A neutralizing mouse monoclonal antibody specifically recognizing IL-24 and its use

Abstract

The application discloses a neutralizing mouse monoclonal antibody specifically recognizing IL-24 and an application thereof, and relates to the technical field of biology. The neutralizing mouse monoclonal antibody comprises a VH CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, a VH CDR2 with an amino acid sequence as shown in SEQ ID NO. 7, a VH CDR3 with an amino acid sequence as shown in SEQ ID NO. 8, a VL CDR1 with an amino acid sequence as shown in SEQ ID NO. 9, a VL CDR2 with an amino acid sequence of WAS and a VL CDR3 with an amino acid sequence as shown in SEQ ID NO. 10. The neutralizing mouse monoclonal antibody provided by the application can specifically recognize IL-24, and provides a reliable scientific research tool antibody for mechanism research and target verification of IL-24 related diseases such as inflammation and fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a neutralizing murine monoclonal antibody that specifically recognizes IL-24 and its applications. Background Technology

[0002] Interleukin-24 (IL-24) is a multifunctional cytokine belonging to the IL-10 family. It is primarily secreted by activated fibroblasts, T cells, and macrophages. By binding to two receptor complexes, IL20RA / IL20RB and IL20RB / IL22RA1, it activates downstream signaling pathways such as JAK1-STAT3 and MAPK, participating in multiple physiological and pathological processes including immune regulation, inflammatory responses, tissue repair, and tumorigenesis and development. Studies have confirmed that IL-24 can induce a type I interferon response and exert antiviral effects in influenza virus infection. It also mediates pro-inflammatory effects in autoimmune diseases and fibrotic diseases, making it an important target for research on the mechanisms of immune diseases and potential interventions.

[0003] Current research tools targeting mouse IL-24 have significant shortcomings: commercially available antibodies are mainly polyclonal, exhibiting large batch-to-batch variations, insufficient specificity, and a tendency to cross-react with cytokines in the same family as IL-20; the few reported monoclonal antibodies are mostly only applicable to detection scenarios such as immunoblotting and ELISA, and neutralizing antibodies with definite receptor-blocking activity are extremely scarce, generally suffering from low affinity and unstable in vivo neutralization effects, severely limiting the progress of in vitro and in vivo functional studies of IL-24 and the validation of its disease target. Therefore, obtaining high-affinity, high-specificity, and definite neutralizing monoclonal antibodies against mouse IL-24 has significant scientific research value and application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a neutralizing murine monoclonal antibody that specifically recognizes IL-24 and its applications, thereby addressing the problems existing in the prior art. The neutralizing murine monoclonal antibody provided by this invention can specifically recognize IL-24, filling the technical shortcomings of insufficient affinity and poor specificity of existing anti-mouse IL-24 neutralizing antibodies. It can be used to construct IL-24 detection products and as an IL-24 activity inhibitor, providing a reliable research tool antibody for the study of the mechanisms and target validation of IL-24-related inflammation, fibrosis, and other diseases.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a neutralizing murine monoclonal antibody that specifically recognizes IL-24, comprising VH CDR1 with the amino acid sequence shown in SEQ ID NO.6, VH CDR2 with the amino acid sequence shown in SEQ ID NO.7, VH CDR3 with the amino acid sequence shown in SEQ ID NO.8, VL CDR1 with the amino acid sequence shown in SEQ ID NO.9, VLCDR2 with the amino acid sequence WAS, and VL CDR3 with the amino acid sequence shown in SEQ ID NO.10.

[0006] Furthermore, the amino acid sequence of the heavy chain variable region of the neutralizing murine monoclonal antibody is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3.

[0007] The present invention also provides a gene encoding the above-mentioned neutralizing murine monoclonal antibody.

[0008] Furthermore, the coding gene includes a light chain variable region coding gene and a heavy chain variable region coding gene; The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.4; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.5.

[0009] The present invention also provides a recombinant expression vector comprising the above-described coding gene.

[0010] The present invention also provides a recombinant non-plant host cell, comprising the above-described recombinant expression vector.

