A hybridoma cell strain 2F8 secreting monoclonal antibody of calmodulin 2, monoclonal antibody and preparation and application

Abstract

The application belongs to the technical field of biology, and discloses a hybridoma cell strain 2F8 secreting a calmodulin 2 monoclonal antibody, a specific monoclonal antibody, and preparation and application. The hybridoma cell strain 2F8 secreting the calmodulin 2 monoclonal antibody is classified and named as 2F8, and is preserved in the China General Microbiological Culture Collection Center on April 9, 2025, with a preservation number of CGMCC No.46343. The application also provides the calmodulin 2 monoclonal antibody secreted by the hybridoma cell strain 2F8 and a polynucleotide encoding the calmodulin 2 monoclonal antibody. The antibody has specific recognition ability for the calmodulin 2, and is suitable for immunological detection, in particular, Western-blot and immunohistochemical detection. The antibody can be used for preparing a reagent, a kit or other immunological detection product for detecting the calmodulin 2, and has practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a hybridoma cell line 2F8 that secretes a monoclonal antibody against calmodulin 2, the specific monoclonal antibody, and its preparation and application. Background Technology

[0002] Calponin-2 (CNN2), a member of the calponins protein family, is a highly conserved protein that plays a crucial role in various biological processes, including muscle contraction and cell migration. Located on chromosome 19 (19p13.3) in the human genome, CNN2 contains seven exons. Its N-terminus is the Calponin homologous motif, the CH domain, which, as part of the actin-binding region, regulates actin binding. The highly conserved middle region inhibits smooth muscle myosin MgATPase activity and myosin kinetics. The variable C-terminal region has inhibitory regulatory functions, determining the subcellular distribution and potential physiological functions of CNN2. Furthermore, CNN2 is widely expressed in smooth muscle and non-muscle cells, participating in the regulation of various biological functions by modulating the dynamic changes of the actin cytoskeleton.

[0003] Studies have shown that plaque status in patients with coronary artery disease may affect the expression level of calmodulin 2, and elevated calmodulin 2 levels are associated with in-stent restenosis after percutaneous coronary intervention. Research reports that in atherosclerotic ApoE... - / - In a mouse model, calcified aortic valves showed higher calmodulin 2 expression than healthy aortic valves, and the deletion of the calmodulin 2 gene slowed the progression of aortic valve calcification in mice. Calcitonin 2 can influence the progression of atherosclerosis (AS) by regulating the function of macrophages and vascular smooth muscle cells (VSMCs). Specific knockout of calmodulin 2 in myeloid cells reduced macrophage infiltration and alleviated ApoE. - / - The development of AS lesions in mice. The above evidence suggests that calmodulin 2 has significant potential applications in the diagnosis, treatment, and prognostic assessment of cardiovascular diseases. Therefore, developing monoclonal antibodies with high affinity, high specificity, and high sensitivity is of great importance for studying the function and mechanism of calmodulin 2 in cardiovascular diseases. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a hybridoma cell line 2F8 that secretes a monoclonal antibody against calponin-2 (CNN2).

[0005] Another object of the present invention is to provide a calmodulin 2 monoclonal antibody secreted by the above-mentioned hybridoma cell line 2F8 or its passaged cell lines. The calmodulin 2 monoclonal antibody of the present invention can specifically bind to calmodulin 2 and can be used as an immunoassay reagent in the preparation of in vitro diagnostic kits.

[0006] Another object of the present invention is to provide the application of the above-mentioned calmodulin 2 monoclonal antibody in the preparation of a kit.

[0007] The objective of this invention is achieved through the following solution:

[0008] A hybridoma cell line secreting a monoclonal antibody against calmodulin 2, classified as hybridoma cell line (Mus musculus)2F8, was deposited on April 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46343.

[0009] The present invention also provides a monoclonal antibody against calmodulin 2, named MAb-2F8, which is secreted by the hybridoma cell line 2F8 or its passaged cell line with accession number CGMCC No. 46343.

