A monoclonal antibody against ogt and a preparation method and application thereof

Abstract

This invention relates to the fields of biotechnology and immunochemistry, specifically to an anti-OGT monoclonal antibody, its preparation method, and its applications. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO. 10~SEQ ID NO. 12; the light chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO. 13~SEQ ID NO. 15. This anti-OGT monoclonal antibody can accurately identify OGT expression. Immunohistochemical detection of various tissues has shown that this monoclonal antibody can effectively detect OGT protein expression on tumor cells, and it can be applied to detection and screening fields such as immunohistochemistry (IHC), flow cytometry, and Western blotting.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and immunochemistry, specifically to an anti-OGT monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Human O-linked N-acetylglucosamine transferase (OGT) is a multidomain enzyme with a molecular weight of approximately 110-150 kDa. It is the only intracellular transferase that catalyzes O-GlcNAc glycosylation modification. OGT is widely expressed in all nucleated cells in normal tissues, mainly located in the nucleus and cytoplasm, and participates in basal metabolism and signal transduction. However, under pathological conditions, the activity and expression level of OGT are significantly abnormal in various solid tumors, such as breast cancer, ovarian cancer, prostate cancer, and colorectal cancer, and are closely related to tumor proliferation, metastasis, and drug resistance.

[0003] OGT influences protein stability, interaction networks, and subcellular localization by dynamically regulating the O-GlcNAc modification of thousands of substrate proteins (such as transcription factors, nucleoporins, and kinases), thereby participating in key processes such as gene transcription, cellular stress response, and metabolic reprogramming. Its dysfunction has been proven to be directly associated with diabetic complications, neurodegenerative diseases, and malignant progression of cancer.

[0004] Given the central role of OGT in diseases and the wide range of its substrate modifications, screening for highly sensitive and specific anti-OGT antibodies is crucial for accurately elucidating the O-GlcNAc modification network and developing therapeutic strategies targeting the OGT / OGA pathway. Summary of the Invention

[0005] To address the shortcomings and deficiencies of the existing technologies, this invention provides a monoclonal antibody against OGT with broad application prospects. This anti-OGT monoclonal antibody can accurately identify OGT expression. Immunohistochemical assays of various tissues have shown that this monoclonal antibody can effectively detect OGT protein expression on tumor cells, and it can be applied in detection and screening fields such as immunohistochemistry (IHC), flow cytometry, and Western blotting. Furthermore, this invention also relates to the nucleotide sequence encoding this anti-OGT monoclonal antibody, the recombinant plasmid or expression vector, the preparation method, and the application of the anti-OGT monoclonal antibody in OGT protein detection methods or devices.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a monoclonal antibody against OGT, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region; wherein, The heavy chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10~SEQ ID NO.12, respectively; The light chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.15, respectively.

[0007] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4.

[0008] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5.

[0009] Secondly, the present invention provides a coding gene that can be used to encode the monoclonal antibody described in the present invention. The coding genes include genes encoding the heavy chain variable region and genes encoding the light chain variable region; wherein... The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.2; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.3.

[0010] Thirdly, the present invention provides a nucleic acid molecule containing the coding gene described in the present invention.

[0011] Fourthly, the present invention provides an expression vector or recombinant plasmid containing the nucleic acid molecule described in the present invention.

[0012] Fifthly, the present invention provides a method for preparing an anti-OGT monoclonal antibody as described in the present invention, the method comprising: transfecting cells with the expression vector or recombinant plasmid described in the present invention, culturing the cells after transfection, collecting the cell supernatant and purifying it to obtain the anti-OGT monoclonal antibody.

[0013] Sixthly, the present invention provides the application of a monoclonal antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid based on the anti-OGT monoclonal antibody described in the present invention in the preparation of an OGT detection tool.

[0014] Preferably, the tools include reagents, kits, test strips, and antibody chips.

[0015] In a seventh aspect, the present invention provides an OGT detection kit, the OGT detection kit comprising the anti-OGT monoclonal antibody and immunocytochemical detection reagents described in the present invention.

