A mouse-derived OPN protein neutralizing antibody and its application

Abstract

The present invention relates to a mouse-derived OPN protein neutralizing antibody and its application, belonging to the field of biomedicine technology. The antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain signal peptide being shown as SEQ ID NO.1; the amino acid sequence of the heavy chain variable region being shown as SEQ ID NO.2; the amino acid sequence of the heavy chain constant region being shown as SEQ ID NO.3; the amino acid sequence of the light chain signal peptide being shown as SEQ ID NO.4; the amino acid sequence of the light chain variable region being shown as SEQ ID NO.5; and the amino acid sequence of the light chain constant region being shown as SEQ ID NO.6. The present invention provides an OPN protein neutralizing antibody for use in preparing a diagnostic kit for diseases associated with OPN protein, as well as for use in preparing an antibody drug that blocks the function of an OPN molecule.

Description

Technical Field

[0001] The present invention relates to a mouse-derived OPN protein neutralizing antibody and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] OPN (osteopontin, OPN; also known as secreted pyrophosphate protein, secretedphosphoprotein 1, SPP1) is a negatively charged glycosylated phosphoprotein with a relative molecular mass of about 32kDa. It is expressed in many tissues; in bones, it is produced by both osteoblasts that produce matrix and osteoclasts that can resorb bone. The production of OPN is regulated by cytokines such as TGF-β (transforming growth factor-β), EGF (epidermal growth factor), PDGF (platelet-derived growth factor) and IL-2 (interleukin-2), and has a chemotactic effect on smooth muscle cells, macrophages and other cells. OPN is a receptor α v β3-mediated proteins play a major role in various cascades of damage, exerting their stimulatory effects on target cells and causing damage to the body under various pathological conditions. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a mouse-derived OPN protein neutralizing antibody and the application of the OPN protein neutralizing antibody.

[0004] In order to achieve the purpose of solving the above problems, the technical solution adopted by the present invention is to provide a mouse-derived OPN protein neutralizing antibody, which includes a heavy chain and a light chain, the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3; the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.6; and the antibody is an IgG2A antibody.

[0005] The present invention provides an application of a mouse-derived OPN protein neutralizing antibody in preparing a diagnostic kit for diseases associated with the OPN protein.

[0006] The present invention provides an application of a mouse-derived OPN protein neutralizing antibody in the preparation of an antibody drug that blocks the function of OPN molecules.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] The OPN protein neutralizing antibody provided by the present invention:

[0009] ① High specificity: It binds specifically to OPN protein with strong binding force and has less binding to unrelated antigens.

[0010] ② Low side effects: Based on the broad expression spectrum of OPN protein in vivo, the heavy chain of this antibody is selected as IgG2A type. This subtype is mainly used to neutralize antigens, and its complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) are relatively weak. The FC segment of the antigen-antibody complex is less likely to mediate cellular immunity, and the cell killing ability is low, thus avoiding the impact on target cells and antigen-secreting cells.

[0011] ③Wide scope of application: Suitable for in vivo and in vitro research and treatment experiments on blocking OPN protein. DETAILED DESCRIPTION

[0012] In order to make the present invention more clearly understood, preferred embodiments are described in detail as follows:

[0013] The present invention provides a mouse-derived OPN protein neutralizing antibody.

[0014] Antibodies consist of heavy and light chains.

[0015] The amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3;

[0016] The amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.6; and the antibody is an IgG2A antibody.

[0017] The present invention provides an application of a mouse-derived OPN protein neutralizing antibody in preparing a diagnostic kit for diseases associated with the OPN protein.

[0018] The present invention provides an application of a mouse-derived OPN protein neutralizing antibody in the preparation of an antibody drug that blocks the function of OPN molecules.