[0010] Furthermore, the calmodulin-2 monoclonal antibody of the present invention was secreted by the hybridoma cell line 2F8 with accession number CGMCC No. 46343.

[0011] Furthermore, the calmodulin 2 monoclonal antibody of the present invention is an IgG1 Kappa subtype monoclonal antibody.

[0012] Furthermore, the calmodulin 2 monoclonal antibody of the present invention can specifically recognize calmodulin 2.

[0013] Furthermore, the calmodulin-2 monoclonal antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:5 and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:6.

[0014] Furthermore, a polynucleotide encoding the calmodulin 2 monoclonal antibody, wherein the heavy chain variable region of the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:3, and the light chain variable region has a nucleotide sequence as shown in SEQ ID NO:4.

[0015] The calmodulin 2 monoclonal antibody of the present invention can specifically bind to calmodulin 2 and can be used as an immunoassay reagent to prepare in vitro diagnostic kits.

[0016] The present invention also provides the use of the hybridoma cell line that secretes calmodulin 2 monoclonal antibody in the preparation of the kit.

[0017] The present invention also provides the application of the hybridoma cell line that secretes calmodulin 2 monoclonal antibody in the preparation of a kit for detecting calmodulin 2.

[0018] The present invention also provides the application of the above-mentioned calmodulin 2 monoclonal antibody in the preparation kit.

[0019] The present invention also provides the application of the above-mentioned calmodulin 2 monoclonal antibody in the preparation of a kit for detecting calmodulin 2.

[0020] The present invention also provides the application of the above-mentioned polynucleotides in the preparation kit.

[0021] The present invention also provides the application of the above-mentioned polynucleotides in the preparation of a kit for detecting calmodulin 2.

[0022] The present invention also provides a kit comprising the hybridoma cell line described above, or the calmodulin-2 monoclonal antibody described above, or the polynucleotide described above.

[0023] Furthermore, the kit described is an immunoassay kit.

[0024] Furthermore, the aforementioned immunoassays include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence assay, and immunohistochemical assay.

[0025] Furthermore, the kit may be a colloidal gold immunoassay kit, a chemiluminescence immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, or a fluorescence immunoassay kit.

[0026] Furthermore, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0027] This invention provides the application of the above-mentioned hybridoma cell line, calmodulin 2 monoclonal antibody or polynucleotide in the preparation of an immunoassay kit for labeling human calmodulin 2 in normal and diseased tissues.

[0028] Furthermore, the tissue includes carotid plaque tissue, prostate tissue, colorectal tissue, liver tissue, etc.

[0029] This invention provides the application of the above-mentioned hybridoma cell lines, calmodulin 2 monoclonal antibodies or polynucleotides in the preparation of immunoassay reagents, kits, calmodulin 2 detection reagents, cardiovascular disease diagnostic reagents, and cardiovascular disease diagnostic kits.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] The hybridoma cell line 2F8 of this invention is characterized by its high efficiency and stable secretion of calmodulin-2 monoclonal antibody. The calmodulin-2 monoclonal antibody secreted by the hybridoma cell line 2F8 of this invention specifically recognizes both recombinant calmodulin-2 protein and calmodulin-2 from human aortic smooth muscle cells. Western blot results show no non-specific bands, indicating reliable detection results. In cellular immunofluorescence and immunohistochemical staining, the calmodulin-2 monoclonal antibody of this invention effectively and specifically recognizes calmodulin-2 protein, with accurate staining localization, clear staining without non-specific staining, and a clean background. This indicates that the monoclonal antibody can be used in Western blot, immunofluorescence, and immunohistochemical detection techniques, and has practical application value. The monoclonal antibody of this invention is independently developed and prepared. Hybridoma cell lines that stably produce calmodulin 2 monoclonal antibodies have been screened. It has the advantages of low cost and easy promotion and application. It can be used to prepare kits for detecting calmodulin 2, or to prepare immunological detection products for detecting calmodulin 2 expression, such as Western blot, immunofluorescence, and immunohistochemistry, and has practical application value. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The image shows the protein electrophoresis results of the purified recombinant calmodulin 2 antigen of this invention; where lane M is the protein marker and rCNN2 is the purified recombinant antigen.

