stability and regulate immune function by degrading complex carbohydrates. *Ruminococcus lactaris*, *Ruminococcus gnavus*, *Ruminococcus bromii*, *Ruminococcus callidus*, and *Ruminococcus champanellensis* are among the major species of *Ruminococcus* in the human gut, playing a key role in maintaining host intestinal health.

[0003] Ruminococci play a crucial role in complex carbohydrate metabolism. Active rumenococci are among the important bacteria found to colonize the infant gut early. They utilize a unique sialic acid metabolic pathway to metabolize human milk oligosaccharides (HMOs) and mucins, providing a competitive strategy for early colonization and allowing them to adapt well to the intestinal environment and promote growth. 16SRNA gene sequencing analysis revealed that the abundance of active rumenococci is influenced by different infant feeding methods, affecting normal infant weight development. Ruminococci bryonicus are often enriched in populations fed high-resistant starch diets. They contribute significantly to intestinal metabolism by optimizing the utilization of resistant starch through the formation of amyloid bodies to release energy. Lactococci are abundant in the gut microbiota of mothers of macrosomic infants (birth weight ≥4000g) and may be considered a potential predictor of macrosomia. Ruminococci chapatis produce a complex cellulose system, digesting cellulose through various cellulases. Furthermore, the relationship between *Ruminococcus* and obesity and metabolic syndrome is frequently explored. The abundance of *Lactococcus rumeniformis* is associated with obesity and circadian rhythm disorders. *Lactococcus lactis* enhances altered carbohydrate metabolism pathways in individuals with high plasma choline levels, thereby promoting glucose regulation and may be one of the key species influencing the development of type 2 diabetes. In patients with severe acute pancreatitis, the abundance of beneficial bacteria, including *Lactococcus lactis*, is reduced; the abundance of these species may help in the early identification of severe acute pancreatitis. Berberine exerts its hypoglycemic effect by inhibiting the deoxycholine biotransformation of *Ruminococcus brevicornu*. *Ruminococcus* is closely related to disease; *Lactococcus rumeniformis* is highly associated with inflammatory bowel disease and colorectal cancer, and often serves as a potential biomarker in patients with Crohn's disease. The abundance of *Lactococcus brevicornu* is significantly reduced in patients with colorectal cancer, and primary membranous nephropathy is positively correlated with the abundance of *Lactococcus lactis*. Other studies have shown that Ruminococcus spp. may be associated with brain and neurological disorders, including depression and autism, possibly due to their role in regulating host immune responses and neurotransmitters.

[0004] Given that *Ruminococcus* spp. possesses the ability to digest resistant starch and cellulose, and that there is limited research on the preparation of antibodies against *Ruminococcus*-specific proteins, this invention selects membrane proteins associated with starch degradation, constructs key proteins, and prepares corresponding antibodies for the analysis of *Ruminococcus* spp. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an antibody targeting rumenococcal bacteria-specific proteins and its applications. This invention uses the outer membrane proteins glgA, glgB, GH18, and pulA of rumenococci to immunize mice, respectively. Monoclonal antibodies 3F, 6D, 10E, and 2C are prepared using hybridoma technology. These monoclonal antibodies are then conjugated to magnetic beads, and rumenococcal bacteria are enriched based on the magnetic bead antibody conjugates. The magnetic bead antibody conjugates of this invention capture rumenococci with high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to enrich rumenococci in feces, and can be used for the identification of rumenococcal metabolites, as well as for qualitative and quantitative studies of rumenococci.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0007] This invention provides a monoclonal antibody against a Ruminococcus spp.-specific protein, wherein the monoclonal antibody is any one of monoclonal antibody 3F, monoclonal antibody 6D, monoclonal antibody 10E, and monoclonal antibody 2C, wherein...

[0008] The monoclonal antibody 3F includes a 3F heavy chain variable region and a 3F light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0009] The monoclonal antibody 6D includes a 6D heavy chain variable region and a 6D light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0010] The monoclonal antibody 10E includes a 10E heavy chain variable region and a 10E light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0011] The monoclonal antibody 2C includes a 2C heavy chain variable region and a 2C light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0012] Furthermore, in the monoclonal antibody 3F, the 3F heavy chain variable region includes three complementarity-determining regions (CDR-H), namely:

[0013] 3F-CDR-H1: VMFSTQC;

[0014] 3F-CDR-H2:NPUMVPCKGMEWDLQ;

[0015] 3F-CDR-H3: AEYAYVWQ;

[0016] The 3F light chain variable region includes three complementary determinant regions (CDR-Ls):

[0017] 3F-CDR-L1: TPSARQMERMQDASMK;

[0018] 3F-CDR-L2: RETPK;

[0019] 3F-CDR-L3: FKQSWYTS;

[0020] In the monoclonal antibody 6D, the 6D heavy chain variable region includes three complementarity-determining regions (CDR-H), namely:

[0021] 6D-CDR-H1: KPFMWR;

[0022] 6D-CDR-H2:AYQKDREISERRDYMRFIK;

[0023] 6D-CDR-H3: DWRSFDIMQ;

[0024] The 6D light chain variable region includes three complementarity-determining regions (CDR-Ls):

[0025] 6D-CDR-L1: CPYSDPARE;

[0026] 6D-CDR-L2: YMEKRLQP;

[0027] 6D-CDR-L3: MPEPMYELI;

