Anti-MAK16-IgG1 monoclonal subtype antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-MAK16-IgG1 monoclonal subtype antibody as well as a preparation method and application thereof, and belongs to the technical field of drugs for immune diseases. According to the invention, the monoclonal antibody which is high in concentration and purity and is resistant to MAK16 protein is successfully prepared, and the monoclonal antibody is a monoclonal IgG1 subtype antibody, namely an anti-MAK16-IgG1 monoclonal subtype antibody; the titer is 160000; compared with RAW264.7 cells of an IgG1 + LPS co-stimulation induction group, the expression quantity of IL-6 and IL-10 of the RAW264.7 cells which are subjected to co-stimulation induction for 24 hours and 48 hours by adopting the anti-MAK16-IgG1 monoclonal subtype antibody prepared by the invention and LPS is remarkably reduced, and the difference is more obvious as time is prolonged; and the effective dose of the anti-MAK16-IgG1 monoclonal subtype antibody is not less than 50 [mu] g / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunological disease drugs, in particular to an anti-MAK16-IgG1 monoclonal subtype antibody and a preparation method and application thereof. BACKGROUND

[0002] When MAK16 gene mutates, it will make Saccharomyces cerevisiae show temperature-sensitive growth, and plays a key role in the maturation process of 25S rRNA and 5.8S rRNA of Saccharomyces cerevisiae. Studies have shown that MAK16 protein plays an important role in the biosynthesis of 60S ribosome subunit, and has extensive interaction with the rest of the proteins on the 60S ribosome subunit.

[0003] Interleukin 6 (IL-6) is a multifunctional cytokine that can exhibit pro-inflammatory and anti-inflammatory activities depending on the immune environment in the body. When the body is infected or tissue is damaged, IL-6 is rapidly produced and participates in host defense. On the other hand, excessive secretion of IL-6 can cause acute and chronic systemic inflammatory response and cytokine storm. The hexameric complex of IL-6 (IL-6 / IL-6R / gp130) executes various biological effects by activating different signal transduction mechanisms, including JAK / STAT3, Ras / MAPK, PI3K–PKB / Akt, etc., leading to chronic immune diseases such as cancer, rheumatoid arthritis, inflammatory bowel disease, etc. Studies have shown that in the preclinical stage of systemic lupus erythematosus (SLE), accompanied or preceded by the appearance of various autoantibodies, IL-6 levels can be detected in serum, and serum IL-6 levels are also elevated in active SLE patients, neuropsychiatric SLE, and high cardiovascular disease risk lupus patients.

[0004] Similar to the immune function of IL-6, interleukin 10 (IL-10) also has both anti-inflammatory and pro-inflammatory properties in immune responses: on the one hand, it can inhibit the expression and / or function of inflammatory cytokines such as IFN-γ, TNF, IL-1, IL-6, etc. in monocytes and T cells under certain conditions, on the other hand, it can also stimulate the proliferation and differentiation of autoreactive B cells to form plasma cells, thereby releasing immunoglobulins and other factors that promote immune inflammatory responses. Studies have shown that in lupus model mice, continuous administration of blocking anti-IL-10 antibodies at birth can delay the occurrence of autoantibodies and the appearance of symptoms such as proteinuria and glomerulonephritis.

[0005] Systemic lupus erythematosus (SLE) is a chronic, diffuse connective tissue disease caused primarily by an abnormally activated immune system that attacks the body's own tissues. While the specific cause of SLE remains unclear, its symptoms vary widely, often including fever, photosensitivity, rash, enlarged lymph nodes, muscle and joint pain, headaches, and fatigue. Because SLE can damage organs throughout the body, it can lead to a variety of complications, including kidney damage and neuropsychiatric symptoms.

[0006] Treatment of systemic lupus erythematosus typically relies on multiple therapies, including medication, phototherapy, and lifestyle changes. Commonly used medications include nonsteroidal anti-inflammatory drugs, antimalarial drugs, immunosuppressants, and corticosteroids. Currently, there is no complete cure. Summary of the Invention

[0007] The purpose of the present invention is to provide an anti-MAK16-IgG1 monoclonal subtype antibody and its preparation method and application, so as to provide a candidate monoclonal antibody for the treatment of immune diseases, enrich the types of drugs for the treatment or early prevention of autoimmune diseases such as SLE, and contribute to the early conquest of autoimmune diseases.

