Antibody of bifidobacterium lactis specific protein, antibody conjugated magnetic bead and application

Abstract

The application discloses a Bifidobacterium lactis specific protein antibody, an antibody conjugated magnetic bead and application. The antibody is a monoclonal antibody BL1, comprising a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 3 and 4 respectively. The antibody conjugated magnetic bead is coupled by the monoclonal antibody BL1 and activated carboxyl magnetic beads. The antibody conjugated magnetic bead can specifically enrich Bifidobacterium lactis, has high enrichment efficiency, simple enrichment operation, and can be applied to identification of metabolic products of Bifidobacterium lactis, and 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 Bifidobacterium lactis-specific protein, antibody-conjugated magnetic beads, and their applications. Background Technology

[0002] Bifidobacterium lactis is a subtype or variant of Bifidobacterium, usually compared and classified with Bifidobacterium animalis. Bifidobacterium is a Gram-positive anaerobic bacterium widely distributed in the intestines of humans and animals, and is an important component of the intestinal microecology. Bifidobacterium lactis is a pleomorphic bacterium, appearing Y-shaped, V-shaped, curved, or spoon-shaped, with its typical morphological characteristic being a branched bacterium. Bifidobacterium lactis (or Bifidobacterium animalis) does not form spores, is non-motile, and is an obligate anaerobe; its optimal fermentation temperature is 35–40℃, and its optimal growth pH is 6.7–7. This bacterium is an important physiological bacterium in the intestines of humans and animals, participating in a series of physiological processes such as immunity, nutrition, digestion, and protection.

[0003] Bifidobacterium lactis is isolated from the infant gut and has been widely described and applied in numerous studies. It possesses many of the functions of probiotics, including improving gut health and enhancing the immune system. Furthermore, Bifidobacterium lactis frequently appears on lists of bacteria that can be used in food, further demonstrating its important status as a type of Bifidobacterium.

[0004] Different strains of Bifidobacterium lactis, such as BL-99, Bi-07, and V9, each have their own unique health benefits. For example, BL-99, screened from the intestines of healthy infants, has been proven to help maintain the gut microbiota; Bi-07 performs well in regulating gut microbiota and enhancing immune function; while V9 is a strain with excellent probiotic properties isolated from the intestines of healthy Mongolian children on the Inner Mongolian grasslands.

[0005] Bifidobacterium lactis, as an important probiotic, has shown significant health benefits in many aspects, including regulating gastrointestinal function, enhancing immunity, improving allergy symptoms, and promoting nutrient absorption. The application of different strains of Bifidobacterium lactis further expands its potential in the medical and healthcare fields.

[0006] There are few studies on the preparation of specific antibodies against Bifidobacterium lactis target proteins. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an antibody for a Bifidobacterium lactis-specific protein, antibody-conjugated magnetic beads, and their applications. This invention uses the outer membrane protein PG2011B_1472 of Bifidobacterium lactis to immunize mice, prepares a monoclonal antibody BL1 using hybridoma technology, and then conjugates the monoclonal antibody BL1 with magnetic beads. Based on the antibody-conjugated magnetic beads, Bifidobacterium lactis is enriched. The antibody-conjugated magnetic beads of this invention capture Bifidobacterium lactis with high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to enrich Bifidobacterium lactis in feces, and can be used for the identification of Bifidobacterium lactis metabolites, as well as for qualitative and quantitative studies of Bifidobacterium lactis.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0009] This invention provides a monoclonal antibody against a Bifidobacterium lactis-specific protein, wherein the monoclonal antibody is monoclonal antibody BL1, and monoclonal antibody BL1 includes a heavy chain variable region and a light chain variable region.

[0010] The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody BL1 is shown in SEQ ID NO: 3;

[0011] The amino acid sequence of the variable region of the light chain of the monoclonal antibody BL1 is shown in SEQ ID NO: 4.

