Antibodies to rectal specific proteins, magnetic bead antibody conjugates and uses thereof
By preparing monoclonal antibodies against E. rectum-specific proteins and magnetic bead conjugates, the problem of efficient enrichment of E. rectum in existing technologies has been solved, achieving efficient enrichment and metabolite analysis while reducing costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MEI YI TIAN BIOLOGICAL MEDICINE WUHAN CO LTD
- Filing Date
- 2024-12-31
- Publication Date
- 2026-07-03
AI Technical Summary
Existing technologies lack efficient methods for enriching Escherichia coli, especially since there is limited research on monoclonal antibodies targeting its specific proteins, making it difficult to achieve efficient enrichment of Escherichia coli and identification of its metabolites.
Monoclonal antibodies 4D3F6H7 and 5C8E7D6, which are specific proteins of E. rectum, were prepared and conjugated with magnetic beads to form magnetic bead antibody conjugates for the specific capture and enrichment of E. rectum.
It achieves efficient enrichment of E. rectum, improves enrichment efficiency, and can be used for qualitative and quantitative analysis of its metabolites, reducing costs and eliminating reliance on expensive instruments.
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Figure CN122325596A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to an antibody, magnetic bead antibody conjugate, and their applications against a protein specific to Escherichia coli. Background Technology
[0002] The close relationship between the gut microbiota and human health is widely recognized. They play crucial roles in maintaining intestinal barrier function, immune regulation, inflammation control, and energy metabolism regulation. Furthermore, the gut microbiota interacts with the central nervous system (CNS) through the gut-microbe axis, directly or indirectly influencing brain function and behavior. Among these, *Eubacterium*, as a core member of the gut microbiota, has attracted widespread attention due to its unique physiological functions, phylogenetic diversity, and taxonomic significance with multiple phenotypes and genotypes. *Eubacterium rectale*, as a representative bacterium of the *Eubacterium* genus, has garnered attention due to its metabolites and functional characteristics. Recent studies have found that its abundance variations not only participate in regulating immune function and influencing the occurrence and development of various systemic diseases, but may also be associated with neuropsychiatric disorders.
[0003] *Eubacterium rectum* is a Gram-positive, obligate anaerobic bacterium that produces short-chain fatty acids (SCFAs), particularly butyrate, by breaking down carbohydrates such as dietary fiber. Butyrate is an important energy source for colonic epithelial cells and plays a crucial role in maintaining gastrointestinal health, regulating the immune system, and suppressing inflammatory responses. Studies have found that the abundance of *Eubacterium rectum* is closely related to dietary fiber intake; a decrease in dietary fiber intake may lead to lower butyrate levels, and butyrate levels are associated with intestinal diseases (such as inflammatory bowel disease) and systemic diseases (such as non-small cell lung cancer). *Eubacterium rectum* also plays an important role in anti-tumor immunity; butyrate-producing bacteria are significantly reduced in the gut microbiota of patients with primary gastrointestinal B-cell lymphoma. Further research found that *Eubacterium rectum* was significantly enriched in melanoma patients who responded to anti-PD1 immunotherapy, and high abundance of *Eubacterium rectum* was significantly associated with longer patient survival. This finding reveals the possible regulatory role of *Eubacterium rectum* in the tumor microenvironment and its potential impact on anti-tumor immunotherapy. Furthermore, butyrate also plays a role in signal transduction along the gut-brain axis. For example, in autistic patients, reduced abundance of butyrate-producing bacteria is associated with gastrointestinal dysfunction and neurodevelopmental disorders, making them more susceptible to digestive tract diseases such as diarrhea, constipation, bloating, and gastrointestinal dysfunction. Based on the "Lunar Palace 365" experiment, a correlation analysis between gut microbiota and psychological changes revealed that *Escherichia coli* is one of four potential psychobiotics, and its abundance is significantly positively correlated with changes in positive emotions and significantly negatively correlated with the evolution of negative emotions.
