Monoclonal antibody of lactobacillus fermentum specific protein and its application

Abstract

The application discloses a kind of Lactobacillus fermentum specific protein monoclonal antibody and its application.The monoclonal antibody of the application is any one of monoclonal antibody 9C4 and monoclonal antibody 7F9, the amino acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 9C4 are respectively shown as SEQ ID NO:5 and SEQ ID NO:6, and the amino acid sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 7F9 are respectively shown as SEQ ID NO:7 and SEQ ID NO:8.The Lactobacillus fermentum of the application can be used to enrich in fecal separation solution, can be used for strain identification sequencing, and can be used for the identification of Lactobacillus fermentum metabolite and qualitative and quantitative research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against a Lactobacillus fermentum-specific protein and its applications. Background Technology

[0002] The gut microbiota plays a crucial role in maintaining the host's intestinal physiology and immune homeostasis. Lactobacillus, a recognized probiotic, is widely used in fermented foods due to its ability to ferment and produce a distinctive flavor. Lactobacillus is abundant in the animal gastrointestinal tract and is an essential beneficial bacterium for host metabolic activities. It enhances the body's antioxidant capacity and participates in antibacterial and antitumor physiological functions. Therefore, it is often used in animal production to improve the health of poultry and promote their growth and reproduction. Numerous studies have shown that lactic acid bacteria are beneficial to human gut health, especially in the prevention and treatment of irritable bowel syndrome, enteritis, bacterial infections, diarrhea, and various gastrointestinal diseases, demonstrating high therapeutic potential.

[0003] *Lactobacillus fermentum* is a heterofermentative lactobacillus. As a Gram-positive bacterium, it exhibits strong adhesion in the gastrointestinal tract and possesses antibacterial activity against pathogens. Studies have shown that *Lactobacillus fermentum* can maintain the gut microbiota by competitively rejecting pathogens, reducing the incidence of gastrointestinal diseases caused by pathogenic bacteria, regulating the immune system, and lowering the risk of bladder cancer. *Lactobacillus fermentum* alleviates colitis by regulating the gut microbiota and strengthening the intestinal mucosal barrier; its extracellular vesicles may play a key role in this regulatory process. Furthermore, a specific strain of *Lactobacillus fermentum*, KBL375, possesses significant immunomodulatory capabilities and can alleviate atopic dermatitis-like symptoms by promoting serotonin synthesis through regulation of gut microbiota and metabolome composition.

[0004] The surface proteins of gut microbiota possess the ability to recognize and adhere to host cells, acting as a bridge between the microbiota and the host gut. They promote bacterial adhesion and colonization, and prevent pathogen invasion. Furthermore, surface proteins can regulate the host's immune response and enhance the body's immune defense capabilities. Therefore, understanding the surface proteins of *Lactobacillus fermentum* is crucial for understanding its probiotic mechanisms and biological characteristics, making research on *Lactobacillus fermentum* surface proteins of significant importance. However, research on the preparation of *Lactobacillus fermentum* surface proteins is limited, especially regarding antibody preparation. Based on this, this invention selected the *Lactobacillus fermentum* surface-specific proteins Csp (cell surface protein) and Mucbp (mucin-binding protein) as research subjects, constructed vectors, and synthesized specific proteins and antibodies, providing a solid foundation for future research. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a monoclonal antibody against a specific protein of *Lactobacillus fermentum* and its application. This invention uses the specific proteins Csp and Mucbp of *Lactobacillus fermentum* to immunize mice, and then prepares monoclonal antibodies 9C4 and 7F9 respectively using hybridoma technology. These antibodies can be used to enrich *Lactobacillus fermentum* in feces, for strain identification and sequencing, and for the identification and qualitative and quantitative study of *Lactobacillus fermentum* metabolites.

[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 Lactobacillus fermentum-specific protein, wherein the monoclonal antibody is either monoclonal antibody 9C4 or monoclonal antibody 7F9, wherein...

[0008] Monoclonal antibody 9C4 includes a 9C4 heavy chain variable region and a 9C4 light chain variable region. The 9C4 heavy chain variable region includes three complementarity-determining regions (CDRs):

[0009] 9C4-CDR-H1: WATEP;

[0010] 9C4-CDR-H2:TLRPIEVRWSFMEAMLLGS;

[0011] 9C4-CDR-H3: TVCMWYYQN;

[0012] The 9C4 light chain variable region includes three complement-determining regions (CDRs), namely:

[0013] 9C4-CDR-L1: TRQIAKVEPLVS;

[0014] 9C4-CDR-L2: PQGKTER;

[0015] 9C4-CDR-L3: YEKVNIRWL;

[0016] Monoclonal antibody 7F9 includes a 7F9 heavy chain variable region and a 7F9 light chain variable region. The 7F9 heavy chain variable region includes three complementarity-determining regions (CDRs):

[0017] 7F9-CDR-H1: GYNQF;

[0018] 7F9-CDR-H2:YNRSPQHTGTKCTYWMV;

[0019] 7F9-CDR-H3: TLVRMG;

[0020] The 7F9 light chain variable region includes three complementary determinant regions (CDRs):

[0021] 7F9-CDR-L1: MLRKAIWTEQS;

[0022] 7F9-CDR-L2: GSMTKFY;

[0023] 7F9-CDR-L3: WGNVINVTR.

