Anti-human il-9 protein rabbit monoclonal antibody and use thereof

Abstract

The application belongs to the technical field of IL-9 protein, and particularly relates to an anti-human IL-9 protein rabbit monoclonal antibody and application thereof. The anti-human IL-9 protein rabbit monoclonal antibody is characterized in that CDR1 is SSNAMG; CDR2 is VITSSGTIFYTSWAKG; CDR3 is GGYDGVGGDYAFLGI; CDR1 is QASQSIGSSLS; CDR2 is EASTLAS; and CDR3 is QSYYYSGISSYAFT. The application provides a rabbit monoclonal antibody capable of specifically recognizing and combining with human IL-9 protein, and the rabbit monoclonal antibody is used for immunodetection, biomedical research and research on an IL-9 signal path, and has high specificity and affinity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of IL-9 protein, and particularly relates to an anti-human IL-9 protein rabbit monoclonal antibody and application thereof. BACKGROUND

[0002] Interleukin-9 (IL-9) is a pleiotropic cytokine produced in different amounts in different immune cells. These include mast cells, NKT cells, Th2, Th17, Treg cells and Th9 cells, the latter of which produces the largest amount. It is a 14kD glycoprotein composed of 144 amino acids and an 18-amino acid signal peptide. IL-9R, as a receptor for interleukin-9 (IL-9), is located on the cell membrane and is part of the cytokine family that shares a common gamma chain receptor, including IL-2, IL-4, IL-7, IL-15 and IL-21. In addition to the common gamma chain, the IL-9 receptor is also composed of IL-9R alpha.

[0003] As an important cytokine, IL-9 has a variety of biological functions. It can maintain the long-term growth of TH cells without IL-2 and IL-4, showing a unique growth support effect. In addition, IL-9 can also promote the growth of certain myeloid leukemia cell lines (such as MOTE), which suggests that it may be involved in the regulation of hematopoietic processes. In addition, IL-9 can also significantly promote the growth and activity of mast cells, further demonstrating its important role in the immune system. More importantly, IL-9 plays a key role in a variety of diseases, including allergic inflammation and tumor immunity, providing a new perspective and target for the research and treatment of related diseases.

[0004] In the technology of monoclonal antibodies, rabbit monoclonal antibodies are of great interest due to their high affinity and specificity. However, existing technical solutions such as mouse-rabbit heterohybridoma and rabbit-rabbit hybridoma methods have stability problems. Hybridomas are prone to lose antibody genes, leading to unstable antibody production. In addition, although rabbit monoclonal antibodies have advantages, existing technologies cannot guarantee that all antibodies have ideal affinity and specificity, which may be related to antibody library diversity and screening methods. The preparation process involves complex cell culture, genetic manipulation and screening steps, which are technically difficult and challenging to implement key technologies. Therefore, the preparation of rabbit monoclonal antibodies still needs to be further improved and optimized. SUMMARY

[0005] The present application provides a monoclonal antibody of IL and application thereof.

[0006] The present application provides a monoclonal antibody of IL and application thereof.

[0007] TECHNICAL SCHEME

[0008] The application discloses an anti-human IL-9 protein rabbit monoclonal antibody, and relates to the field of medicine.

[0009] The anti-human IL-9 protein rabbit monoclonal antibody is characterized in that the amino acid sequence of the heavy chain is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain is shown in SEQ ID NO. 2.

[0010] The anti-human IL-9 protein rabbit monoclonal antibody is characterized in that the amino acid sequence of the antibody is shown in SEQ ID NO. 3.

[0011] The application discloses an anti-human IL-9 protein rabbit monoclonal antibody, and relates to the field of medicine.

[0012] The application discloses an anti-human IL-9 protein rabbit monoclonal antibody, and relates to the field of medicine.

[0013] The application discloses an anti-human IL-9 protein rabbit monoclonal antibody, and relates to the field of medicine.