[0008] To achieve the above object, the present invention provides a method for preparing an anti-MAK16-IgG1 monoclonal antibody, comprising the following steps:

[0009] S1. Prepare MAK16 protein as an antigen;

[0010] S2. Immunize mice with antigens, prepare mouse polyclonal antiserum, and detect the titer;

[0011] S3, cell electrofusion and cell subcloning and titer detection;

[0012] S4, sequencing hybridoma cells;

[0013] S5. Prepare mouse monoclonal antibodies, and obtain anti-MAK16-IgG1 monoclonal subtype antibodies after purification.

[0014] Preferably, in S1, the gene sequence of MAK16 is subcloned into the prokaryotic expression vector pET-21a(+), and then the competent cells are transformed to induce protein expression. After crude protein extraction, the protein is purified and the purified protein is used as the antigen.

[0015] Preferably, the cells used for cell electrofusion in S3 are: B cells of the mouse with the highest titer detected in S2 and non-secreting SP2 / 0 myeloma cells.

[0016] Preferably, cell subcloning in S3 is performed twice. First, hybridoma cells in good growth state are collected and prepared into a single cell suspension. After culturing for 3 days, single clones are selected. After culturing for 8 days, wells with OD values ​​consistent with positive serum are selected for subcloning again.

[0017] An anti-MAK16-IgG1 monoclonal subtype antibody prepared by the method for preparing the anti-MAK16-IgG1 monoclonal subtype antibody as described above.

[0018] A use of the anti-MAK16-IgG1 monoclonal subtype antibody as described above in the preparation of a drug for treating immune diseases.

[0019] Preferably, the immune disease includes rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus.

[0020] Therefore, the present invention provides an anti-MAK16-IgG1 monoclonal subtype antibody and its preparation method and application, and its specific technical effects are as follows:

[0021] (1) The present invention successfully prepared a high-concentration, high-purity, monoclonal antibody against MAK16 protein, which is a monoclonal IgG1 subtype antibody - anti-MAK16-IgG1 monoclonal subtype antibody; the titer is 160,000;

[0022] (2) Compared with the RAW264.7 cells in the mouse IgG1 antiserum + LPS co-stimulation induction group, the IL-6 and IL-10 expression levels of the RAW264.7 cells after 24h and 48h of co-stimulation induction using the anti-MAK16-IgG1 monoclonal subtype antibody prepared by the present invention + LPS were significantly reduced, and the difference became more obvious as time prolonged; the effective dose of the anti-MAK16-IgG1 monoclonal subtype antibody was not less than 50 μg / mL.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0025] Figure 1 This is the SDS-PAGE detection result of the MAK16 protein in Example 1 of the present invention;

[0026] Figure 2 is the titer detection result of the mouse polyclonal antiserum in the embodiment 1 of the present application;

[0027] Figure 3 is the ELISA detection result of the secondary subcloning cell antibody titer in the embodiment 1 of the present application, wherein a and b are the binding conditions of the hybridoma cell secreted antibody and antigen in each well; c is the Ig2a clone with relatively high expression amount; d is the Ig2b clone with relatively high expression amount;

[0028] Figure 4 is the SDS-PAGE and ELISA detection result of the monoclonal antibody in the embodiment 1 of the present application; wherein a is the SDS-PAGE detection result; b is the ELISA detection result;

[0029] Figure 5 is the SDS-PAGE and ELISA detection result of the anti-MAK16-IgG1 monoclonal subtype antibody after purification in the embodiment 1 of the present application; wherein a is the SDS-PAGE detection result; b is the ELISA detection result;

[0030] Figure 6 is the change of IL-6, IL-10 secretion amount after the anti-MAK16-IgG1 monoclonal subtype antibody acts for different time in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0031] Figure 7 is the change of IL-6, IL-10 gene expression level after the anti-MAK16-IgG1 monoclonal subtype antibody acts for different time in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0032] Figure 8 is the change of IL-6, IL-10 secretion amount after the antibody acts for 48h in different doses in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0033] Figure 9 is the change of IL-6, IL-10 gene expression level after the antibody acts for 48h in different doses in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0034] Figure 10 is the p-STAT3 / STAT3, p-AKT / AKT ratio and Western blot representative graph after the antibody acts for different time in the embodiment 3 of the present application; wherein a is the p-STAT3 / STAT3 ratio; b is the p-AKT / AKT ratio; c is the Western blot representative graph;

