Hybridoma cell strain 2F8 secreting calmodulin 2 monoclonal antibody, monoclonal antibody, preparation and application

Abstract

The invention belongs to the technical field of biology, and discloses a hybridoma cell strain 2F8 capable of secreting a calmodulin 2 monoclonal antibody, a specific monoclonal antibody, and preparation and application of the specific monoclonal antibody. The hybridoma cell strain secreting the calmodulin 2 monoclonal antibody, provided by the invention, is classified and named as 2F8, and is preserved in the China General Microbiological Culture Collection Center on April 9, 2025, and the preservation number is CGMCC No.46343. The invention also provides a calmodulin 2 monoclonal antibody secreted by the calmodulin 2 monoclonal antibody and polynucleotide for coding the calmodulin 2 monoclonal antibody. The antibody disclosed by the invention has specific recognition capability on calmodulin 2, and is suitable for immunological detection, especially Western-blot and immunohistochemical detection. The method can be used for preparing reagents, kits or other immunological detection products for detecting calmodulin 2, and has practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a hybridoma cell line 2F8 secreting a monoclonal antibody against calponin-2, a specific monoclonal antibody, and their preparation and application. Background Art

[0002] Calponin-2 (CNN2) is a member of the calponins protein family, a highly conserved protein, and plays an important role in various biological processes such as muscle contraction and cell migration. Calponin-2 is located on chromosome 19 (19p13.3) in the human genome and contains 7 exons. The N-terminal of calponin-2 is the Calponin homology motif, namely the CH domain, which, as part of the actin-binding region, regulates the binding to actin; the middle region is highly conserved and has the function of inhibiting the MgATPase activity of smooth muscle myosin and the generation of myosin power; the C-terminal variable region has an inhibitory regulatory function, determining the subcellular distribution and potentially different physiological functions of calponin-2. In addition, calponin-2 is widely expressed in smooth muscle and non-muscle cells and participates in the regulation of different biological functions by regulating the dynamic changes of the actin cytoskeleton.

[0003] Research shows that the plaque situation in patients with coronary heart disease may affect the expression level of calponin-2 in the body, and the increase in the calponin-2 level in patients is related to in-stent restenosis after percutaneous coronary intervention. Research reports that in atherosclerotic ApoE - / - In a mouse model, the calcified aortic valve shows higher calponin-2 expression than the healthy aortic valve, and the deletion of the calponin-2 gene can slow down the process of aortic valve calcification disease in mice. Calponin-2 can affect the disease process of atherosclerosis (AS) by regulating the functions of macrophages and vascular smooth muscle cells (VSMCs). Specific knockout of calponin-2 in myeloid cells can reduce macrophage infiltration and alleviate the development of AS lesions in ApoE - / - mice. The above evidence indicates that calponin-2 has great potential application value in the diagnosis, treatment, and prognosis evaluation of cardiovascular diseases. Therefore, the development of monoclonal antibodies with high affinity, high specificity, and high sensitivity is of great significance for the study of the function and mechanism of action of calponin-2 in cardiovascular diseases. Summary of the Invention

[0004] In order to overcome the above-mentioned disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a hybridoma cell line 2F8 secreting a monoclonal antibody against calponin-2 (Calponin-2, CNN2).

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

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

[0007] The object of the present invention is achieved by the following scheme:

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

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

[0010] Further, the monoclonal antibody against calmodulin 2 of the present invention is secreted by the above-mentioned hybridoma cell line 2F8 with the deposit number CGMCC No. 46343.

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

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

[0013] Further, the monoclonal antibody against calmodulin 2 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.

[0014] Further, a polynucleotide encodes the monoclonal antibody against calmodulin 2. In the polynucleotide, the heavy chain variable region has the nucleotide sequence shown in SEQ ID NO: 3, and the light chain variable region has the nucleotide sequence shown in SEQ ID NO: 4.

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

[0016] The present invention also provides the application of the above-mentioned hybridoma cell line secreting calmodulin 2 monoclonal antibody in the preparation of a kit.