[0012] Furthermore, the monoclonal antibody BL1 heavy chain variable region includes four heavy chain backbone regions FR-H and three heavy chain complementarity-determining regions CDR-H, the four heavy chain backbone regions FR-H being FR-H1, FR-H2, FR-H3 and FR-H4, and the three heavy chain complementarity-determining regions CDR-H being CDR-H1, CDR-H2 and CDR-H3;

[0013] The amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are shown in SEQ ID NO: 5 to 8, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO: 9 to 11, respectively.

[0014] The monoclonal antibody BL1 light chain variable region includes four light chain backbone regions FR-L and three light chain complementarity-determining regions CDR-L. The four light chain backbone regions FR-L are FR-L1, FR-L2, FR-L3 and FR-L4, and the three light chain complementarity-determining regions CDR-L are CDR-L1, CDR-L2 and CDR-L3.

[0015] The amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 are shown in SEQ ID NO: 12-15, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 16-18, respectively.

[0016] Furthermore, the monoclonal antibody BL1 is prepared from a hybridoma cell line.

[0017] This invention also provides a method for preparing a hybridoma cell line, comprising the following steps:

[0018] (1) The optimized nucleotide sequence of protein PG2011B_1472 was transformed into E. coli BL21, expressed, and purified by sonication to obtain the purified protein.

[0019] (2) Mix the purified protein with Freund's adjuvant, emulsify it and then immunize the mice;

[0020] (3) Then, spleen cells from immunized mice were fused with myeloma cells SP2 / 0, and hybridoma cell lines were obtained through screening.

[0021] Furthermore, the optimized nucleotide sequence of the protein GMA92_05645 codon is shown in SEQ ID NO: 2.

[0022] Furthermore, the amino acid sequence of the protein GMA92_05645 is shown in SEQ ID NO: 1.

[0023] The present invention also provides the application of the monoclonal antibody in the preparation of antibody-conjugated magnetic beads.

[0024] This invention also provides a method for preparing antibody-conjugated magnetic beads, comprising the following steps:

[0025] (1) Dilute monoclonal antibody BL1 to 1.5-2.5 mg / mL using morpholine ethanesulfonic acid buffer to obtain diluted monoclonal antibody BL1;

[0026] (2) The magnetic beads were activated by carboxyl groups to obtain an activated carboxyl magnetic bead solution;

[0027] (3) The diluted monoclonal antibody BL1 is coupled with the activated carboxyl magnetic bead solution to obtain antibody-coupled magnetic beads, wherein the particle size of the activated carboxyl magnetic beads is 10-30 μm and the molar ratio of monoclonal antibody BL1 to activated carboxyl magnetic beads is 1:5-10.

[0028] Further, the monoclonal antibody BL1 is diluted to 2 mg / mL;

[0029] The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of monoclonal antibody BL1 to activated carboxyl magnetic beads is 1:5.

[0030] The present invention also provides an application of antibody-conjugated magnetic beads prepared by the above preparation method in the enrichment of Bifidobacterium lactis.

[0031] The beneficial effects of this invention are:

[0032] 1. This invention provides a specific antibody BL1 against the outer membrane protein of Bifidobacterium lactis cell wall. Western blotting (WB) confirmed that the BL1 antibody specifically binds to Bifidobacterium lactis. This antibody can be used to enrich Bifidobacterium lactis in feces and can be used for species identification and sequencing, as well as for the identification and qualitative and quantitative studies of Bifidobacterium lactis metabolites.

[0033] 2. This invention utilizes antibody-conjugated magnetic beads to capture Bifidobacterium lactis, which has high specificity, high sensitivity, and can improve enrichment efficiency.

[0034] 3. The method for enriching Bifidobacterium lactis in this invention is simple, does not require expensive instruments such as flow cytometers, reduces costs, and is conducive to widespread application. Attached Figure Description

[0035] Figure 1 Here is an electrophoresis image of the purified monoclonal antibody BL1;

[0036] Figure 2 This is a Western blot (WB) result of the monoclonal antibody BL1. Detailed Implementation

[0037] 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.