[0024] Furthermore, in the monoclonal antibody 9C4, the amino acid sequence of the 9C4 heavy chain variable region is shown in SEQ ID NO: 5, and the amino acid sequence of the 9C4 light chain variable region is shown in SEQ ID NO: 6;

[0025] The monoclonal antibody 7F9 has the following amino acid sequence: heavy chain variable region as shown in SEQ ID NO: 7, and light chain variable region as shown in SEQ ID NO: 8.

[0026] The present invention also provides a method for preparing the monoclonal antibody, comprising the following steps:

[0027] (1) The protein codon-optimized nucleotide sequence is transformed into Escherichia coli BL21 or, expressed, and purified by sonication to obtain purified protein; wherein, the protein is either protein Csp or protein Mucbp; the amino acid sequence of protein Csp is shown in SEQ ID NO: 1, and the protein Csp codon-optimized nucleotide sequence is shown in SEQ ID NO: 2;

[0028] The amino acid sequence of the protein Mucbp is shown in SEQ ID NO: 3, and the nucleotide sequence of the protein Mucbp after codon optimization is shown in SEQ ID NO: 4;

[0029] (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 lines were obtained by detection and screening. The hybridoma cell lines were either hybridoma cell lines 9C4 that recognize the Csp protein or hybridoma cell lines 7F9 that recognize the Mucbp protein.

[0030] (3) Monoclonal antibody 9C4 was prepared by hybridoma cell line 9C4, and monoclonal antibody 7F9 was prepared by hybridoma cell line 7F9.

[0031] The present invention also provides the application of the monoclonal antibody described above in the enrichment or isolation of Lactobacillus fermentum.

[0032] The present invention also provides a kit for immunoprecipitation protein analysis, the kit comprising antibody-conjugated magnetic beads containing the aforementioned monoclonal antibody.

[0033] Furthermore, the preparation method of the antibody-conjugated magnetic beads is as follows:

[0034] Protein A / G magnetic bead suspension was magnetically separated, the supernatant was removed, monoclonal antibody was added, and the mixture was incubated. After washing, the supernatant was removed to obtain antibody-incubated Protein A / G magnetic beads. Then, Couple buffer (20x) and disuccinimide succinate were added, and the mixture was incubated to terminate the coupling. Finally, the magnetic beads were washed to obtain antibody-conjugated magnetic beads. The monoclonal antibody was either monoclonal antibody 9C4 or monoclonal antibody 7F9. The working concentration of the monoclonal antibody was 100–150 μg / mL.

[0035] Furthermore, the kit also includes a lysis buffer containing an inhibitor, wherein the lysis buffer is a mixture of Protease Inhibitor Cocktail (100X) and Lysis Buffer at a volume ratio of 1:100 to 120.

[0036] Furthermore, the volume ratio of the ProteaseInhibitor Cocktail (100X) to the Lysis Buffer is 1:100.

[0037] The present invention also provides an application of the kit described herein in the detection of Lactobacillus fermentum.

[0038] The present invention also provides a method for immunoprecipitation protein analysis using the aforementioned kit, comprising the following steps:

[0039] (1) Collect the sample to be tested, culture the bacteria, centrifuge to collect the precipitate, add pre-cooled lysis buffer containing inhibitor, lyse, centrifuge to collect the supernatant, and obtain crude protein extract.

[0040] (2) Add the crude protein extract to the antibody-conjugated magnetic beads and incubate; separate magnetically, remove the supernatant, wash, and obtain the protein magnetic bead antibody conjugate;

[0041] (3) The protein magnetic bead antibody conjugate was added to 2×SDS-PAGE loading buffer, boiled, magnetically separated, and the supernatant was collected as the protein antibody conjugate. The protein antibody conjugate was detected by Western blot. If the result was positive, the sample contained Lactobacillus fermentum.

[0042] Alternatively, if the result is negative, then the sample does not contain Lactobacillus fermentum.

[0043] The principle of this invention:

[0044] The reasons for selecting proteins Csp and Mucbp in this invention are as follows:

[0045] Cell surface proteins (Csp) act as adhesion factors, mediating the recognition and adhesion between bacteria and the intestinal mucosa. They also function as receptors for signal transduction, influencing immune responses and participating in metabolic regulation, playing a crucial role in maintaining intestinal homeostasis. Activation of mucin-binding protein (Mucbp) helps bacteria adhere to the host's intestinal mucosal surface, promoting adhesion and thus inhibiting pathogen colonization. Mucbp also interacts with mucins, enhancing mucosal barrier function.

[0046] The beneficial effects of this invention are:

[0047] 1. This invention prepares recombinant proteins of outer membrane proteins of Lactobacillus fermentum and their antibodies. Western blot is used to verify whether the protein and antibody can bind specifically, and the target protein is separated by immunoprecipitation. It can be applied to the analysis of Lactobacillus fermentum proteins and immune layers, as well as to the analysis of substances that interact with it.