[0004] Given the complex role of *E. rectum* in various diseases, developing specific monoclonal antibodies against this bacterium is crucial for achieving efficient enrichment of *E. rectum*. Research on the preparation of specific antibodies against *E. rectum* target proteins is limited. S-layer proteins and LPXTG-like proteins, due to their location on the bacterial surface and unique structural characteristics, can be recognized by the host immune system and elicit an immune response, making them key antigen candidates for Gram-positive bacteria. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an antibody, a magnetic bead antibody-conjugate, and their applications for E. rectum-specific proteins. This invention uses LPTSG-like protein (CSX02_09140) and S-layer protein (CSX02_05975) from E. rectum to immunize mice. Monoclonal antibodies 4D3F6H7 and 5C8E7D6 are prepared using hybridoma technology. These monoclonal antibodies are then conjugated with magnetic beads, and E. rectum is enriched based on the magnetic bead antibody-conjugate. The magnetic bead antibody-conjugate of this invention captures E. rectum with high specificity and sensitivity, and can improve enrichment efficiency. It can be applied to enrich E. rectum in feces, for the identification of E. rectum metabolites, and for qualitative and quantitative studies of E. rectum.
[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 protein specific to *E. rectum*, wherein the monoclonal antibody is either monoclonal antibody 4D3F6H7 or monoclonal antibody 5C8E7D6, wherein...
[0008] The monoclonal antibody 4D3F6H7 includes a 4D3F6H7 heavy chain variable region and a 4D3F6H7 light chain variable region, whose amino acid sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
[0009] The monoclonal antibody 5C8E7D6 includes a 5C8E7D6 heavy chain variable region and a 5C8E7D6 light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
[0010] Furthermore, in the monoclonal antibody 4D3F6H7, the 4D3F6H7 heavy chain variable region includes three complementarity-determining regions (CDRs), namely:
[0011] 4D3F6H7-CDR-H1:KGVQF;
[0012] 4D3F6H7-CDR-H2:GPINPDBAKQYMARCPF;
[0013] 4D3F6H7-CDR-H3: MLSRVPD;
[0014] The 4D3F6H7 light chain variable region includes three complementary determinant regions (CDRs), namely:
[0015] 4D3F6H7-CDR-L1:GAGRITNPKEQIM;
[0016] 4D3F6H7-CDR-L2: DQFSTLD;
[0017] 4D3F6H7-CDR-L3: WNDGF;
[0018] The monoclonal antibody 5C8E7D6 includes three complementarity-determining regions (CDRs) in its heavy chain variable region:
[0019] 5C8E7D6-CDR-H1: ELCKP;
[0020] 5C8E7D6-CDR-H2:LSCLICDNRYTMATET;
[0021] 5C8E7D6-CDR-H3:GRFHKSPDKMQWN;
[0022] The 5C8E7D6 light chain variable region includes three complementary determinant regions (CDRs), namely:
[0023] 5C8E7D6-CDR-L1: QRCYTRTMKLWS;
[0024] 5C8E7D6-CDR-L2:QDYWVBR;
[0025] 5C8E7D6-CDR-L3: PTDER.
[0026] Furthermore, the monoclonal antibody is prepared from a hybridoma cell line.
[0027] This invention also provides a method for preparing a hybridoma cell line, comprising the following steps:
[0028] (1) The nucleotide sequence with optimized protein codons was transformed into Escherichia coli BL21, expressed, and purified by sonication to obtain purified protein; wherein, the protein is either protein CSX02_09140 or protein CSX02_05975;
[0029] (2) The purified protein was mixed with Freund's adjuvant, emulsified and then used to immunize mice. Then, the spleen cells 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 4D3F6H7 or hybridoma cell lines 5C8E7D6.
[0030] Furthermore, when the protein is protein CSX02_09140, the nucleotide sequence of protein CSX02_09140 after codon optimization is shown in SEQ ID NO: 2;
[0031] When the protein is protein CSX02_05975, the optimized nucleotide sequence of protein CSX02_05975 codons is shown in SEQ ID NO: 4.
[0032] Furthermore, the amino acid sequence of the protein CSX02_09140 is shown in SEQ ID NO: 1, and the amino acid sequence of the protein CSX02_05975 is shown in SEQ ID NO: 3.