[0038] Example 1

[0039] Protein recombinant expression, purity and concentration detection

[0040] The amino acid sequence of Bifidobacterium lactis cell wall outer membrane protein PG2011B_1472 is shown in SEQ ID NO: 1, and the codon-optimized nucleotide sequence is shown in SEQ ID NO: 2.

[0041] The gene PG2011B_1472 was transformed into *E. coli* BL21 to express the protein PG2011B_1472. After sonication and centrifugation, the supernatant was purified using a nickel column chromatography method. The protein A280 concentration was measured using a micro spectrophotometer, and the protein purity was analyzed by SDS-PAGE. The results are shown in Table 1. The purity of the purified protein PG2011B_1472 reached 92%, which is relatively high.

[0042] Table 1. Protein concentration and purity results after purification.

[0043] protein name Purification Tag A280 concentration SDS-PAGE protein purity PG2011B_1472 His 1.3mg / ml 92％

[0044] Example 2

[0045] Mouse immune and antiserum titers and hybridoma cell fusion

[0046] 1. Mouse immunization

[0047] (1) After mixing protein PG2011B_1472 with Freund's adjuvant, emulsify the mixture in a mixer. The initial immunization dose was 50 μg protein / mouse, with Freund's complete adjuvant, and the interval between the first and second immunizations was 3 weeks. The dose for the second to fourth immunizations was 50 μg protein / mouse, with Freund's incomplete adjuvant, and the interval between the second and second immunizations was 2 weeks.

[0048] (2) Take blood from the tail vein of mice and detect the serum antibody titer. Mice with a titer of 1:10000 or higher are generally selected for fusion.

[0049] (3) Three days before hybridoma cell fusion, mice were immunized with 100 μg protein per mouse without adjuvant. The immunization method is shown in Table 2.

[0050] Table 2. Mouse Immunization Schedule

[0051] Number of immunizations Immune sites adjuvant Immunization dose 1 subcutaneous Freund's complete adjuvant 50μg 2 abdominal cavity Freund's incomplete adjuvant 50μg 3 abdominal cavity Freund's incomplete adjuvant 50μg 4 abdominal cavity Freund's incomplete adjuvant 50μg 5 abdominal cavity No adjuvants 100μg

[0052] 2. Hybridoma cell fusion

[0053] (1) After blood was collected from the eyeballs of mice after immunization, spleen cell suspension was prepared from mice with good immunization effect and washed with PBS. SP2 / 0 cells were then mixed in at a ratio of spleen cells:SP2 / 0 = 10:1. After centrifugation at 1000 rpm for 5 min, the mixed cells were drained and the cell clumps were loosened by tapping.

[0054] (2) Add 1 mL of PEG1450 to a 37°C water bath. After adding the PEG1450, react in a 37°C water bath for 2 min. Then slowly add 20 mL of RPMI-1640 stop solution along the tube wall.

[0055] (3) After cell fusion is terminated, centrifuge at 800 rpm for 5 min and aspirate any residual liquid. Resuspend the cells in DMEM complete medium containing HAT and transfer them to 96-well cell culture plates using a multichannel pipette. Cell colonies can generally be observed 3 days after fusion, and the medium should be changed and tested on day 7.

[0056] (4) Based on cell growth, when the colony size reaches approximately 1 / 4 of the bottom area of ​​the well, it is considered for detection. Take 100 μL of supernatant and perform detection using the indirect ELISA method. Select positive wells with high OD values ​​and good colony status for subcloning.

[0057] (5) Using HT medium, the selected positive cell colonies were diluted to 1 cell / well using the limiting dilution method. The cells were then seeded into 96-well cell culture plates. Once the monoclonal cells reached a medium size and a density of approximately 102, the cells were allowed to grow. 4 The titer can be detected by taking more than one cell; then take the positive cell wells again and repeat the subcloning screening. After all the cell supernatants in the microwells are positive, perform the same subcloning again until all positive results are obtained again. This confirms that a positive hybridoma cell line has been screened. In this example, two cell lines were selected, and the cell line numbers were BL1 and BL2.