[0028] In the monoclonal antibody 10E, the 10E heavy chain variable region includes three complementarity-determining regions (CDR-H), namely:

[0029] 10E-CDR-H1: NPRQA;

[0030] 10E-CDR-H2: NMYQPTSTIEEYSVTL;

[0031] 10E-CDR-H3:YADEEWDCYOLQDESGPW;

[0032] The 10E light chain variable region includes three complementary determinant regions (CDR-Ls), namely:

[0033] 10E-CDR-L1:LKAYSEGNNDFEMTR;

[0034] 10E-CDR-L2: VGIAEMY;

[0035] 10E-CDR-L3: TMQNGCRVA;

[0036] In the monoclonal antibody 2C, the 2C heavy chain variable region includes three complementarity-determining regions (CDR-H), namely:

[0037] 2C-CDR-H1: ENPGQ;

[0038] 2C-CDR-H2:AYPESACNPSSIQCVEY;

[0039] 2C-CDR-H3:MYPLCNDAWGKSERVT;

[0040] The 2C light chain variable region includes three complementary determinant regions (CDR-Ls), namely:

[0041] 2C-CDR-L1: CMKPRGQEKYT;

[0042] 2C-CDR-L2: PWRTITA;

[0043] 2C-CDR-L3: TRCPMNLDG.

[0044] Furthermore, the monoclonal antibody is prepared from a hybridoma cell line.

[0045] This invention also provides a method for preparing a hybridoma cell line, comprising the following steps:

[0046] (1) The codon-optimized nucleotide sequence was transformed into Escherichia coli BL21, expressed, and purified by sonication to obtain the purified protein; wherein the protein is any one of glgA, glgB, GH18 and pulA.

[0047] (2) The purified protein was mixed with Freund's adjuvant, emulsified and then used to immunize mice. Then, the spleen lymphocytes of the immunized mice were fused with myeloma cells SP2 / 0 and the hybridoma cell line was obtained by detection and screening. The hybridoma cell line is any one of hybridoma cell 3F, hybridoma cell 6D, hybridoma cell 10E and hybridoma cell 2C.

[0048] Furthermore, when the protein is protein glgA, the codon-optimized nucleotide sequence of protein glgA is shown in SEQ ID NO: 5;

[0049] When the protein is protein glgB, the codon-optimized nucleotide sequence of protein glgB is shown in SEQ ID NO: 6;

[0050] When the protein is protein GH18, the codon-optimized nucleotide sequence of protein GH18 is shown in SEQ ID NO: 7;

[0051] When the protein is protein pulA, the codon-optimized nucleotide sequence of protein pulA is shown in SEQ ID NO: 8.

[0052] Furthermore, the amino acid sequence of the protein glgA is shown in SEQ ID NO: 1, the amino acid sequence of the protein glgB is shown in SEQ ID NO: 2, the amino acid sequence of the protein GH18 is shown in SEQ ID NO: 3, and the amino acid sequence of the protein pulA is shown in SEQ ID NO: 4.

[0053] The present invention also provides the application of the monoclonal antibody in the preparation of magnetic bead antibody conjugates.

[0054] This invention also provides a method for preparing magnetic bead antibody conjugates, comprising the following steps:

[0055] (1) Dilute the monoclonal antibody using MES buffer;

[0056] (2) The magnetic beads were activated by carboxyl groups to obtain activated carboxyl magnetic beads;

[0057] (3) The monoclonal antibody from step (1) is conjugated with activated carboxyl magnetic beads to obtain magnetic bead antibody conjugates. The magnetic bead antibody conjugates are magnetic bead antibody conjugate 3F, magnetic bead antibody conjugate 6D, magnetic bead antibody conjugate 10E and magnetic bead antibody conjugate 2C.

[0058] 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.

[0059] Furthermore, 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.

[0060] The present invention also provides the application of a magnetic bead antibody conjugate in the enrichment or isolation of Ruminococcus bacteria, wherein the magnetic bead antibody conjugate is any one of magnetic bead antibody conjugate 3F, magnetic bead antibody conjugate 6D, magnetic bead antibody conjugate 10E and magnetic bead antibody conjugate 2C.

[0061] The principle of this invention:

[0062] The reasons for selecting outer membrane proteins glgA, glgB, GH18, and pulA in this invention are as follows:

[0063] Glycogen synthase A (glgA) plays a crucial role in glycogen synthesis, utilizing ADP-glucose to synthesize α-1-4-glycosidic bonds. 1,4-alpha-glucan branching enzyme (glgB) cleaves the α-1-4-glycosidic bonds in starch molecules and attaches them to the acceptor chain as α-1-6-glycosidic bonds, participating in starch synthesis and altering starch structure. Glycoside hydrolases family 18 (GH18) digests various food and host carbohydrates, as well as mucins, to adapt to the environment. Pullulanase (pulA) hydrolyzes α-1-6-glycosidic bonds in dextran, amylose, and glycogen, as well as α- and β-limited dextrins in amylose and glycogen.

[0064] The beneficial effects of this invention are:

[0065] 1. This invention utilizes magnetic bead antibody conjugates to capture Ruminococcus bacteria, which has high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to the analysis of Ruminococcus metabolites, as well as their qualitative and quantitative analysis.

[0066] 2. This invention utilizes outer membrane proteins glgA, glgB, GH18, pulA, and corresponding antibodies to more accurately analyze the presence and activity of Ruminococcus bacteria in the gut microbiota, thereby providing a new perspective for understanding their role in host health.