[0033] The present invention also provides the application of the monoclonal antibody in the preparation of magnetic bead antibody conjugates.
[0034] This invention also provides a method for preparing magnetic bead antibody conjugates, comprising the following steps:
[0035] (1) Dilute the monoclonal antibody using MES buffer;
[0036] (2) The magnetic beads were activated by carboxyl groups to obtain activated carboxyl magnetic beads;
[0037] (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 either magnetic bead antibody conjugate 4D3F6H7 or magnetic bead antibody conjugate 5C8E7D6.
[0038] 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.
[0039] 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.
[0040] The present invention also provides the application of a magnetic bead antibody conjugate in the enrichment or isolation of Escherichia coli, wherein the magnetic bead antibody conjugate is any one or a combination of two of magnetic bead antibody conjugate 4D3F6H7 and magnetic bead antibody conjugate 5C8E7D6.
[0041] The beneficial effects of this invention are:
[0042] 1. This invention utilizes magnetic bead antibody conjugates to capture E. rectum, which has high specificity and sensitivity and can improve enrichment efficiency. It can be applied to the analysis of E. rectum metabolites, as well as their qualitative and quantitative analysis.
[0043] 2. This invention obtains specific antibodies that recognize E. rectum LPXTG-like protein (CSX02_09140) and S-layer protein (CSX02_05975), respectively. The antibodies are conjugated with magnetic beads for the enrichment or isolation of E. rectum. They can bind to specific sites of E. rectum and form a synergistic effect, thereby improving the enrichment efficiency by enriching E. rectum from the magnetic bead antibody conjugate and the specific protein target.
[0044] 3. The method for enriching E. rectum 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
[0045] Figure 1 SDS-PAGE electrophoresis images of purified proteins CSX02_09140 and CSX02_05975;
[0046] Figure 2 The graph shows the titer results of monoclonal antibodies 4D3F6H7 and 5C8E7D6.
[0047] Figure A shows the titer determination results of monoclonal antibody 4D3F6H7, and Figure B shows the titer determination results of monoclonal antibody 5C8E7D6.
[0048] Figure 3 SDS-PAGE electrophoresis images of purified monoclonal antibodies 4D3F6H7 and 5C8E7D6;
[0049] Figure 4 The image shows the Western blot (WB) results for monoclonal antibodies 4D3F6H7 and 5C8E7D6. Detailed Implementation
[0050] 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.
[0051] Example 1
[0052] Recombinant protein expression and purity detection
[0053] 1. Protein sequence
[0054] (1) The amino acid sequence of LXTG-like protein CSX02_09140 (UniProt accession number: A0A2G3E1J7) 722~960aa of E. rectum is shown in SEQ ID NO: 1, and the nucleotide sequence after codon optimization is shown in SEQ ID NO: 2.
[0055] (2) The amino acid sequence of the N-terminus 23-589aa of E. rectum S-layer protein CSX02_05975 (UniProt accession number: A0A2G3E415) is shown in SEQ ID NO: 3, and the nucleotide sequence after codon optimization is shown in SEQ ID NO: 4.
[0056] 2. Recombination of the target gene into the pET-28a vector
[0057] The full genome sequences of the target genes (CSX02_09140 and CSX02_0597) were obtained and introduced into the pET-28a vector to construct the recombinant expression vectors pET-28a-CSX02_09140 and pET-28a-CSX02_0597. DNA sequencing confirmed the successful construction of these recombinant expression vectors.
[0058] 4. Protein expression and purification
[0059] The recombinant expression vectors pET-28a-CSX02_09140 and pET-28a-CSX02_0597 were transformed into Escherichia coli BL21(DE3) competent cells, respectively. The cells were plated on LB solid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C. Single colonies were picked and cultured overnight at 37°C and 200 rpm in 10 mL of LB liquid medium containing 100 μg / mL kanamycin. Then, 10 mL of the bacterial culture was added to TB medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm until the OD of the bacterial culture reached 0.6–0.8. 0.2 mM IPTG was then added to induce the expression of the target protein. The cells were then cultured in shake flasks at 16°C and 200 rpm for 12 h, followed by further incubation at 8000 rpm. The precipitate was collected by centrifugation for 30 minutes. After resuspending, the precipitate was sonicated and the supernatant was subjected to nickel column chromatography to elute impurities and purify the target proteins (CSX02_09140 and CSX02_0597). Protein purity was analyzed by SDS-PAGE. Figure 1 As shown, proteins CSX02_09140 and CSX02_0597 have high purity.