[0058] 3. Antiserum titer detection

[0059] The antibody titer was detected by indirect ELISA, as shown in Table 3. The BL1 cell line with the highest antibody titer was selected for ascites preparation and subsequent antibody purification and expression. The isotype of the monoclonal antibody BL1 was identified using an antibody isotype identification kit, and the results are shown in Table 4. The isotype of monoclonal antibody BL1 was IgG1. This embodiment successfully obtained the monoclonal antibody BL1.

[0060] Table 3. Titer determination of two monoclonal antibodies

[0061]

[0062]

[0063] Table 4. Isotype determination of monoclonal antibody BL1

[0064] Cell line number Subtype BL1 IgG1

[0065] Example 3

[0066] Preparation of ascites fluid, purification of monoclonal antibody BL1 and concentration determination

[0067] 1. One week before inoculation with hybridoma cells, BALB / c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant per mouse, followed by inoculation with 5 × 10⁶ cells per mouse. 6 Positive hybridoma cells were collected, and ascites fluid was collected 7–12 days later to determine the titer of monoclonal antibody BL1.

[0068] 2. Centrifuge the collected ascites fluid at 10,000 rpm for 10 min to remove cellular components and other precipitates. Collect the supernatant and test the titer of the monoclonal antibody BL1. Aliquot the supernatant and store at -80℃ for later use.

[0069] 3. Load the monoclonal antibody BL1 sample to be purified onto a Protein A-agarose affinity chromatography column at a flow rate of 0.5 mL / min to allow the monoclonal antibody BL1 to bind to Protein A. Finally, elute with elution buffer to obtain the monoclonal antibody BL1. Identify its purity using SDS-PAGE. Figure 1 As shown in Table 5, the concentration of monoclonal antibody BL1 was determined using the NanoDrop method. The purified monoclonal antibody BL1 showed high purity.

[0070] Table 5 Concentration of monoclonal antibody BL1

[0071] Antibody name A280 concentration BL1 5.2 mg / mL

[0072] Example 4

[0073] Cell line sequencing

[0074] 1. Culture hybridoma cells BL1, lyse them, and extract total RNA and mRNA from the lysates. Use random hexamer primers (5'-Pd(NNNNNN)-3'N=G,A,T, or C) to reverse transcribe the mRNA into cDNA, then perform two rounds of nested PCR: First-strand cDNA is amplified using the first-strand cDNA as a template; the forward primer is a sequence complementary to the corresponding heavy and light chain leader sequences, and the reverse primer is a sequence within the constant region of the heavy and light chains. The second-round PCR uses the following forward and reverse primers for the heavy and light chains:

[0075] Heavy chain forward primer: CGGCCCAGCCGGCC;

[0076] Heavy chain reverse primer: TGAACCGCCTCCACC;

[0077] Light chain forward primer: GGTTCCACTGGT;

[0078] Light chain reverse primer: GTGCAGCATCAGC.

[0079] The PCR amplification program was as follows: denaturation at 94℃ for 2 min; denaturation at 94℃ for 20 s, annealing at 58℃ for 20 s, extension at 72℃ for 60 s, for 40 PCR cycles; final extension at 72℃ for 5 min.

[0080] 2. The second round of amplification yielded the gene product containing restriction enzyme sites (EcoRI and HindIII), which was ligated into the pMD19-T cloning vector. Sequencing and analysis then yielded the variable region sequences of the BL1 light and heavy chains of the monoclonal antibody. The forward and reverse primers for the heavy and light chains in the second round of PCR were:

[0081] Heavy chain forward primer: TGAATTCCGGCCCAGCCGGCC;

[0082] Heavy chain reverse primer: TAAGCTTTGAACCGCCTCCACC;

[0083] Light chain forward primer: TGATTCGGTTCCACTGGT;

[0084] Light chain reverse primer: TAAGCTTGTGCAGCATCAGC.