[0011] The present invention also provides the use of the above-mentioned encoding gene, recombinant expression vector or recombinant non-plant host cell in the preparation of the above-mentioned neutralizing murine monoclonal antibody.

[0012] The present invention also provides the use of the above-mentioned neutralizing murine monoclonal antibody in (1) or (2): (1) Preparation of detection products for mouse-derived IL-24; (2) Preparation of an active inhibitor of mouse IL-24.

[0013] The present invention also provides a detection product for murine IL-24, comprising the above-mentioned neutralizing murine monoclonal antibody.

[0014] The present invention also provides an activity inhibitor of murine IL-24, the active ingredient of which includes the above-mentioned neutralizing murine monoclonal antibody.

[0015] The present invention discloses the following technical effects: This invention yields a neutralizing murine monoclonal antibody, 1A6-13-8, that specifically recognizes murine IL-24. ELISA verification showed that this antibody specifically binds to murine IL-24 and exhibits no cross-reactivity with IL-20 and IL-22, demonstrating excellent specificity. Affinity assays showed that the antibody possesses high binding activity and strong binding capacity. Receptor competition ELISA confirmed that this antibody can dose-dependently block the binding of IL-24 to the receptor complex, achieving an inhibition rate of 89.4% at a concentration of 2 μg / mL, demonstrating outstanding neutralizing activity at the molecular level.

[0016] Cellular function experiments showed that the antibody itself is non-cytotoxic and does not directly affect fibroblast proliferation; it can effectively neutralize the IL-24-induced fibroblast proliferation-promoting effect, bringing cell proliferation levels back to near the basal state, and exerting a clear neutralizing and blocking effect at the cellular level.

[0017] This invention fills the technical gaps in existing anti-mouse IL-24 neutralizing antibodies, which suffer from insufficient affinity and poor specificity. It can be used to construct IL-24 detection products and as an IL-24 activity inhibitor, providing a reliable scientific research tool antibody for mechanism research and target validation of IL-24-related inflammation, fibrosis and other diseases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The graph shows the results of affinity verification for different antibodies; Figure 2 The image shows the CCK-8 assay results of the effect of 1A6-13-8 on the proliferation capacity of fibroblasts. Figure 3 This is an immunofluorescence staining observation of the effect of 1A6-13-8 on the proliferation capacity of fibroblasts. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 1. Preparation of recombinant IL-24 immunogen The 27-160 aa region of mouse IL-24 protein was selected as the antigenic region, which covers the core epitope of IL-24 binding to the receptor. The amino acid sequence is: QEFRSGSCQVTGVVLPELWEAFWTVKNTVQTQDDITSIRLLKPQVLRNVSGAESCYLAHSLLKFYLNTVFKNYHSKIAKFKVLRSFSTLANNFIVIMSQLQPSKDNSMLPISESAHQRFLLFRRAFKQLDTEVA (SEQ ID NO.1). The coding gene was codon optimized using E. coli, and the recombinant plasmid was constructed into the His-tagged prokaryotic expression vector pSmart-I (Navigation; V013438).

[0026] The recombinant plasmid was transformed into E. coli Rosetta (DE3) competent cells, and single colonies were picked and inoculated into LB medium containing kanamycin and cultured at 37°C and 220 rpm until OD500. 600 =0.4~0.6; Add IPTG to a final concentration of 1.0 mM, induce at 37℃ for 4 h; collect bacterial samples for SDS-PAGE detection, and the target protein was successfully expressed.

[0027] The recombinant strain was transferred to 400 mL LB medium at a 1% inoculum. After IPTG induction, the bacterial cells were collected by centrifugation at 4500 rpm, resuspended in 1×PBS, and then sonicated on ice (3 s sonication, 3 s pause, 100 times). The cells were then centrifuged at 12000 rpm and 4℃ for 15 min, the supernatant was discarded, and the precipitate was washed three times with 1×PBS. The precipitate was dissolved in denaturing buffer containing 8 M urea, incubated at room temperature for 1 h, and then centrifuged to collect the supernatant, which was the inclusion body protein solution. SDS-PAGE analysis showed that the target protein was mainly distributed in inclusion bodies.