[0017] The present invention also provides the application of the above-mentioned hybridoma cell line secreting 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 of a 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 polynucleotide in the preparation of a kit.

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

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

[0023] Furthermore, the kit is an immunoassay-based kit.

[0024] Furthermore, the immunoassay includes enzyme-linked immunosorbent assay, Western blot assay, immunofluorescence assay, immunohistochemical assay, etc.

[0025] Furthermore, the kit can be a colloidal gold immunoassay kit, a chemiluminescence 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 kit.

[0027] The present 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 tissues and diseased tissues.

[0028] Furthermore, the tissues include carotid plaque tissues, prostate tissues, colorectal tissues, liver tissues, etc.

[0029] The present invention provides the application of the above-mentioned hybridoma cell line, calmodulin 2 monoclonal antibody or polynucleotide in the preparation of immunoassay reagents, kits, reagents for detecting calmodulin 2, diagnostic reagents for cardiovascular diseases, diagnostic kits for cardiovascular diseases.

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

[0031] The hybridoma cell line 2F8 of the present invention has the characteristics of efficiently and stably secreting the calponin 2 monoclonal antibody. The calponin 2 monoclonal antibody secreted by the hybridoma cell line 2F8 of the present invention has specific recognition ability for both recombinant calponin 2 protein and calponin 2 in human aortic smooth muscle cells. The Western blot color development result has no non-specific bands, and the detection result is reliable. In the cell immunofluorescence detection and immunohistochemical detection of tissues, the calponin 2 monoclonal antibody of the present invention can effectively and specifically recognize the calponin 2 protein, with accurate staining localization, clear staining and no non-specific staining, and a clean background, indicating that this monoclonal antibody can be used in the applications of Western blot, immunofluorescence and immunohistochemistry detection techniques, and has practical application value. The monoclonal antibody of the present invention is independently developed and prepared, and a hybridoma cell line that stably produces the calponin 2 monoclonal antibody is screened out. It has the advantages of low cost and easy popularization and application, and can be used to prepare a kit for detecting calponin 2, or for preparing immunological detection products such as Western blot, immunofluorescence, immunohistochemistry, etc. for detecting the expression of calponin 2, and has practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0033] Figure 1 It is the protein electrophoresis result of the purified recombinant calponin 2 antigen of the present invention; among them, lane: M is the protein marker; rCNN2 is the purified recombinant antigen.

[0034] Figure 2 It is the protein electrophoresis result of the purified monoclonal antibody of the present invention; among them, lane: M is the protein marker; MAb-2F8 is the purified monoclonal antibody.

[0035] Figure 3 It is the identification result of the immunoglobulin subtype of the monoclonal antibody of the present invention.

[0036] Figure 4 It is the immunoblot analysis result of the monoclonal antibody of the present invention; among them, lane: M is the protein marker; rCNN2 is the recombinant calponin 2 of the present invention; HASMC is the human aortic smooth muscle cell sample.

[0037] Figure 5This is the result of immunofluorescence identification of the monoclonal antibody of the present invention.

[0038] Figure 6 This is a comparative diagram of immunohistochemical staining results of human carotid plaque tissue.

[0039] Figure 7 This is a diagram of immunohistochemical staining results of tumor tissue. Detailed implementation manners

[0040] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. The materials involved in the following embodiments can be obtained from commercial channels without special instructions. The methods are conventional methods without special instructions.

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

[0042] According to the amino acid sequence of Calponin-2 numbered Q99439 in the Uniprot database (https: / / www.uniprot.org), the 1-283 amino acid fragment was selected, and 10 histidines were added at the end, that is, the sequence is:

[0043] MSSTQFNKGPSYGLSAEVKNRLLSKYDPQKEAELRTWIEGLTGLSIGPDFQKGLKDGTILCTLMNKLQPGSVPKINRSMQNWHQLENLSNFIKAMVSYGMNPVDLFEANDLFESGNMTQVQVSLLALAGKAKTKGLQSGVDIGVKYSEKQERNFDDATMKAGQCVIGLQMGTNKCASQSGMTAYGTRRHLYDPKNHILPPMDHSTISLQMGTNKCASQVGMTAPGTRRHIYDTKLGTDKCDNSSMSLQMGYTQGANQSGQVFGLGRQIYDPKYCPQGTVADGAHHHHHHHHHH (SEQ ID NO:1), a total of 293 amino acids.