[0085] 3. The amino acid sequence of the variable region of the heavy chain of monoclonal antibody BL1 is shown in SEQ ID NO: 3:

[0086]

[0087] Note: The underlined region indicates the complementarity-determining region (CDR-H) of the BL1 heavy chain, and the bolded region indicates the BL1 heavy chain backbone region (FR-H). The amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 are shown in SEQ ID NO: 5, 6, 7, and 8, respectively; the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO: 9, 10, and 11, respectively.

[0088] FR-H1:EVKLQESGAELMKPGASVKISCKATGYTFS;

[0089] CDR-H1: HNSAD;

[0090] FR-H2: WVKQRPGHGLEWIG;

[0091] CDR-H2: EVAFACWYCYNYDRMRA;

[0092] FR-H3:KATFTADSSSNTAYMQLSSLTSEDSAVYYCAR;

[0093] CDR-H3: KAIIHMEN;

[0094] FR-H4: WGQGTTVTVSS.

[0095] The amino acid sequence of the variable region of the light chain of the monoclonal antibody BL1 is shown in SEQ ID NO: 4.

[0096]

[0097] Note: The underlined region indicates the complementarity-determining region (CDR-L) of the BL1 light chain, and the bolded region indicates the BL1 light chain backbone region (FR-L). The amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 are shown in SEQ ID NO: 12, 13, 14, and 15, respectively; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 16, 17, and 18, respectively.

[0098] FR-L1:DIQLTQSPAIMSASPGEKVTMTC;

[0099] CDR-L1: RASSSFTTTCQG;

[0100] FR-L2: WYQQKSGASPKLWIY;

[0101] CDR-L2: SCNSTAQ;

[0102] FR-L3: GVPARFSGSGSGTSYSLTISSVEAEDAATYYC;

[0103] CDR-L3: SQCNGYPLT;

[0104] FR-L4: FGSGTKLGIK.

[0105] Example 5

[0106] Preparation of antibody-conjugated magnetic beads

[0107] 1. Dilute monoclonal antibody BL1

[0108] Replace the monoclonal antibody BL1 buffer with 15mM MES buffer (pH 6.0), and dilute the monoclonal antibody BL1 to 2mg / mL with MES buffer to obtain the diluted monoclonal antibody BL1.

[0109] 2. Activation of carboxyl groups on the surface of magnetic beads

[0110] (1) After mixing the magnetic beads, take 100 μL of Mag COOH magnetic beads (70113-5, Suzhou Beaver Biotechnology) into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, wash twice with 200 μL of MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20), and then remove the supernatant.

[0111] (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).

[0112] After the above steps, the carboxyl groups on the surface of the magnetic beads are 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; it is recommended to perform coupling immediately).

[0113] 3. Covalent coupling of magnetic beads and monoclonal antibody BL1

[0114] (1) Take 200 μg of diluted monoclonal antibody BL1 and mix it with 100 μL of activated carboxyl magnetic bead solution (10 μm in diameter). After coupling at 25 °C for 1 h, place it at 4 °C overnight. Keep the magnetic beads in suspension during coupling (you can use a vertical mixer to invert and mix them).

[0115] (3) Magnetic separation: aspirate the supernatant and simultaneously detect the remaining antibody content in the supernatant. Calculate the amount and concentration of the magnetic bead-coupled antibody. Wash the magnetic beads 2 to 3 times with physiological saline and resuspend them with physiological saline to obtain antibody-coupled magnetic beads, i.e., BL1 magnetic beads.

[0116] Example 6

[0117] Bifidobacterium lactis enrichment

[0118] 1. Add 5 grams of feces to physiological saline at a ratio of 1:5 (for example, add 25 mL of physiological saline to 5 grams of feces), filter through gauze, and collect the pre-treated fecal microbial solution.