[0014] Beneficial effects

[0015] The application is dedicated to the development of a rabbit monoclonal antibody specific to human IL-9, and the sequence of the monoclonal antibody is a brand-new sequence and has not been reported in the literature. Specifically, through a carefully designed immunization program, and through affinity experiments, the antibody M30903-1C3 and M30903-2G1 with high affinity and high titer are screened out, the antibody exhibits specificity and affinity to IL-9, provides a powerful tool for in-depth study of the IL-9 / IL-9R signal pathway, and opens up new possibilities for the diagnosis and treatment of IL-related diseases. The antibody M30903-2G1 has effects on rheumatoid arthritis and leukemia. IL-9 plays an important role in asthma, allergic diseases, inflammatory bowel diseases, autoimmune diseases, cancers and parasitic infections and the like. As the IL-9 monoclonal antibody, inhibiting IL-9 can become a potential strategy for treating these diseases. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The application discloses an anti-human IL-9 protein rabbit monoclonal antibody, and relates to the field of medicine.

[0017] Figure 2Figure 1 is the result of antibody purification, wherein A is M30903-1C3; B is M30903-2G1;

[0018] Figure 3 Figure 2 is the result of Western Blot detection of IL-9 monoclonal antibody IL-9R monoclonal antibody binding to cell IL-9 / IL-9R. Wherein A is M30903-1C3; B is M30903-2G1;

[0019] Figure 4 Figure 3 is the result of flow detection of IL-9 monoclonal antibody on THP-1 cell M1 / M2 polarization; A IL-9 antibody on IL-9 inhibition B IL-9 antibody on macrophage M1 polarization C IL-9 antibody on macrophage M2 polarization

[0020] Figure 5 Figure 4 is the result of transwell detection of IL-9 monoclonal antibody on RA patient synovial fibroblast-like cell migration ability. DETAILED DESCRIPTION

[0021] Example 1 Antibody preparation

[0022] 1. Product structure

[0023] Nucleotide sequence of immunogen IL-9 gene: gene ID: 3578

[0024] (1) The amino acid sequence of antigen IL9 His is as follows:

[0025]

[0026] His: HHHHHH*

[0027] (2) Amino acid sequence of monoclonal antibody (M30903-2G1)

[0028]

[0029] 2G1-VH:

[0030]

[0031] 2G1-VL:

[0032]

[0033] The CDR sequence of the heavy chain: CDR1 is SSNAMG, (such as SEQ ID NO. 4); CDR2 is VITSSGTIFYTSWAKG (such as SEQ ID NO. 5), CDR3 is GGYDGVGGDYAFLGI (such as SEQ ID NO. 6);

[0034] CDR sequences of the light chain: CDR1 is QASQSIGSSLS (as SEQ ID NO. 7); CDR2 is EASTLAS (as SEQ ID NO. 8); CDR3 is QSYYYSGISSYAFT (as SEQ ID NO. 9);

[0035] 2. Experimental steps

[0036] 2.1 Preparation of antigen

[0037] (1) PCR amplification reaction system

[0038]

[0039]

[0040] The template sequence is shown in gene ID: 3578;

[0041] (2) PCR amplification reaction conditions

[0042]

[0043] Electrophoretic detection and recovery of amplified fragments

[0044] 2.2 Enzymatic digestion of vector and target gene

[0045]

[0046] 37°C for 1-2h; electrophoretic detection and recovery of the digestion product

[0047] 23 Connection of vector and target gene

[0048]

[0049] 16°C, 0.5-1h

[0050] 2.4 Transformation

[0051] (1) Add the DNA fragment to be transformed to the tube containing the TOP10 competent cells (25ng of DNA is needed for 50μL of competent cells), the volume should not exceed 5% of the competent cells, gently rotate several times to mix the contents, and ice bath for 30min.

[0052] (2) Place the centrifuge tube mixture in a circulating water heated to 42°C, heat shock for 90s, do not shake the tube.

[0053] (3) Quickly transfer the tube to the ice bath to cool the cells for 1-2min.

[0054] (4) Add 200 μl SOC liquid medium to each tube, and warm the medium to 37°C in a water bath. Then transfer the tube to a shaker set at 37°C, and incubate at 220 rpm for 45 min to allow the cells to recover and express the resistance marker gene encoded by the plasmid.

[0055] (5) Transfer the transformed competent cells of appropriate volume (200 μl for each 90 mm plate) to LB medium containing the corresponding antibiotic.

