Hybridoma cell strain secreting monoclonal antibody of canine OSMR beta and application thereof

Abstract

This invention discloses a hybridoma cell line that secretes a canine OSMRβ monoclonal antibody and its applications. The monoclonal antibody binds to the extracellular terminal region of canine OSMRβ cells, exhibiting high affinity and specificity, and can be used for immunofluorescence detection, flow cytometry, and ELISA. The monoclonal antibody of this invention shows significant application potential in the development of canine OSMRβ detection reagents and diagnostic reagents for diseases and symptoms related to canine OSMRβ.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and immunology, specifically to a hybridoma cell line that secretes a canine OSMRβ monoclonal antibody and its applications. Background Technology

[0002] OSMRβ is a member of the interleukin-6 receptor family. OSMRβ is distributed across various epithelial and immune cells, including mast cells, macrophages, eosinophils, keratinocytes, endothelial cells, hepatocytes, peripheral neuronal subsets, dorsal root ganglion neurons, and some epithelial cells (such as those in the breast, skin, and lung). Furthermore, OSMRβ is expressed in more than 20 tumor cell types, including sarcoma, melanoma, glioma, breast cancer, prostate cancer, ovarian cancer, and cervical cancer.

[0003] OSMRβ, together with gp130, forms the heterodimer receptor for its major ligand, oncostatin M (OSM). Upon binding to OSM, OSMRβ primarily signals via the JAK / STAT pathway, while also activating signaling pathways such as MAPK / ERK and PI3K / AKT, inducing the transcription and expression of various cytokines, chemokines, and other regulatory factors. OSM–OSMRβ signaling plays a crucial role in inflammation, hematopoiesis, and development. Numerous studies have found that OSM-OSMRβ signaling plays a significant role in promoting tumor growth, such as pancreatic ductal adenocarcinoma, prostate cancer, ovarian cancer, breast cancer, and brain cancer, and is one of the important factors in cancer progression. Therefore, OSMRβ can serve as a target for cancer therapy. Recently, researchers have developed monoclonal antibodies targeting OSMRβ, which inhibit STAT3 signaling induced by OSM-OSMRβ, thereby suppressing ovarian cancer growth.

[0004] Furthermore, OSMRβ can also co-form the heterodimeric receptor for IL-31 with IL-31RA. After IL-31 binds to both receptor proteins, it can trigger downstream signal transduction, such as activation of the JAK / STAT and PI3K / AKT signaling pathways. The activation of these signaling pathways results in the production of large amounts of cytokines, such as IL-1α, CCL17, and CCL2, leading to cell migration and differentiation, nerve fiber elongation and branching, epidermal thickening, and itching. Studies have found that the signal transduction induced by the binding of IL-31 to these two receptors is involved in the development and progression of diseases such as pruritic skin lesions, atopic dermatitis, type II inflammatory response, inflammatory bowel disease, breast cancer, type I hypersensitivity reactions, and airway hypersensitivity. Summary of the Invention

[0005] The primary objective of this invention is to provide a hybridoma cell line that secretes a canine OSMRβ monoclonal antibody. This hybridoma cell, named 38223-1-1 / 2O2, was deposited on December 9, 2021, at the China General Microbiological Culture Collection Center (address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China), with accession number CGMCC No. 45007.

[0006] The second objective of this invention is to provide a canine OSMRβ monoclonal antibody produced by the above-mentioned hybridoma cell line for use in immunofluorescence detection, flow cytometry detection, and ELISA detection of canine OSMRβ protein.

[0007] In a first aspect, the present invention provides a hybridoma cell line that secretes a canine OSMRβ monoclonal antibody, named 38223-1-1 / 2O2, with accession number CGMCC No. 45007.

[0008] The present invention also provides a mouse monoclonal antibody that binds to canine OSMRβ, said monoclonal antibody being secreted by the hybridoma cell line.

[0009] The application of the monoclonal antibody in the preparation of reagents for the detection or diagnosis of canine OSMRβ or canine OSMRβ-related diseases. Preferably, the detection method is immunofluorescence detection, flow cytometry, or ELISA.

[0010] Specifically, the canine OSMRβ-related diseases are tumors, preferably sarcomas, melanomas, gliomas, breast cancers, prostate cancers, ovarian cancers, and cervical cancers.

[0011] This invention also provides the application of the hybridoma cells described above in a detection or diagnostic reagent for canine OSMRβ or canine OSMRβ-related diseases. Preferably, the detection method is immunofluorescence detection, flow cytometry, or ELISA. Specifically, the canine OSMRβ-related diseases are tumors, preferably sarcomas, melanomas, gliomas, breast cancer, prostate cancer, ovarian cancer, and cervical cancer.