[0034] Figure 2 The following are the protein electrophoresis results of the purified monoclonal antibody of this invention; wherein, lane M is the protein marker; MAb-2F8 is the purified monoclonal antibody.

[0035] Figure 3 The results show the immunoglobulin subtype identification of the monoclonal antibody of this invention.

[0036] Figure 4 The results of immunoblotting analysis of the monoclonal antibody of this invention are shown below; lane M is the protein marker; rCNN2 is the recombinant calmodulin 2 of this invention; HASMC is a human aortic smooth muscle cell sample.

[0037] Figure 5The results are the results of cell immunofluorescence identification of the monoclonal antibody of this invention.

[0038] Figure 6 Comparison of immunohistochemical staining results of human carotid artery plaque tissue.

[0039] Figure 7 This is a diagram showing the results of immunohistochemical staining of tumor tissue. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0041] Example 1: Expression and purification of recombinant human calmodulin-2 immunogen

[0042] Based on the Calponin-2 amino acid sequence numbered Q99439 in the Uniprot database (https: / / www.uniprot.org), a fragment of amino acids 1-283 was selected, and 10 histidine residues were added to the end, resulting in the following sequence:

[0043] MSSTQFNKGPSYGLSAEVKNRLLSKYDPQKEAELRTWIEGLTGLSIGPDFQKGLKDGTILCTLMNKLQPGSVPKINRSMQNWHQLENLSNFIKAMVSYGMNPVDLFEANDLFESGNMTQVQVSLLALAGKAKTKGLQSGVDIGVKYSEK QERNFDDATMKAGQCVIGLQMGTNKCASQSGMTAYGTRRHLYDPKNHILPPMDHSTISLQMGTNKCASQVGMTAPGTRRHIYDTKLGTDKCDNSSMSLQMGYTQGANQSGQVFGLGRQIYDPKYCPQGTVADGAHHHHHHHHHH(SEQID NO: 1), a total of 293 amino acids.

[0044] After optimizing the human codons, the sequence is:

[0045] ATGTCCTCCACCCAGTTTAACAAGGGCCCCAGCTACGGCCTGTCCGCTGAGGTGAAAAACAGACTGCTGAGCAAGTACGACCCCCAGAAGGAGGCCGAGCTGAGAACATGGATCGAGGGCCTGACCGGCCTGTCTATCGGACCTGATTTCCAGAAGGGCCTGAAGGACGGCACCATCCTGTGCACACTGATGAACAAGCTGCAGCCTGGCAGCGTGCCTAAGATCAACAGAAGCATGCAGAACTGGCACCAGCTGGAGAATCTGTCCAATTTTATCAAGGCTATGGTTAGCTACGGCATGAACCCTGTGGACCTGTTTGAGGCCAACGATCTGTTTGAGTCCGGCAACATGACCCAGGTGCAGGTGAGCCTGCTGGCTCTGGCTGGAAAGGCTAAGACCAAGGGCCTGCAGAGCGGCGTTGATATCGGCGTTAAGTACTCCGAGAAGCAGGAGAGAAACTTCGACGACGCCACAATGAAGGCCGGCCAGTGTGTGATCGGCCTGCAAATGGGCACCAATAAGTGTGCCAGCCAGTCCGGCATGACAGCCTACGGAACCAGGAGGCACCTGTACGATCCCAAGAACCACATCCTGCCCCCTATGGATCACAGCACAATCAGCCTGCAGATGGGCACAAACAAGTGTGCCTCCCAGGTGGGCATGACAGCTCCTGGAACCAGAAGGCACATCTACGATACCAAGCTGGGCACAGATAAGTGCGACAATAGCAGCATGTCCCTGCAGATGGGATACACACAGGGCGCCAATCAGAGCGGCCAGGTTTTTGGCCTGGGCAGACAGATCTACGATCCTAAGTACTGTCCTCAGGGCACCGTGGCCGACGGAGCTCACCACCACCACCATCATCACCACCACCACTGA(SEQ ID NO:2)。