[0028] Purification was performed using a Ni-Smart affinity chromatography column. The column was equilibrated with deionized water and denaturation buffer in sequence. After loading the sample, impurities were removed with washing buffer containing 10 mM imidazole, and the target protein was eluted with elution buffer containing 100 mM imidazole. The elution peak was collected, and the purity of the target protein after purification was ≥85%.

[0029] The eluent was placed in a dialysis bag and refolded by a 6 M→4 M→3 M→2 M→1 M→0 M urea gradient, with each stage lasting 2 h. The solution was then replaced with storage buffer (1×PBS, 1 mM DTT, 0.1 M arginine, 0.25 M NaCl, 10% glycerol, pH 7.4). After ultrafiltration and concentration, recombinant IL-24 protein was obtained. The protein concentration was determined to be 0.8 mg / mL using the BCA method. The protein was then aliquoted and stored at -80°C.

[0030] 2. Evaluation of animal immunization and serum titer Five 6-week-old female Balb / c mice, numbered MS11644-1 to MS11644-5, were selected. For the initial immunization, recombinant IL-24 protein was diluted to 0.5 mg / mL, emulsified thoroughly with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple sites on the back at a dose of 50 μg / mouse. Subsequent booster immunizations were administered every 2 weeks, using Freund's incomplete adjuvant emulsification at the same dose as the initial immunization.

[0031] Serum titer assay: Blood was collected from the orbital rimus on day 7 after the fourth immunization, and serum was separated. The titer was determined using an indirect ELISA method: 96-well microplates were coated with 1 μg / mL IL-24 protein and incubated overnight at 4°C; blocked with 1% BSA-PBST at 37°C for 1 h; serially diluted mouse serum (1:250 to 1:1024000) was added, and incubation was performed at 37°C for 30 min; after washing with PBST, 1:20000 diluted HRP-goat anti-mouse secondary antibody was added, and incubation was performed at 37°C for 45 min; TMB color development was performed for 10 min, and the assay was terminated with 1 M sulfuric acid. OD values ​​were then read. 450 value.

[0032] All five mice produced high-titer antibodies, and the OD value was still significantly higher than that of the negative control (OD≈0.03 in the PBS group) when diluted to 1:1024000. Among them, the MS11644-2 mouse had the best titer and was selected for shock immunization and cell fusion.

[0033] 3. Cell fusion and hybridoma screening Shock immunization: Three days before fusion, MS11644-2 mice were subjected to intraperitoneal and tail vein shock immunization with 100 μg of adjuvant-free IL-24 protein.

[0034] Cell fusion: Mouse spleens were aseptically harvested and ground to prepare a spleen cell suspension; mixed with logarithmically growing SP2 / 0 myeloma cells at a ratio of 5:1, centrifuged at 1200 rpm for 3 min and the supernatant was discarded; 1 mL of 50% PEG-1500 was added dropwise over 1 min in a 37°C water bath and allowed to stand for 30 s; fusion was terminated by slowly adding IMDM medium, centrifuged and resuspended in HAT complete medium, seeded into 96-well plates containing feeder cells and cultured at 37°C with 5% CO2.

[0035] Primary and secondary screening: HT medium was replaced on day 7 after fusion, and indirect ELISA was used for primary screening on day 8, yielding a total of 34 positive clones. The positive clones from the primary screening were then validated through secondary screening, and 13 positive hybridomas with strong binding signals and good cell condition were selected for monoclonalization.

[0036] 4. Hybridoma cell monoclonalization Two rounds of subcloning were performed using the limiting dilution method: First subcloning: Positive hybridomas were serially diluted and seeded into 96-well plates at 50, 10, and 1 cells per well, and cultured in HT medium. After clone formation, the supernatant was collected, and binding activity and receptor competitive activity were detected simultaneously. Positive single clones were selected for the second subcloning.