[0012] The present invention also provides a kit containing the aforementioned monoclonal antibody for detecting canine OSMRβ or canine OSMRβ-related diseases. Preferably, the canine OSMRβ-related disease is a tumor, preferably sarcoma, melanoma, glioma, breast cancer, prostate cancer, ovarian cancer, or cervical cancer.

[0013] Compared with existing technologies, the present invention has the following advantages: the monoclonal antibody can bind to the extracellular terminal region of canine OSMRβ cells, exhibiting high affinity and specificity, and can be applied to immunofluorescence detection, flow cytometry detection, and ELISA detection. The monoclonal antibody of the present invention has significant application prospects in the development of canine OSMRβ detection reagents and diagnostic reagents for diseases and symptoms related to canine OSMRβ. Attached Figure Description

[0014] Figure 1 The canine OSMRβ prepared in this invention is subjected to Superdex... TM Purification results of 200 10 / 300 GL molecular sieve.

[0015] Figure 2 The monoclonal antibodies secreted from the ascites fluid of mice after the hybridoma cell line prepared in this invention are analyzed by Superdex. TM Purification results of 200 10 / 300 GL molecular sieve.

[0016] Figure 3 The results show the affinity of the monoclonal antibody prepared in this invention to canine OSMRβ as determined by BLI assay. Specifically, a and c represent the affinity assay results of 38223-1-1 / 2O2 with canine and feline OSMRβ, respectively, while b and d represent the affinity assay results of the negative control antibody with canine and feline OSMRβ, respectively.

[0017] Figure 4 The results show the binding of the monoclonal antibody prepared in this invention to canine OSMRβ expressed on the cell membrane, as determined by immunofluorescence.

[0018] Figure 5 The results show the binding of the monoclonal antibody prepared in this invention to canine OSMRβ expressed on the cell membrane, as determined by cell flow cytometry.

[0019] Figure 6 The results show the binding of the monoclonal antibody prepared in this invention to canine OSMRβ immobilized on an ELISA plate, as determined by the ELISA method. Detailed Implementation

[0020] It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Furthermore, unless otherwise specified, all reagents used in the following embodiments are commercially available or can be synthesized according to methods described herein or known to the art. Reaction conditions not listed are also readily available to those skilled in the art.

[0021] Example 1: Expression and purification of canine OSMRβ

[0022] Canine OSMRβ antigen for the preparation of hybridoma cells secreting canine OSMRβ monoclonal antibodies. In this embodiment, the amino acid sequence of the protein is shown in SEQ ID NO:1:

[0023] .

[0024] In this embodiment, the DNA fragment encoding the protein is shown in SEQ ID NO.2:

[0025]

[0026] The preparation method of canine OSMRβ protein includes the following steps:

[0027] 1. After codon optimization of canine OSMRβ (amino acids 28-734, sequence as shown in SEQ ID NO: 1) expressed in 293T cells (as shown in SEQ ID NO: 2), a signal peptide, 6HIS amino acid and TEV restriction site were added to the N-terminus, and a TEV restriction site and STREP tag were added to the C-terminus. The resulting plasmid was inserted into the pCI vector to obtain pCI-dogOSMRβ-ecto recombinant plasmid.

[0028] 2. The recombinant plasmid pCI-dogOSMRβ-ecto was transfected into 293T cells using the transfection reagent polyethyleneimine (PEI). 4-6 hours after transfection, the cells were washed 1-2 times with physiological saline, then DMEM medium without serum was added. Cell supernatant was collected 96 hours after transfection. Enrichment and purification were performed using Strep affinity chromatography. Subsequently, the cells were processed using Superdex. TM Further purification using 200 10 / 300 GL molecular sieves yielded the target protein, canine OSMRβ, in a homogeneous form. Figure 1 The theoretical molecular weight of the tagged canine OSMRβ extracellular fragment protein is 81.1 kDa. Because this extracellular fragment protein has multiple glycosylation sites, the protein expressed by 293T cells is located above 100 kDa on SDS-PAGE. Figure 1 ).

[0029] Example 2: Preparation and purification of mouse monoclonal antibodies

[0030] Purified canine OSMRβ was used as the antigen, and female BALB / c mice were subcutaneously immunized at multiple sites five times over 6-8 weeks. The immunization dose was 30 μg / immunization, with the first four immunizations spaced 14 days apart. Freund's complete adjuvant was used for the first immunization; Freund's incomplete adjuvant was used for the second to fourth immunizations. Seven days after the third immunization, tail vein blood was collected to detect serum antibody titers, and mice with the highest titers were selected for fusion. Three days before fusion, a sprint immunization was performed, i.e., the fifth immunization, in which 100 μg of immunogen was injected into the peritoneal cavity of the mice. Spleen cells from these mice were fused with myeloma cells SP2 / 0 at a ratio of 10:1, and selected and cultured using HAT selection medium. Hybridoma cell lines were obtained using the limiting dilution method. Canine OSMRβ was coated in ELISA plates at pH 9.6 carbonate buffer (25 ng / well), and antigen-binding monoclonal cell lines were screened using ELISA.