[0046] The calmodulin 2 gene was constructed into the multiple cloning site Nhe I and Pme I of the eukaryotic expression vector pcDNA3.1 and transformed into DH5α competent engineered bacteria. After cloning and amplification, the accuracy of the eukaryotic expression plasmid was finally confirmed by PCR and sequencing.

[0047] A large quantity of endotoxin-free eukaryotic expression plasmid pcDNA3.1-CNN2 was extracted. Suspension HEK293F cells in good growth condition and in logarithmic growth phase were then divided into groups at a cell density of 1.5 × 10⁻⁶ cells / cells. 6 HEK293F cells were seeded at 100 mL SMM 293-TII medium (Beijing Yiqiao Shenzhou). The recombinant plasmid pcDNA3.1-CNN2 was transfected into HEK293F cells using EZ Trans high-efficiency transfection reagent (Heyuan Liji (Shanghai) Biotechnology Co., Ltd.). SMM 293-ENH cell culture enhancer (Beijing Yiqiao Shenzhou) and feed were added every two days. After 5 days of continuous cell expression, the cell culture supernatant was collected by centrifugation at 5000 rpm for 10 min.

[0048] Recombinant proteins were purified using His-tag protein purification magnetic beads (IDA-Ni) (Beaver Nanoparticles). After pretreatment with the magnetic beads, cell supernatant was incubated with the beads for 30 min. The supernatant was discarded, and the beads were washed for 10 min. The washing buffer was discarded, and the recombinant protein was eluted with elution buffer. 2 μg of the recombinant protein was prepared and subjected to SDS-PAGE electrophoresis. After Coomassie brilliant blue staining, the molecular weight and purity were analyzed. Figure 1 As shown, a clear protein band was observed at approximately 36 kDa, and the purity of recombinant calmodulin 2 was good. Recombinant calmodulin 2 was collected, aliquoted, and stored at -80°C for subsequent mouse immunization.

[0049] Example 2: Preparation of monoclonal antibody against human calmodulin 2

[0050] 1. Immunization of mice by recombinant calmodulin 2

[0051] Recombinant calmodulin 2 from Example 1 was fully emulsified with Freund's complete adjuvant (Sigma) and used for the initial immunization of three 6-8 week old female BALB / c mice (Guangzhou Southern Medical University Animal Technology Development Co., Ltd.). 100 μg / mouse was administered subcutaneously at multiple sites in the abdomen. A booster immunization was performed every two weeks, with recombinant CNN2 protein fully emulsified with Freund's incomplete adjuvant (Sigma) and 100 μg / mouse administered subcutaneously at multiple sites in the abdomen. Seven days after the third immunization, the serum anti-CNN2 protein titer was detected by indirect enzyme-linked immunosorbent assay (ELISA), and mice with the highest serum titer were selected. Recombinant calmodulin 2 was diluted with physiological saline and administered as a pulse immunization at 100 μg / mouse via intraperitoneal injection, resulting in the final immunized BALB / c mice.

[0052] 2. Preparation and screening of hybridoma cells

[0053] Five days after the initial immunization, mice were euthanized by cervical dislocation, and their spleens were aseptically removed and prepared into a spleen cell suspension. Logarithmic growth phase myeloma cells SP2 / 0 were taken and thoroughly mixed with mouse spleen cells at a ratio of 1:5. After centrifugation, the supernatant was discarded. Cell fusion was performed using the standard polyethylene glycol (Sigma) method: 1 mL of preheated 37°C 50% polyethylene glycol was slowly added dropwise over 1 min, mixing constantly. The fusion was terminated by adding preheated 37°C serum-free 1640 medium. After centrifugation and discarding the supernatant, the cells were resuspended thoroughly in RPMI 1640 complete medium containing HAT. The fused cells were seeded at 200 μL / well into 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator.