[0056] (6) Invert the plate, and incubate at 37°C. After 12-16 hours, bacterial colonies can be observed.

[0057] 2.5 Colony PCR verification

[0058] After bacterial colonies grow on the plate, randomly pick several colonies for colony PCR verification to detect the transformants.

[0059] 2.6 Sequencing verification

[0060] Send the positive clones to the sequencing platform of the company for sequencing verification.

[0061] 3. Protein expression

[0062] Use the mammalian HEK293 cell expression system for expression test. Transfect the target gene IL9-His into HEK293 cells for protein expression and purification test.

[0063] 3.1 Expression conditions

[0064] - HEK293 cell density: 1.5-2.0 x 10 6 cells / mL

[0065] - Incubation temperature: 37°C

[0066] - Incubation rotation speed: 130 rpm / min

[0067] - Incubation sampling time: Day 6

[0068] 3.2 Purification scheme

[0069] - Collect the culture medium supernatant, centrifuge at high speed, pass through a 0.22 μm filter membrane, and combine the supernatant with Ni 2+ resin to purify the target protein.

[0070] - Resin: Ni 2+ resin.

[0071] - Binding and equilibration resin solution: PBS, pH = 7.4.

[0072] - Washing and elution solution:

[0073] *Wash: PBS, pH = 7.4, 10 mM imidazole

[0074] *Elution: PBS, pH = 7.4, 300 mM imidazole

[0075] - Collect the culture medium supernatant, centrifuge at high speed, filter through 0.22 um membrane, and purify the target protein by binding the supernatant to Protein A resin.

[0076] - Resin: Protein A resin.

[0077] - Binding and equilibration resin solution: PBS, pH = 7.4.

[0078] - Washing and elution solution:

[0079] *Wash: PBS, pH = 7.4

[0080] *Elution: 0.1 M glycine, pH = 3.0

[0081] *Neutralization: 1 M Tris-Cl, pH = 8.5

[0082] 3.3 Test results

[0083] See Figure 1 and Table 1, the protein antigen IL9-His is obtained.

[0084] Table 1 is the detection result of IL9-His

[0085]

[0086] 4. Animal immunization

[0087] 4.1 Treatment method

[0088] The primary antigen is the protein antigen IL9-His mixed and emulsified with an equal volume of Freund's complete adjuvant, and the secondary, tertiary, fourth, fifth, and sixth antigens are mixed and emulsified with an equal volume of Freund's incomplete adjuvant.

[0089] 4.2 Animals

[0090] Healthy female New Zealand white rabbits, 4 months old, 2.1 kg, were selected.

[0091] 4.2 Immunization

[0092] Primary immunization: the immunization antigen is Freund's complete adjuvant + immunogen;

[0093] Secondary immunization: the immunization antigen is Freund's incomplete adjuvant + immunogen;

[0094] Three immunization: the antigen for immunization is Freund's incomplete adjuvant + immunogen;

[0095] Four immunization: the antigen for immunization is Freund's incomplete adjuvant + immunogen;

[0096] Take small blood for elisa detection; take 1 mL of antiserum for elisa

[0097] Five immunization: the antigen for immunization is Freund's incomplete adjuvant + immunogen;

[0098] Final release: take whole blood from the carotid artery, take serum after standing at 4℃ overnight, and freeze store. Take spleen cells, and freeze store in liquid nitrogen.

[0099] Immunization cycle table:

[0100] Primary immunization: the antigen for immunization is Freund's complete adjuvant + protein antigen 0.5 mg;

[0101] Second immunization: the antigen for immunization is Freund's incomplete adjuvant + protein antigen 0.25 mg;

[0102] Third immunization: the antigen for immunization is Freund's incomplete adjuvant + protein antigen 0.25 mg;

[0103] Four immunization: the antigen for immunization is Freund's incomplete adjuvant + protein antigen 0.25 mg;

[0104] Take small blood for elisa detection; take 1 mL of antiserum for elisa

[0105] Five immunization: the antigen for immunization is Freund's incomplete adjuvant + protein antigen 0.25 mg;

[0106] Live rabbit final release takes spleen

[0107] 5. Indirect ELISA detection

[0108] 5.1 Coating antigen

[0109] Dilute the antigen to 2 ug / mL with 0.05 mol / L carbonate (PH = 9.6), 100 ul / well, and incubate overnight at 4℃.