[0067] 3. The method for enriching Ruminococcus spp. of this invention is simple, does not require expensive instruments such as flow cytometers, reduces costs, and is conducive to widespread application. Attached Figure Description

[0068] Figure 1 Here are SDS-PAGE images of the four proteins after purification;

[0069] Figure 2 The graph shows the titer determination results of four monoclonal antibodies.

[0070] Figure 3 SDS-PAGE images of four monoclonal antibodies after purification;

[0071] Figure 4 The image shows the results of Western blot identification of the specificity of monoclonal antibodies against Ruminococcus spp. Detailed Implementation

[0072] 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.

[0073] Example 1

[0074] Construction of recombinant protein expression vectors and protein purification

[0075] 1. Search for the protein sequences of the target genes glgA, glgB, GH18, and pulA in *Lactococcus lactis*, *Ruminococcus viridans*, *Ruminococcus brucelli*, *Ruminococcus leptospirae*, and *Ruminococcus chapatis* on uniprot (https: / / www.uniprot.org / ). Perform homology comparison in MEGA software to screen out protein sequence fragments of the target genes glgA, glgB, GH18, and pulA with high intergeneric homology.

[0076] 2. The amino acid sequence of 1-472aa of the outer membrane protein glgA (UniProt accession number U2MBB0) of Ruminococcus spp. is shown in SEQ ID NO: 1, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 5;

[0077] The amino acid sequence of 163–522 amino acids of the outer membrane protein glgB of Ruminococcus spp. (UniProt accession number A0A414P290) is shown in SEQ ID NO: 2, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 6.

[0078] The amino acid sequence of GH18 (UniProt accession number A0A414P1C5) of Ruminococcus outer membrane protein 281–610aa is shown in SEQ ID NO: 3, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 7.

[0079] The amino acid sequence of 118–544 amino acids of the outer membrane protein pulA (UniProt accession number A7B290) of Ruminococcus is shown in SEQ ID NO: 4, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 8.

[0080] 3. The synthesized gene sequence and PET32a vector were double-digested with BamHⅢ and EcoRⅠ. The digested PET32a vector product and the target gene fragment were ligated at 16℃ for 2 h to construct fusion protein expression vectors PET32a-glgA, PET32a-glgB, PET32a-GH18, and PET32a-pulA. The fusion protein expression vectors were transformed into E. coli DH5α competent cells, and 800 μL of LB liquid medium was added. The cells were cultured at 37℃ with shaking for 1 h. The culture medium was then spread onto LB agar plates (containing 100 μg / mL Amp), inverted, and cultured overnight at 37℃. Single colonies were picked for sequencing.

[0081] 4. Extract recombinant plasmids from single colonies that have been verified by sequencing, transform the recombinant plasmids into competent Escherichia coli BL21(DE3) cells, pick single colonies for verification, and inoculate positive bacteria into 10 mL LB medium (containing 100 μg / mL Amp) and culture overnight at 37°C with shaking at 200 rpm.

[0082] 10 mL of overnight cultured bacterial suspension was added to LB medium (containing 100 μg / mL Amp) at a ratio of 1:100 and incubated at 37°C with shaking at 200 rpm until the OD of the bacterial suspension reached 0.6–0.8. Then, 0.5 mM IPTG was added to induce the expression of the target protein. After incubation at 16°C with shaking at 200 rpm for 16 h, the bacterial cells were collected by centrifugation at 4°C and 12000 rpm for 5 min. Bacterial lysis buffer was added to resuspend the bacterial cells, and the suspension was intermittently sonicated on ice until the suspension was clear and non-viscous. The supernatant was bound to an affinity column to elute impurities and purify the target protein. Protein purity was analyzed by SDS-PAGE, and protein concentration was determined by the BCA method. Figure 1 As shown in the figure. This embodiment successfully obtained the purified target proteins glgA, glgB, GH18, and pulA.

[0083] Example 2

[0084] Mouse immune and antiserum titers and hybridoma cell fusion

[0085] 1. Mouse immunization

[0086] (1) Take the purified proteins glgA, glgB, GH18, and pulA as antigens and dilute them with physiological saline to 1 μg / μL (prepared according to 50 μL per injection). Mix them with Freund's adjuvant and emulsify them in a mixer.

[0087] (2) Healthy female Balb / c mice at 8 weeks of age were selected as immune recipients. The initial immunization dose was 100 μL protein per mouse, with Freund's complete adjuvant as the adjuvant. On day 21, mice in good condition were selected for a booster immunization. For the second to fourth immunizations, the dose was 50 μL protein per mouse, with an interval of 14 days, and Freund's incomplete adjuvant as the adjuvant. Each adjuvant and antigen were prepared and used immediately. The immunization method is shown in Table 1.

[0088] Table 1. Mouse Immunization Schedule

[0089] Number of immunizations Immune sites adjuvant Immunization dose 1 Subcutaneous + peritoneal Freund's complete adjuvant 50μL+50μL 2 abdominal cavity Freund's incomplete adjuvant 50μL 3 abdominal cavity Freund's incomplete adjuvant 50μL 4 abdominal cavity Freund's incomplete adjuvant 50μL 5 abdominal cavity No adjuvants 50μL

[0090] 2. Antiserum titer detection

[0091] Blood was collected from the tail vein of immunized mice to detect serum antibody titers. Purified proteins glgA, glgB, GH18, and pulA were diluted to a concentration of 10 μg / mL and coated onto ELISA plates, 100 μL per well, and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 200 μL of blocking buffer for 2 h. After washing three times with PBST, serially diluted serum was added, and the plates were incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of HRP-labeled rabbit anti-mouse secondary antibody (1:5000 dilution) was added per well, and the plates were incubated at 37°C for 1 h. After washing three times with PBST, chromogenic solutions A and B were added, and the plates were incubated at 37°C for 10 min. The incubation was terminated with stop solution, and the OD value was measured using an ELISA reader.