[0054] Ten days after fusion culture, hybridoma cell clones were selected and labeled with cell line numbers. The culture supernatant of the hybridoma cells was detected using indirect ELISA. The supernatant from positive wells was discarded, and the medium was replaced with RPMI 1640 complete medium containing HA. Two days later, the culture supernatant was collected again for indirect ELISA. Hybridoma cell clones showing strong positive reactions were selected, and clonal culture was performed using limiting dilution to obtain hybridoma cell lines with a 100% positive rate and stable secretion of monoclonal antibodies. Monoclonal cell lines with high positive values ​​and large cell clusters were selected for amplification culture and cryopreserved in liquid nitrogen. The hybridoma cell line (2F8) was deposited at the China General Microbiological Culture Collection Center on April 9, 2025, with accession number CGMCC No. 46343.

[0055] 3. Expression and purification of monoclonal antibodies against human calmodulin 2

[0056] Hybridoma cell line (2F8) was expanded and cultured to the logarithmic growth phase, then cultured at 5.0 × 10⁻⁶ cells / year. 5Cells / mL were inoculated into shake flasks containing L-glutamine in CDM4 medium (Hyclone, USA) and incubated in suspension at 37°C, 5% CO2, and 100 rpm on a shaker. Cell Boost was added daily starting the next day. TM 5. Feed (Hyclone, USA). After 5 days of culture, the cell supernatant was collected by centrifugation, filtered through a 0.22 μm filter membrane, and stored at 4°C for later use. Antibody purification was performed using Protein A Sepharose affinity chromatography: Protein A packing material was loaded into a purification column and connected to a Bio-rad atmospheric pressure purification system. The column was equilibrated with 5 column volumes of equilibration buffer. The cell supernatant to be purified was slowly loaded into the column, and washed to baseline with 10 column volumes of equilibration buffer. The antibody was eluted with elution buffer, and the antibody peak was collected. The purified antibody was neutralized with neutralization buffer (1.0 M Tris-HCl solution, pH 8.8), aliquoted, and stored at -20°C for later use. The monoclonal antibody secreted by hybridoma cell line 2F8 was named MAb-2F8. After preparing the purified antibody sample, protein gel electrophoresis was performed, and the purity and size of the monoclonal antibody were identified after Coomassie brilliant blue staining. Results are shown below. Figure 2 The purified MAb-2F8 monoclonal antibody obtained by this invention has a purity of over 90%. The heavy chain and light chain molecular weights of this monoclonal antibody are approximately 50 kDa and 28 kDa, respectively.

[0057] Hybridoma cell line 2F8 was sent to Nanjing Mingyan Biotechnology Co., Ltd. for monoclonal antibody variable region gene and amino acid sequence analysis. Total RNA was extracted from the hybridoma cells, and cDNA was obtained through reverse transcription. The heavy chain and light chain variable region genes were amplified using specific primers, ligated into cloning vectors, transformed into plating, and positive clones were selected for gene sequencing. The nucleotide sequences of the heavy chain variable region (SEQ ID NO:3) and light chain variable region (SEQ ID NO:4) of the monoclonal antibody prepared in this invention were obtained, and the amino acid sequences of the heavy chain variable region (SEQ ID NO:5) and light chain variable region (SEQ ID NO:6) were deduced.