[0035] Figure 11 These are representative graphs of the p-STAT3 / STAT3 and p-AKT / AKT ratios and Western blots after 40 minutes of treatment with different doses of antibodies in Example 3 of the present invention; wherein a is the p-STAT3 / STAT3 ratio; b is the p-AKT / AKT ratio; and c is a representative graph of Western blot. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0037] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0038] The instruments, equipment, and reagents used in the examples were all commercially available. Serum samples from 30 SLE patients in the experimental group with high levels of anti-MAK16 (both the chip and ELISA test values ​​were greater than the corresponding critical values) and serum samples from 15 SLE patients in the disease control group with low levels of anti-MAK16 (both the chip and ELISA test values ​​were less than the corresponding critical values) were obtained from clinical biological samples collected by the First Affiliated Hospital of Anhui Medical University. Serum samples from the 20 healthy control groups were obtained from biological samples of healthy people undergoing physical examinations collected by the First Affiliated Hospital of Anhui Medical University. The experimental group patients were roughly matched with the healthy control group and the disease control group in terms of gender and age. Mouse RAW264.7 cells were obtained from the American Type Culture Collection (ATCC).

[0039] The experiments in the examples were approved by the Ethics Committee of Anhui Medical University (Ethics Approval No.: 20180078) before implementation, and all patients and control group people signed informed consent before participating in the study.

[0040] The method steps not described in detail in the examples are all conventional techniques in the art.

[0041] Example 1

[0042] Preparation of anti-MAK16-IgG1 monoclonal antibody, the specific steps are as follows:

[0043] (1) Prepare antigens.

[0044] 1) The amino acid sequence information of the target gene MAK16 retrieved from UniProt (as shown in SEQ ID NO. 1) was sent to a company for codon optimization and gene synthesis. The synthesis product was subcloned into a prokaryotic expression vector pET-21a(+), and after gene sequencing confirmed that the sequence in the inserted vector was correct, the recombinant prokaryotic expression vector pET-21a(+)-MAK16 was obtained.

[0045] SEQ ID NO. 1:

[0046] MQSDDVIWDTLGNKQFCSFKIRTKTQSFCRNEYSLTGLCNRSSCPLANSQYAT

[0047] IKEEKGQCYLYMKVIERAAFPRRLWERVRLSKNYEKALEQIDENLIYWPRFIR

[0048] HKCKQRFTKITQYLIRIRKLTLKRQRKLVPLSKKVERREKRREEKALIAAQLD

[0049] NAIEKELLERLKQDTYGDIYNFPIHAFDKALEQQEAESDSSDTEEKDDDDDDE

[0050] EDVGKREFVEDGEVDESDISDFEDMDKLDASSDEDQDGKSSSEEEEEKALSA

[0051] KHKGKMPLRGPLQRKRVYVEIEYEQETEPVAKAKTT

[0052] 2) The recombinant prokaryotic expression vector pET-21a(+)-MAK16 was transformed into BL21 competent cells, and 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used for induction at 37°C for 5 h, and the induction was expressed as an inclusion body.

[0053] 3) The protein was subjected to crude extraction, and the expressed protein was purified by nickel column. The purified sample was dialyzed into phosphate buffered saline (PBS) and concentrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the expression results of MAK16 protein. The results are shown in FIG. 1. Figure 1As shown, the desired MAK16 protein target antigen was obtained.

[0054] (2) Antigen immunization of mice. All mice were housed in a barrier system using sterilized pelleted feed and autoclaved drinking water. Five BALB / c mice (SPF grade) were ear-tagged and immunized with purified MAK16 recombinant protein according to the immunization process (three immunizations of mice, first blood collection, fourth immunization of mice, second blood collection, and final booster immunization).

[0055] (3) Preparation of mouse polyclonal antiserum samples:

[0056] 1) After the third and fourth immunizations, approximately 20 μL of blood was collected from the tail vein of the mice.

[0057] 2) Place the blood sample in a 4°C refrigerator overnight.

[0058] 3) Then centrifuge at 4°C and 10,000 g for 10 min, transfer the yellow serum layer to a new test tube, and store in a -20°C refrigerator.

[0059] 4) The positive serum obtained above was diluted at a ratio of 1:1000, 1:5000, 1:20000, 1:40000, 1:80000, and 1:160000, and PBS and negative serum controls were set up. The serum titer was then detected by ELISA.