[0016] Preferably, the immunocytochemical detection reagent includes HRP-labeled secondary antibody, EDTA repair solution, catalase blocking solution, DAB concentrate, DAB buffer, hematoxylin and blueing solution.

[0017] In the above technical solution, the anti-OGT monoclonal antibody of the present invention has high specificity and high sensitivity in binding to OGT protein molecules. It can specifically recognize and detect the expression of OGT protein on cells and shows positive high expression when detecting OGT protein. Therefore, the monoclonal antibody can be applied to the fields of detection and screening such as immunohistochemistry (IHC), flow cytometry, and Western blotting, which is beneficial to obtaining accurate assessment and detection results.

[0018] The 7-D12-B1 anti-OGT monoclonal antibody prepared in this invention has the characteristics of good specificity and strong positive signal, which makes it easier to score in IHC staining and more accurate in detecting and distinguishing cancer.

[0019] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0020] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 The images show the immunohistochemical detection results of the 7-D12-B1 anti-OGT monoclonal antibody prepared in this invention and commercially available antibodies in ovarian cancer and breast cancer tissues; a) is the immunohistochemical detection result of the 7-D12-B1 clone OGT antibody in ovarian cancer tissue; b) is the immunohistochemical detection result of the commercially available antibody in ovarian cancer tissue; c) is the immunohistochemical detection result of the 7-D12-B1 clone OGT antibody in breast cancer tissue; d) is the immunohistochemical detection result of the commercially available antibody in breast cancer tissue.

[0021] Figure 2 The 7-D12-B1 anti-OGT monoclonal antibody prepared for this invention was used as the primary antibody, and the expression level of OGT protein in six common cell lines was detected by Western blotting.

[0022] Figure 3 The 7-D12-B1 anti-OGT monoclonal antibody prepared for this invention was used as the primary antibody, and the expression level of OGT protein in wild-type / KD stable cell lines was detected by Western blotting. Detailed Implementation

[0023] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] In a first aspect, the present invention provides a monoclonal antibody against OGT, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region; wherein, The heavy chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10~SEQ ID NO.12, respectively; SEQ ID NO.10: GFAFSRYW; SEQ ID NO.11: IDPDSSTIN; SEQ ID NO.12: ASAYYYAGYWYFDV; The light chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.15, respectively.

[0026] SEQ ID NO.13: QSIVHHNGNTY; SEQ ID NO.14: KVS; SEQ ID NO.15: FQGSHASPT.

[0027] In this invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4.

[0028] In this invention, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5.

[0029] SEQ ID NO.4: EVKLLESGGGLVQPGGSLKKSCAASGFAFSRYWLSWVRQAPGKGLEWIGEIDPDSSTINKYTPSLKDKFIISRDNAKNTKYLQMTKVRSEDTALYYCASAYYYAGYWYFDVWGAGTTVTVSS; SEQ ID NO.5: DVLMTKTPLSLPVSLGDQASISCRSSQSIVHHNGNTYLEWYGQKPGQSPKLLIYKVSNRFSGVIKDRFSGSISGTDFTLKISRVEAEDLGVYYCFQGSHASPTFGTGTKLELK.

[0030] The anti-OGT monoclonal antibody (human OGT antibody) of the present invention can be used for immunohistochemical detection, and can identify and detect the expression of OGT protein on tumor cells or immune cells with high specificity and high sensitivity.

[0031] In this invention, the anti-OGT monoclonal antibody is obtained through recombinant expression in *E. coli*. Specifically, the anti-OGT monoclonal antibody provided by this invention is produced through murine hybridoma fusion screening and eukaryotic expression in 293 cells. In preparing the anti-OGT monoclonal antibody, the antigen used to immunize BALB / c mice is a truncated recombinant protein, the sequence of which is shown in SEQ ID NO.1, and it is produced by prokaryotic expression in *E. coli*. After immunization, cell fusion and clonal screening are performed to obtain a positive hybridoma cell line that can efficiently secrete monoclonal antibodies. Molecular cloning technology is used to obtain the nucleotide sequences encoding the heavy and light chain amino acid sequences of the antibody. The nucleotide sequences are constructed on a eukaryotic expression vector, transfected into the 293 cell line using a transfection reagent, and the cell supernatant is collected and purified by Protein G column affinity chromatography to obtain the murine monoclonal antibody. Immunohistochemical detection shows that the antibody specifically recognizes the OGT protein. The anti-OGT monoclonal antibody can recognize recombinant OGT antigen protein and OGT molecules on tumor cells and immune cells; the anti-OGT monoclonal antibody can also be used in immunohistochemical pathological diagnostic agents.