[0060] Example 2
[0061] Mouse immune and antiserum titers and hybridoma cell fusion
[0062] 1. Mouse immunization
[0063] (1) Proteins CSX02_09140 and CSX02_05975 were used as antigens and diluted with physiological saline to 1 μg / μL (prepared according to 50 μL per injection). They were then mixed with Freund's adjuvant and emulsified in a mixer. The initial immunization dose was 100 μL of protein per mouse, with Freund's complete adjuvant as the adjuvant. On day 21, mice in good mental condition were selected for booster immunization.
[0064] (2) For the second to fourth immunizations, the immunization dose is 50 μL protein / mouse, with an interval of 14 days. The adjuvant is Freund's incomplete adjuvant. Each adjuvant and antigen should be prepared and used immediately.
[0065] (3) Blood was collected from the tail vein of mice to detect serum antibody titers. Mice with a titer of 1:20000 or higher were generally selected for antigen pulse immunization. The immunization dose was 50 μL protein per mouse, without adjuvant. Spleen cells from mice could be collected 3–7 days after pulse immunization for cell fusion. The immunization method is shown in Table 1.
[0066] Table 1. Mouse Immunization Schedule
[0067] 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
[0068] 2. Hybridoma cell fusion and antiserum titer detection
[0069] (1) Take mice with good immune response and high serum titer, collect blood from the eyeballs and kill them. Prepare spleen cell suspension and wash it with PBS. Then mix it with SP2 / 0 cells at a ratio of spleen cells:SP2 / 0 = 5:1. Centrifuge at 1000 rpm for 10 min, then drain the mixed cells and gently tap to loosen the cell clumps.
[0070] (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.
[0071] (3) After cell fusion is terminated, centrifuge at 1000 rpm for 10 min and discard the supernatant. Resuspend the cells in 100 mL of complete medium containing 20% FBS and HAT, and transfer the medium to 96-well cell culture plates containing feeder cells using a multichannel pipette. Incubate at 37°C in a 5% CO2 incubator. Observe the cell status 8–10 days after fusion, and change the medium using complete medium containing 20% FBS and HT.
[0072] (4) Based on cell growth, when the colony size reaches approximately 1 / 4 of the well bottom area, detection can be considered. 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.
[0073] (5) Using HT medium, the selected cells 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 had grown to a medium size and reached a density of approximately 10, the cells were allowed to mature. 4 Titer can be detected with more than one cell. Then, positive cells are taken again and subcloning is repeated. When all cell supernatants in the microwells are positive, hybridoma monoclonal cell lines are obtained after three subcloning processes. The hybridoma cells with cell numbers 4D3F6H7 (recognition protein CSX02_09140) and 5C8E7D6 (recognition protein CSX02_05975) are identified.
[0074] The titer test results of the two monoclonal antibodies are as follows: Figure 2 As shown, the antibody titers detected by indirect ELISA all reached 1:320000 or higher.
[0075] Example 3
[0076] Monoclonal antibody purification and monoclonal antibody subtype identification
[0077] 1. Monoclonal antibody purification
[0078] (1) Positive hybridoma cells 4D3F6H7 and 5C8E7D6 were cultured separately and then collected. The cell concentration was adjusted to 10⁻⁶. 6 Approximately [number] cells / mL.
[0079] (2) Unimmunized mice were selected and injected intraperitoneally with 0.5 mL of sterile paraffin oil. Seven days later, 1 mL of hybridoma cells 4D3F6H7 and 5C8E7D6 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 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, elution was performed with elution buffer to obtain monoclonal antibodies 4D3F6H7 and 5C8E7D6. The purity was identified by SDS-PAGE. Figure 3 As shown, purified monoclonal antibodies 4D3F6H7 and 5C8E7D6 were successfully obtained.