[0048] 2. This invention establishes a method for enriching Lactobacillus fermentum through antibody and protein-specific precipitation reaction. It utilizes two monoclonal antibodies to capture the surface proteins of Lactobacillus fermentum, which is highly specific and can be used for the study of Lactobacillus fermentum-related proteins, the analysis of Lactobacillus fermentum metabolites, and their qualitative and quantitative analysis.

[0049] 3. This invention obtains specific recombinant proteins and specific antibodies of Lactobacillus fermentum, which is beneficial for further research on the function and mechanism of action of Lactobacillus fermentum surface proteins in the intestinal environment and is of great significance for the development of novel intestinal microbial products. Attached Figure Description

[0050] Figure 1 Figure 1 shows the SDS-PAGE results of proteins Csp and Mucbp.

[0051] Figure 2 The image shows the specificity detection results for monoclonal antibodies 9C4 and 7F9. Detailed Implementation

[0052] 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.

[0053] Example 1

[0054] Construction and purification of expression vectors for proteins Csp and Mucbp

[0055] 1. Construction of vectors pET28a-Csp and pET28a-Mucbp

[0056] The target amino acid sequence of protein Csp (Uniprot accession number A0A1D7ZXA8) was selected as shown in SEQ ID NO: 1. The nucleotide sequence obtained by E. coli codon usage preference analysis is shown in SEQ ID NO: 2. The whole gene was synthesized and cloned into the pET28a vector to construct the recombinant expression vector pET28a-Csp.

[0057] The target amino acid sequence of the protein Mucbp (Uniprot accession number A0A1L7GSW6) was selected as shown in SEQ ID NO: 3. The nucleotide sequence obtained by E. coli codon usage preference analysis is shown in SEQ ID NO: 4. The whole gene was synthesized and cloned into the pET28a vector to construct the recombinant expression vector pET28a-Mucbp.

[0058] The recombinant expression vector pET28a-Csp was introduced into Escherichia coli DH5α competent cells, and positive clones were screened using LB solid medium containing 50 μg / mL kanamycin. After culturing at 37°C for 24 h, a single clone strain Csp containing the vector pET28a-Csp was obtained.

[0059] The recombinant expression vector pET28a-Mucbp was introduced into Escherichia coli DH5α competent cells, and positive clones were screened using LB solid medium containing 50 μg / mL kanamycin. After culturing at 37°C for 24 h, a single clone strain Mucbp containing the vector pET28a-Mucbp was obtained.

[0060] 2. High expression of protein

[0061] (1) Select the monoclonal strain Csp obtained above for amplification in LB liquid medium and verify by sequencing.

[0062] (2) Extract plasmids from the monoclonal strains that have been verified by sequencing, transfer the plasmids into Escherichia coli BL21(DE3) competent cells, incubate overnight at 37°C, and then pick positive clones to be inoculated into LB liquid medium containing 50 μg / mL kanamycin for activation.

[0063] (3) The optimal induction parameters were determined by controlling a single variable, and the IPTG induction concentration was determined to be 0.5 mmol / L, the induction time was 20 h, and the temperature was 16 °C.

[0064] (4) Expand the culture according to the determined induction parameters, expand the culture liquid with an inoculum of 5%, and culture overnight according to the above-mentioned optimal induction conditions.

[0065] 3. Protein purification

[0066] (1) After overnight culture, collect the bacterial culture and centrifuge at 8000 rpm for 30 min at 4℃ to collect the bacterial cells. The bacterial precipitate is resuspended in equilibration buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Mimidazole, pH=8.0). Add protease inhibitor at a volume ratio of 1%, sonicate (5 s on, 2 s off) for 15 min, collect the solution, and centrifuge at 8000 rpm for 30 min at 4℃ to collect the supernatant.

[0067] (2) The supernatant was filtered through a 0.22 μm filter membrane. The target protein was purified by His Trap™ HP affinity chromatography column. The column was equilibrated with equilibration buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH=8.0). The supernatant after filtration was injected at a flow rate of 0.5 mL / min and washed with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH=8.0) until no background value was detected.

[0068] (3) Elute with elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM Mimidazole, pH=8.0) and collect the eluent in fractions. The collected protein eluent is then filtered and concentrated using an ultrafiltration tube. The concentration of the target protein Csp is determined using a BCA protein quantification kit, and the purity of the protein Csp is analyzed by SDS-PAGE.

[0069] The results are as follows Figure 1 As shown, this embodiment successfully obtained the purified protein Csp.

[0070] Purified Mucbp protein was prepared using a single-clone strain of Mucbp through high-level protein expression and purification. The concentration of the target Mucbp protein was determined using a BCA protein quantification kit, and the purity of the Mucbp protein was analyzed by SDS-PAGE. The results are as follows: Figure 1 As shown, this embodiment successfully obtained the purified protein Mucbp.

[0071] Example 2

[0072] Preparation of monoclonal antibodies

[0073] 1. Animal immunization

[0074] (1) Select healthy female Balb / c mice that are 6 to 8 weeks old for the first immunization. The purified protein Csp (100 μg protein / mouse) is used as the antigen and emulsified with Freund's complete adjuvant (1:1 volume). The mixture is vortexed for 20 min to form a stable water-in-oil emulsion. After sufficient emulsification, it is used for the first immunization via subcutaneous injection in the back of the neck.