[0058] The nucleotide sequence of the heavy chain variable region is as follows:

[0059] GAGGTGCAGCTTGTTGAGACTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTTACGTTCAATACCAATTCCATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAAAGTGATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAACATTCTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGCATTACTGTGTGAGAGATCGAACTGGGGGGGGATTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO:3);

[0060] The nucleotide sequence of the light chain variable region is:

[0061] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:4);

[0062] The amino acid sequence of the heavy chain variable region is:

[0063] EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNSMNWVRQAPGKGLEWVARIRSKS DNYATYYADSVKDRFTISRDDSQNILYLQMNNLKTEDTAMHYCVRDRTGGGFAYWGQG TLVTVSA(SEQ ID NO:5);

[0064] The heavy chain variable region contains 120 amino acids in total. The number of amino acids in the four domains of its FR are 25, 17, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 10 and 11, respectively. The corresponding amino acid sequences of the regions of HCDR1, HCDR2 and HCDR3 are GFTFNTNS, IRSKSDNYAT and VRDRTGGGFAY, respectively.

[0065] The amino acid sequence of the light chain variable region is as follows:

[0066] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNL ESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK(SEQ ID NO:6);

[0067] The light chain variable region contains 111 amino acids in total. The number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the three domains of LCDR are 10, 3 and 9, respectively. The corresponding amino acid sequences of the regions of LCDR1, LCDR2 and LCDR3 are KSVSTSGYSY, LAS and QHSRELPLT, respectively.

[0068] Example 3: Characterization of monoclonal antibodies against human calmodulin 2

[0069] 1. Subtype identification

[0070] Immunoglobulin subtypes of purified monoclonal antibodies were identified using a mouse monoclonal antibody subtype identification kit (Proteintech). Monoclonal antibody samples were diluted to 200 ng / mL with 1×PBST, and 50 μL / well was added to each well coated with anti-heavy chain IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, and light chain Kappa, Lambda antibodies, respectively. Then, 50 μL / well of diluted goat anti-mouse IgA+IgM+IgG-HRP was added to each well. The mixture was gently stirred, sealed with a sealing film, and incubated at room temperature for 1 h. The liquid in the wells was discarded, and the plate was washed three times with 1×PBST and patted dry on absorbent paper. 100 μL / well of the prepared chromogenic solution was added to each well, and the plate was incubated at room temperature in the dark for 15 min. Finally, 100 μL / well of stop solution was added, and the OD value at 450 nm was read using a microplate reader. The results showed (…). Figure 3 The monoclonal antibody of this invention is an IgG1 type immunoglobulin with a Kappa chain light chain.

[0071] 2. Antibody affinity assay

[0072] The purified recombinant calmodulin 2 from Example 1 was diluted to 2 μg / mL with 0.05 M carbonate buffer (pH 9.6), and 100 μL was added to each well of a polystyrene microplate. The plate was incubated overnight at 4°C. The plate was washed 5 times with PBST and patted dry on absorbent paper. Blocking buffer was added at 200 μL per well, and the plate was incubated at 37°C for 1 hour. The plate was washed 5 times with PBST and patted dry on absorbent paper. Take the purified monoclonal antibody (200 μg / mL) from Example 2 and dilute it with 1×PBS at dilutions of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 102400, and 204800. Use PBS as a blank control. Add 100 μL of the monoclonal antibody at different dilutions to the coated ELISA plate and incubate at 37°C for 1 h. Wash the plate 5 times with PBST and pat dry on absorbent paper. Take HRP-labeled goat anti-mouse secondary antibody (Invitrogen) diluted 1:10000 and add 100 μL to the reaction wells. Incubate at room temperature for 1 h. Wash the plate 5 times with PBST and pat dry on absorbent paper. Add 100 μL of the prepared TMB chromogenic solution (Beyotime) to each well and incubate at room temperature in the dark for 15 min. Add 100 μL of stop solution to each well and measure the OD using a microplate reader. 450 The dissociation constant Kd of the antibody was calculated using the "GraphPad Prism" software to automatically fit the curve. The calculated dissociation constant Kd of the monoclonal antibody of this invention was 2.2 × 10⁻⁶. -9 M.