[0080] 2. Monoclonal antibody subtype identification
[0081] The isotypes of two monoclonal antibodies were identified using an antibody isotype identification kit. Antigen dilution buffer was diluted to 10 μg / mL, and the substrate was coated onto an ELISA plate. The plate was blocked at 37°C for 2 h. 10 μL of purified and diluted monoclonal antibody 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) 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, chromogenic reaction was performed. The incubation was terminated with stop solution. The isotype results are shown in Table 2. The isotype of monoclonal antibody 4D3F6H7 was IgG2b, and the isotype of monoclonal antibody 5C8E7D6 was IgG1.
[0082] Table 2. Isotype determination of the two monoclonal antibodies
[0083] serial number Subtype Monoclonal antibody 4D3F6H7 IgG2b Monoclonal antibody 5C8E7D6 IgG1
[0084] Example 4
[0085] Cell line sequencing
[0086] 1. Culture hybridoma cells 4D3F6H7 and 5C8E7D6, lyse them, and extract total RNA and mRNA from the lysate. Use random hexamer primers (5'-Pd(NNNNNN)-3'N=G,A,T or C) to reverse transcribe mRNA to synthesize cDNA, and then perform two rounds of nested PCR: amplify with the first-strand cDNA as a template, the forward primer is a sequence complementary to the corresponding heavy and light chain leader sequences, and the reverse primer is a sequence in the constant region of the heavy and light chains.
[0087] Heavy chain forward primer:
[0088] CAGGTCCAGGTGAAGCARTC;
[0089] Heavy chain reverse primer:
[0090] CGAGGAAACGGTGACCGTGGT;
[0091] Light chain forward primer:
[0092] AGTCATTCGGATCCTTACCGTTTGATTTCCAGCTTGGTGC;
[0093] Light chain reverse primer:
[0094] GAGACTGGGTGAGCTCGATGTCCGATCCGCCACCGCCAGATC CGCCTCCGC.
[0095] The PCR amplification program was as follows: 95℃ for 2 min; 94℃ for 30 s, 45℃ for 30 s, 72℃ for 45 s, 7 PCR cycles; 94℃ for 30 s, 65℃ for 30 s, 72℃ for 45 s, 29 PCR cycles; 72℃ for 10 min.
[0096] 2. The second round of amplification yielded the gene product with restriction enzyme sites (EcoRI and NcoI), which was ligated into the pMD19-T cloning vector. Then, through sequencing and analysis, the variable region sequences of the antibody light chain and heavy chain were obtained.
[0097] Heavy chain forward primer:
[0098] TGAATTCCAGGTCCAGGTGAAGCARTC;
[0099] Heavy chain reverse primer:
[0100] TAAGCTTCGAGGAAACGGTGACCGTGGT;
[0101] Light chain forward primer:
[0102] TGAATTCAGTCATTCGGATCCTTACCGTTTGATTTCCAGCTTG GTGC;
[0103] Light chain reverse primer:
[0104] TAAGCTTGAGACTGGGTGAGCTCGATGTCCGATCCGCCACCG CCAGATCCGCCTCCGC.
[0105] 3. The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 4D3F6H7 is shown in SEQ ID NO: 5:
[0106] QIQLQQSGADLVRPGALVKLSCKASGFNIK KGVQF WVKQRPEQGLEWIG GPINPDBAKQYMARCPF KASITADTSSNTAYLQLSSLTSEDTAVYYCIR MLSRVPD WGQGTTLTVSS
[0107] Note: The bold and underlined regions are the complementarity-determining regions (CDR-H) of the heavy chain of monoclonal antibody 4D3F6H7, and the rest are the heavy chain backbone regions (FR-H) of monoclonal antibody 4D3F6H7.
[0108] 4D3F6H7-FR-H1: QIQLQQSGADLVRPGALVKLSCKASGFNIK;
[0109] 4D3F6H7-CDR-H1:KGVQF;
[0110] 4D3F6H7-FR-H2: WVKQRPEQGLEWIG;
[0111] 4D3F6H7-CDR-H2:GPINPDBAKQYMARCPF;
[0112] 4D3F6H7-FR-H3:KASITADTSSNTAYLQLSSLTSEDTAVYYCIR;
[0113] 4D3F6H7-CDR-H3: MLSRVPD;
[0114] 4D3F6H7-FR-H4: WGQGTTLTVSS.