[0110] 5.2 Wash plate

[0111] Wash three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0112] 5.3 Blocking

[0113] Add 5% skimmed milk powder (PBST) 150 μL blocking solution per well, and block at 37℃ for 60 minutes.

[0114] 5.4 Wash plate

[0115] The plate was taken out and washed three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0116] 5.5 plus primary antibody

[0117] The antiserum was diluted 1:1000, then serially diluted, and incubated at 37°C for 1 hour.

[0118] 5.6 Washing the plate

[0119] The plate was taken out and washed three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0120] 5.7 Add secondary antibody

[0121] Horseradish enzyme-conjugated goat anti-rabbit IgG (H+L), Catalog No. 116154, Antiserum Company: Jackson. Dilute 1:8000, incubate at 37°C for 45 minutes.

[0122] 5.8 Washing the plate

[0123] The plate was taken out and washed five times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0124] 5.9 color rendering

[0125] Add 100 μL / well of substrate solution (TMB), react for 5-10 minutes, and finally add 100 μL of 2 mol / L sulfuric acid to terminate the reaction.

[0126] 5.10 OD value measurement

[0127] The OD value was measured at a wavelength of 450 nm using a microplate reader (Kehua ST-360).

[0128] 6. B cell collection

[0129] 6.1 Collect peripheral blood B cells;

[0130] 6.2 Wash the cells twice with 50 mL of wash solution each time, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and collect B cells.

[0131] 7. Single B Cell Screening

[0132] 7.1 Screen B cells using specific antibodies / antigens to identify positive B cells.

[0133] 7.2 Count the positive B cells after screening, perform limiting dilution, plate them in a 96-well plate, and add culture medium for incubation. After about 15 days of incubation, collect the supernatant and perform ELISA analysis.

[0134] 8. Experimental Results

[0135] 8.1 Single B cell screening results

[0136] Two antibodies M30903-2G1 and M30903-1C3 were screened out

[0137] Table 2: IL9 96-well plate supernatant Elisa verification results (TBM color development)

[0138] B1 1 2 3 4 5 6 7 8 9 10 11 12 A 0.088 0.085 0.084 0.088 0.08 0.085 0.076 0.079 0.075 0.078 0.082 0.078 B 0.086 0.08 0.079 0.074 0.071 0.07 0.073 0.071 0.073 0.074 0.306 0.076 C 0.086 0.077 0.079 0.077 0.152 0.077 0.07 0.074 0.071 0.071 0.071 0.077 D 0.077 0.075 0.073 0.071 0.077 0.07 1.095 0.067 0.072 0.099 0.071 0.084 E 0.085 0.077 0.078 0.082 0.079 0.074 0.073 0.073 0.074 0.071 0.071 0.083 F 0.084 0.075 0.074 0.074 0.07 0.073 0.074 0.073 0.072 0.069 0.069 0.082 G 0.092 0.146 0.078 0.08 0.074 0.078 0.077 0.076 0.075 0.074 0.075 0.091 H 0.095 0.087 0.081 0.083 0.078 0.077 0.081 0.08 0.081 0.08 0.062 2.117

[0139] Wherein: G2: M30903-2G1; H11 is: negative control; H12 is: positive control (rabbit polyclonal serum)

[0140] Table 3: IL9 96-well plate supernatant Elisa verification results (TBM color development)