[0060] (4) ELISA test of antiserum titer:

[0061] 1) Dilute the purified MAK16 recombinant protein with PBS and adjust the protein concentration to 5 mg / mL.

[0062] 2) Add 100 μL of 5 mg / mL antigen sample to each well of a 96-well plate and coat overnight at 4°C.

[0063] 3) Wash three times with PBST (PBS buffer containing 0.5% Tween 20).

[0064] 4) Block with 5% BSA at room temperature for 1 h.

[0065] 5) Repeat step 3) and wash 3 times.

[0066] 6) Take 100 μL of the diluted positive serum (1:1000, 1:5000, 1:20000, 1:40000, 1:80000, 1:160000 dilution) as well as PBS and negative serum control and add them to the ELISA plate coated with the corresponding antigen.

[0067] 7) Incubate at 37°C for 1 hour.

[0068] 8) Repeat step 3) and wash 3 times.

[0069] 9) Add 100 μL of 1:20,000 diluted HRP-labeled anti-mouse IgG to each well.

[0070] 10) Incubate at 37°C for 1 hour.

[0071] 11) Repeat step 3) and wash 3 times.

[0072] 12) Add 100 μL of TMB color development solution to each well and develop the color for 15 minutes.

[0073] 13) Add 50 μL of stop solution to stop the color development reaction and detect using a microplate reader.

[0074] The results are as follows Figure 2 As shown, B4 mice have better antibody titers against target antigens. Therefore, B4 mice were selected for pulse immunization, and their immune B cells were taken for cell electrofusion and titer detection.

[0075] (5) Cell electrofusion:

[0076] 1) The mouse with the highest titer was killed by cervical dislocation, and the spleen was obtained under sterile conditions. A single-cell suspension of B cells was prepared and mixed with non-secreting SP2 / 0 myeloma cells at a 1:1 ratio. Cell fusion was performed using the Bio-rad Gene electroporation system.

[0077] 2) After electrofusion, all cells were immediately suspended in complete culture medium (DMEM, 20% FBS and HAT) and seeded into 96-well plates.

[0078] 3) About 10 days after fusion, the medium was changed to HT medium. After two days of culture, 100 μL of the supernatant was collected and tested by ELISA. The corresponding wells with OD values ​​consistent with the positive serum (1:1000) were selected for subcloning.

[0079] (6) Cell subcloning:

[0080] 1) Collect hybridoma cell lines with good growth status and prepare single cell suspension.

[0081] 2) Count the cells using a hemocytometer, take 100 cells, and dilute to 10 mL.

[0082] 3) Take a 96-well plate and add 100 μL of cell suspension to each well, and finally add the volume to 200 μL.

[0083] 4) Place in a 37°C incubator and label.

[0084] 5) After 3 days of continuous culture, single clones were selected under a microscope.

[0085] 6) After culturing for 7 days, replace the culture medium with fresh one.

[0086] 7) On the 8th day of culture, the cell supernatant was detected by ELISA, and the wells with OD values ​​consistent with the positive serum (1:1000) were selected for subcloning again.

[0087] After cell electrofusion and secondary subcloning, the cells were plated in a 96-well plate. The hybridoma cells in each well secreted antibodies, and the binding with the antigen was as follows: Figure 3 A and Figure 3 As shown in B, select Figure 3 C and Figure 3 The relatively high expression levels of Ig2a and Ig2b clones are shown in Figure D. At this point, the cells in each well may still be polyclonal or monoclonal. Further microscopic examination is performed to confirm monoclonal cells, and wells containing only a single clone are selected for subsequent experiments.

[0088] (7) Preparation and purification of mouse monoclonal antibodies. The hybridoma cells determined by subcloning titer detection were sterilized under sterile conditions to obtain approximately 1×10 6 Add 100 hybridoma cells to a 1.5 mL centrifuge tube and centrifuge at 250 g / min at 4°C for 10 minutes. Remove the supernatant. Wash three times with 1× PBS. Resuspend the hybridoma cells in 500 μL of 1× PBS and inject the cells into the abdomen of a mouse with a 1 mL syringe. Wait 7-10 days to collect ascites. Use ammonium sulfate precipitation and protein G affinity chromatography to purify the antibody. Analyze the antibody titer by SDS-PAGE and ELISA.