[0032] Secondly, the present invention provides a coding gene that can be used to encode the monoclonal antibody described in the present invention.

[0033] In a preferred embodiment of the present invention, the coding gene includes a gene encoding the heavy chain variable region and a gene encoding the light chain variable region; wherein, The DNA sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.2; The DNA sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.3.

[0034] SEQ ID NO.2: GAAGTGAAACTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGAAAAAAAGCTGCGCGGCGAGCGGCTTTGCGTTTAGCCGCTATTGGCTGAGCTTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGATTGGCGAAATTGATCCGGATAGCAGCACCATTAACAAATAT ACCCGAGCCTGAAAGATAAATTTATTATTAGCCGCGATAACGCGAAAAACACCAAATATCTGCAGATGACCAAAGTGCGCAGCGAAGATACCGCGCTGTATTATTGCGCGAGCGCGTATTATTATGCGGGCTATTGGTATTTTGATGTGTGGGGCGCGGGCACCACCGTGACCGTGAGCAGC; SEQ ID NO.3: GATGTGCTGATGACCAAAACCCCGCTGAGCCTGCCGGTGAGCCTGGGCGATCAGGCGAGCATTAGCTGCCGCAGCAGCCAGAGCATTGTGCATCATAACGGCAACACCTATCTGGAATGGTATGGCCAGAAACCGGGCCAGAGCCCGAAACTGCTGATTTATAAAGTGAG CAACCGCTTTAGCGGCGTGATTAAAGATCGCTTTAGCGGCAGCATTAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATCTGGGCGTGTATTATTGCTTTCAGGGCAGCCATGCGAGCCCGACCTTTGGCACCGGCACCAAACTGGAACTGAAA.

[0035] Thirdly, the present invention provides a nucleic acid molecule containing the coding gene described in the present invention.

[0036] Fourthly, the present invention provides an expression vector or recombinant plasmid containing the nucleic acid molecule described in the present invention.

[0037] Fifthly, the present invention provides a method for preparing an anti-OGT monoclonal antibody as described in the present invention, the method comprising: transfecting cells with the expression vector or recombinant plasmid described in the present invention, culturing the cells after transfection, collecting the cell supernatant and purifying it to obtain the anti-OGT monoclonal antibody.

[0038] In a preferred embodiment of the present invention, the preparation method includes the following steps: (1) Immunization of animals: First, the molecular sequence of OGT protein is analyzed. Based on the structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure of OGT, a suitable amino acid sequence is selected and used to prepare recombinant protein by prokaryotic expression, which is used as an immunogen to immunize mice; the sequence is the amino acid sequence shown in SEQ ID No.1; (2) Preparation of hybridoma cell lines: lymphocytes from immunized mice were electrofused with sp2 / 0 cells, and after clonal screening, serum titers were detected by ELISA to obtain a positive stable hybridoma cell line that can efficiently secrete antibodies. Total RNA was isolated from the hybridoma cell line. (3) Obtaining antibody sequences: Total RNA was reverse transcribed into cDNA, and the nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were obtained by PCR amplification using specific primers; (4) Antibody expression and purification: The nucleotide sequence was cloned into the expression vector, and the cultured cells were transiently transfected using the transfection method. After culture, the supernatant was collected and purified using Protein G to obtain an antibody with a purity >95%.

[0039] SEQ ID NO.1: MGSSHHHHHHSSGLVPRGSHMFQGLAELAHREYQAGDFEAAERHCMQLWRQEPDNTGVLLLLSSIHFQCRRLDRSAHFSTLAIKQNPLLAEAYSNLGNVYKERGQLQEAIEHYRHALRLKPDFIDGYINLAAALVAAGDMEGAVQAYVSALQYNPDLYCVRSDLGNLLKALG.

