Anti-human IL-9 protein rabbit monoclonal antibody and application thereof

Abstract

The invention belongs to 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 (Sequence-Specific Nucleic Acid Monoclonal Growth); the CDR2 is VITSSGTIFYTSWAKG, and the CDR2 is The CDR3 is GGYDGVGGDYAFLGI, and the CDR3 is GGYDGV The CDR1 is QASQSIGSSLS (Quantitative Associated The CDR2 is EASTLAS (Ethylene-Activated Staphylococcus And the CDR3 is QSYYYSGISSYAFT, and the CDR3 is The invention provides a rabbit monoclonal antibody capable of specifically recognizing and combining with human IL-9 protein, which is used for immunodetection, biomedical research and research aiming at an IL-9 signal channel, and has high specificity and affinity.

Description

Technical Field

[0001] The present invention belongs to IL-9 protein, and specifically relates to anti-human IL-9 protein rabbit monoclonal antibody and application thereof. Background Art

[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 producing the largest amount. It is a 14kD glycoprotein consisting of 144 amino acids and an 18 amino acid signal peptide. IL-9R, the receptor for interleukin-9 (IL-9), is located on the cell membrane and is part of a family of cytokines that share a common γ chain receptor, including IL-2, IL-4, IL-7, IL-15, and IL-21. The IL-9 receptor consists of IL-9Rα in addition to the common γ chain.

[0003] As an important cytokine, IL-9 has multiple biological functions. It can maintain the long-term growth of TH cells in the absence of 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), a characteristic that suggests that it may be involved in the regulation of the hematopoietic process. 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 new perspectives and targets for the research and treatment of related diseases.

[0004] In monoclonal antibody technology, rabbit monoclonal antibodies have attracted much attention due to their high affinity and specificity. However, existing technical solutions such as mouse-rabbit heterologous hybridoma and rabbit-rabbit hybridoma methods have stability problems, and hybridomas are prone to lose antibody genes, resulting in unstable antibody production. In addition, although rabbit monoclonal antibodies have advantages, it is difficult to ensure that all antibodies have ideal affinity and specificity with existing technology, which may be related to the diversity of antibody libraries and screening methods. The preparation process involves complex cell culture, gene manipulation and screening steps, which is technically difficult, and the implementation of key technologies is also challenging. Therefore, the preparation of rabbit monoclonal antibodies still needs further improvement and optimization. Summary of the invention

[0005] Purpose of the invention:

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

[0007] Technical Solution

[0008] The anti-human IL-9 protein rabbit monoclonal antibody is characterized in that it includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is: CDR1 is SSNAMG; CDR2 is VITSSGTIFYTSWAKG; CDR3 is GGYDGVGGDYAFLGI; the amino acid sequence of the light chain variable region is: CDR1 is QASQSIGSSLS; CDR2 is EASTLAS; CDR3 is QSYYYSGISSYAFT.

[0009] The anti-human IL-9 protein rabbit monoclonal antibody is characterized in that the amino acid sequence of its heavy chain is shown in SEQ ID NO.1; and the amino acid sequence of its 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 of the anti-human IL-9 protein rabbit monoclonal antibody in the preparation of drugs for treating IL-9 related diseases.

[0012] The application is characterized in that the diseases include asthma, allergic diseases, inflammatory bowel disease, autoimmune diseases, cancer and parasitic infections.

[0013] The anti-human IL-9 protein rabbit monoclonal antibody is used in the preparation of a reagent for detecting IL-9.

[0014] Beneficial Effects

[0015] The present invention is committed to developing rabbit monoclonal antibodies specific for human IL-9. The sequence of the monoclonal antibody is a completely new sequence and has not been reported in the literature. Specifically: through a carefully designed immunization program, and through affinity experiments, high-affinity, high-titer antibodies M30903-1C3 and M30903-2G1 were screened. The antibodies show specificity and affinity for IL-9, which provides a powerful tool for in-depth research on the IL-9 / IL-9R signaling pathway, and also opens up new possibilities for the diagnosis and treatment of IL-related diseases. Among them, the antibody M30903-2G1 has effects on rheumatoid arthritis and leukemia. IL-9 plays an important role in a variety of diseases such as asthma, allergic diseases, inflammatory bowel disease, autoimmune diseases, cancer and parasitic infections. As an IL-9 monoclonal antibody, inhibiting IL-9 may become a potential strategy for the treatment of these diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of antigen IL-9 protein expression and purification;

[0017] Figure 2The antibody purification results are shown in Figure 1, where A is M30903-1C3; B is M30903-2G1;

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

[0019] Figure 4 Flow cytometry analysis of the effect of IL-9 monoclonal antibody on M1 / M2 polarization of THP-1 cells; A. The inhibitory effect of IL-9 antibody on IL-9; B. The effect of IL-9 antibody on M1 polarization of macrophages; C. The effect of IL-9 antibody on M2 polarization of macrophages