[0019] Example 1

[0020] 1. Synthesis and verification of OPN recombinant protein:

[0021] A. HEK293 cells were transfected with the plasmid and the mouse SPP1 protein sequence from the Uniprot database was used: MRLAVICFCLFGIASSLPVKVTDSGSSEEKLYSLHPDPIATWLVPDPSQKQNLLAPQNAVSSEEKDDFKQETLPSNSNESHDHMDDDDDDDDDDGDHAESEDSVDSDESDESHHSDESDETVTASTQADTFTPIVPTV DVPNGRGDSLAYGLRSKSRSFQVSDEQYPDATDEDLTSHMKSGESKESLDVIPVAQLLSMPSDQDNNGK GSHESSQLDEPSLETHRLEHSKESQESADQSDVIDSQASSKASLEHQSHKFHSHKDKLVLDPKSKEDDRYLKFRISHELESSSSEVN (wherein positions 1-16 are a signal peptide) to synthesize OPN recombinant protein.

[0022] B. Verify the potency of OPN recombinant protein:

[0023] a) Peritoneal macrophage extraction:

[0024] 1. 6-8 week old male C57B6 mice were depilated from the abdomen, killed by cervical dislocation, and immersed in 75% ethanol for 10 seconds.

[0025] 2. Take out the mouse, drain it, and place it supinely on a stainless steel square tray with its limbs fixed.

[0026] 3. Draw 6 mL of normal saline into the abdominal cavity with a syringe and gently massage the mouse's abdomen for 150 seconds to allow the liquid to flow fully in the abdominal cavity.

[0027] 4. Use forceps to lift the mouse's lower abdominal skin, tilt the animal slightly to one side, cut the abdominal skin, and then make a 30mm long incision in the muscle layer. Use a pipette to aspirate the intraperitoneal fluid.

[0028] 5. The aspirated fluid was transferred to a 15 ml centrifuge tube through a 70 μm filter. The collected peritoneal lavage fluid was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and complete RPMI-1640 cell culture medium was added for resuspending.

[0029] 6. Inoculate into a non-TC treated 100 mm dish and culture for 4 hours.

[0030] 7. Aspirate the culture medium, rinse once with PBS, add 2 ml of 4°C pre-cooled 2.5% EDTA, and let it stand on ice for 10 minutes for digestion. Under the microscope, some cells may be floating. Add 3 ml of RPMI-1640 culture medium and repeatedly pipette until most of the cells fall off.

[0031] 8. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in complete RPMI-1640 cell culture medium to 1 x 10 6 / ml concentration.

[0032] b) Grouping:

[0033] 1. The total volume of each well is 500 μL;

[0034] 2. Blank control group: 500 μL serum-free RPMI-1640 medium;

[0035] 3. 1 nmol / L OPN recombinant protein group: 5 nmol / L OPN protein in serum-free RPMI-1640 medium, cultured for 24 h;

[0036] 4. 5 nmol / L OPN recombinant protein group: 5 nmol / L OPN protein + serum-free RPMI-1640 medium, culture for 12 / 24 / 48 h;

[0037] 5. 20 nmol / L OPN recombinant protein group: 5 nmol / L OPN protein in serum-free RPMI-1640 medium, cultured for 24 h;

[0038] 6. LPS stimulation group: 30 ng / ml LPS + serum-free RPMI-1640 medium, culture for 24 h;

[0039] 7. OPN protein + LPS stimulation group: 5 nmol / L OPN recombinant protein + 30 ng / ml LPS + serum-free RPMI-1640 medium, culture for 24 h;

[0040] c) Co-cultivation process and detection indicators:

[0041] 1. Inoculate the extracted peritoneal macrophage cell dilution into a 24-well plate, 2*10 per well. 6 cells + 500 μL complete RPMI-1640 medium.

[0042] 2. After 12 hours of culture, remove the complete culture medium from the plate and wash once with 1 ml of PBS solution.

[0043] 3. Add each group in the group list to the well plate separately.

[0044] 4. After 24 hours of culture, aspirate the cell supernatant in the well plate.

[0045] 5. Centrifuge the collected cell supernatant at 4°C, 1000g for 20 min.

[0046] 6. Aspirate the supernatant after centrifugation, avoiding the cell pellet attached to the wall.

[0047] 7. Use IL-10 and IL-12 ELISA detection kits to detect the cytokine concentration in the supernatant.

[0048] 8. IL-10 testing, across nine biological replicates, showed a 30.53% decrease in the mean level of the OPN protein + LPS stimulation group compared to the LPS stimulation group, with a statistical p-value less than 0.05. IL-12 testing, across nine biological replicates, showed a 37.10% increase in the mean level of the 5 nmol / L OPN recombinant protein group compared to the blank control group, with a statistical p-value less than 0.05. This indicates that the OPN recombinant protein titer meets the required standards.