[0092] 3. Hybridoma cell fusion

[0093] (1) Mice with a titer of 1:20000 or higher are generally selected for antigen shock immunization without adjuvant. 3-7 days after shock immunization, mouse spleen cells can be collected for cell fusion. Mice with good immunization effect and high serum titer are selected, and blood is collected from the eyeballs for sacrifice. Splenic lymphocyte suspension is prepared, washed with PBS, and mixed with SP2 / 0 cells at a ratio of spleen lymphocytes:SP2 / 0 = 5:1. After centrifugation at 1000 rpm for 10 min, the mixed cells are drained and the cell clumps are loosened by gentle tapping.

[0094] (2) Add 1 mL of 50% PEG preheated at 37℃. After adding, react in a 37℃ water bath for 1 min. Then slowly add 40 mL of RPMI-1640 stop solution along the tube wall.

[0095] (3) After cell fusion was terminated, the cells were centrifuged at 1000 rpm for 10 min and the supernatant was discarded. The cells were resuspended in 100 mL of complete culture medium containing 20% ​​FBS and HAT, and transferred to 96-well cell culture plates containing feeder cells using a multichannel pipette. The cells were cultured in a carbon dioxide incubator at 37°C and 5% CO2.

[0096] (4) Observe the cell status 8 to 10 days after fusion, and change the medium using a complete culture medium containing 20% ​​FBS and HT.

[0097] (5) Based on cell growth, observe the size of the colonies. When the colonies reach approximately 1 / 4 of the well bottom area, consider detection. Take 100 μL of supernatant and perform detection using the indirect ELISA method. Select positive cell lines with high OD values, high titers, and good specificity for subcloning. Dilute the selected cells to 1 cell / well using HT medium via limiting dilution. Seed the cells in a 96-well cell culture plate and wait for the monoclonal cells to grow to a medium size and a density of approximately 102. 4Titer detection was performed on cells of 100 or more. Positive cells were then collected again for a second subcloning screening. Once all cell supernatants in the microwells were found to be positive, hybridoma cell lines were obtained through three subcloning processes: 3F for glgA recognition protein, 6D for glgB recognition protein, 10E for GH18 recognition protein, and 2C for pulA recognition protein. The titer results for the four monoclonal antibodies are as follows: Figure 2 As shown, the monoclonal antibody 3F recognizing glgA, the monoclonal antibody 6D recognizing glgB, the monoclonal antibody 10E recognizing GH18, and the monoclonal antibody 2C recognizing pulA all achieved antibody titers of over 1:320000.

[0098] Example 3

[0099] Monoclonal antibody purification and monoclonal antibody subtype identification

[0100] 1. Monoclonal antibody purification

[0101] Hybridoma cells 3F, 6D, 10E, and 2C were cultured separately and then collected, with the cell concentration adjusted to 102. 6 Cells / mL were selected from unimmunized mice, and 0.5 mL of sterile paraffin oil was injected intraperitoneally. Seven days later, 1 mL of hybridoma cells 3F, 6D, 10E, and 2C were injected, respectively. After one week of feeding, ascites fluid was collected from the mice's peritoneal cavity. The supernatant was collected by centrifugation at 10,000 rpm for 15 min, filtered, and the cell supernatant sample to be purified was loaded onto a Protein A affinity chromatography column at a flow rate of 0.5 mL / min to allow the antibody to bind to Protein A. Finally, the antibody was eluted with elution buffer, and its purity was identified by SDS-PAGE. Figure 3 As shown, purified monoclonal antibodies 3F, 6D, 10E, and 2C were successfully obtained.

[0102] 2. Identification of monoclonal antibody subtypes

[0103] Antibody subtype identification kits were used to identify the subtypes of four monoclonal antibodies. The antigens were diluted to 10 μg / mL (antigens were protein glgA, protein glgB, protein GH18, or protein pulA), coated with substrate onto an ELISA plate, blocked at 37°C for 2 h, 10 μL of purified and diluted antibody was added, and incubated at 37°C for 1 h. After washing three times, HRP-labeled anti-mouse (IgG1 / IgG2a / IgG2b / IgG3 / IgM / IgA) antibody was added, and incubated at 37°C for 1 h. The plate was then washed three times with PBST, and finally, chromogenic reaction was performed. The incubation was terminated with stop solution. The subtype results are shown in Table 2. Monoclonal antibody 3F was subtype IgG2b, monoclonal antibody 6D was subtype IgG2b, monoclonal antibody 10E was subtype IgG1, and monoclonal antibody 2C was subtype IgG2b.

[0104] Table 2. Isotype determination of monoclonal antibodies

[0105]

[0106]

[0107] Example 4

[0108] Western blot identification of antibodies against Ruminococcus spp.