[0020] Figure 5 Transwell was used to detect the effect of IL-9 monoclonal antibody on the migration ability of synovial fibroblasts from RA patients. 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 the 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 sequences of the heavy chain: CDR1 is SSNAMG (such as SEQ ID NO.4); CDR2 is VITSSGTIFYTSWAKG (such as SEQ ID NO.5), and CDR3 is GGYDGVGGDYAFLGI (such as SEQ ID NO.6);

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

[0035] 2. Experimental Procedure

[0036] 2.1 Antigen preparation

[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] Electrophoresis detection and recovery of amplified fragments

[0044] 2.2 Enzyme digestion of vector and target gene

[0045]

[0046] 37℃1-2h; electrophoresis detection and recovery of enzyme digestion products

[0047] 23. Ligation of vector and target gene

[0048]

[0049] 16℃, 0.5-1h

[0050] 2.4 Conversion

[0051] (1) Add the DNA fragment to be transformed into the tube containing TOP10 competent cells (50 μL competent cells require 25 ng DNA). The volume should not exceed 5% of the competent cells. Gently swirl several times to mix the contents and place on ice for 30 minutes.

[0052] (2) Place the mixture in the centrifuge tube in circulating water heated to 42°C and heat shock for 90 seconds without shaking the tube.

[0053] (3) Quickly transfer the tube to an ice bath and allow the cells to cool for 1 to 2 minutes.

[0054] (4) Add 200 μL SOC liquid culture medium to each tube, warm the medium to 37°C using a water bath, and then transfer the tube to a shaker set at 37°C and culture 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 an appropriate volume (up to 200 μL per 90 mm plate) of the transformed competent cells to LB medium containing the corresponding antibiotics.

[0056] (6) Invert the plate and incubate at 37°C. Bacterial plaques will appear after 12 to 16 hours.

[0057] 2.5 Colony PCR verification

[0058] When colonies grow on the plate, randomly pick several of them and perform colony PCR verification to detect transformants.

[0059] 2.6 Sequencing verification

[0060] The positive clones were sent to our company's sequencing platform for sequencing verification.

[0061] 3. Protein Expression

[0062] The mammalian HEK293 cell expression system was used for expression testing. The target gene IL9-His was transfected into HEK293 cells for protein expression and purification testing.

[0063] 3.1 Expression conditions

[0064] -HEK293 cell density: 1.5-2.0x10 6 Pieces / mL

[0065] -Cultivation temperature: 37℃

[0066] -Cultivation speed: 130rmp / min

[0067] -Cultivation sampling time: Day 6

[0068] 3.2 Purification scheme

[0069] - Collect the culture medium supernatant, centrifuge at high speed, filter through 0.22um filter membrane, and separate the supernatant with Ni 2+ Purify the target protein by resin binding.

[0070] -Resin: Ni 2+ Resin.

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

[0072] -Washing and elution solutions:

[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 0.22um filter membrane, and combine the supernatant with Protein A resin to purify the target protein.

[0076] -Resin: Protein A resin.

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

[0078] -Washing and elution solutions:

[0079] *Wash: PBS, pH=7.4

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

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

[0082] 3.3 Test Results

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

[0084] Table 1 shows the results of IL9-His detection

[0085]

[0086] 4. Animal immunization

[0087] 4.1 Treatment

[0088] The primary immunization antigen is the protein antigen IL9-His which is mixed and emulsified with an equal volume of Freund's complete adjuvant. The secondary, tertiary, quadruple, penta- and hexa-immunization 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 and 2.1 kg, were selected.

[0091] 4.2 Immunity

[0092] First immunization: The antigen used for immunization is Freund's complete adjuvant + immunogen;

[0093] Second immunization: The antigen used for immunization is Freund's incomplete adjuvant + immunogen;

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

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

[0096] Take a small amount of blood for ELISA test; take 1mL of antiserum for ELISA test

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

[0098] Final release: whole blood was collected from the carotid artery, and serum was collected and cryopreserved after standing at 4°C overnight. Spleen cells were collected and cryopreserved in liquid nitrogen.

[0099] Immunity periodic table:

[0100] First immunization: The immunization antigen is Freund's complete adjuvant + protein antigen 0.5 mg;

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

[0102] Three immunizations: The antigen used for immunization is Freund's incomplete adjuvant + protein antigen 0.25 mg;

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

[0104] Take a small amount of blood for ELISA test; take 1mL of antiserum for ELISA test

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

[0106] Spleen removal from living rabbits

[0107] 5. Indirect ELISA test

[0108] 5.1 Coating Antigen

[0109] The antigen was diluted to 2 ug / mL with 0.05 mol / L carbonate (PH=9.6), 100 ul / well, and incubated at 4°C overnight.

[0110] 5.2 Washing plate

[0111] The plate was taken out and washed three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0112] 5.3 Closure

[0113] 150 μL of blocking solution (5% skim milk powder (PBST)) was added to each well and blocked at 37°C for 60 minutes.

[0114] 5.4 Washing 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 diluted in multiples, and incubated at 37°C for 1 hour.