[0141] B2 1 2 3 4 5 6 7 8 9 10 11 12 A 0.119 0.111 0.099 0.093 0.081 0.076 0.072 0.068 0.071 1.113 0.085 0.097 B 0.09 0.076 0.074 0.079 0.071 0.071 0.072 0.072 0.07 0.075 0.083 0.09 C 0.282 0.081 0.077 0.091 0.072 0.071 0.075 0.072 0.073 0.262 0.082 0.097 D 0.082 0.072 0.075 0.073 0.066 0.069 0.074 0.069 0.071 0.069 0.089 0.096 E 0.085 0.076 0.07 0.079 0.071 0.071 0.073 0.172 0.074 0.076 0.089 0.103 F 0.082 0.071 0.336 0.071 0.069 0.071 0.071 0.081 0.073 0.069 0.085 0.319 G 0.084 0.08 0.073 0.076 0.073 0.074 0.074 0.08 0.082 0.087 0.099 0.122 H 0.097 0.081 0.08 0.077 0.079 0.078 0.074 0.082 0.081 0.08 0.084 2.206

[0142] Wherein: C1: M30903-1C3; H11 is: negative control; H12 is: positive control (rabbit polyclonal serum)

[0143] 8.2 Single B cell sequencing

[0144] After extracting RNA from single B cells, reverse transcription and amplification of heavy chain and light chain sequences were performed, and constructed into recombinant antibody expression vectors.

[0145] 9. Recombinant antibody expression

[0146] 9.1 Expression and purification test

[0147] The mammalian HEK293 cell expression system was used for expression test. The target gene was transfected into HEK293 cells for protein expression and purification test.

[0148] 9.2 Expression conditions

[0149] -HEK293 cell density: 1.5-2.0x10 6 cells / mL

[0150] - Incubation temperature: 37℃

[0151] - Incubation rotation speed: 130rmp / min

[0152] - Incubation sampling time: Day6

[0153] 9.3 Purification scheme

[0154] - Collect the culture medium supernatant, centrifuge at high speed, pass through 0.22 urn filter membrane, and the supernatant is combined with Pratein A resin to purify the target protein.

[0155] - Resin: Pratein A resin.

[0156] - Binding and equilibration resin solution: PBS, pH=7.4.

[0157] - Washing and elution solution:

[0158] * Wash: PBS, pH=7.4

[0159] * Elution: 0.1 M glycine, pH=3.0

[0160] * Neutralization: 1 M Tris-Cl, pH=8.5

[0161] 9.4 Test results

[0162] The purification results are as follows Figure 2 and shown in Table 4: the antibody titer after purification was still 0.338 at 128K, and the antibody titer was estimated to be >512K.

[0163] Table 4: Recombinant antibody expression results

[0164]

[0165]

[0166] Note: 100 ng / well of protein antigen was coated.

[0167] Example 2 WB detection of IL-9 monoclonal antibody binding ability

[0168] 1. Sample preparation: Jurkat cells were collected and lysed using RIPA buffer to extract proteins. The protein concentration was then determined to ensure that the protein concentrations of each sample were consistent. After determining the concentration, the protein samples were mixed with the loading buffer and boiled for 5-10 minutes to denature the proteins.

[0169] 2. SDS-PAGE electrophoresis: A 10% concentration of SDS-polyacrylamide gel was prepared, and the treated protein samples were added to the gel wells along with protein molecular weight markers (Marker). Electrophoresis was performed in a fast electrophoresis buffer at a constant voltage (150 v) to separate the proteins according to their molecular weights.

[0170] 3. Membrane transfer: The gel, PVDF membrane, filter paper, and sponge pad were assembled in order in the membrane transfer device. Membrane transfer was performed in a fast membrane transfer buffer at a constant current (400 mA) to transfer the proteins from the gel to the membrane.

[0171] 4. Blocking Put the membrane into blocking buffer, incubate at room temperature for 15 min to block the non-specific binding sites on the membrane.

[0172] 5. Primary antibody incubation: Put the membrane into the diluted primary antibody solution, incubate at 4℃ overnight or at room temperature for 1-2 hours. Then wash the membrane with TBST buffer for 3 times, PBS for once, each for 7 minutes to remove the unbound primary antibody.

[0173] 6. Secondary antibody incubation: Put the membrane into the diluted secondary antibody solution (skim milk dilution), incubate at room temperature for 1 hour. Then wash the membrane with TBST buffer for 3 times, PBS for once, each for 7 minutes to remove the unbound primary antibody.

[0174] 7. Detection: Add the ECL chemiluminescence substrate evenly to the membrane, use the chemiluminescence imaging system to detect the signal. Adjust the exposure time according to the signal intensity to obtain the protein band image.