[0075] (2) The first booster immunization was performed 21 days apart. Protein (50 μg / mouse) and Freund's incomplete adjuvant were prepared in a 1:1 volume ratio. The booster immunization was performed every 14 days thereafter. Protein (50 μg / mouse) and Freund's incomplete adjuvant were prepared in a 1:1 volume ratio. The adjuvant and antigen were prepared and used immediately each time. Seven days after the fourth immunization, tail vein blood was collected from mice, and the antiserum titer was detected by indirect ELISA.

[0076] 2. Indirect ELISA

[0077] The purified protein Csp was diluted to a concentration of 10 μg / mL and coated onto an ELISA plate, 100 μL per well, and incubated overnight at 4°C. The next day, the plate was washed three times with PBST, diluted 2-fold with 5% skim milk powder, and added to the plate. The plate was blocked at 37°C for 2 h. After washing three times with PBST, serially diluted mouse tail vein blood was added, 100 μL per well, and incubated at 37°C for 1 h. After washing three times with PBST, HRP-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (1:5000 dilution) was added, 100 μL per well, and incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of TMB was added for color development, and the plate was incubated at 37°C for 10 min. The incubation was terminated with 2M H₂SO₄ stop solution, and the OD value at 450 nm was measured using a microplate reader.

[0078] 3. Establishment of monoclonal hybridoma cell lines

[0079] (1) Mice with good immunization effect and high serum titer were euthanized by blood collection from their eyeballs, and a portion of the serum solution was used as a negative control. Spleen cell suspension was prepared and washed with PBS, and then SP2 / 0 cells were mixed in at a ratio of spleen lymphocytes:SP2 / 0 = 5:1.

[0080] (2) Cell membrane fusion was mediated by 50% PEG-1500 (1 mL for 1 min), and the reaction was terminated with RPMI-1640 medium. After centrifugation at 1000 rpm for 10 min, the supernatant was discarded, and the fused cells were resuspended in HAT selective medium (containing 20% ​​FBS) and seeded into 96-well plates containing feeder cells. The cells were then cultured in a 5% CO2 incubator.

[0081] (3) Observe the cell status 8-10 days after fusion, and change the medium to HT complete medium containing 20% ​​FBS. Observe the colony size and take 100 μL of supernatant for detection using the indirect ELISA method. Select positive cell lines with high OD value, high titer and good specificity for subcloning. Perform three limiting dilutions on the initially screened positive wells for subcloning, and finally obtain a hybridoma cell line that stably secretes monoclonal antibodies, which is numbered as hybridoma cell 9C4, recognizing protein Csp.

[0082] Using the protein Mucbp, hybridoma cells 7F9 that recognize the protein Mucbp were screened through animal immunization, indirect ELISA, and the establishment of monoclonal hybridoma cell lines.

[0083] 4. Preparation and purification of monoclonal antibodies using the antibody-induced ascites fluid method.

[0084] Unimmunized female Balb / c mice aged 6–8 weeks were intraperitoneally injected with 0.5 mL of sterile liquid paraffin oil to establish a microenvironment for ascites production. One week later, the mice were injected with 1 mL of hybridoma cells 9C4(10) 6 The mice were fed for one week and their abdominal cavity was observed to be dilated. Ascites was collected aseptically from the significantly distended abdominal cavity of the mice, yielding 9C4 ascites.

[0085] Unimmunized female Balb / c mice aged 6–8 weeks were intraperitoneally injected with 0.5 mL of sterile liquid paraffin oil to establish a microenvironment for ascites production. One week later, the mice were injected with 1 mL of hybridoma cells 7F9(10) 6 The mice were fed for one week and their abdominal cavity was observed to be dilated. Ascites was collected aseptically from the significantly distended abdominal cavity of the mice to obtain 7F9 ascites.

[0086] 5. Monoclonal antibody purification

[0087] (1) Dilute the 9C4 ascites fluid 3 to 4 times with 0.06M HCl buffer (pH=4) and adjust the pH to 4.5.

[0088] (2) Slowly add caprylic acid to the solution (add 25 μL of caprylic acid per mL of diluted ascites), stir at room temperature for 30 min, and centrifuge at 8000 rpm for 30 min.

[0089] (3) After filtering the supernatant, add 0.1M PBS buffer at a volume ratio of 9:1, adjust the pH to 7.4, and let stand at 4℃ for 30 min.

[0090] (4) Add 0.28g of ammonium sulfate to each mL of the mixed solution, stir for 30 min, let stand at 4℃ for 2 h, and centrifuge at 8000 rpm for 20 min.

[0091] (5) Collect the precipitate and dissolve it in ascites fluid mixture diluted 10 times with 0.01M PBS buffer. Dialyze overnight and collect the antibody by centrifugation to remove impurities.

[0092] (6) The purity of the purified antibody was detected by SDS-PAGE protein electrophoresis. The results showed that the purity of the obtained monoclonal antibody 9C4 reached more than 95%.

[0093] The purified monoclonal antibody 7F9 was prepared using 7F9 ascites fluid and the above steps. The purity was detected by SDS-PAGE protein electrophoresis and reached over 95%.

[0094] (7) The titer of the purified antibody was detected by indirect ELISA. The results showed that the titers of the two monoclonal antibodies were both above 1:128000.