[0037] Second subcloning: Limiting dilution was repeated to ensure <1 cell per well; supernatant activity was assessed again after culture, ultimately yielding 12 stable monoclonal hybridoma cell lines secreting antibodies from 6 sources. The binding activities of the 12 clones were compared, and the results are as follows: Figure 1 As shown, strain 1A6-13-8 had the highest OD value in its supernatant and the best cell growth status, and was selected as the core cell line for expanded culture and cryopreservation.

[0038] Example 2 Monoclonal antibody variable region sequencing: 1A6-13-8 hybridoma cells were collected, total RNA was extracted using the Trizol method, and cDNA was synthesized by reverse transcription. Using cDNA as a template, gene fragments of the heavy chain variable region (VH) and light chain variable region (VL) were amplified by PCR using mouse antibody variable region universal primers.

[0039] The PCR product was cloned into a T vector, transformed into E. coli, and positive clones were selected for sequencing. Bioinformatics analysis confirmed that the sequence was a functional antibody variable region gene, with no stop codon and the correct reading frame.

[0040] The neutralizing monoclonal antibody secreted by the 1A6-13-8 cell line has the following amino acid sequence in the heavy chain variable region: EVQLQQSGPELVKPGTSVKVSCKASGYSFTDYNIYWVKQSHGKSLEWIGYIDPYNDDTSYNQKFKGKATLTVDKSSSTAFMHLNSLTSEDSAVYYCTRWEITDPWYFDVWGAGTTVTVSS (SEQ ID NO.2).

[0041] The amino acid sequence of the light chain variable region is: DIVMTQSHKFMSISVGDRASIMCKASQDVGTAVSWYQQKPGQSPKALTYWASRRHTGVPDRFTGSGSGTDFTLIIGNVQSEDLAAYFCQQYSSYPYTFGGGTKLEIK (SEQ ID NO.3); The nucleotide sequence of the gene encoding the heavy chain variable region is: GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGACTTCAGTGAAGGTATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATTTACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTACAATGATGATACT AGTTATAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACGGCCTTCATGCATCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTATTGTACAAGATGGGAGATTACGGACCCTTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG (SEQ ID NO.4); The nucleotide sequence of the gene encoding the light chain variable region is: GACATTGTGATGACCCAGTCTCACAAATTCATGTCCATATCAGTAGGAGACAGGCCAGCATTATGTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTATCATGGTATCAACAGAAACCAGGACAATCTCCTAAAGCACTGACCTACTGGGCAT CCCGCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCATCATTGGCAATGTGCAGTCTGAAGACTTGGCAGCTTATTTCTGTCAGCAATATAGCAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC (SEQ ID NO.5).

[0042] In the heavy chain variable region, VH CDR1-3 are as follows: VH CDR1: GYSFTDYN (SEQ ID NO.6); VH CDR2: IDPYNDDT (SEQ ID NO.7) VH CDR3: TRWEITDPWYFDV (SEQ ID NO. 8).

[0043] In the variable region of the light chain, VL CDR1-3 are as follows: VL CDR1: QDVGTA (SEQ ID NO.9); VL CDR2: WAS; VL CDR3: QQYSSYPYT (SEQ ID NO.10) Example 3 Antibody specific cross-reactivity verification: Coating: Recombinant mouse IL-24, recombinant mouse IL-20, and recombinant mouse IL-22 proteins (MCE) were diluted to 100 ng / mL with carbonate coating buffer. 100 μL of each protein was added to a 96-well microplate and coated overnight at 4°C. Blank wells (coating buffer only) were set up as blank controls.

[0044] Blocking: Discard the coating solution, wash 3 times with PBST; add 200 μL of 1% BSA-PBST to each well, and block at 37℃ for 1 h.

[0045] Antibody incubation: Discard the blocking buffer and wash three times with PBST; add 100 μL of the purified 1A6-13-8 monoclonal antibody in a gradient of 1 μg / mL, 0.2 μg / mL, and 0.04 μg / mL to each well and incubate at 37°C for 30 min; set up a negative control group: use unrelated isotype mouse IgG antibodies for gradient incubation at the same concentration.