[0109] 1. Sample preparation: Take 2 mL of the identified Ruminococcus spp. bacteria. 4 Add 100 μL of protein per mL to a centrifuge tube, add 200 μL of RIPA lysis buffer to extract total protein, centrifuge at 10000 rpm for 5 min, add an equal volume of 2× loading buffer, boil in water for 5 min, aliquot and store at -20℃.

[0110] 2. Electrophoresis: Prepare SDS-PAGE gels according to the standard protein electrophoresis method, load 15 μL of sample into each well, and run at a constant voltage of 200V for 30 min.

[0111] 3. Transfer: Use a wet transfer apparatus to transfer the protein in the gel into a PDVF membrane. Transfer at a constant current of 150mA for 20-30 minutes.

[0112] 4. Blocking: Remove the membrane and wash it three times with PBST for 5 minutes each time (shaking on a horizontal shaker); remove the membrane and immerse it in blocking solution at 37°C for 2 hours.

[0113] 5. Primary antibody incubation: Remove the membrane and wash it three times with PBST for 5 minutes each time (shaking on a horizontal shaker); remove the membrane and immerse it in the primary antibody dilution solution diluted with 5% skim milk powder at 37°C for 1 hour (the primary antibody is monoclonal antibody 3F, monoclonal antibody 6D, monoclonal antibody 10E or monoclonal antibody 2C, and is generally diluted at 1:1000).

[0114] 6. Secondary antibody incubation: Remove the membrane and wash it three times with PBST for 5 minutes each time (shaking on a horizontal shaker); remove the membrane and immerse it in secondary antibody dilution solution diluted with 5% skim milk powder at 37°C for 1 hour; (the secondary antibody is rabbit anti-mouse-HRP, diluted at 1:5000).

[0115] 7. Color development: ECL color development reaction.

[0116] 8. Data Reading: The molecular weight of the target band on the membrane was analyzed using a fully automated chemiluminescence analyzer. Results are as follows: Figure 4 As shown, the monoclonal antibodies 3F, 6D, 10E, or 2C prepared using the outer membrane proteins of Ruminococcus bacteria can capture this bacterium with high specificity.

[0117] Example 5

[0118] Cell line sequencing

[0119] 1. Hybridoma cells 3F, 6D, 10E, and 2C 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, orC). 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.

[0120] Heavy chain forward primer: CGGCCCAGCCGGCC;

[0121] Heavy chain reverse primer: TGAACCGCCTCCACC;

[0122] Light chain forward primer: GGTTCCACTGGT;

[0123] Light chain reverse primer: GTGCAGCATCAGC

[0124] 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.

[0125] The second round of amplification produced the gene product with restriction enzyme sites (EcoRI and HindIII), which was then ligated into the pMD19-T cloning vector. Sequencing and analysis were then used to obtain the variable region sequences of the antibody light chain and heavy chain.

[0126] Heavy chain forward primer: TGAATTCCGGCCCAGCCGGCC;

[0127] Heavy chain reverse primer: TAAGCTTTGAACCGCCTCCACC;

[0128] Light chain forward primer: TGATTCGGTTCCACTGGT;

[0129] Light chain reverse primer: TAAGCTTGTGCAGCATCAGC.

[0130] 2. The amino acid sequence of the variable region of the 3F heavy chain of the monoclonal antibody is shown in SEQ ID NO: 9:

[0131] Note: The bold and underlined regions are the complementarity-determining regions (CDR-H) of the monoclonal antibody 3F heavy chain, while the rest are the backbone regions (FR-H) of the monoclonal antibody 3F heavy chain.

[0132] SVQHQQSGTMLARPSACVKMSCKASGYSFT VMFSTQC REWV KQRPGQGIEPIA NPUMVPCKGMEWDL Q KAKLTAVTSKQTAYMEW SNLTNEDSAVFYCTR AEYAYVWQ EGQGTSVTVSS.

[0133] 3F-FR-H1:SVQHQQSGTMLARPSACVKMSCKASGYSFT;

[0134] 3F-CDR-H1: VMFSTQC;

[0135] 3F-FR-H2: REWVKQRPGQGIEPIA;

[0136] 3F-CDR-H2:NPUMVPCKGMEWDLQ;

[0137] 3F-FR-H3:KAKLTAVTSKQTAYMEWSNLTNEDSAVFYCTR;

[0138] 3F-CDR-H3: AEYAYVWQ;

[0139] 3F-FR-H4: EGQGTSVTVSS.

[0140] The amino acid sequence of the variable region of the 3F light chain of the monoclonal antibody is shown in SEQ ID NO: 10.

[0141] Note: The bold and underlined regions are the complementarity-determining regions (CDR-L) of the 3F light chain of the monoclonal antibody, while the rest are the backbone regions (FR-L) of the 3F light chain of the monoclonal antibody.

[0142] DVLMTQTPLSLPVSLGQASISC TPSARQMERMQDASMK FYLQ KPQSPKLLIY RETPK GVPFSGSGSGTDFLKISRVEEDLGVPSSYYC F KQSWYTS FGGGTEIKRA.

[0143] 3F-FR-L1: DVLMTQTPLSLPVSLGQASISC;

[0144] 3F-CDR-L1: TPSARQMERMQDASMK;

[0145] 3F-FR-L2: FYLQKPQSPKLLIY;

[0146] 3F-CDR-L2: RETPK;

[0147] 3F-FR-L3: GVPFSGSGSGTDFLKISRVEEDLGVPSSYYC;

[0148] 3F-CDR-L3: FKQSWYTS;

[0149] 3F-FR-L4: FGGGTEIKRA.