[0095] Example 3

[0096] Monoclonal antibody subtype identification

[0097] Two monoclonal antibodies were identified using an antibody subtype identification kit.

[0098] 1. Dilute the purified protein Csp to 10 μg / mL as antigen and coat it onto an ELISA plate. Block overnight at 4°C. Add the purified and diluted monoclonal antibody 9C4, incubate at 37°C for 1 h, wash three times, then add HRP-labeled anti-mouse (IgG1 / IgG2a / IgG2b / IgG3 / IgM / IgA / Kappa / Lambda) antibody, incubate at 37°C for 1 h, wash three times with PBST, and finally add TMB for color development. Stop the reaction with 2M H2SO4. Read the OD value at 450 nm using an ELISA reader.

[0099] 2. The purified protein Mucbp was diluted to 10 μg / mL as an antigen and coated onto an ELISA plate, then blocked overnight at 4°C. The purified and diluted monoclonal antibody 7F9 was added, and the plate was incubated at 37°C for 1 h. After washing three times, HRP-labeled anti-mouse (IgG1 / IgG2a / IgG2b / IgG3 / IgM / IgA / Kappa / Lambda) antibody was added, and the plate was incubated at 37°C for 1 h. The plate was then washed three times with PBST, and finally, TMB was added for color development. The incubation was stopped with 2M H2SO4. The OD value at 450 nm was read using an ELISA reader.

[0100] The identification results are shown in Table 1. The heavy chain constant region of monoclonal antibody 9C4 is the IgG2b isotype, and the light chain constant region is the Kappa isotype. The heavy chain constant region of monoclonal antibody 7F9 is the IgG1 isotype, and the light chain constant region is the Kappa isotype.

[0101] Table 1. Isotype determination of monoclonal antibodies

[0102]

[0103] Example 4

[0104] Antibody sequencing

[0105] Sequencing of monoclonal antibodies 9C4 and 7F9 yielded the following results:

[0106] 1. The monoclonal antibody 9C4 includes the 9C4 heavy chain variable region and the 9C4 light chain variable region;

[0107] (1) The 9C4 heavy chain variable region includes three heavy chain complementarity-determining regions (CDR-H) and three heavy chain backbone regions (FR-H), which are as follows:

[0108] 9C4-FR-H1: EVKVEESGGGLVQPGGSMKLSCVASGFTFS;

[0109] 9C4-CDR-H1: WATEP;

[0110] 9C4-FR-H2: WVRQSPEKGLEWVA;

[0111] 9C4-CDR-H2:TLRPIEVRWSFMEAMLLGS;

[0112] 9C4-FR-H3: RFTISRDDSKSSVYLQMNNLRADDTGIYYCRG;

[0113] 9C4-CDR-H3: TVCMWYYQN;

[0114] 9C4-FR-H4:WGQGTSVTVSS.

[0115] The amino acid sequence of the variable region of the 9C4 heavy chain of the monoclonal antibody is shown in SEQ ID NO: 5;

[0116]

[0117] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-H) of the 9C4 heavy chain of the monoclonal antibody, while the underlined regions indicate the FR-H of the 9C4 heavy chain of the monoclonal antibody.

[0118] (2) The 9C4 light chain variable region includes three light chain complementarity determining regions (CDR-L) and three light chain backbone regions (FR-L), which are as follows:

[0119] 9C4-FR-L1: QIVLTQSPAVMSASPGEKVTLTC;

[0120] 9C4-CDR-L1: TRQIAKVEPLVS;

[0121] 9C4-FR-L2: WYQQKPGSPKLWIY;

[0122] 9C4-CDR-L2: PQGKTER;

[0123] 9C4-FR-L3: GVPTRFSGSGSGTSYSLTISNMEAEDAASYFC;

[0124] 9C4-CDR-L3: YEKVNIRWL;

[0125] 9C4-FR-L4:FGSGTKLEIR.

[0126] The amino acid sequence of the variable region of the light chain of the monoclonal antibody 9C4 is shown in SEQ ID NO: 6;

[0127]

[0128] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-L) of the 9C4 light chain of the monoclonal antibody, while the underlined regions indicate the light chain backbone region (FR-L) of the 9C4 light chain of the monoclonal antibody.

[0129] 2. The monoclonal antibody 7F9 includes the 7F9 heavy chain variable region and the 7F9 light chain variable region;

[0130] (1) The 7F9 heavy chain variable region includes three heavy chain complementarity determining regions (CDR-H) and three heavy chain backbone regions (FR-H), which are as follows:

[0131] 7F9-FR-H1:EVQLQQSGAEFVKPGASVKLSCTASGFNIR;

[0132] 7F9-CDR-H1: GYNQF;

[0133] 7F9-FR-H2: WVKQRPEQGLEWIG;

[0134] 7F9-CDR-H2:YNRSPQHTGTKCTYWMV;

[0135] 7F9-FR-H3:KATITADTSSNTAYLQLSSLTSEDTAVYYCTP;

[0136] 7F9-CDR-H3: TLVRMG;

[0137] 7F9-FR-H4:WGQGTPLTVSS.