[0031] Monoclonal antibodies were prepared using retired BALB / c mice. First, 0.4 ml of a specific adjuvant was injected intraperitoneally into the ascites fluid per mouse. 10-14 days after adjuvant injection, hybridoma cells were injected intraperitoneally at a dose of 300,000 cells per mouse. 10-15 days after cell injection, ascites fluid was collected from the mice, and IgG was enriched and purified using Protein A, followed by Superdex assay. TM Further purification using 200 10 / 300 GL molecular sieves yielded homogeneous IgG protein of 38223-1-1 / 2O2 form. Figure 2 ).

[0032] Example 3: Affinity determination of monoclonal antibody with canine OSMRβ

[0033] The affinity of 38223-1-1 / 2O2 for canine and feline OSMRβ was determined using biolayer interferometry (BLI). Mouse IgG was first immobilized using an Anti-Mouse Fc Capture (AMC) chip, and then bound to different concentrations of canine or feline OSMRβ (6.25–200 nM), with binding and dissociation times both around 300 sec. The results showed that the affinity of 38223-1-1 / 2O2 for canine OSMRβ was 2.49 × 10⁻⁶. -9 M ( Figure 3 a) The binding constant is 4.64 × 10 4 (1 / Ms), dissociation constant is 1.16 × 10 -4 (1 / s). It was also found that 38223-1-1 / 2O2 did not bind to cat OSMRβ. Figure 3 c). The negative control antibody did not bind to OSMRβ protein in either dogs or cats. Figure 3 (b, 3d). The BLI results indicate that 38223-1-1 / 2O2 is a canine OSMRβ-specific monoclonal antibody with high affinity for the antigen and a slow dissociation rate.

[0034] Example 4: Immunofluorescence detection of monoclonal antibody binding to canine OSMRβ on cell membrane

[0035] Plasmids pEGFP-dogOSRMβ, pEGFP-catOSRMβ, and pEGFP-humanOSRMβ, expressing canine, feline, and human OSMRβ fused to the C-terminus of EGFP, were transfected into 48-well plates with coverslips at the bottom using PEI. After 4-6 hours post-transfection, the plates were replaced with serum-free DMEM. 48 hours post-transfection, the cells were fixed with 3.7% formaldehyde for 15 minutes. After washing three times with PBS, the cells were blocked with 2% BSA for 1 hour. After washing three times with PBS, diluted monoclonal antibody 38223-1-1 / 2O2 (final concentration 8.3 μg / ml) was added and incubated for 1 hour. The cells were washed three times with PBS, 5 minutes each time. Alexa Fluor 594-labeled goat anti-mouse IgG-A594 (1:1000 dilution) was added and incubated at room temperature for 1 hour. After incubation with secondary antibody for 40 min, DAPI dye was added, and incubation continued for another 20 min. The slides were washed three times with PBS, 5 min each time, and mounted with anti-fluorescence quenching mounting solution before fluorescence confocal imaging. Results showed that 38223-1-1 / 2O2 specifically recognized canine OSMRβ expressed on the cell membrane, but did not bind to feline OSMRβ or human OSMRβ expressed on the cell membrane. Figure 4 ).

[0036] Example 5: Cell flow cytometry detection of monoclonal antibody binding to canine OSMRβ on the cell membrane.

[0037] The plasmids pEGFP-dogOSRMβ and pEGFP-humanOSRMβ, which express canine OSMRβ and human OSMRβ fused to EGFP at the C-terminus, were transfected into 293T cells using PEI. After 4-6 hours post-transfection, the cells were replaced with serum-free DMEM. After 48 hours of culture, transfected cells were digested with trypsin to form single cells, centrifuged at 500 g for 5 min, and the cell pellet was resuspended in DMEM for counting. The cells were then aliquoted into 1*102 96-well plates. 6Cells / well. Add diluted 38223-1-1 / 2O2 monoclonal antibody (final concentration 10.0 μg / ml) or negative control antibody, 100 μl / well. Incubate at room temperature with gentle shaking for 1 hr. Wash twice with DMEM, centrifuging at 500 g for 5 min after each addition of DMEM, and discard the supernatant. Add PE / Cyanine7-labeled goat anti-mouse IgG (1:1000 dilution), 100 μl / well. Incubate at room temperature with gentle shaking for 1 hr. Then wash twice with DMEM, and resuspend the cells in DMEM, 150-200 μl / well. Finally, analyze the resuspended cells by flow cytometry. The results showed that flow cytometry could detect the specific binding of 38223-1-1 / 2O2 monoclonal antibody to canine OSMRβ, but not to human OSMRβ. Figure 5 ).