[0040] Sixthly, the present invention provides the application of a monoclonal antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid based on the anti-OGT monoclonal antibody described in the present invention in the preparation of an OGT detection tool.

[0041] In this invention, the tool can be any form of tool commonly used in the art, including but not limited to reagents, kits, test strips, and antibody chips.

[0042] In a seventh aspect, the present invention provides an OGT detection kit, the OGT detection kit comprising the anti-OGT monoclonal antibody and immunocytochemical detection reagents described in the present invention.

[0043] In a preferred embodiment of the present invention, the immunocytochemical detection reagent includes HRP-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, DAB concentrate, DAB buffer, hematoxylin, and blueing solution. The immunohistochemical detection steps include dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, counterstaining, dehydration, mounting, and microscopic examination.

[0044] The present invention will be described in detail below through embodiments. In the following embodiments, the drugs and pharmaceuticals are all conventional commercially available products. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions.

[0045] Table 1. Amino acid sequences and DNA sequences involved in this invention.

[0046] Example 1 This embodiment describes the preparation and screening of anti-OGT monoclonal antibodies, including the following steps: (1) Antigen preparation The specific sequence of the recombinant protein is shown in SEQ ID NO.1.

[0047] Based on comprehensive bioinformatics analysis, spatial structure prediction, reference to homologous protein information, and consideration of downstream application requirements, we selected Phe21-Gly171 and recombinantly expressed it using an E. coli expression system with an N-His tag. The truncated protein expressed in prokaryotes was used as an immunogen to immunize mice.

[0048] (2) Immunity The recombinant protein (OGT antigen) of SEQ ID NO.1 was mixed with complete Freund's adjuvant (1:1) and emulsified. Five BALB / c mice were immunized subcutaneously. Two weeks later, the OGT antigen containing the above recombinant protein (SEQ ID NO.1) was emulsified with incomplete Freund's adjuvant (1:1) for a second and third immunization. After the three immunizations, blood was collected and serum titers were determined by serial dilution using ELISA. Mice with the highest antibody titer against the SEQ ID NO.1 antigen were selected for the next step of cell fusion.

[0049] (3) Cell fusion Prepare mouse-derived sp2 / 0 myeloma cells in advance, ensuring they are in the logarithmic growth phase at the time of fusion. Obtain spleens from immunized mice and prepare a single-cell suspension of lymphocytes. Mix the mouse spleen lymphocytes with the myeloma cells and perform electrofusion according to the program set on a BTX 2001 LITE cell fusion instrument. After fusion, allow the cells to stand for 4 minutes, then dilute them in 50 ml centrifuge tubes to 40 ml of HAT-containing medium and incubate at 37°C for 1 hour. Then, aliquot the diluted cells into 10 wells of 96-well plates and incubate at 37°C with 5% CO2. Observe the fused cell status in the 96-well plates 6-9 days after fusion, change the medium with HT, and continue incubation at 37°C with 5% CO2.

[0050] (4) Screening and cloning Seven to ten days after fusion, clonal cells were screened using an ELISA test with OGT antigen (SEQ ID NO.1). The corresponding cell line numbers were labeled, and the cells in the positive wells were subjected to limiting dilution until the entire 96-well plate showed a positive result for the ELISA test. Stable monoclonal lines with high positive values ​​were selected to obtain hybridoma cell lines secreting specific monoclonal antibodies, recorded as 7-D12-B1.

[0051] (5) Perform antibody sequencing on the selected hybridoma cell lines. Total RNA was isolated from 7-D12-B1 hybridoma cells according to the TriZol reagent instructions. Following the Vazyme first-strand cDNA synthesis kit instructions, the total RNA was reverse transcribed into cDNA. Using specific primers: heavy chain variable region primers as shown in SEQ ID NO. 6 and SEQ ID NO. 7; light chain variable region primers as shown in SEQ ID NO. 8 and SEQ ID NO. 9, the nucleotide sequences of the heavy and light chain variable regions of the 7-D12-B1 anti-OGT monoclonal antibody were amplified. These sequences were then cloned into a eukaryotic expression vector (Thermo Fisher, pcDNA3.1) for cell transfection.