[0089] SDS-PAGE picture Figure 4 As shown in a, the ELISA test results are as follows Figure 4 As shown in b, a higher concentration of IgG2 subtype antibodies was obtained.

[0090] (8) Hybridoma cell gene sequencing (1-2-d clone). The variable region sequences of VH and VL were amplified according to the standard laboratory operating procedures. The VH and VL variable regions of the 1-2-d clone were cloned into T vectors using standard molecular cloning methods. Blue-white spot screening was performed, and the plaques were selected and shaken for gene sequencing.

[0091] The variable region sequences of the VH and VL of the 1-2-d clone were amplified according to standard operating procedures. The VH and VL variable regions were cloned into T vectors using standard molecular cloning methods. Blue-white screening was performed, and the strains were picked and sequenced. The sequencing results showed that the nucleotide sequence of heavy chain 1 is shown in SEQ ID NO. 2, the nucleotide sequence of light chain 1 is shown in SEQ ID NO. 3, and the nucleotide sequence of light chain 2 is shown in SEQ ID NO. 4. The sequencing results showed that the two light chains differed only by a single amino acid in the CDR1 region (italicized and underlined fonts indicate CDR1, CDR2, and CDR3, respectively; the differential nucleotide sequence in CDR1 of the light chain is indicated by bold italics and underlining).

[0092] SEQ ID NO.2:

[0093]

[0094] SEQ ID NO.3:

[0095]

[0096]

[0097] SEQ ID NO.4:

[0098]

[0099] (9) Expression and purification of target antibodies. Based on the previous hybridoma sequencing results, a mouse IgG1 antibody expression vector was constructed and an endotoxin-free plasmid was prepared for use. Take out the LVTransm transfection reagent and antibody expression vector, thaw them at room temperature, and mix them thoroughly with a micropipette. Take out the phosphate buffered saline (PBS) and equilibrate it to room temperature at room temperature. Use a micropipette to take 2 mL of PBS into different wells of the reaction plate, add 130 μg of antibody expression vector to each well, mix them thoroughly with a micropipette, then add 400 μL of LVTransm to each well, mix them again with a micropipette, and let them stand at room temperature for 10 minutes. Add the DNA / LVTransm complex to a culture flask containing 50 mL of 293F-SVP16 cells, and gently shake the culture flask to mix the complex and cells thoroughly. Place the culture flask in a 37°C incubator containing 5% CO2 and shake at 130 rpm for 6 to 8 hours. Then add 50 mL of fresh FreeStyle TM293 Expression Medium was added and the cell culture flask was returned to the incubator for continued shaking culture. After 7 days of continuous culture, the supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane. The filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein G column and tested by ELISA.

[0100] The results are as follows Figure 5 As shown, it was shown that a higher concentration of IgG1 subtype antibodies was obtained.

[0101] Example 2

[0102] The effects of the anti-MAK16-IgG1 monoclonal subtype antibody prepared in Example 1 on IL-6 and IL-10 were investigated as follows:

[0103] Mouse mononuclear macrophage leukemia cells RAW264.7 purchased from ATCC were cultured in DMEM medium containing 10% FBS in a cell culture incubator at 37° C. and 5% CO 2 .

[0104] In the first stage, RAW264.7 cells were cultured overnight and the cell density was adjusted to 5×10 4 The cells were cultured for 12 h, 24 h, and 48 h, and the supernatant was collected for ELISA detection.

[0105] In the second stage, after overnight culture of RAW264.7 cells, the cell density was adjusted to 5×10 4cells / mL, and the experiment was divided into 8 groups (RAW264.7 group; RAW264.7 + 1 μg / mL LPS group; RAW264.7 + 1 μg / mL LPS + 25 μg / mL mouse IgG1 group; RAW264.7 + 1 μg / mL LPS + 50 μg / mL mouse IgG1 group; RAW264.7 + 1 μg / mL LPS + 100 μg / mL mouse IgG1 group; RAW264.7 + 1 μg / mL LPS + 25 μg / mL anti-MAK16-IgG1 group; RAW264.7 + 1 μg / mL LPS + 50 μg / mL anti-MAK16-IgG1 group). LPS and different doses of antibodies were added to the cells (anti-MAK16-IgG1 group), with 3 replicates per group. After incubation for a period of time based on the optimal time determined in the first stage, the culture supernatant was collected for ELISA detection.