[0115] The amino acid sequence of the light chain variable region of monoclonal antibody 4D3F6H7 is shown in SEQ ID NO: 6.
[0116] DVVVTQTPLSLSVSLGDRASISC GAGRITNPKEQIM WYLQKPGQSPKPLIY DQFSTLD GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC WNDGF IVLPTF
[0117] Note: The bold and underlined regions are the complementarity-determining regions (CDR-L) of the light chain of monoclonal antibody 4D3F6H7, and the rest are the light chain backbone regions (FR-L) of monoclonal antibody 4D3F6H7.
[0118] 4D3F6H7-FR-L1: DVVVTQTPLSLSVSLGDRASISC;
[0119] 4D3F6H7-CDR-L1:GAGRITNPKEQIM;
[0120] 4D3F6H7-FR-L2:WYLQKPGQSPKPLIY;
[0121] 4D3F6H7-CDR-L2: DQFSTLD;
[0122] 4D3F6H7-FR-L3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC;
[0123] 4D3F6H7-CDR-L3: WNDGF;
[0124] 4D3F6H7-FR-L4:IVLPTF.
[0125] 4. The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 5C8E7D6 is shown in SEQ ID NO: 7:
[0126] QSLGESRGGLVTPGTPLTLTCTVSGFSLD ELCKP WVRQAPGKGLQWVG LSCLICDNRYTMATET RFTISKTSTTTVDLKMTSLTAADTATYFCAR GRFHKSPDKMQWN WGQGTLVTVSS
[0127] Note: The bold and underlined regions are the complementarity-determining regions (CDR-H) of the heavy chain of the monoclonal antibody 5C8E7D6, while the rest are the heavy chain backbone regions (FR-H) of the monoclonal antibody 5C8E7D6.
[0128] 5C8E7D6-FR-H1: QSLGESRGGLVTPGTPLTLTCTVSGFSLD;
[0129] 5C8E7D6-CDR-H1: ELCKP;
[0130] 5C8E7D6-FR-H2:WVRQAPGKGLQWVG;
[0131] 5C8E7D6-CDR-H2:LSCLICDNRYTMATET;
[0132] 5C8E7D6-FR-H3:RFTISKTSTTVDLKMTSLTAADTATYFCAR;
[0133] 5C8E7D6-CDR-H3:GRFHKSPDKMQWN;
[0134] 5C8E7D6-FR-H4:WGQGTLVTVSS.
[0135] The amino acid sequence of the variable region of the light chain of monoclonal antibody 5C8E7D6 is shown in SEQ ID NO: 8.
[0136] AVVLTQTASPVSAALGSTVTINC QRCYTRTMKLWS WYQQKPGQAPKRLIY QDYWVBR GVPSRFRGSGSGTQFTLTISDVQCDDAATYYC PTDER GDRRAF
[0137] Note: The bold and underlined regions are the complementarity-determining regions (CDR-L) of the light chain of monoclonal antibody 5C8E7D6, and the rest are the light chain backbone regions (FR-L) of monoclonal antibody 5C8E7D6.
[0138] 5C8E7D6-FR-L1:AVVLTQTASPVSAALGSTVTINC;
[0139] 5C8E7D6-CDR-L1: QRCYTRTMKLWS;
[0140] 5C8E7D6-FR-L2:WYQQKPGQAPKRLIY;
[0141] 5C8E7D6-CDR-L2:QDYWVBR;
[0142] 5C8E7D6-FR-L3:GVPSRFRGSGSGTQFTLTISDVQCDDAATYYC;
[0143] 5C8E7D6-CDR-L3: PTDER;
[0144] 5C8E7D6-FR-L4:GDRRAF.
[0145] Example 5
[0146] Preparation of magnetic bead antibody conjugates
[0147] 1. Monoclonal antibody dilution
[0148] The buffers for monoclonal antibodies 4D3F6H7 and 5C8E7D6 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 4D3F6H7 and antibody dilution 5C8E7D6, respectively.