[0150] 3. The amino acid sequence of the 6D heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 11:

[0151] Note: The bold and underlined regions are the complementarity-determining regions (CDR-H) of the 6D heavy chain of monoclonal antibodies, while the rest are the backbone regions (FR-H) of the 6D heavy chain of monoclonal antibodies.

[0152] QVQLQQSGPELVKPGSVSCKASGYSFN KPFMWR WVKQRPGQ GLEWIG AYQKDREISERRDYMRFIK ALTVDRSSSTAHMQCTLSSPT EDSAVYYCTR DWRSFDIMQ WGAGTTETTVSS.

[0153] 6D-FR-H1: QVQLQQSGPELVKPGSVSCKASGYSFN;

[0154] 6D-CDR-H1: KPFMWR;

[0155] 6D-FR-H2: WVKQRPGQGLEWIG;

[0156] 6D-CDR-H2:AYQKDREISERRDYMRFIK;

[0157] 6D-FR-H3:KALTVDRSSSTAHMQCTLSSPTEDSAVYYCTR;

[0158] 6D-CDR-H3: DWRSFDIMQ;

[0159] 6D-FR-H4:WGAGTTETTVSS.

[0160] The amino acid sequence of the 6D light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 12.

[0161] Note: The bold and underlined regions are the complementarity-determining regions (CDR-L) of the 6D light chain of the monoclonal antibody, while the rest are the backbone regions (FR-L) of the 6D light chain of the monoclonal antibody.

[0162] QIVLTQSPAIMSAFPDGESVTMTC CPYSDPARE WYETQKPGSSP RDLLI YMEKRLQP GVRFSGSGSGTSYSLDTINRLESEDGATYYC MP EPRMYELI FGAGTKLLKR.

[0163] 6D-FR-L1: QIVLTQSPAIMSAFPDGESVTMTC;

[0164] 6D-CDR-L1: CPYSDPARE;

[0165] 6D-FR-L2:WYETQKPGSPRDLLI;

[0166] 6D-CDR-L2: YMEKRLQP;

[0167] 6D-FR-L3: GVRFSGSGSGTSYSLDTINRLESEDGATYYC;

[0168] 6D-CDR-L3: MPEPMYELI;

[0169] 6D-FR-L4:FGAGTKLLKR.

[0170] 4. The amino acid sequence of the variable region of the 10E heavy chain of the monoclonal antibody is shown in SEQ ID NO: 13:

[0171] Note: The bold and underlined regions are the complementarity-determining regions (CDR-H) of the 10E heavy chain of the monoclonal antibody, while the rest are the backbone regions (FR-H) of the 10E heavy chain of the monoclonal antibody.

[0172] EVQLVESGMGGLVQPFGDLRKLCAPSGFTFS NPRQA WVRQAP GKGLEWDS NMYQPTSTIEEYSVTL RFTISPDNAHNSLYLLMNSLRA EWTAVYYVAR YADEEWDCYOLQDESGPW WGRGTLVSTVSF.

[0173] 10E-FR-H1: EVQLVESGMGGLVQPFGDLRKLCAPSGFTFS;

[0174] 10E-CDR-H1: NPRQA;

[0175] 10E-FR-H2: WVRQAPGKGLEWDS;

[0176] 10E-CDR-H2: NMYQPTSTIEEYSVTL;

[0177] 10E-FR-H3:RFTISPDNAHNSLYLLMNSLRAEWTAVYYVAR;

[0178] 10E-CDR-H3:YADEEWDCYOLQDESGPW;

[0179] 10E-FR-H4:WGRGTLVSTVSF.

[0180] The amino acid sequence of the variable region of the light chain of monoclonal antibody 10E is shown in SEQ ID NO: 14.

[0181] Note: The bold and underlined regions are the complementarity-determining regions (CDR-L) of the 10E light chain of the monoclonal antibody, while the rest are the backbone regions (FR-L) of the 10E light chain of the monoclonal antibody.

[0182] DVVMTQTPLSLPVSLGDQASISC LKAYSEGNNDFEMTR WYLQ KPGQSPKLLIY VGIAEMY GVPDRFSGSGSCTDFTLKISRVAEDLGVY FC TMQNGCRVA FGGGTKLEIK.

[0183] 10E-FR-LI:DVVMTQTPLSLPVSLGDQASISC;

[0184] 10E-CDR-L1:LKAYSEGNNDFEMTR;

[0185] 10E-FR-L2:WYLQKPGQSPKLLIY;

[0186] 10E-CDR-L2: VGIAEMY;

[0187] 10E-FR-L3:GVPDRFSGSGSCTDFTLKISRVAEDLGVYFC;

[0188] 10E-CDR-L3: TMQNGCRVA;

[0189] 10E-FR-L4:FGGGTKLEIK.

[0190] 5. The amino acid sequence of the variable region of the 2C heavy chain of the monoclonal antibody is shown in SEQ ID NO: 15:

[0191] Note: The bold and underlined regions are the complementarity-determining regions (CDR-H) of the monoclonal antibody 2C heavy chain, while the rest are the backbone regions (FR-H) of the monoclonal antibody 2C heavy chain.