[0106] After overnight culture of RAW264.7 cells, the cell density was adjusted to 5 × 10 4 cells / mL. After stimulation and induction by mixed culture of LPS, antibodies and cells in different groups at the two stages of the ELISA test, cellular RNA was extracted and the expression levels of IL-6 and IL-10 genes were detected by RT-qPCR. Each experiment was repeated three times.

[0107] The results of the first stage ELISA and RT-qPCR tests are shown in Tables 1 and 2. Figure 6-Figure 7 As shown, after 12h, 24h, and 48h of co-stimulation with LPS (1μg / mL), LPS (1μg / mL) + mouse IgG1 (100μg / mL), or LPS (1μg / mL) + anti-MAK16-IgG1 (100μg / mL), the expression of IL-6 and IL-10 increased over time in mouse mononuclear macrophage leukemia cells RAW264.7. However, after 24h and 48h of co-stimulation, the expression of IL-6 and IL-10 in the anti-MAK16-IgG1 stimulation group decreased compared with the control group (P<0.05), and the difference became more obvious as time went on.

[0108] Table 1 ELISA detection of IL-6 and IL-10 secretion after different antibody treatment times

[0109]

[0110] Table 2 RT-qPCR detection of IL-6 and IL-10 gene expression levels after antibody treatment for different time periods

[0111]

[0112] The results of the second-stage ELISA and RT-qPCR tests are shown in Tables 3-4 and Figure 8-Figure 9 As shown in the data, after 48 h of co-stimulation with LPS (1 μg / mL) and mouse IgG1 or anti-MAK16-IgG1 at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, the expression levels of IL-6 and IL-10 did not change significantly; with the increase of the dose of anti-MAK16-IgG1, the expression levels of IL-6 and IL-10 showed a downward trend; after stimulation with anti-MAK16-IgG1 at doses of 50 μg / mL and 100 μg / mL, the expression levels of IL-6 and IL-10 were both lower than those in the control group with the same dose (P<0.05).

[0113] Table 3 ELISA detection of IL-6 and IL-10 secretion after 48 hours of treatment with different doses of antibodies

[0114]

[0115]

[0116] Table 4 RT-qPCR detection of IL-6 and IL-10 gene expression levels after 48 hours of treatment with different doses of antibodies

[0117] Group IL-6 IL-10 RAW264.7 1±0.06 1±0.06 RAW264.7+LPS 9.12±0.4 7.73±0.38 RAW264.7+mouse IgG1 (25 μg / mL)+LPS 10.28±0.46 7.59±0.66 RAW264.7+mouse IgG1 (50 μg / mL)+LPS 10.67±0.38 7.86±0.29 RAW264.7+mouse IgG1 (100 μg / mL)+LPS 9.84±0.36 7.52±0.37 RAW264.7+anti-MAK16-IgG1(25μg / mL)+LPS 7.79±0.34 6.85±0.29 RAW264.7+anti-MAK16-IgG1(50μg / mL)+LPS 5.17±0.34 5.53±0.20 RAW264.7+anti-MAK16-IgG1(100μg / mL)+LPS 2.82±0.19 4.78±0.29

[0118] Example 3

[0119] Western blot was used to investigate the effect of the anti-MAK16-IgG1 monoclonal subtype antibody prepared in Example 1 on the intracellular p-STAT3 / STAT3 and p-AKT / AKT levels. The group settings were the same as those in the first stage of Example 2. The protein concentrations of the test samples are shown in Table 5.

[0120] Table 5 Protein concentration of each test sample

[0121]

[0122]

[0123] The results are shown in Table 6 and Figure 10The experimental data were processed and statistically analyzed, and it was found that after different antibodies and LPS (1 μg / mL) were used to stimulate RAW264.7 cells for different time periods, the expression levels of STAT3 and AKT did not change significantly. After 40 minutes of stimulation, the expression levels of their phosphorylated products p-STAT3 and p-AKT reached high values ​​in the anti-MAK16-IgG1 (100 μg / mL) group (experimental group) and the mouse IgG1 (100 μg / mL) control group, and then the expression levels showed a decreasing trend. The expression levels of p-STAT3 and p-AKT in the experimental group were lower than those in the mouse IgG1 control group at different stimulation time periods (P < 0.05), but there was no difference in expression between the LPS (1 μg / mL) control group and the mouse IgG1 (100 μg / mL) + LPS (1 μg / mL) control group (Table 6, Figure 10 After 40 min of co-stimulation with different doses of different antibodies and LPS, the expression levels of STAT3 and AKT in RAW264.7 cells did not change significantly. The expression levels of p-STAT3 and p-AKT in the experimental group showed a downward trend with the increase of anti-MAK16-IgG1 dose. At the highest dose of antibody, the expression levels of p-STAT3 and p-AKT in the experimental group were lower than those in the mouse IgG1 control group (P<0.05) (Table 7, Figure 11 ).