[0052] (6) Cell transfection and screening Prepare the 293F cells to be transfected in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 6-well plates, 2 ml per well, at a density of 0.5 × 10⁻⁶ cells / well. 6Cells / ml. The eukaryotic expression vector was mixed with lipo2000 at a ratio of 1:3 and added to the prepared 293F cells, which were then cultured in a shaker at 37°C and 5% CO2. After 3-5 days of culture, the transfected cell supernatant was subjected to ELISA to screen positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunohistochemical detection. If the immunohistochemical detection was positive, the detected antibody sequence was confirmed to be correct.

[0053] (7) Preparation and purification of monoclonal antibodies on cells The confirmed positive expression vector was used to transfect large numbers of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4-8°C.

[0054] Ultimately, the heavy chain variable region nucleotide sequence of the 7-D12-B1 anti-OGT monoclonal antibody is encoded by the DNA sequence shown in SEQ ID NO.2, and the light chain variable region nucleotide sequence of the 7-D12-B1 anti-OGT monoclonal antibody is encoded by the DNA sequence shown in SEQ ID NO.3.

[0055] The obtained base sequence was translated into an amino acid sequence and analyzed. The amino acid sequence of the heavy chain variable region of the 7-D12-B1 anti-OGT monoclonal antibody is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region of the 7-D12-B1 anti-OGT monoclonal antibody is shown in SEQ ID NO.5. Example 2 This embodiment demonstrates immunohistochemical detection using the 7-D12-B1 anti-OGT monoclonal antibody of the present invention. The results are shown below. Figure 1 The specific method is as follows: (1) Sample preparation: Ovarian cancer and breast cancer tissue sections that have been fixed in formalin and embedded in paraffin are baked in a 60℃ constant temperature oven for 1-2 hours and stored for later use; (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 min each time; (3) Hydration of sections: The sections were hydrated by soaking in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes in sequence, and then rinsed twice with purified water for 3 minutes each time. (4) Antigen retrieval: It is recommended to use the high temperature heat retrieval method for 3 min (if using an automatic retrieval instrument, the high temperature retrieval at 98℃ for 20 min can be set). After the slides are naturally cooled to room temperature, the tissue to be tested is circled with an immunohistochemical pen and rinsed twice with purified water for 3 min each time. (5) Inactivation of endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocking agent to completely cover the tissue, incubate at room temperature for 10 min, rinse twice with purified water for 3 min each time, and rinse once with PBST. (6) Primary antibody incubation: Add 100 μL of 0.5 μg / mL 7-D12-B1 anti-OGT monoclonal antibody to completely cover the tissue, incubate in a 37℃ incubator for 1 h, and wash with PBST 3 times for 5 min each time; (7) Secondary antibody incubation: Perform secondary antibody incubation according to the instructions of the DAB staining solution kit of the secondary antibody staining system used. After incubation, rinse the slides with PBST 3 times for 5 minutes each time, and rinse with purified water once. (8) DAB staining: Prepare DAB staining solution according to the instructions of the DAB staining solution kit. Drop an appropriate amount of the prepared DAB staining solution to completely cover the tissue. Stop staining when the color does not deepen. Rinse 3 times with purified water. (9) Hematoxylin counterstaining: Counterstain the sections according to the operating steps and suggestions in the instructions of the hematoxylin manufacturer, and rinse with PBST or tap water to return to blue; (10) Dehydration and clearing: Soak in 70%, 85%, 95%, 100%, and 100% graded alcohols sequentially for 3 minutes each time; clear with xylene twice for 5 minutes each time; (11) Mounting: Mount the sample with neutral resin.

[0056] Replace “7-D12-B1 anti-OGT monoclonal antibody” in step (6) with “commercially available antibody (Hefei Shanben Biotechnology Co., Ltd., catalog number: 1325)”, keeping all other steps unchanged. The result is as follows. Figure 1 As shown.