[0119] 2. Take the pre-treated fecal microbial solution, add 0.1 mg of BL1 magnetic beads, mix and incubate at 37°C for 0.5 h, separate the BL1 magnetic beads using a magnetic rack, and remove the microorganisms and supernatant that are not bound to the BL1 magnetic beads.

[0120] 3. The BL1 magnetic beads (labeled magnetic beads) contaminated with Bifidobacterium lactis were then 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 cleave the Fc and Fab of the mouse monoclonal antibody, thus separating the magnetic beads from the Bifidobacterium lactis. The BL1 magnetic beads were then collected using a magnetic rack, and the supernatant was the Bifidobacterium lactis suspension. The Bifidobacterium lactis was diluted and added to a hemocytometer for counting under a microscope. The results are shown in Table 6. The yield of Bifidobacterium lactis enriched by the BL1 magnetic beads of this invention was 1.3 * 10⁻⁶. 5CFU / mL, the BL1 magnetic beads of the present invention can efficiently enrich Bifidobacterium lactis in fecal samples.

[0121] Table 6. Yield of BL1 magnetic bead-enriched bacteria

[0122] Serial Number Combination type Yield 1 BL1 <![CDATA[1.3*10 5 ]]>

[0123] Example 7

[0124] Culture and sequencing of Bifidobacterium lactis

[0125] 1. Dilute the Bifidobacterium lactis isolated in Example 6 to 10. 3 ~10 4 The bacteria were cultured at 1 / mL using Delman-Rogosa-Sharp (MRS) medium at 37°C and pH 6.8, with fermentation carried out at 0% to 10% glucose concentration. Single colony characteristics of *Bifidobacterium lactis*: pleomorphic bacteria, exhibiting Y-shaped, V-shaped, curved, or scraper-like forms.

[0126] 2. Select 20 single colonies and use them as templates for PCR amplification. The upstream primer sequence for PCR is 5'-AGAGTTTGATCCTGGCTCAGPCR-3', and the downstream primer sequence is 5'-GGTTACCTTGTTACGACTT-3'.

[0127] 3. PCR reaction system: DNA template (10ng / μL) 1μL, forward and reverse primers (10μmol / L) 2μL each, 10×PCR Buffer 5μL, dNTPs (2.5mmol / L) 4μL, Taq enzyme (5U / μL) 0.5μL, ddH2O 35.5μL.

[0128] PCR reaction conditions: 94℃ for 10 min; 94℃ for 1 min, 56℃ for 1 min, 72℃ for 25 s, 72℃ for 10 min, 30 cycles.

[0129] 4. After the reaction, agarose gel electrophoresis was performed to identify the results. The target band was recovered, purified, and sequenced using nucleotides. The sequencing results were then compared with BLAST in the NCBI database. The results showed that the 16S rRNA gene sequences of the 20 colonies had 97% homology with Bifidobacterium lactis (strain number: ASM22496v2, GenBank: GCA_000224965.2). Therefore, the isolate was identified as Bifidobacterium lactis.

[0130] Example 8

[0131] Western blot identification of Bifidobacterium lactis antibodies

[0132] 1. Sample preparation: Take 10g of Bifidobacterium lactis identified in Example 7. 4 Add 1 / mL of protein to a 2mL centrifuge tube, add 200μL of RIPA lysis buffer to extract total protein, centrifuge at 10000rpm for 5min, add an equal volume of 2×loading buffer, boil in water for 5min, aliquot and store at -20℃.

[0133] 2. Electrophoresis: Prepare SDS-PAGE gels according to the standard protein electrophoresis method, load 15 μL of sample into each well, and perform electrophoresis at a constant voltage of 200V for 30 min.

[0134] 3. Transfer: Use a wet transfer apparatus to transfer the protein sample in the gel into a PDVF membrane. Transfer at a constant current of 150mA for 20-30 minutes.