[0049] 2. In vivo immunization using recombinant proteins:

[0050] 1. Six-week-old Balb / c mice were immunized with OPN recombinant protein at multiple sites. The OPN recombinant protein concentration was 0.25 mg / ml and the dose was 50 μg / mouse. The mice were injected subcutaneously and intraperitoneally.

[0051] 2. The interval between the 1st to 3rd immunizations was 15 days, and the interval between the 3rd and 4th immunizations was 7 days. After four immunizations with the same method, mouse serum was collected 7 days after the 4th immunization.

[0052] 3. Serum titer detection:

[0053] 1. Use a 96-well ELISA plate pre-coated with OPN recombinant protein (1 μg / mL*50 μL, overnight at 4°C), and block with 100 μL of 1% BSA at 37°C for 1 hour.

[0054] 2. Dilute the immune mouse serum at 1:300 with PBS buffer, then dilute three-fold to 1:218700 in 7 groups for indirect ELISA. 50 μL per well, incubate at 37°C for 45 min.

[0055] 3. Use horseradish peroxidase-conjugated goat anti-mouse IgG (GAM-HRP), 50 μL per well, incubate at 37°C for 45 min.

[0056] 4. The OD value is the difference between the readings at 450nm and 630nm on a microplate reader. Mice with an OD450nm value > 1.0 and a titer > 72,900 were selected for the fusion stage.

[0057] 4. Fusion hybridoma screening:

[0058] A. Construction of fusion hybridomas:

[0059] 1. Extract the spleen of the fused mouse and add it to a homogenizer containing RPMI-1640 medium. After homogenizing the spleen, aspirate the upper cell suspension.

[0060] 2. Centrifuge the suspension at 1200 rpm for 5 minutes, discard the supernatant, add 5 ml of RPMI-1640 medium and mix well. Repeat twice, resuspend and place in the incubator for later use.

[0061] 3. Take the pre-prepared SP2 / 0 myeloma cells and add them at a ratio of tumor cells to spleen cells of 1:10. Centrifuge horizontally at 1200 rpm for 5 minutes, aspirate and discard the supernatant.

[0062] 4. Place the mixed cell pellet in a 37°C water bath, slowly add 1 mL of 37°C 50% PEG to the mixed cells, gently stir, slowly add 50 mL of RPMI-1640 along the wall to the fused cells, and invert to mix.

[0063] 5. Centrifuge for 5 minutes at 1200 rpm, discard the supernatant, resuspend in 20 ml of RPMI-1640 complete medium containing 1% HAT, and seed into 6-well plates for screening. After 7 days, cell colonies will be observed. Select appropriate cell clusters and transfer them to 96-well culture plates.

[0064] 6. After transfer, when the colonies grow to 2 / 3 of the size of the culture well, extract the supernatant and perform antibody detection.

[0065] B. Detection of fusion hybridoma supernatant titer:

[0066] 1. Centrifuge the cell supernatant at 1000 g for 20 min at 4°C.

[0067] 2. Aspirate the supernatant after centrifugation, avoiding the cell pellet attached to the wall.

[0068] 3. Follow the same procedure as serum titer testing.

[0069] 4. After testing, positive fusion hybridomas are selected and entered into monoclonal screening.

[0070] 5. Positive hybridoma monocloning and screening:

[0071] 1. Using the limiting dilution method, dilute the positive fusion hybridoma to a cell concentration of 50-60 cells / mL.

[0072] 2. Add 0.1 mL of diluent to each well of a 96-well culture plate and inoculate two rows. Dilute the remaining cell suspension in HT culture medium in multiple ratios and inoculate two more rows, and so on, until each well contains half or one cell.

[0073] 3. After dilution, there is only a single cell in each well of the 96-well plate.

[0074] 4. After 7 days of growth, a single cell will grow into a monoclonal hybridoma. Monoclonal hybridomas derived from the same positive hybridoma are called subclones.

[0075] 5. Perform ELISA test on the supernatant of each subclone again, following the same procedure as the serum titer test.