[0073] 3. Antibody specificity detection

[0074] The recombinant calmodulin-2 purified in Example 1 and human aortic smooth muscle cells were prepared separately, and the antigen-binding specificity of the monoclonal antibody of this invention was detected by immunoblotting. Electrophoresis was performed on a 10% SDS-PAGE protein gel, and the charged gel proteins were transferred to a PVDF membrane using a Bio-Rad transfer device. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and washed three times with TBST. The monoclonal antibody purified in Example 2 was added at a 1:2000 dilution, incubated overnight at 4°C, and washed three times with TBST for 8 minutes each time. HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) was added, incubated at room temperature for 1 hour, and washed three times with TBST for 8 minutes each time. ECL ultrasensitive chromogenic solution (Biosharp) was added, and chemiluminescence image data were acquired using a ChemiDoc MP all-around imaging system (BIO-RAD). The results are as follows: Figure 4As shown, the monoclonal antibody secreted by the hybridoma cells prepared in this invention can specifically recognize recombinant calmodulin 2 and calmodulin 2 in the natural sample, and no other impurities were observed, indicating that the monoclonal antibody prepared in this invention has good specificity and can be applied to immunoblotting experiments.

[0075] Example 4: Application of monoclonal antibodies against human calmodulin 2

[0076] 1. Immunofluorescence detection

[0077] Human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) were seeded into 24-well cell culture plates with climbing slides and cultured to the appropriate density. The plates were then washed three times with PBS. Fixation was performed with 4% paraformaldehyde at room temperature for 30 min, followed by three washes with PBS. Permeabilization was performed with 0.1% Triton X-100 at room temperature for 15 min, followed by three washes with PBS. Blocking was performed with 5% BSA at room temperature for 30 min, and the blocking solution was discarded. The purified monoclonal antibody from Example 2 was diluted to 2 μg / mL with 5% BSA and added to the climbing slides. The slides were incubated at room temperature for 2 h, followed by three washes with PBS. DyLight 594-labeled goat anti-mouse IgG fluorescent secondary antibody (Invitrogen) was diluted 1:500 with 5% BSA and added to the climbing slides. The slides were incubated at room temperature for 1 h, followed by three washes with PBS. Anti-fluorescence attenuation mounting medium (containing DAPI) was added, and the climbing slides were mounted upside down on a glass slide. Observation and photography were performed using an inverted fluorescence microscope. Microscopic examination results showed ( Figure 5 The monoclonal antibody secreted by the hybridoma cell line 2F8 of this invention can effectively recognize calmodulin 2 (red fluorescence) in cells. The red fluorescence is mainly distributed in the cytoplasm, and the staining results are clear with a clean background, indicating that the monoclonal antibody prepared by this invention can be used for cell immunofluorescence staining with good specificity.

[0078] 2. Immunohistochemical analysis

[0079] Carotid plaque tissue sections were dewaxed twice in environmentally friendly tissue clearing and dewaxing agent (Beyotime), 15 min each time. They were then graded hydration in 100%, 100%, 95%, 85%, and 75% ethanol, 5 min each time, and finally rinsed with tap water for 5 min. Heat antigen retrieval was performed using citrate buffer. After natural cooling to room temperature, the sections were washed three times with PBS, 3 min each time. After drying, endogenous catalase in the tissue was inactivated with 3% hydrogen peroxide and incubated at room temperature in the dark for 15 min. The sections were washed three times with PBS, 3 min each time. Blocking was performed with 5% BSA and incubated at room temperature for 30 min. After drying, an immunohistochemical pen was used to draw circles around the tissue, and monoclonal antibodies secreted by the hybridoma cell line 2F8 of this invention (1 μg / mL) and commercially available anti-human calcitonin 2 rabbit polyclonal antibodies (Proteintech) were added, respectively, and incubated overnight at 4°C. The sections were washed five times with PBS, 3 min each time. Add enzyme-labeled goat anti-mouse / anti-rabbit IgG secondary antibody (Gene Tech) and incubate at room temperature for 30 min. Wash 5 times with PBS, 3 min each time. Add DAB chromogenic solution (Gene Tech) and develop color in the dark for 3-5 min (observe under a light microscope and control the color development time). Stop the color development by immersing in tap water and rinse for 10 min. Hematoxylin counterstaining, differentiation, and blue reversion. Dehydrate sections sequentially in 75%, 85%, 95%, 100%, and 100% alcohol gradients for 3 min each, and finally clear in environmentally friendly tissue clearing and dewaxing agent for 3 min. Mount with neutral resin and observe and photograph under a microscope. Microscopic results show ( Figure 6 The monoclonal antibody secreted by hybridoma cells 2F8 of this invention can detect calmodulin 2 in plaque tissue by IHC method. The staining is clear and there is no non-specific staining. The background is clean and consistent with the detection results of commercially available antibodies on tissue sections. The staining localization is accurate and it shows specific cytoplasmic expression in the tissue, indicating that the specificity of this antibody in tissue is comparable to that of commercially available antibodies. It can be used for immunohistochemical detection.