[0135] 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 or 4°C overnight.

[0136] 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 a monoclonal antibody BL1 dilution buffer diluted with 1% casein at 37°C for 1 hour; the monoclonal antibody BL1 is the primary antibody, and the primary antibody is diluted to 1:1000.

[0137] 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 a secondary antibody dilution solution diluted with 1% casein at 37°C for 1 hour; the secondary antibody is goat anti-mouse-HRP, diluted to 1:5000.

[0138] 7. Color development: DAB color reaction.

[0139] Data reading: The molecular weight and net optical density of the target band on the membrane were analyzed using a gel image processing system.

[0140] The results are as follows Figure 2 As shown, the present invention obtains a specific antibody BL1 for the outer membrane protein of Bifidobacterium lactis cell wall. After Western blotting, the specific antibody BL1 can specifically bind to Bifidobacterium lactis.

[0141] 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 Bifidobacterium lactis-specific protein, characterized in that: The monoclonal antibody is monoclonal antibody BL1, which includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody BL1 is shown in SEQ ID NO: 3; The amino acid sequence of the variable region of the light chain of the monoclonal antibody BL1 is shown in SEQ ID NO:

4.

2. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody BL1 heavy chain variable region includes four heavy chain backbone regions FR-H and three heavy chain complementarity-determining regions CDR-H. The four heavy chain backbone regions FR-H are FR-H1, FR-H2, FR-H3 and FR-H4, and the three heavy chain complementarity-determining regions CDR-H are CDR-H1, CDR-H2 and CDR-H3. The amino acid sequences of FR-H1, FR-H2, FR-H3 and FR-H4 are shown in SEQ ID NO: 5 to 8, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO: 9 to 11, respectively. The monoclonal antibody BL1 light chain variable region includes four light chain backbone regions FR-L and three light chain complementarity-determining regions CDR-L. The four light chain backbone regions FR-L are FR-L1, FR-L2, FR-L3 and FR-L4, and the three light chain complementarity-determining regions CDR-L are CDR-L1, CDR-L2 and CDR-L3. The amino acid sequences of FR-L1, FR-L2, FR-L3 and FR-L4 are shown in SEQ ID NO: 12-15, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 16-18, respectively.

3. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody BL1 was prepared from a hybridoma cell line.

4. A method for preparing a hybridoma cell line, characterized in that: Includes the following steps: (1) The optimized nucleotide sequence of protein PG2011B_1472 was transformed into E. coli BL21, expressed, and purified by sonication to obtain the purified protein; (2) Mix the purified protein with Freund's adjuvant, emulsify it and then immunize the mice; (3) Then, spleen cells from immunized mice were fused with myeloma cells SP2 / 0 and the hybridoma cell lines were obtained by screening.

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

2.

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

1.

7. The use of the monoclonal antibody as described in claim 1 in the preparation of antibody-conjugated magnetic beads.

8. A method for preparing antibody-conjugated magnetic beads, characterized in that: Includes the following steps: (1) Dilute monoclonal antibody BL1 to 1.5-2.5 mg / mL using morpholine ethanesulfonic acid buffer to obtain diluted monoclonal antibody BL1; (2) The magnetic beads were activated by carboxyl groups to obtain an activated carboxyl magnetic bead solution; (3) The diluted monoclonal antibody BL1 is coupled with the activated carboxyl magnetic bead solution to obtain antibody-coupled magnetic beads, wherein the particle size of the activated carboxyl magnetic beads is 10-30 μm and the molar ratio of monoclonal antibody BL1 to activated carboxyl magnetic beads is 1:5-10.

9. The preparation method according to claim 8, characterized in that: The monoclonal antibody BL1 was diluted to 2 mg / mL; The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of monoclonal antibody BL1 to the activated carboxyl magnetic bead solution is 1:

5.

10. The application of antibody-conjugated magnetic beads prepared by the method described in claim 8 in the enrichment of Bifidobacterium lactis.

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