[0038] Example 6: ELISA assay of the binding activity of monoclonal antibody to the extracellular domain of canine OSMRβ secreted by 293T cells.

[0039] First, purified canine OSMRβ extracellular fragment protein was immobilized on ELISA plates using carbonate coating buffer at pH 9.6, 200 ng / well, and incubated overnight at 4°C. The ELISA plates were then placed at room temperature and shaken for 30 min to allow them to return to room temperature. The liquid in the ELISA plates was discarded, and the plates were washed three times with PBS, 5 min each time. Next, blocking buffer (PBS + 3% BSA) was added, and the plates were incubated on a shaker for 1 hour. The plates were then washed three times with PBS, 5 min each time. Subsequently, 100 μl / well of 38223-1-1 / 2O2 monoclonal antibody at different dilutions was added, and the plates were shaken on a shaker at room temperature for 1 hour. The plates were then washed three times with PBST (0.05% Tween-20 + PBS), 5 min each time. Finally, 1:2000 dilution of HRP-labeled goat anti-mouse IgG was added, and the plates were shaken on a shaker at room temperature for 1 hour. After washing the plates three times with PBST, TMB was added for color development for 5 min. The reaction was terminated by adding 2 M H2SO4, and the readings were taken using a microplate reader. The results showed that 38223-1-1 / 2O2 specifically binds to canine OSMRβ, EC50. 50 96.96 ng / ml ( Figure 6 ). <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A hybridoma cell line secreting canine OSMRβ monoclonal antibody and its application <160> 2 <210> 1 <211> 707 <212> PRT <213> Dog (Canis lupus familiaris) <400> 1 EPLPWAPESLKVSINSTHQCLHLQWSVHNLAYHQELKMVFQIEISRIKTSNVIWVENYSTTVKGNQLLHWSWESQLPLECAHFIRMSAVDDATTPEQRFWSNWSSWEEVDVQNSLGHEPLFVFPKDKLVEEGSNVTICYVSRSQQNNISCYLEGVRMQGQQLDPNVSMFNLHNV AFIRETGTNIYCKVDRGDDIKGIVLFVSKILEEPKDFSCETRDFQTLSCTWDPGRDTGLLKQLPQSYTLFESFSGKKTLCKHKSWCNWQVASESQEMYNFTLTAENYLRKRSVHILFNLTHRVHPMAPFNVLFKDVSVTNATMTWKVHSTGNYYTLLCQVELYGEGKVIQKHNVSVK VNGELVLSGLEPDTEYSAQVRCANANHFKWSEWTRQNFTTVEAAPSEAPDVWRNVKSVQGYCVVTLFWKPLSRLQANGEILFYNIVVEDLDPSGLQLLSVPAPANRTELTLDQRAYQIHVTANNSVGTSPASVLVVSGDPGDEEVEEERVKGTEDGFSLSWKPQPGNITGYIVEW CDRPRDPLCDLQWKHLGPNTTSTVISSDAFRPGVRYNFRIYGISTEMVPYLLEKKTGYSQELAPSDNPVRVMSNLTSHSFTLSWKDYSTESQPSFIRGYHVYLKSKAGQCHPGSEKAVLSDDSVCCKYKIDDPKQKMFVWGNLQPESFYEFLVTPYSAVGEGPHGAFTKVTTPDEYS 707 <210> 2 <211> 2121 <212> DNA <213> Dog (Canis lupus familiaris) <400> 2

Claims

1. A hybridoma cell line capable of secreting canine OSMRβ monoclonal antibody, named 38223-1-1 / 2O2, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 9, 2021, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 45007.

2. A mouse monoclonal antibody that binds to canine OSMRβ, said monoclonal antibody being secreted by the hybridoma cell line of claim 1.

3. The use of the monoclonal antibody as described in claim 2 in the preparation of a detection or diagnostic reagent for canine OSMRβ or canine OSMRβ-related diseases, wherein the canine OSMRβ-related diseases are sarcoma, melanoma, glioma, breast cancer, prostate cancer, ovarian cancer, or cervical cancer.

4. The application as described in claim 3, characterized in that, The detection method is immunofluorescence detection, flow cytometry, or ELISA.

5. The use of the hybridoma cells as described in claim 1 in the preparation of a detection or diagnostic reagent for canine OSMRβ or canine OSMRβ-related diseases, wherein the canine OSMRβ-related diseases are sarcoma, melanoma, glioma, breast cancer, prostate cancer, ovarian cancer, or cervical cancer.

6. The application as described in claim 5, characterized in that, The detection methods are immunofluorescence detection, flow cytometry, or ELISA detection.

7. A kit for detecting canine OSMRβ or canine OSMRβ-related diseases containing the monoclonal antibody as described in claim 2, wherein the canine OSMRβ-related diseases are sarcoma, melanoma, glioma, breast cancer, prostate cancer, ovarian cancer, or cervical cancer.

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