[0080] 3. Immunohistochemical detection in different tumor tissues

[0081] Paraffin sections of tissues from different tumors (prostate cancer, liver cancer, and colorectal cancer) were obtained. Immunohistochemical staining was performed on the tissue sections using the monoclonal antibody MAb-2F8 of this invention, following the same experimental procedures as in Example 4.2. The experimental results are as follows: Figure 7 As shown in the figure, the immunohistochemical results indicate that the monoclonal antibody of this invention accurately stained and localized in various tumor tissue sections, with clear staining and a clean background, and no non-specific staining. This demonstrates that the monoclonal antibody of this invention, when applied to immunohistochemical analysis, can effectively detect the expression level and location of calmodulin 2 in tumor tissues. Combined with the results of Western blotting and immunofluorescence, this indicates that the monoclonal antibody MAb-2F8 secreted by hybridoma cells 2F8 has good efficacy and specificity in recognizing human calmodulin 2 antigen.

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A hybridoma cell line that secretes a monoclonal antibody against calmodulin-2, characterized in that... Classified as 2F8, it was deposited on April 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46343.

2. A monoclonal antibody against calmodulin-2, characterized in that... Named MAb-2F8, it is secreted by the hybridoma cell line 2F8 or its passaged cell line, which has the accession number CGMCC No. 46343 according to claim 1.

3. The calmodulin-2 monoclonal antibody according to claim 2, characterized in that: The calmodulin 2 monoclonal antibody is an IgG1 Kappa subtype monoclonal antibody.

4. The calmodulin-2 monoclonal antibody according to claim 2, characterized in that: The calmodulin-2 monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

6.

5. A polynucleotide, characterized in that... The polynucleotide encodes the calmodulin-2 monoclonal antibody according to any one of claims 2-4, wherein the heavy chain variable region of the polynucleotide is a nucleotide sequence as shown in SEQ ID NO:3, and the light chain variable region is a nucleotide sequence as shown in SEQ ID NO:

4.

6. The use of the hybridoma cell line of claim 1, the calmodulin-2 monoclonal antibody of any one of claims 2-4, or the polynucleotide of claim 5 in the preparation kit.

7. The use of the hybridoma cell line of claim 1, the calmodulin 2 monoclonal antibody of any one of claims 2-4, or the polynucleotide of claim 5 in the preparation of a kit for detecting calmodulin 2.

8. A reagent kit, characterized in that: The kit comprises the hybridoma cell line that secretes calmodulin 2 monoclonal antibody as described in claim 1, or the calmodulin 2 monoclonal antibody as described in any one of claims 2-4, or the polynucleotide as described in claim 5.

9. The use of the hybridoma cell line of claim 1, the calmodulin 2 monoclonal antibody of any one of claims 2-4, or the polynucleotide of claim 5 in the preparation of an immunoassay kit for labeling human calmodulin 2 in normal and diseased tissues.

10. The use of the hybridoma cell line of claim 1, the calmodulin-2 monoclonal antibody of any one of claims 2-4, or the polynucleotide of claim 5 in the preparation of immunoassay reagents.

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