[0138] The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 7F9 is shown in SEQ ID NO: 7;

[0139] EVQLQQSGAEFVKPGASVKLSCTASGFNIRGYNQFWVKQRPEQGLEWIGYNRSPQHTGTKCTYWMVKAT ITADTSSNTAYLQLSSLTSEDTAVYYCTPTLVRMGWGQGTPLTVSS

[0140] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-H) of the 7F9 heavy chain of the monoclonal antibody, while the underlined regions indicate the heavy chain backbone region (FR-H) of the 7F9 monoclonal antibody.

[0141] (2) The 7F9 light chain variable region includes three light chain complementarity determining regions (CDR-L) and three light chain backbone regions (FR-L), which are as follows:

[0142] 7F9-FR-L1: DIVMTQSQKFMSTSVGDRVSITC;

[0143] 7F9-CDR-L1: MLRKAIWTEQS;

[0144] 7F9-FR-L2:WYQQKPGQSPKLLIY;

[0145] 7F9-CDR-L2: GSMTKFY;

[0146] 7F9-FR-L3:GVPDRFTGSGSGTDFTLTISNMQSEDLADYFC;

[0147] 7F9-CDR-L3: WGNVINVTR;

[0148] 7F9-FR-L4:FGSGTKLEIK.

[0149] The amino acid sequence of the variable region of the light chain of monoclonal antibody 7F9 is shown in SEQ ID NO: 8;

[0150] DIVMTQSQKFMTSVGDRVSITCMLRKAIWTEQSWYQQKPGQSPKLLIYGSMTKFYGVPDRFTGSGSGT DFTLTISNMQSEDLADYFCWGNVINVTRFGSGTKLEIK

[0151] Note: The bold and underlined regions indicate the complementarity-determining region (CDR-L) of the 7F9 light chain of the monoclonal antibody, while the underlined regions indicate the light chain backbone region (FR-L) of the 7F9 monoclonal antibody.

[0152] Example 5

[0153] Purity and specificity analysis of Lactobacillus fermentum antibodies

[0154] 1. Take 2 mL of the identified Lactobacillus fermentum 10 4Add 1 / mL of protein to a centrifuge tube, add 200 μL of LRIPA 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℃ to obtain the sample.

[0155] 2. Prepare the SDS-PAGE gel according to the SDS-PAGE two-step gel kit. Take the sample obtained in step 1, load 10 μL of sample into each well, and maintain constant voltage at 80V for 20 min, then at 200V for 50 min. After electrophoresis, cut the gel according to the position of the pre-stained marker and cut the PDVF membrane to an appropriate size.

[0156] 3. Activate the PDVF membrane by immersing it in methanol for 1 min. Stack the membranes in the following order: anode plate - filter paper - SDS - PAGE gel - PDVF membrane - filter paper - cathode plate. Perform electrotransfer using a wet transfer apparatus at a constant current of 200 mA for 75 min to transfer the protein from the gel into the PDVF membrane.

[0157] 4. After successful transfer, remove the PDVF membrane and wash it three times in PBST solution, 5 minutes each time. Pour out the PBST solution and place the PDVF membrane in blocking buffer for 2 hours.

[0158] 5. Remove the PDVF membrane and wash it three times with PBST for 5 minutes each time. After washing, pour out the PBST solution and immerse the PDVF membrane in a solution of primary antibody diluted with 5% skim milk powder (1:2000 dilution). Incubate at 37°C for 1 hour. The primary antibody is monoclonal antibody 9C4.

[0159] 6. Pour out the primary antibody and wash three times with PBST solution for 5 min each time. Pour out the PBST solution and add the secondary antibody (the secondary antibody is anti-mouse-HRP, diluted at a ratio of 1:5000) diluted with 5% skim milk powder. Incubate at 37°C for 1 h.

[0160] 7. After the secondary antibody incubation, wash three times with PBST for 5 minutes each time, and develop with ECL. Finally, analyze the molecular weight of the target band on the PDVF membrane using a fully automated chemiluminescence analyzer.

[0161] The monoclonal antibody 7F9 was used as the primary antibody for specificity analysis through the above steps.

[0162] Monoclonal antibody 9C4 can specifically react with protein Csp, and monoclonal antibody 7F9 can specifically react with protein Mucbp. The specific bands are as expected.

[0163] Example 6

[0164] Analysis of antibody affinity for Lactobacillus fermentum

[0165] The antigen (protein Csp) was coated at gradient concentrations of 0.25 μg / mL and 0.5 μg / mL, 100 μL / well, and incubated overnight at 4°C. Serial dilutions were then performed at ratios of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and Negative Control. The affinity of the antigen and antibody was determined using an indirect ELISA method (the antibody used was monoclonal antibody 9C4).

[0166] The formula for calculating the antibody affinity constant Ka is as follows:

[0167] Where n is antigen[Ag]t / [Ag']t, [Ag]t and [Ag']t are two different concentrations of the coating antigen ([Ag]t is 0.25 μg / mL, [Ag']t is 0.5 μg / mL), [Ab′]t and [Ab]t are the antibody concentrations corresponding to the 50% maximum OD450 value at each coating antigen concentration, and Ka is in L / mol.

[0168] The antibody affinity constant was determined using the protein Mucbp as an antigen and the monoclonal antibody 7F9 through the steps described above.