[0057] Depend on Figure 1 The results showed that OGT protein exhibited specific cell membrane staining in ovarian and breast cancer tissues. The 7-D12-B1 anti-OGT monoclonal antibody of this invention has higher specificity and a stronger positive signal than currently commercially available antibodies, making it easier to score in IHC staining and more accurate in detecting and differentiating cancers.

[0058] Example 3 This embodiment describes the determination of the affinity of the 7-D12-B1 anti-OGT monoclonal antibody of the present invention. The determination method is as follows: (1) Remove the labeled OGT recombinant protein from 4℃ and allow it to return to room temperature. Dilute to a concentration of 1 μg / ml and add 100 μL / well to a 96-well microplate. Incubate overnight at 4℃, then block with 2% BSA overnight at 4℃. (2) The 7-D12-B1 anti-OGT monoclonal antibody with an initial concentration of 1 mg / mL and the commercially available antibody were diluted to 1k, 2k, 4k, 8k, 16k, 32k, 64k and 128k respectively to set up a total of 8 concentration gradients for comparison; (3) Add the diluted antibody at 100 μL / well to a 96-well microplate containing peptides, cover with a sealing film, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium. (4) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry. (5) Dilute HRP-labeled goat anti-mouse IgG according to the instructions for use of the secondary antibody, add 100 μL / well to the microplate, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium; (6) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry. (7) Add 100 μL of TMB colorimetric solution to each well and react at room temperature for 6 minutes; (8) After the reaction is complete, add 2M H2SO4 at a rate of 50μL / well to stop the color development; (9) Read the OD value at 450 nm on the microplate reader, organize the data, and analyze the results as shown in Table 2 below.

[0059] Table 2 shows the statistical results of the titer detection of the 7-D12-B1 anti-OGT monoclonal antibody prepared in this invention and commercially available antibodies at eight different concentration gradients.

[0060]

[0061] As shown in Table 2, in the eight concentration gradient experiments, the 7-D12-B1 anti-OGT monoclonal antibody of this invention has a stronger affinity and higher sensitivity for OGT protein molecules compared with commercially available antibodies. It can still achieve a higher OD value under lower antibody concentration conditions, which can save experimental and detection costs.

[0062] Example 4 In this embodiment, the 7-D12-B1 anti-OGT monoclonal antibody was used as the primary antibody. Western blotting was employed to detect the OGT protein expression levels in six common cell lines, verifying its ability to recognize OGT protein. The method is as follows: (1) Six cell lines with different OGT expression levels, namely HT-1080, HepG2, Hela, H9c2, NIH / 3T3 and PC3, were selected for culture. Proteins were extracted, lysates were prepared, gels were prepared, samples were spotted, electrophoresed, and membranes were transferred. The PVDF membrane was activated with methanol for 1 min, washed twice with pure water, and then washed three times with TBST. Blocking: The membrane was placed in a blocking solution prepared with PBST containing 5% skim milk and shaken at room temperature for 1 h. (2) Primary antibody incubation: Dilute 7-D12-B1 anti-OGT monoclonal antibody at a ratio of 1:5000 to 5 ml of antibody dilution solution, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: Dilute HRP-anti-mouse IgG with antibody diluent at a ratio of 1:5000, mix well, add to membrane strip, and shake at room temperature for 1 hour; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix developer A and developer B in a 1:1 ratio and develop using a developing system; The results are as follows Figure 2 As shown, where, Lane 1: HT-1080 represents human fibrosarcoma cell lysate; band size: 110 kDa.

[0063] Lane 2: HepG2 represents human liver cancer cell lysate; band size: 110 kDa.

[0064] Lane 3: Hela represents human cervical cancer cell lysate; band size: 110 kDa.

[0065] Lane 4: H9C2 represents rat cardiomyocyte lysate; band size: 110 kDa.

[0066] Lane 5: NIH / 3T3 indicates mouse embryonic fibroblast lysate; band size: 110 kDa.