[0149] 2. Activation of carboxyl groups on the surface of magnetic beads
[0150] (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.
[0151] (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).
[0152] 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).
[0153] 3. Covalent coupling of magnetic beads and antibodies
[0154] (1) Take 200 μg of antibody dilution 4D3F6H7 and antibody dilution 5C8E7D6 and mix them with 100 μL of the above activated carboxyl magnetic beads (10 μm in diameter, with a molar ratio of antibody to magnetic beads 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).
[0155] (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 with physiological saline 2 to 3 times, resuspend them with physiological saline to obtain magnetic bead antibody conjugate 4D3F6H7 and magnetic bead antibody conjugate 5C8E7D6.
[0156] Example 6
[0157] Enrichment of Eubacterium rectum
[0158] 1. Take the magnetic bead antibody conjugate 4D3F6H7 and magnetic bead antibody conjugate 5C8E7D6 prepared in Example 5, and mix them in a 1:1 mass ratio to obtain a magnetic bead mixture.
[0159] 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.
[0160] 3. Take the pre-treated fecal microbial solution and add 0.1 mg of magnetic bead antibody conjugate 4D3F6H7, magnetic bead antibody conjugate 5C8E7D6, or the above magnetic bead mixture, 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.
[0161] 4. Then, the magnetic beads (labeled magnetic beads) bound to E. rectum are resuspended with physiological saline. The labeling removal reagent, i.e., 0.05% papain mixed with the labeled magnetic beads, is used to incubate at 37°C for 0.5 h to cut the Fc and Fab of the mouse monoclonal antibody, so that the magnetic beads and E. rectum are separated. The magnetic beads are then collected with a magnetic rack, and the supernatant is the E. rectum suspension.
[0162] 5. After diluting the *E. rectum* 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 conjugate in enriching *E. rectum*. The results are shown in Table 3, demonstrating that the effect of two magnetic bead antibody conjugates is greater than that of one.
[0163] Table 3. Types and yields of magnetic bead antibody conjugate combinations
[0164] Serial Number Combination type Yield 1 Magnetic bead antibody conjugate 4D3F6H7 <![CDATA[1.3*10 5 ]]> 2 Magnetic bead antibody conjugate 5C8E7D6 <![CDATA[1.1*10 5 ]]> 3 Magnetic bead antibody conjugate 4D3F6H7 + magnetic bead antibody conjugate 5C8E7D6 <![CDATA[1.7*10 5 ]]>
[0165] Example 7
[0166] Culture and sequencing of Eubacterium rectum
[0167] 1. Dilute the *E. rectum* isolated in Example 6 to 10... 3 ~10 4 The colony count was 1 / mL, then spread onto YCFC medium and anaerobic cultured at 37°C, 80% nitrogen, 12% carbon dioxide and 8% hydrogen, pH=6.7 for 3 days. The colony morphology was then observed.
[0168] 2. Select 20 single colonies and use them as templates for PCR amplification. Select the upstream primer sequence as 5'-ggcagcagtggggaatattg-3' and the downstream primer sequence as 5'-tcagcgtcagttatcgtcca-3'.
[0169] PCR reaction system: DNA template (10 ng / μL) 1 μL; upstream primer (10 μmol / L) 2 μL; downstream primer (10 μmol / L) 2 μL; Premix Taq 9.5 μL, ddH2O 35.5 μL; total system 50 μL.
[0170] PCR reaction conditions: 94℃ for 10 min; 94℃ for 1 min, 60℃ for 1 min, 72℃ for 25 s, 30 PCR cycles; 72℃ for 10 min.
[0171] 3. 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 the NCBI database using BLAST. The results showed that the 16S rDNA gene sequence of the 20 colonies had 94% homology with Escherichia coli (NCBI accession number: AY804152). Therefore, the isolate was identified as Escherichia coli.
[0172] Example 8
[0173] Western blot identification of E. rectum antibodies
[0174] 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℃.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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 4D3F6H7 or monoclonal antibody 5C8E7D6, and the primary antibody is generally diluted at 1:1000).