[0169] The results showed that the antibody affinity constant Ka of the monoclonal antibody 9C4 against protein Csp was 4.96 × 10⁻⁶. 9 L / mol, the antibody affinity constant Ka for the monoclonal antibody 7F9 against protein Mucbp is 2.34 × 10⁻⁶. 10 L / mol, all of which are high-affinity antibodies.

[0170] Example 7

[0171] A kit for immunoprecipitation protein analysis

[0172] This embodiment provides a kit for immunoprecipitation protein analysis, comprising antibody-conjugated magnetic beads containing monoclonal antibody 9C4 or monoclonal antibody 7F9 and lysis buffer containing inhibitors.

[0173] 1. Preparation of Protein A / G magnetic bead suspension (20 mg / mL, Beyotime Biotechnology Co., Ltd.): Place 40 μL of Protein A / G magnetic beads (Beyotime Biotechnology Co., Ltd.) in a centrifuge tube, wash twice with 1 mL of Couple buffer (1x) (Beyotime Biotechnology Co., Ltd.), and suspend the magnetic beads in 1 mL of Couple buffer (1x) to obtain Protein A / G magnetic bead suspension.

[0174] 2. Preparation of lysis buffer containing inhibitor: Add Protease Inhibitor Cocktail (100X) (Beyotime Biotechnology Co., Ltd.) to Lysis Buffer (Beyotime Biotechnology Co., Ltd.) at a volume ratio of 1:100. Prepare fresh before use.

[0175] 3. The preparation method of antibody-conjugated magnetic beads is as follows:

[0176] Protein A / G magnetic bead suspension was magnetically separated. After discarding the supernatant, 500 μL of monoclonal antibody (working concentration 100 μg / mL) was added and incubated at room temperature for 1 h. The beads were washed three times with Couple buffer (1x) (Beyotime Biotechnology Co., Ltd.) and the supernatant was removed to obtain antibody-incubated Protein A / G magnetic beads. The monoclonal antibody was either monoclonal antibody 9C4 or monoclonal antibody 7F9. 20 μL of Couple buffer (20x) (Beyotime Biotechnology Co., Ltd.) and 32 μL of 1 mM chemical cross-linking agent disuccinimide octanoate (DSS) were added to the antibody-incubated Protein A / G magnetic beads. The mixture was diluted to 400 μL with ultrapure water and incubated at room temperature for 1 h. The supernatant was then removed. 400 μL of Acid Elution Buffer (Beyotime Biotechnology Co., Ltd.) was added and incubated for 5 min to terminate the coupling. The magnetic beads were washed twice with 1 mL of lysis buffer containing inhibitors and the supernatant was removed to obtain antibody-conjugated magnetic beads.

[0177] Example 8

[0178] Methods for immunoprecipitation protein analysis

[0179] This embodiment uses the kit from Example 7 to perform immunoprecipitation protein analysis, including the following steps:

[0180] (1) Bacterial lysis and sample preparation: Collect the sample to be tested and culture the bacteria. Centrifuge at 14000g for 15 min at 4℃ to collect the precipitate, add 1 mL of pre-cooled lysis buffer containing inhibitor, and lyse at 4℃ for 30 min, shaking once every 10 min to ensure complete lysis. Centrifuge at 14000g for 15 min at 4℃ and collect the supernatant, which is the crude protein extract.

[0181] (2) Protein and magnetic bead incubation: Add the crude protein extract to the antibody-conjugated magnetic beads and incubate overnight at 4°C. Magnetic separation is performed, the supernatant is removed, and the mixture is washed three times with 1 mL of lysis buffer containing inhibitors to obtain the protein-magnetic bead antibody conjugate.

[0182] (3) Detection: The protein magnetic bead antibody conjugate was added to 100 μL of 2×SDS-PAGE loading buffer for sample preparation, boiled at 100℃ for 10 min, magnetically separated, and the supernatant was collected as the protein antibody conjugate. The protein antibody conjugate was detected by Western blot.

[0183] Western blot results showed that, due to the specific reaction between monoclonal antibody 9C4 and protein Csp, and the specific reaction between monoclonal antibody 7F9 and protein Mucbp, the sample was rich in Lactobacillus fermentum. Therefore, both monoclonal antibody 9C4 and monoclonal antibody 7F9 of the present invention can be used to detect whether a sample contains Lactobacillus fermentum and to enrich Lactobacillus fermentum.

[0184] In summary, this invention prepares recombinant proteins of *Lactobacillus fermentum* outer membrane proteins and their monoclonal antibodies. Western blotting verifies the specific binding between the proteins and antibodies, and immunoprecipitation separates the target proteins. This method can be applied to the analysis of *Lactobacillus fermentum* outer membrane proteins and immunoassays, as well as to the analysis of substances interacting with them. Furthermore, this invention establishes a method for enriching *Lactobacillus fermentum*-specific proteins through antibody-protein specific precipitation reactions. Two monoclonal antibodies are used to capture surface proteins of *Lactobacillus fermentum*, exhibiting high specificity and applicability to the study of *Lactobacillus fermentum*-related proteins. This method can be applied to the analysis of *Lactobacillus fermentum* metabolites, as well as their qualitative and quantitative analysis.