[0046] Secondary antibody incubation: Wash 5 times with PBST; add 1:20000 diluted HRP-labeled goat anti-mouse IgG secondary antibody, 100 μL / well, and incubate at 37℃ for 45 min.

[0047] Colorimetric termination: Wash 5 times with PBST; add 100 μL of TMB colorimetric solution and incubate at room temperature in the dark for 10 min; add 50 μL of 1 MH2SO4 to terminate the reaction, and read the OD value using a microplate reader. 450 Absorbance value.

[0048] Experimental results showed that at a working antibody concentration of 1 μg / mL, the 1A6-13-8 antibody coated mouse IL-24 wells OD 450 The OD values ​​reached 1.42±0.08; while those of mice coated with IL-20 and IL-22 were 1.42±0.08. 450 The values ​​were 0.11±0.03 and 0.09±0.02, respectively, which were not statistically different from the OD values ​​of the blank control well (0.08±0.02).

[0049] The results showed that the 1A6-13-8 monoclonal antibody of the present invention can specifically recognize mouse IL-24 protein, and there is no obvious cross-reactivity with mouse IL-20 and mouse IL-22, which are homologous to the IL-10 family, indicating good antibody specificity.

[0050] Example 4 Receptor competition ELISA for quantitative detection of antibody neutralizing activity: ELISA plate coating: Recombinant mouse IL-24 protein was diluted to 100 ng / mL, 100 μL / well, and coated onto the ELISA plate overnight at 4°C.

[0051] Blocking: Wash plate with PBST, then block with 1% BSA-PBST at 37°C for 1 h.

[0052] Antibody gradient pre-incubation: Purified 1A6-13-8 monoclonal antibody was serially diluted to the following concentrations: 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, and 0 μg / mL. Isotype-independent mouse IgG was used as a negative control. Different concentrations of 1A6-13-8 monoclonal antibody were pre-mixed with a fixed concentration of recombinant IL20RA-IL20RB receptor fusion protein and pre-incubated at 37°C for 30 min.

[0053] Competitive binding: Add the pre-incubation mixture to the wells of the blocked ELISA plate, 100 μL / well, and incubate at 37°C for 1 h.

[0054] Detection: Wash plate thoroughly with PBST; add biotin-labeled antibody against receptor fusion protein, incubate, then add streptavidin-HRP; develop with TMB, stop with sulfuric acid, and read OD. 450 .

[0055] Using the 0 μg / mL antibody group as the maximum binding signal, the receptor binding inhibition rate at each antibody concentration was calculated; the dose-inhibition curve was fitted using GraphPad Prism, and the half-inhibition concentration (IC50) was calculated. 50 .

[0056] Inhibition rate (%) = (1 - OD value of experimental group / OD value of antibody control group) × 100% Experimental results showed that the 1A6-13-8 antibody exhibited a dose-dependent blocking effect on IL-24 / receptor binding: as the antibody concentration increased, the inhibition rate of IL-24 binding to the receptor gradually increased; when the antibody concentration reached 2 μg / mL, the receptor binding inhibition rate reached 89.4%. The negative control, the same subtype IgG, showed almost no inhibitory effect on IL-24 binding to the receptor at all tested concentrations, with inhibition rates all below 8%.

[0057] The results showed that the 1A6-13-8 monoclonal antibody can competitively block the interaction between IL-24 and its receptor complex at the molecular level, and has strong in vitro neutralizing activity.

[0058] Example 5 Previous studies have shown that IL-24 significantly enhances the proliferation of fibroblasts. In this invention, mouse fibroblasts were stimulated with recombinant IL-24 protein (MCE) for one day, while 1A6-13-8 was added for neutralization. The specific experimental method is as follows: Cell seeding: Primary mouse fibroblasts were seeded into cell culture plates at a density of 5 × 10⁶ cells / year. 3 Cells / well, cultured in DMEM medium containing 10% fetal bovine serum overnight at 37°C with 5% CO2, until cells adhere.