[0179] 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).
[0180] 7. Color development: ECL color development reaction.
[0181] 8. Data Reading: The molecular weight of the target band on the membrane is analyzed using a fully automated chemiluminescence analyzer processing system.
[0182] The results are as follows Figure 4As shown, the monoclonal antibodies 4D3F6H7 and 5C8E7D6 prepared by the present invention using the LPXTG-like protein and S-layer protein of E. rectus can capture this bacterium with high specificity.
[0183] 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 Eubacterium rectale, characterized in that: The monoclonal antibody is either monoclonal antibody 4D3F6H7 or monoclonal antibody 5C8E7D6, wherein... The monoclonal antibody 4D3F6H7 includes a 4D3F6H7 heavy chain variable region and a 4D3F6H7 light chain variable region, whose amino acid sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The monoclonal antibody 5C8E7D6 includes a 5C8E7D6 heavy chain variable region and a 5C8E7D6 light chain variable region, the amino acid sequences of which are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
2. The monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody 4D3F6H7 includes three complementarity-determining regions (CDRs) in its heavy chain variable region: 4D3F6H7-CDR-H1:KGVQF; 4D3F6H7-CDR-H2:GPINPDBAKQYMARCPF; 4D3F6H7-CDR-H3: MLSRVPD; The 4D3F6H7 light chain variable region includes three complementary determinant regions (CDRs), namely: 4D3F6H7-CDR-L1:GAGRITNPKEQIM; 4D3F6H7-CDR-L2: DQFSTLD; 4D3F6H7-CDR-L3: WNDGF; The monoclonal antibody 5C8E7D6 includes three complementarity-determining regions (CDRs) in its heavy chain variable region: 5C8E7D6-CDR-H1: ELCKP; 5C8E7D6-CDR-H2:LSCLICDNRYTMATET; 5C8E7D6-CDR-H3:GRFHKSPDKMQWN; The 5C8E7D6 light chain variable region includes three complementary determinant regions (CDRs), namely: 5C8E7D6-CDR-L1:QRCYTRTMKLWS; 5C8E7D6-CDR-L2:QDYWVBR; 5C8E7D6-CDR-L3: PTDER.
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 nucleotide sequence with optimized protein codons was transformed into Escherichia coli BL21, expressed, and purified by sonication to obtain purified protein; wherein, the protein is either protein CSX02_09140 or protein CSX02_05975; (2) The purified protein was mixed with Freund's adjuvant, emulsified and then used to immunize mice. Then, the spleen cells 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 4D3F6H7 or hybridoma cell lines 5C8E7D6.
5. The preparation method according to claim 4, characterized in that: When the protein is protein CSX02_09140, the optimized nucleotide sequence of protein CSX02_09140 codons is shown in SEQ ID NO: 2; When the protein is protein CSX02_05975, the optimized nucleotide sequence of protein CSX02_05975 codons is shown in SEQ ID NO:
4.
6. The preparation method according to claim 4, characterized in that: The amino acid sequence of the protein CSX02_09140 is shown in SEQ ID NO: 1, and the amino acid sequence of the protein CSX02_05975 is shown in SEQ ID NO:
3.
7. The use of the monoclonal antibody of claim 1 in the preparation of magnetic bead antibody conjugates.
8. A method for preparing a magnetic bead antibody conjugate, characterized in that: Includes the following steps: (1) Dilute the monoclonal antibody of claim 1 using MES buffer; (2) 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 either magnetic bead antibody conjugate 4D3F6H7 or magnetic bead antibody conjugate 5C8E7D6. The activated carboxyl magnetic beads have a particle size of 10–30 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:5–10.
9. The preparation method according to claim 8, characterized in that: The activated carboxyl magnetic beads have a particle size of 10 μm, and the molar ratio of the monoclonal antibody to the activated carboxyl magnetic beads is 1:
5.
10. The application of a magnetic bead antibody conjugate in the enrichment or isolation of *E. rectum*, characterized in that: The magnetic bead antibody conjugate is any one or a combination of two of magnetic bead antibody conjugate 4D3F6H7 and magnetic bead antibody conjugate 5C8E7D6.