[0076] 6. Observe cells that test positive with ELISA under a microscope. If they appear as single cell clusters, they can be directly transferred to a 24-well plate for expanded culture. If they do not appear as single cell clusters or cannot be confirmed, the cell clusters can be dispersed, the limiting dilution method can be repeated, and subcloning assays can be performed again.

[0077] 7. All hybridomas are expanded to 24-well plates for culture until they are monoclonal.

[0078] 6. Second screening of monoclonal hybridomas:

[0079] After culturing the expanded monoclonal antibodies for 7 days, perform ELISA testing again according to the same procedure as the serum titer detection. The positive monoclonal hybridomas are retained and expanded to T25 culture flasks for culture.

[0080] VII. Antigen-antibody specific binding verification

[0081] 1. Extract the supernatant of the 12 selected monoclonal hybridoma cells and centrifuge the supernatant at 1000g at 4°C for 20 minutes. Aspirate the supernatant after centrifugation, avoiding the adherent cell pellet.

[0082] 2. Use a 96-well microtiter plate pre-coated with OPN recombinant protein (1 μg / mL*50 μL, overnight at 4°C) and a 96-well microtiter plate pre-coated with a His-tagged control protein (1 μg / mL*50 μL, overnight at 4°C). Block the plates with 100 μL of 1% BSA at 37°C for 1 h.

[0083] 3. Add 50 μL of extracted serum to each well, perform duplicate assays for each serum, and incubate at 37°C for 45 min.

[0084] 4. Use horseradish peroxidase-conjugated goat anti-mouse IgG (GAM-HRP), 50 μL per well, incubate at 37°C for 45 min.

[0085] 5.OD value is the difference between the readings of the ELISA plate at 450nm and 630nm wavelengths in the ELISA reader.

[0086] 6. In two biological replicates, the mean OD value of the group coated with OPN recombinant protein was statistically significant compared to the group coated with the control protein, also containing a His tag, with a p-value less than 0.05. This demonstrates that the recombinant antibody binds specifically to the OPN recombinant protein, and that the binding site is unrelated to the His tag added during the construction of the OPN recombinant protein.

[0087] 8. Neutralization capability verification:

[0088] A. Peritoneal macrophage extraction;

[0089] 1. 6-8 week old male C57B6 mice were depilated from the abdomen, killed by cervical dislocation, and immersed in 75% ethanol for 10 seconds.

[0090] 2. Take out the mouse, drain it, and place it supinely on a stainless steel square tray with its limbs fixed.

[0091] 3. Draw 6 mL of normal saline into the abdominal cavity with a syringe and gently massage the mouse's abdomen for 150 seconds to allow the liquid to flow fully in the abdominal cavity.

[0092] 4. Use tweezers to lift the mouse's lower abdominal skin, tilt the animal slightly to one side, cut the abdominal skin, and then make a wound about 30 mm long in the muscle layer. Use a pipette to aspirate the fluid in the abdominal cavity.

[0093] 5. The aspirated fluid was transferred to a 15 ml centrifuge tube through a 70 μm filter. The collected peritoneal lavage fluid was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and complete RPMI-1640 cell culture medium was added for resuspending.

[0094] 6. Inoculate until the non-TC treatment reaches 100 mm for 4 h.

[0095] 7. Aspirate the culture medium, rinse once with PBS, add 2 ml of 4°C pre-cooled 2.5% EDTA, and incubate on ice for 10 min for digestion. When some cells are visible floating under the microscope, add 3 ml of RPMI-1640 culture medium and pipette repeatedly until most of the cells are detached.

[0096] 8. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in complete RPMI-1640 cell culture medium to 1 x 10 6 / ml concentration.

[0097] B. Antigen-antibody neutralization

[0098] 1. Thaw each neutralizing antibody serum in a 37°C water bath;

[0099] 2. Mix 50 μL of serum and 7.5 μL of 320 nmol / L OPN recombinant protein diluent.

[0100] 3. Centrifuge with vortex for 10 seconds and incubate at 37°C for 1 hour.

[0101] 4. Number each group.