[0059] Group settings, a total of 4 groups: Group 1: Blank control group, basal culture medium only, without IL-24, without 1A6-13-8 monoclonal antibody; Group 2: Antibody alone: ​​1A6-13-8 monoclonal antibody was added to a final concentration of 2 μg / mL, without IL-24 recombinant protein; Group 3: IL-24 stimulation group: Added with mouse recombinant IL-24 protein at a final concentration of 100 ng / mL; Group 4: Neutralization experimental group: IL-24 protein at a final concentration of 100 ng / mL + 1A6-13-8 monoclonal antibody at a final concentration of 2 μg / mL were added simultaneously; Each group was set up with 3 replicates. The IL-24 protein and antibody were pre-incubated at 37°C for 30 min before being added to the cell wells.

[0060] Cell culture: Cells in each group were cultured at 37℃ and 5% CO2 for 24 h.

[0061] Detection: The number of viable cells was detected using a CCK-8 assay kit, and the OD was measured. 450 Cell proliferation levels were statistically analyzed. Immunofluorescence staining was used to observe cell proliferation status: DAPI stained the cell nucleus (blue), and phalloidin labeled the fibroblast cytoskeleton (green).

[0062] like Figure 2 As shown, there was no statistically significant difference between the antibody-only group and the blank control group, indicating that the antibody itself has no direct toxicity to fibroblasts and does not directly inhibit cell proliferation; cell proliferation was significantly upregulated in the IL-24-stimulated group; and cell proliferation was significantly inhibited in the IL-24+1A6-13-8 neutralization group. Cellular level results confirm that the 1A6-13-8 antibody can effectively neutralize the biological function of IL-24, eliminate the IL-24-induced fibroblast proliferation-promoting effect, and possesses strong cellular level neutralizing activity.

[0063] like Figure 3As shown, the proliferation level of fibroblasts was low in the blank control group and the antibody-only group; IL-24 stimulation significantly increased the number of fibroblasts and enriched the cytoskeleton, confirming that IL-24 significantly promoted fibroblast proliferation; while the cell proliferation level in the IL-24+1A6-13-8 neutralization group fell back to near the level of the blank control group.

[0064] The above results indicate that recombinant IL-24 protein can significantly enhance the proliferation of fibroblasts. The addition of 1A6-13-8 blocked the proliferative effect of IL-24 on fibroblasts, while 1A6-13-8 alone had no significant inhibitory effect on the proliferation of fibroblasts.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A neutralizing murine monoclonal antibody that specifically recognizes IL-24, characterized in that, Including VH CDR1 with amino acid sequence as shown in SEQ ID NO.6, VH CDR2 with amino acid sequence as shown in SEQ ID NO.7, VH CDR3 with amino acid sequence as shown in SEQ ID NO.8, VL CDR1 with amino acid sequence as shown in SEQ ID NO.9, VLCDR2 with amino acid sequence of WAS and VL CDR3 with amino acid sequence as shown in SEQ ID NO.

10.

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

3.

3. The encoding gene of a neutralizing murine monoclonal antibody as described in claim 1 or 2.

4. The encoding gene according to claim 3, characterized in that, The coding genes include light chain variable region coding genes and heavy chain variable region coding genes; The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.4; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.

5.

5. A recombinant expression vector, characterized in that, Includes the coding gene as described in claim 3 or 4.

6. A recombinant non-plant host cell, characterized in that, Includes the recombinant expression vector as described in claim 5.

7. The use of the encoding gene as described in claim 3 or 4, the recombinant expression vector as described in claim 5, or the recombinant non-plant host cell as described in claim 6 in the preparation of the neutralizing murine monoclonal antibody as described in claim 1.

8. The use of a neutralizing murine monoclonal antibody as described in claim 1 or 2 in (1) or (2): (1) Preparation of detection products for mouse-derived IL-24; (2) Preparation of an active inhibitor of mouse IL-24.

9. A detection product for rodent-derived IL-24, characterized in that, Includes the neutralizing murine monoclonal antibody as described in claim 1 or 2.

10. An inhibitor of murine IL-24 activity, characterized in that, The active ingredient includes the neutralizing murine monoclonal antibody as described in claim 1 or 2.

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