[0175] Results: As shown in Figure 3 , both M30903-1C3 and M30903-2G1 antibodies showed strong binding ability to IL-9.

[0176] Example 3 Flow detection of the effect of IL-9 monoclonal antibody on THP-1 cell (human monocytic leukemia cell line) M1 / M2 polarization.

[0177] 1. Cell preparation: THP-1 cells were inoculated in culture plates, and 0.1 μl of PMA was added to each 1 ml of RPMI-1640 medium containing 10% FBS to induce M0 state for 48 hours. M1 polarization inducer (LPS: 100 ng / mL, IFN-γ: 20 ng / mL) and M2 polarization inducer (IL-4: 20 ng / mL) were added to the M0 state THP-1 cells. Incubate in a 37℃, 5% CO2 incubator for 24-48 hours. The cells are polarized into M1 type macrophages and M2 type macrophages.

[0178] 2. Cell staining

[0179] 2.1 Surface marker staining: The cell suspension was divided into ep tubes, about 100 μL per tube. Add CD86, CD206 fluorescently labeled antibodies, mix well and incubate in the dark, 4℃ for 30 minutes. Add 1-2 mL of PBS buffer, centrifuge (1200-1500 rpm, 5 minutes), and discard the supernatant.

[0180] 2.2 Intracellular protein staining: After surface staining, fix the cells with a fixative and incubate at 4℃ for 15 minutes. Add a membrane breaker and incubate at room temperature for 10 minutes. Add CD68 fluorescently labeled antibody, incubate in the dark, 4℃ for 30 minutes.

[0181] 3. Machine detection and analysis: Turn on the flow cytometer, warm up the laser and calibrate the instrument. Load the sample into the machine, collect data. Then analyze the data using flow data analysis software (e.g. FlowJo).

[0182] Results: M30903-2G1 affected both M1 / M2 polarization of THP-1 cells.

[0183] Example 4. Transwell assay to detect the effect of IL-9 monoclonal antibody on the migration ability of FLS.

[0184] 1. Cell preparation: Digest and collect cells, wash with PBS for 1-2 times.

[0185] 2. Cell inoculation: Place the Transwell chamber into a 24-well plate. Add medium containing 10% FBS into the lower chamber (inside the 24-well plate) as a chemoattractant, usually 600 μL in volume. Add cell suspension into the upper chamber, usually 100-200 μL in volume. Gently shake the chamber to distribute the cells evenly.

[0186] 3. Cell culture: Place the 24-well plate into a 37°C, 5% CO2 incubator for 24 hours.

[0187] 4. Fixation and staining: Remove the Transwell chamber, gently wash with PBS for 2 times to remove non-migrated or non-invasive cells.

[0188] 4.1 Fix the cells: Place the chamber into 4% paraformaldehyde for 20-30 minutes at room temperature.

[0189] 4.2 Staining: Place the chamber into 0.1% crystal violet solution for 20 minutes at room temperature, then wash with PBS.

[0190] 5. Observation and counting: Remove the chamber membrane from the Transwell chamber and place it on a glass slide. Observe and take pictures using a microscope. Randomly select multiple fields of view (usually at least 5) and count the number of migrated or invasive cells.

[0191] Results: Only M30903-2G1 inhibited the migration of RA-FLS (fibroblast-like synoviocytes from rheumatoid arthritis (RA) patients) in an inflammatory environment, while M30903-1C3 had no effect.

Claims

1. An anti-human IL-9 protein rabbit monoclonal antibody, characterized in that: It is composed of a heavy chain and a light chain, wherein: the amino acid sequence of the heavy chain is shown in SEQ ID NO.1; the amino acid sequence of the light chain is shown in SEQ ID NO.

2.

2. The anti-human IL-9 protein rabbit monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the antibody is shown in SEQ ID NO.

3.

3. Use of the anti-human IL-9 protein rabbit monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating IL-9 related diseases, wherein the disease is rheumatoid arthritis.

4. Use of the anti-human IL-9 protein rabbit monoclonal antibody according to claim 1 or 2 in the preparation of a reagent for detecting IL-9.

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