[0185] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against a Lactobacillus fermentum-specific protein, characterized in that: The monoclonal antibody is either monoclonal antibody 9C4 or monoclonal antibody 7F9, wherein... Monoclonal antibody 9C4 includes a 9C4 heavy chain variable region and a 9C4 light chain variable region. The 9C4 heavy chain variable region includes three complementarity-determining regions (CDRs): 9C4-CDR-H1: WATEP; 9C4-CDR-H2:TLRPIEVRWSFMEAMLLGS; 9C4-CDR-H3: TVCMWYYQN; The 9C4 light chain variable region includes three complement-determining regions (CDRs), namely: 9C4-CDR-L1: TRQIAKVEPLVS; 9C4-CDR-L2: PQGKTER; 9C4-CDR-L3: YEKVNIRWL; Monoclonal antibody 7F9 includes a 7F9 heavy chain variable region and a 7F9 light chain variable region. The 7F9 heavy chain variable region includes three complementarity-determining regions (CDRs): 7F9-CDR-H1: GYNQF; 7F9-CDR-H2:YNRSPQHTGTKCTYWMV; 7F9-CDR-H3: TLVRMG; The 7F9 light chain variable region includes three complementary determinant regions (CDRs): 7F9-CDR-L1: MLRKAIWTEQS; 7F9-CDR-L2: GSMTKFY; 7F9-CDR-L3: WGNVINVTR.

2. The monoclonal antibody according to claim 1, characterized in that: In the monoclonal antibody 9C4, the amino acid sequence of the heavy chain variable region of 9C4 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of 9C4 is shown in SEQ ID NO: 6; The monoclonal antibody 7F9 has the following amino acid sequence: heavy chain variable region as shown in SEQ ID NO: 7, and light chain variable region as shown in SEQ ID NO:

8.

3. A method for preparing the monoclonal antibody according to claim 1 or 2, characterized in that: Includes the following steps: (1) The protein codon-optimized nucleotide sequence was transformed into Escherichia coli BL21, expressed, and purified by sonication to obtain purified protein; wherein, the protein is either protein Csp or protein Mucbp; the amino acid sequence of protein Csp is shown in SEQ ID NO: 1, and the protein Csp codon-optimized nucleotide sequence is shown in SEQ ID NO: 2; The amino acid sequence of the protein Mucbp is shown in SEQ ID NO: 3, and the nucleotide sequence of the protein Mucbp after codon optimization is shown in SEQ ID NO: 4; (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 lines were obtained by detection and screening. The hybridoma cell lines were either hybridoma cell lines 9C4 that recognize the Csp protein or hybridoma cell lines 7F9 that recognize the Mucbp protein. (3) Monoclonal antibody 9C4 was prepared by hybridoma cell line 9C4, and monoclonal antibody 7F9 was prepared by hybridoma cell line 7F9.

4. The use of the monoclonal antibody according to claim 1 or 2 in the enrichment or isolation of Lactobacillus fermentum.

5. A kit for immunoprecipitation protein analysis, characterized in that: The kit includes antibody-conjugated magnetic beads containing the monoclonal antibody as described in claim 1 or 2.

6. The reagent kit according to claim 5, characterized in that: The preparation method of the antibody-conjugated magnetic beads is as follows: Protein A / G magnetic bead suspension was magnetically separated, the supernatant was removed, monoclonal antibody was added, and the mixture was incubated. After washing, the supernatant was removed to obtain antibody-incubated Protein A / G magnetic beads. Then, Couple buffer (20x) and disuccinimide succinate were added, and the mixture was incubated to terminate the coupling. Finally, the magnetic beads were washed to obtain antibody-conjugated magnetic beads. The monoclonal antibody was either monoclonal antibody 9C4 or monoclonal antibody 7F9. The working concentration of the monoclonal antibody was 100–150 μg / mL.

7. The reagent kit according to claim 6, characterized in that: The kit also includes a lysis buffer containing an inhibitor, wherein the lysis buffer is a mixture of Protease Inhibitor Cocktail (100X) and Lysis Buffer at a volume ratio of 1:100 to 120.

8. The reagent kit according to claim 7, characterized in that: The volume ratio of the Protease Inhibitor Cocktail (100X) to the Lysis Buffer is 1:

100.

9. The application of the kit according to any one of claims 5 to 8 in the detection of Lactobacillus fermentum.

10. A method for immunoprecipitation protein analysis using the kit according to any one of claims 5 to 8, characterized in that: Includes the following steps: (1) Collect the sample to be tested, culture the bacteria, centrifuge to collect the precipitate, add pre-cooled lysis buffer containing inhibitor, lyse, centrifuge to collect the supernatant, and obtain crude protein extract. (2) Add the crude protein extract to the antibody-conjugated magnetic beads and incubate; Magnetic separation, removal of supernatant, washing, and obtaining protein magnetic bead antibody conjugates; (3) The protein magnetic bead antibody conjugate was added to 2×SDS-PAGE loading buffer, boiled, magnetically separated, and the supernatant was collected as the protein antibody conjugate. The protein antibody conjugate was detected by Western blot. If the result is positive, the sample contains Lactobacillus fermentum. Alternatively, if the result is negative, then the sample does not contain Lactobacillus fermentum.

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