[0102] C. Grouping:

[0103] 1. The total volume of each well is 500 μL

[0104] 2. Blank control group: 500 μL serum-free RPMI-1640 medium

[0105] 3. OPN protein stimulation group: 5 nmol / L OPN protein + serum-free RPMI-1640 medium;

[0106] 4. OPN protein neutralization and stimulation group: antigen-antibody neutralization solution + serum-free RPMI-1640 medium;

[0107] 5. LPS stimulation group: 30 ng / ml LPS + serum-free RPMI-1640 medium;

[0108] 6. OPN protein neutralization + LPS stimulation group: antigen-antibody neutralization solution + 30 ng / ml LPS + serum-free RPMI-1640 medium;

[0109] D. Co-culture process and functional testing indicators

[0110] 1. Inoculate the extracted peritoneal macrophage cell dilution into a 24-well plate, 2*10 per well. 6 cells + 500 μL complete RPMI-1640 medium.

[0111] 2. After the cells adhere to the wall for 12 hours, remove the complete culture medium in the plate and wash once with 1 ml of PBS solution.

[0112] 3. Add each group in the group list to the well plate separately.

[0113] 4. After 24 hours of culture, aspirate the cell supernatant in the well plate.

[0114] 5. Centrifuge the collected cell supernatant at 4°C, 1000g for 20 min.

[0115] 6. Aspirate the supernatant after centrifugation, avoiding the cell pellet attached to the wall.

[0116] 7. Use IL-10 and IL-12 ELISA detection kits to detect the cytokine concentration in the supernatant.

[0117] 9. Select effective antibodies and perform genetic sequencing of hybridomas.

[0118] In this example, we selected the antibody clone 2G6-7, which has been functionally validated to have a strong OPN blocking effect. The following are the amino acid and base sequences of the variable regions of the obtained light and heavy chains.

[0119] Heavy chain variable region base sequence:

[0120] GAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCATGAAACTCTCCTG TGTTGCCTCTGGAATCACTTTTAGTGACTACTGGATGAACTGGATCCGCCAGTCTCCAGAGAAAGGACTGGAGTGGGTAGCACAAATTAGGAACAAACCTTATAATTATGAAACATTTTTATTCAGATTCTGTGAAAGG CAGATTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTCCCTGCAAATGAACCACTTAAGAGCTGACGACATGGGAATATATTACTGTTCATGTAACTGGGACGGGACCTGGTTTGCTTACTGGGGCCAAGGGAC TCTGGTCACTGTCTCTGCA

[0121] Light chain variable region base sequence:

[0122] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTC TTGCAGATCTGGTCAGAGCCTTGTACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAACCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAG TGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAACTACACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0123] Heavy chain variable region amino acid sequence; SEQ ID NO.2:

[0124] EVKLDETGGGLVQPGRPMKLSCVASGITFSDYWMNWIRQSPEKGLEWVAQIRNKPYNYETFYSDS VKGRFTISRDDSKSSVSLQMNHLRADDMGIYYCSCNWDGTWFAYWGQGTLVTVSA

[0125] Light chain variable region amino acid sequence; SEQ ID NO.5::

[0126] DVVMTQTPLSLPVSLGDQASISCRSGQSLVHSNGNTYLHWYLQKPGQSPNLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPYTFGGGTKLEIK

[0127] 10. Insert the variable region base sequence to synthesize a neutralizing antibody with an IgG2A heavy chain;

[0128] The sequenced variable region base sequence was inserted into a plasmid containing a vector sequence with restriction enzyme cleavage sites and modified promoters and terminators. This plasmid already contained the signal peptide and base sequence for the modified heavy chain to IgG2A type and the light chain to kappa type, as well as the base sequence for the fixed region. The base sequence used in the modified plasmid and the corresponding amino acid sequence are as follows:

[0129] Heavy chain base sequence:

[0130] Signal peptide:

[0131] GCGGCCGCCACCATGTACTTGGGACTGAGCTGTGTATTCATTGTTTTTCTCTTAAAAGGTGTCCAGTGT

[0132] Fixed area:

[0133] GCTAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTC GGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGT GACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGC ACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTG GTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGT CAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGT CACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGAGTCTAGA

[0134] Light chain base sequence:

[0135] Signal peptide:

[0136] GCGGCCGCCACCATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT

[0137] Fixed area:

[0138] AGAGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGG TGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCAT GAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGATTCTAGA

[0139] Heavy chain amino acid sequence:

[0140] SEQ ID NO.1: Signal peptide:

[0141] MYLGLSCVFIVFLLKGVQC

[0142] SEQ ID NO.3: Fixed region:

[0143] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLS SSVVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFK CKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0144] Light chain amino acid sequence:

[0145] SEQ ID NO.4: Signal peptide:

[0146] MKLPVRLLVLMFWIPASSS

[0147] SEQ ID NO.6: Fixed region:

[0148] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDST YSMSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0149] The plasmid is transfected into antibody-expressing cells to express specific OPN neutralizing antibodies with modified heavy chains of the IgG2A type.

[0150] OPN protein is widely localized and plays a major role in various cascades of damage, stimulating target cells and causing damage to the body. v OPN binds to α3 of endothelial cells and inhibits osteoclast resorption. v Binding to β3 receptors inhibits angiogenesis.

[0151] Neutralizing antibodies against OPN can block the effects of OPN on various cell types and alter the regulation of cytokine secretion by target cells after OPN acts on them. These antibodies have extensive applications in scientific research and serve as the foundation for the validation and production of clinical-grade therapeutic neutralizing antibodies against OPN. Currently, there are no commercially available neutralizing antibodies against OPN for scientific research.

[0152] Example 2

[0153] In vitro OPN protein blocking experiment:

[0154] 1. Extraction of peritoneal macrophages;

[0155] 1. 6-8 week old male C57B6 mice were depilated from the abdomen, killed by cervical dislocation, and immersed in 75% ethanol for 10 seconds.

[0156] 2. Take out the mouse, drain it, and place it supinely on a stainless steel square tray with its limbs fixed.

[0157] 3. Draw 6 mL of normal saline into the abdominal cavity with a syringe and gently massage the mouse's abdomen for 150 seconds to allow the liquid to flow fully in the abdominal cavity.

[0158] 4. Use forceps to lift the mouse's lower abdominal skin, tilt the animal slightly to one side, cut the abdominal skin, and then make a 30mm long incision in the muscle layer. Use a pipette to aspirate the intraperitoneal fluid.

[0159] 5. The aspirated fluid was transferred to a 15 ml centrifuge tube through a 70 μm filter. The collected peritoneal lavage fluid was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and complete RPMI-1640 cell culture medium was added for resuspending.

[0160] 6. Inoculate into non-TC treated 100 mm cell culture dishes and let stand for 4 hours.

[0161] 7. Aspirate the culture medium, rinse once with PBS, add 2 ml of 4°C pre-cooled 2.5% EDTA, and incubate on ice for 10 min for digestion. When some cells are visible floating under the microscope, add 3 ml of RPMI-1640 culture medium and pipette repeatedly until most of the cells are detached.

[0162] 8. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend in complete RPMI-1640 cell culture medium to 1 x 10 6 / ml concentration.

[0163] 2. Antigen-antibody neutralization;

[0164] 1. Thaw each neutralizing antibody serum in a 37°C water bath;

[0165] 2. Use a ratio of 50 μL serum + 7.5 μL 320 nmol / L OPN recombinant protein diluent;

[0166] 3. Centrifuge with vortex for 10 seconds and incubate at 37°C for 1 hour.

[0167] 4. Number each group.

[0168] 3. Grouping situation;

[0169] 1. The total volume of each well is 500 μL;

[0170] 2. Blank control group: 500 μL serum-free RPMI-1640 medium;

[0171] 3. OPN protein stimulation group: 5 nmol / L OPN protein + serum-free RPMI-1640 medium;

[0172] 4. OPN protein neutralization and stimulation group: antigen-antibody neutralization solution + serum-free RPMI-1640 medium;

[0173] 5. LPS stimulation group: 30 ng / ml LPS + serum-free RPMI-1640 medium;

[0174] 6. OPN protein neutralization + LPS stimulation group: antigen-antibody neutralization solution + 30 ng / ml LPS + serum-free RPMI-1640 medium;

[0175] IV. Co-cultivation process and detection indicators:

[0176] 1. Inoculate the extracted peritoneal macrophage cell dilution into a 24-well plate, 2*10 per well. 6 cells + 500 μL complete RPMI-1640 medium.