[0192] VERRTESGTGLVKPGGILRVSNAASAFTFS ENPGQ WVRQAYGK GLEWVV AYPESACNPSSIQCVEY RFTISRDNAKNSAYLQMNSLRAEDTAVYYCAG MYPLCNDAWGKSERVT WGQFTLVTMVS.

[0193] 2C-FR-H1:VERRTESGTGLVKPGGILRVSNAASAFTFS;

[0194] 2C-CDR-H1: ENPGQ;

[0195] 2C-FR-H2: WVRQAYGKGLEWVV;

[0196] 2C-CDR-H2:AYPESACNPSSIQCVEY;

[0197] 2C-FR-H3:RFTISRDNAKNSAYLQMNSLRAEDTAVYYCAG;

[0198] 2C-CDR-H3:MYPLCNDAWGKSERVT;

[0199] 2C-FR-H4: WGQFTLVTMVS.

[0200] The amino acid sequence of the variable region of the 2C light chain of the monoclonal antibody is shown in SEQ ID NO: 16.

[0201] Note: The bold and underlined regions are the complementarity-determining regions (CDR-L) of the 2C light chain of the monoclonal antibody, while the rest are the backbone regions (FR-L) of the 2C light chain of the monoclonal antibody.

[0202]

[0203] Example 5

[0204] Preparation of magnetic bead antibody conjugates

[0205] 1. Monoclonal antibody dilution

[0206] The buffers for monoclonal antibody 3F, monoclonal antibody 6D, monoclonal antibody 10E, and monoclonal antibody 2C were replaced with 15mM MES buffer at pH 6.0, and the antibodies were diluted to 2mg / mL with MES buffer to obtain antibody dilution 3F, antibody dilution 6D, antibody dilution 10E, and antibody dilution 2C, respectively.

[0207] 2. Activation of carboxyl groups on the surface of magnetic beads

[0208] (1) After mixing the magnetic beads, take 100 μL of MagCOOH 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.

[0209] (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).

[0210] After the above steps, the carboxyl groups on the surface of the magnetic beads have been activated, resulting in activated carboxyl magnetic beads, which 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).

[0211] 3. Covalent coupling of magnetic beads and antibodies

[0212] (1) Take 200 μg of antibody dilution 3F, antibody dilution 6D, antibody dilution 10E and antibody dilution 2C and mix them with 100 μL of the above activated carboxyl magnetic beads (diameter 10 μm, antibody to magnetic beads molar ratio of 1:5). React at 25°C for 2 h, or couple at 25°C for 1 h and then let stand at 4°C overnight. Keep the magnetic beads in suspension during coupling (they can be mixed by inverting using a vertical mixer).

[0213] (2) Magnetic separation, aspirate the supernatant and simultaneously detect the remaining antibody content in the supernatant, calculate the amount and concentration of magnetic bead-conjugated antibodies, wash the magnetic beads 2 to 3 times with physiological saline, resuspend them with physiological saline to obtain magnetic bead antibody conjugate 3F, magnetic bead antibody conjugate 6D, magnetic bead antibody conjugate 10E and magnetic bead antibody conjugate 2C.

[0214] Example 6

[0215] Ruminococcus spp. enrichment

[0216] 1. Take the magnetic bead antibody conjugate 3F, magnetic bead antibody conjugate 6D, magnetic bead antibody conjugate 10E and magnetic bead antibody conjugate 2C prepared in Example 5 for later use.

[0217] 2. Add 5g of feces to physiological saline at a ratio of 1:5 (for example, add 25mL of physiological saline to 5g of feces), filter through gauze, and collect the pre-treated fecal microbial solution.

[0218] 3. Take the pre-treated fecal microbial solution and add 0.1 mg of magnetic bead antibody conjugate 3F, magnetic bead antibody conjugate 6D, magnetic bead antibody conjugate 10E and magnetic bead antibody conjugate 2C respectively. Mix and incubate at 37°C for 0.5 h. Use a magnetic rack to separate the magnetic beads and remove the unbound microorganisms and supernatant.

[0219] 4. Then, the magnetic beads (labeled magnetic beads) contaminated with Ruminococcus bacteria were resuspended in physiological saline. An antibody label removal reagent, namely 0.05% papain mixed with the labeled magnetic beads, was used to incubate at 37°C for 0.5 h to cut the Fc and Fab of the mouse monoclonal antibody, thereby separating the magnetic beads from the Ruminococcus bacteria. The magnetic beads were then collected using a magnetic rack, and the supernatant was the Ruminococcus bacteria suspension.

[0220] 5. After diluting the Ruminococcus spp. suspension, the cells were added dropwise to a hemocytometer and counted under a microscope. The yield was used to calculate the effectiveness of the magnetic bead antibody conjugates in enriching Ruminococcus spp. The results are shown in Table 3. Magnetic bead antibody conjugates 3F, 6D, 10E, and 2C were able to enrich Ruminococcus spp. and achieve high enrichment yields.