[0067] Lane 6: PC3 represents human prostate cancer cell lysate; band size: 110 kDa.

[0068] Depend on Figure 2 The results showed that, in the swimming lane, the 7-D12-B1 anti-OGT monoclonal antibody could specifically recognize OGT protein in six cell lysates with a molecular weight of about 110 kDa, indicating that the 7-D12-B1 anti-OGT monoclonal antibody of the present invention has good specificity and can accurately recognize OGT protein.

[0069] Example 5 In this embodiment, the 7-D12-B1 anti-OGT monoclonal antibody of the present invention was used as the primary antibody. Western blotting was used to detect the expression level of OGT protein in wild-type / KD stable cell lines, verifying its specificity for OGT protein. The method is as follows: (1) Prepare wild-type HSHC cell line and OGT knockdown cell lysate, prepare gel, spot, electrophoresis, transfer membrane. PVDF membrane needs to be activated. Activate with methanol for 1 min, wash the membrane twice with pure water and then wash it three times with TBST. Block: Place the membrane in blocking solution prepared with PBST containing 5% skim milk and shake at room temperature for 1 h. (2) Primary antibody incubation: Dilute 7-D12-B1 antibody at a ratio of 1:5000 to 5 ml of antibody dilution solution, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: Dilute HRP-anti-mouse IgG with antibody diluent at a ratio of 1:5000, mix well, add to membrane strip, and shake at room temperature for 1 hour; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix developer A and developer B in a 1:1 ratio and develop using a developing system; the results are as follows. Figure 3 As shown, where, Lane 1: HSHC represents a haploid human cell lysate developed by Hefei Shanben, with Hsp90α as the internal reference protein; band size: 110 kDa.

[0070] Lane 2: OGT knockdown cell line lysate; band size: 110 kDa.

[0071] Depend on Figure 3 The results showed that the 7-D12-B1 anti-OGT monoclonal antibody of the present invention had a signal in lane 1 with a molecular weight of 110 kDa, and no signal in lane 2, indicating that the expression level of OGT knockdown cell line protein was significantly reduced. The signal recognized by the 7-D12-B1 anti-OGT monoclonal antibody of the present invention was also significantly reduced, indicating that the 7-D12-B1 anti-OGT monoclonal antibody of the present invention can recognize OGT protein with high specificity.

[0072] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0073] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0074] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A monoclonal antibody against OGT, characterized in that, The monoclonal antibody includes a heavy chain variable region and a light chain variable region; wherein... The heavy chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10~SEQ ID NO.12, respectively; The light chain variable region includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.15, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

5.

3. A gene encoding a gene, characterized in that, The encoding gene can be used to encode the monoclonal antibody of claim 1; The coding genes include genes encoding the heavy chain variable region and genes encoding the light chain variable region; wherein... The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.2; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.

3.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains the encoding gene as described in claim 3.

5. An expression vector or recombinant plasmid, characterized in that, The expression vector or recombinant plasmid contains the nucleic acid molecule as described in claim 4.

6. A method for preparing an anti-OGT monoclonal antibody as described in claim 1 or 2, characterized in that, The preparation method includes: transfecting cells with the expression vector or recombinant plasmid as described in claim 5, culturing cells after transfection, collecting cell supernatant and purifying it to obtain the anti-OGT monoclonal antibody.

7. The use of a monoclonal antibody against OGT according to claim 1 or 2, or the encoding gene according to claim 3, or the nucleic acid molecule according to claim 4, or the expression vector or recombinant plasmid according to claim 5 in the preparation of an OGT detection tool.

8. The application according to claim 7, characterized in that, The tools include reagents, kits, test strips, and antibody chips.

9. An OGT detection kit, characterized in that, The OGT detection kit includes the anti-OGT monoclonal antibody and immunocytochemical detection reagent as described in claim 1 or 2.

10. The OGT detection kit according to claim 9, characterized in that, The immunocytochemical detection reagents include HRP enzyme-labeled secondary antibody, EDTA repair solution, catalase blocking solution, DAB concentrate, DAB buffer, hematoxylin, and blueing solution.

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