[0118] 5.6 Washing 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-labeled goat anti-rabbit IgG (H+L), catalog number: 116154, antiserum company: Jackson. 1:8000 dilution, incubate at 37℃ 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 an enzyme 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 a volume of 50 mL / 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 the 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, add culture medium and culture them. After 15 days of culture, take the supernatant for Elisa test.

[0134] 8. Experimental Results

[0135] 8.1 Single B cell screening results

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

[0137] Table 2: IL9 96-well plate supernatant Elisa validation results (TBM colorimetric)

[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] Among them: G2: M30903-2G1; H11: negative control; H12: positive control (rabbit polyclonal antibody serum)

[0140] Table 3: IL9 96-well plate supernatant Elisa validation results (TBM colorimetric)

[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] Among them: C1: M30903-1C3; H11: negative control; H12: positive control (rabbit polyclonal antibody serum)

[0143] 8.2 Single B Cell Sequencing

[0144] After RNA is extracted from single B cells, it is reverse transcribed and the heavy and light chain sequences are amplified and constructed into an expression vector for expressing recombinant antibodies.

[0145] 9. Recombinant Antibody Expression

[0146] 9.1 Expression and purification test

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

[0148] 9.2 Expression Conditions

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

[0150] -Cultivation temperature: 37℃

[0151] -Cultivation speed: 130rmp / min

[0152] -Cultivation sampling time: Day 6

[0153] 9.3 Purification scheme

[0154] - Collect the culture medium supernatant, centrifuge at high speed, filter through 0.22urn filter membrane, and combine the supernatant 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 solutions:

[0158] *Wash: PBS, pH=7.4

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

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

[0161] 9.4 Test Results

[0162] The purification results are as follows Figure 2 As shown in Table 4: The titer of the purified antibody was tested by ELISA, and the titer was still 0.338 at 128K. The antibody titer was estimated to be higher than 512K.

[0163] Table 4: Recombinant antibody expression results

[0164]

[0165]

[0166] Note: Coat with 100 ng / porin antigen.

[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 concentration of each sample was consistent. After the concentration was measured, the protein sample was mixed with the loading buffer and boiled for 5-10 minutes to denature the protein.

[0169] 2. SDS-PAGE electrophoresis: Prepare 10% SDS-polyacrylamide gel, add the treated protein sample into the gel well, and add protein molecular weight markers. Perform electrophoresis at a constant voltage (150V) in a fast electrophoresis buffer to separate proteins according to their molecular weight.

[0170] 3. Transfer: Assemble the gel, PVDF membrane, filter paper and sponge pad into the transfer device in order. Transfer the membrane at a constant current (400mA) in the fast transfer buffer to transfer the protein from the gel to the membrane.

[0171] 4. Blocking Place the membrane in blocking buffer and incubate at room temperature for 15 minutes to block nonspecific binding sites on the membrane.

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

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

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

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

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

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

[0178] 2. Cell Staining

[0179] 2.1 Surface marker staining: Dispense the cell suspension into eppendorf tubes, about 100 μL per tube. Add CD86 and CD206 fluorescent labeled antibodies, mix well and incubate in the dark at 4°C 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°C for 15 minutes. Add a membrane permeabilizer and incubate at room temperature for 10 minutes. Add CD68 fluorescently labeled antibody and incubate at 4°C for 30 minutes in the dark.

[0181] 3. On-machine detection and analysis: Turn on the flow cytometer, preheat the laser and calibrate the instrument. Load the sample onto the machine and collect data. Then use flow cytometry data analysis software (such as FlowJo) to analyze the data.

[0182] Results: M30903-2G1 had effects on 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 and wash 1-2 times with PBS.

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

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

[0187] 4. Fixation and staining: Remove the Transwell chamber and gently wash twice with PBS to remove non-migrated or non-invaded cells.

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

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

[0190] 5. Observation and counting: Remove the chamber membrane from the Transwell chamber and place it on a 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 invaded cells.

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

Claims

1. Anti-human IL-9 protein rabbit monoclonal antibody, characterized in that: It includes a heavy chain variable region and a light chain variable region, The amino acid sequence of the heavy chain variable region is as follows: CDR1 is SSNAMG, as shown in SEQ ID NO.4; CDR2 is VITSSGTIFYTSWAKG, as shown in SEQ ID NO.5; CDR3 is GGYDGVGGDYAFLGI, as shown in SEQ ID NO.6; CDR1 is QASQSIGSSLS, as shown in SEQ ID NO.7; CDR2 is EASTLAS, as shown in SEQ ID NO.8; CDR3 is QSYYYSGISSYAFT, as shown in SEQ ID NO.

9.

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

2.

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

3.

4. Use of the anti-human IL-9 protein rabbit monoclonal antibody according to any one of claims 1 to 3 in the preparation of drugs for treating IL-9 related diseases.

5. The use according to claim 4, characterized in that: Such diseases include asthma, allergic diseases, inflammatory bowel disease, autoimmune diseases, cancer and parasitic infections.

6. Use of the anti-human IL-9 protein rabbit monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent for detecting IL-9.

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