[0221] Table 3. Types and yields of magnetic bead antibody conjugate combinations

[0222] Serial Number Combination type Yield 1 Magnetic bead antibody conjugate 3F <![CDATA[1.2*10 5 <!-- 11 -->]]> 2 Magnetic bead antibody conjugate 6D <![CDATA[1.0*10 5 ]]> 3 Magnetic bead antibody conjugate 10E <![CDATA[1.5*10 5 ]]> 4 Magnetic bead antibody conjugate 2C <![CDATA[1.2*10 5 ]]>

[0223] 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 to a protein specific for bacteria of the genus Ruminococcus, characterized in that: The monoclonal antibody is any one of monoclonal antibody 3F, monoclonal antibody 6D, monoclonal antibody 10E, and monoclonal antibody 2C, wherein... The monoclonal antibody 3F includes a 3F heavy chain variable region and a 3F light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The monoclonal antibody 6D includes a 6D heavy chain variable region and a 6D light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The monoclonal antibody 10E includes a 10E heavy chain variable region and a 10E light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The monoclonal antibody 2C includes a 2C heavy chain variable region and a 2C light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

2. The monoclonal antibody of claim 1, wherein: In the monoclonal antibody 3F, the 3F heavy chain variable region includes three complementarity-determining regions (CDR-H), namely: 3F-CDR-H1: VMFSTQC; 3F-CDR-H2:NPUMVPCKGMEWDLQ; 3F-CDR-H3: AEYAYVWQ; The 3F light chain variable region includes three complementary determinant regions (CDR-Ls): 3F-CDR-L1: TPSARQMERMQDASMK; 3F-CDR-L2: RETPK; 3F-CDR-L3: FKQSWYTS; In the monoclonal antibody 6D, the 6D heavy chain variable region includes three complementarity-determining regions (CDR-H), namely: 6D-CDR-H1: KPFMWR; 6D-CDR-H2:AYQKDREISERRDYMRFIK; 6D-CDR-H3: DWRSFDIMQ; The 6D light chain variable region includes three complementarity-determining regions (CDR-Ls): 6D-CDR-L1: CPYSDPARE; 6D-CDR-L2: YMEKRLQP; 6D-CDR-L3: MPEPMYELI; In the monoclonal antibody 10E, the 10E heavy chain variable region includes three complementarity-determining regions (CDR-H), namely: 10E-CDR-H1: NPRQA; 10E-CDR-H2: NMYQPTSTIEEYSVTL; 10E-CDR-H3:YADEEWDCYOLQDESGPW; The 10E light chain variable region includes three complementary determinant regions (CDR-Ls), namely: 10E-CDR-L1:LKAYSEGNNDFEMTR; 10E-CDR-L2: VGIAEMY; 10E-CDR-L3: TMQNGCRVA; In the monoclonal antibody 2C, the 2C heavy chain variable region includes three complementarity-determining regions (CDR-H), namely: 2C-CDR-H1: ENPGQ; 2C-CDR-H2:AYPESACNPSSIQCVEY; 2C-CDR-H3:MYPLCNDAWGKSERVT; The 2C light chain variable region includes three complementary determinant regions (CDR-Ls), namely: 2C-CDR-L1: CMKPRGQEKYT; 2C-CDR-L2: PWRTITA; 2C-CDR-L3: TRCPMNLDG.

3. The monoclonal antibody of claim 1, wherein: The monoclonal antibody was prepared from a hybridoma cell line.

4. A method of producing a hybridoma cell line, characterized by: Includes the following steps: (1) The protein was transformed into Escherichia coli BL21 with a codon-optimized nucleotide sequence, expressed, and purified by sonication to obtain a purified protein; wherein the protein is any one of glgA, glgB, GH18 and pulA. (2) The purified protein was mixed with Freund's adjuvant, emulsified and then used to immunize mice. Then, the spleen lymphocytes of the immunized mice were fused with myeloma cells SP2 / 0 and the hybridoma cell line was obtained by detection and screening. The hybridoma cell line is any one of hybridoma cell 3F, hybridoma cell 6D, hybridoma cell 10E and hybridoma cell 2C.

5. The method of claim 4, wherein: When the protein is protein glgA, the codon-optimized nucleotide sequence of protein glgA is shown in SEQ ID NO: 5; When the protein is protein glgB, the codon-optimized nucleotide sequence of protein glgB is shown in SEQ ID NO: 6; When the protein is protein GH18, the codon-optimized nucleotide sequence of protein GH18 is shown in SEQ ID NO: 7; When the protein is protein pulA, the codon-optimized nucleotide sequence of protein pulA is shown in SEQ ID NO:

8.

6. The method of claim 4, wherein: The amino acid sequence of the protein glgA is shown in SEQ ID NO: 1, the amino acid sequence of the protein glgB is shown in SEQ ID NO: 2, the amino acid sequence of the protein GH18 is shown in SEQ ID NO: 3, and the amino acid sequence of the protein pulA is shown in SEQ ID NO:

4.

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 by: Includes the following steps: (1) Dilute the monoclonal antibody of claim 1 using MES buffer; (2) The magnetic beads were activated by carboxyl groups to obtain activated carboxyl magnetic beads; (3) The monoclonal antibody from step (1) is coupled with activated carboxyl magnetic beads to obtain a magnetic bead antibody conjugate. The magnetic bead antibody conjugate is any one of magnetic bead antibody conjugate 3F, magnetic bead antibody conjugate 6D, magnetic bead antibody conjugate 10E and magnetic bead antibody conjugate 2C. The particle size of the activated carboxyl magnetic beads is 10-30 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5-10.

9. The method of claim 8, wherein: 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. Use of a magnetic bead antibody conjugate for enriching or isolating bacteria of the genus Ruminococcus, characterized in that: The magnetic bead antibody conjugate is any one of magnetic bead antibody conjugate 3F, magnetic bead antibody conjugate 6D, magnetic bead antibody conjugate 10E, and magnetic bead antibody conjugate 2C.

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