[0124] Table 6 Ratios of p-STAT3 / STAT3 and p-AKT / AKT after different antibody treatment times

[0125]

[0126]

[0127] Table 7 Ratios of p-STAT3 / STAT3 and p-AKT / AKT after 40 min of treatment with different doses of antibodies

[0128] Group p-STAT3 / STAT3 p-AKT / AKT RAW264.7 0.266±0.039 0.291±0.043 RAW264.7+LPS 0.801±0.093 0.787±0.071 RAW264.7+mouse IgG1 (25 μg / mL)+LPS 0.709±0.076 0.835±0.089 RAW264.7+mouse IgG1 (50 μg / mL)+LPS 0.782±0.083 0.795±0.088 RAW264.7+mouse IgG1 (100 μg / mL)+LPS 0.918±0.097 0.844±0.087 RAW264.7+anti-MAK16-IgG1(25μg / mL)+LPS 0.680±0.073 0.860±0.097 RAW264.7+anti-MAK16-IgG1(50μg / mL)+LPS 0.528±0.068 0.808±0.069 RAW264.7+anti-MAK16-IgG1(100μg / mL)+LPS 0.375±0.048 0.585±0.073

[0129] Therefore, the present invention successfully prepared a high-concentration and high-purity monoclonal antibody against MAK16 protein, which is a monoclonal IgG1 subtype antibody - anti-MAK16-IgG1 monoclonal subtype antibody; the titer is 160,000; compared with the RAW264.7 cells in the IgG1+LPS co-stimulation induction group, the RAW264.7 cells co-stimulated and induced with the anti-MAK16-IgG1 monoclonal subtype antibody prepared by the present invention + LPS for 24 hours and 48 hours, the expression levels of IL-6 and IL-10 were significantly reduced, and the difference became more obvious as time prolonged; the effective dose of the anti-MAK16-IgG1 monoclonal subtype antibody is not less than 50 μg / mL.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an anti-MAK16-IgG1 monoclonal antibody, characterized in that: The steps include: S1. Prepare MAK16 protein as an antigen; S2. Immunize mice with antigens, prepare mouse polyclonal antiserum, and detect the titer; S3, cell electrofusion and cell subcloning and titer detection; S4, sequencing hybridoma cells; S5. Prepare mouse monoclonal antibodies, and obtain anti-MAK16-IgG1 monoclonal subtype antibodies after purification.

2. The method for preparing an anti-MAK16-IgG1 monoclonal antibody according to claim 1, characterized in that: In S1, the gene sequence of MAK16 was subcloned into the prokaryotic expression vector pET-21a(+), and then the competent cells were transformed to induce protein expression. After crude protein extraction, the protein was purified and the purified protein was used as an antigen.

3. The method for preparing an anti-MAK16-IgG1 monoclonal antibody according to claim 1, characterized in that: The cells used for cell electrofusion in S3 are: B cells from the mouse with the highest titer detected in S2 and non-secreting SP2 / 0 myeloma cells.

4. The method for preparing an anti-MAK16-IgG1 monoclonal antibody according to claim 1, characterized in that: Cell subcloning was performed twice in S3. First, hybridoma cells with good growth status were collected and prepared into single cell suspension. After culturing for 3 days, single clones were selected. After culturing for 8 days, wells with OD values ​​consistent with those of positive serum were selected for subcloning again. 5 . An anti-MAK16-IgG1 monoclonal subtype antibody prepared by the method for preparing an anti-MAK16-IgG1 monoclonal subtype antibody according to any one of claims 1 to 4 .

6. Use of the anti-MAK16-IgG1 monoclonal subtype antibody according to claim 5 in the preparation of a drug for treating immune diseases.

7. Use of an anti-MAK16-IgG1 monoclonal antibody according to claim 6 in the preparation of a drug for treating or preventing immune diseases, characterized in that: The immune diseases include rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus.

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