[0177] 2. After the cells adhere to the wall for 12 hours, remove the complete culture medium in the plate and wash once with 1 ml of PBS solution.

[0178] 3. Add each group in the group list to the well plate separately.

[0179] 4. After 24 hours of culture, aspirate the cell supernatant in the well plate.

[0180] 5. Centrifuge the collected cell supernatant at 4°C, 1000g for 20 min.

[0181] 6. Aspirate the supernatant after centrifugation, avoiding the cell pellet attached to the wall.

[0182] 7. Use IL-10 and IL-12 ELISA detection kits to detect the cytokine concentration in the supernatant.

[0183] 8. IL-10 assay: In two biological replicates, the mean IL-10 level in the OPN protein neutralization group was found to be 245.97% higher than that in the OPN protein stimulation group, with a statistically significant p-value less than 0.05. This indicates that the neutralization capacity of the OPN protein neutralizing antibody is satisfactory.

[0184] V. Test results and analysis:

[0185] IL-10 and IL-12 ELISA detection kits were used to detect the cytokine concentrations in the supernatant. Compared with the OPN recombinant protein group, the neutralization group was able to inhibit the secretion of IL-12. At the same time, compared with the OPN+LPS stimulation group, the LPS+neutralization OPN stimulation group was able to restore the upregulated secretion of IL-10.

[0186] Use of OPN protein neutralizing antibodies:

[0187] 1. Storage conditions:

[0188] Store at -20℃, valid for 1 year. According to conventional antibody standards.

[0189] 2. Neutralization capability verification method:

[0190] A. Antigen-antibody neutralization:

[0191] 1. Thaw each neutralizing antibody serum in a 37°C water bath

[0192] 2. Use a ratio of 50 μL serum + 7.5 μL 320 nmol / L OPN recombinant protein diluent;

[0193] 3. Centrifuge with vortex for 10 seconds and incubate at 37°C for 1 hour.

[0194] 4. Number each group.

[0195] B. Grouping

[0196] 1. The total volume of each well is 500 μL;

[0197] 2. Blank control group: 500 μL serum-free RPMI-1640 medium;

[0198] 3. OPN protein stimulation group: 5 nmol / L OPN protein + serum-free RPMI-1640 medium;

[0199] 4. OPN protein neutralization and stimulation group: antigen-antibody neutralization solution + serum-free RPMI-1640 medium;

[0200] 5. LPS stimulation group: 30 ng / ml LPS + serum-free RPMI-1640 medium;

[0201] 6. OPN protein neutralization + LPS stimulation group: antigen-antibody neutralization solution + 30 ng / ml LPS + serum-free RPMI-1640 medium;

[0202] C. Co-culture process and detection indicators:

[0203] 1. Inoculate the extracted peritoneal macrophage cell dilution into a 24-well plate, 2*10 per well. 6 cells + 500 μL complete RPMI-1640 medium.

[0204] 2. After the cells adhere to the wall for 12 hours, remove the complete culture medium in the plate and wash once with 1 ml of PBS solution.

[0205] 3. Add each group in the group list to the well plate separately.

[0206] 4. After 24 hours of culture, aspirate the cell supernatant in the well plate.

[0207] 5. Centrifuge the collected cell supernatant at 4°C, 1000g for 20 min.

[0208] 6. Aspirate the supernatant after centrifugation, avoiding the cell pellet attached to the wall.

[0209] 7. Use IL-10 and IL-12 ELISA detection kits to detect the cytokine concentration in the supernatant.

[0210] D.Test results and analysis:

[0211] IL-10 and IL-12 ELISA detection kits were used to detect the cytokine concentrations in the supernatant. Compared with the OPN recombinant protein group, the neutralization group was able to inhibit the secretion of IL-12. At the same time, compared with the OPN+LPS stimulation group, the LPS+neutralization OPN stimulation group was able to restore the upregulated secretion of IL-10.

[0212] 3. Usage:

[0213] The actual antibody concentration and dosage should be determined by the doubling dilution method according to the specific situation.

[0214] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A mouse-derived OPN protein neutralizing antibody, characterized in that: The antibody comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO.1; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3; the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.6; and the antibody is an IgG2A antibody.