[0044] After human codon optimization, the sequence is:

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

[0046] The above-mentioned calmodulin 2 gene was constructed between the multiple cloning sites Nhe I and Pme I of the eukaryotic expression vector pcDNA3.1 and transformed into the competent engineering bacteria DH5α. After picking clones and amplifying, the accuracy of the eukaryotic expression plasmid was finally confirmed by PCR and sequencing.

[0047] A large amount of endotoxin-free eukaryotic expression plasmid pcDNA3.1-CNN2 was extracted. Suspended HEK293F cells in good growth state and in the logarithmic growth phase were inoculated into a culture flask containing 100 mL of SMM 293-TII medium (Beijing Sino Biological Inc.) at a cell density of 1.5×10 6 cells / mL. The recombinant plasmid pcDNA3.1-CNN2 was transfected into HEK293F cells using the EZ Trans high-efficiency transfection reagent (Heyuan LiJi (Shanghai) Biotechnology Co., Ltd.). The SMM 293-ENH cell culture medium enhancer (Beijing Sino Biological Inc.) 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] The recombinant protein was purified using His-tag protein purification magnetic beads (IDA-Ni) (BeaverNano). After pretreatment of the magnetic beads, the cell supernatant was incubated with the magnetic beads for 30 min. After discarding the supernatant, the magnetic beads were washed for 10 min, the washing solution was discarded, and the recombinant protein was eluted by adding the elution buffer. After preparing a sample with 2 μg of the recombinant protein, SDS-PAGE electrophoresis was performed. After Coomassie brilliant blue staining, its molecular weight and purity were analyzed and identified. As Figure 1 shown, there was a clear protein band at about 36 kDa, and the purity of the recombinant calmodulin 2 was good. The recombinant calmodulin 2 was collected and stored in aliquots at -80 °C in the refrigerator for subsequent mouse immunization.

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

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

[0051] The recombinant calmodulin 2 in Example 1 was fully emulsified with Freund's complete adjuvant (Sigma), and three 6-8-week-old female BALB / c mice (Guangzhou Southern Medical University Animal Science and Technology Development Co., Ltd.) were immunized for the first time. Multiple-point injections were performed subcutaneously on the abdomen of the mice at a dose of 100 μg / mouse. Booster immunizations were performed every 2 weeks. The recombinant CNN2 protein was fully emulsified with Freund's incomplete adjuvant (Sigma) and multiple-point injections were performed subcutaneously on the abdomen of the mice at a dose of 100 μg / mouse. Seven days after the third immunization, the titer of anti-CNN2 protein in the mouse serum was detected by indirect enzyme-linked immunosorbent assay (ELISA), and the mouse with the highest serum titer was selected. The recombinant calmodulin 2 was diluted with normal saline and boost immunization was performed by intraperitoneal injection at a dose of 100 μg / mouse. Finally, the immunized BALB / c mice were obtained.

[0052] 2. Preparation and screening of hybridoma cells

[0053] Five days after the boost immunization, the mice were sacrificed by cervical dislocation. The spleens of the mice were removed under sterile conditions and prepared into spleen cell suspensions. Logarithmic growth phase myeloma cells SP2 / 0 were mixed well with mouse spleen cells at a ratio of 1:5. After centrifugation, the supernatant was discarded. Cell fusion was carried out by the conventional polyethylene glycol method (Sigma): 1 mL of 50% polyethylene glycol preheated at 37°C was slowly added dropwise within 1 minute while mixing, and the addition was terminated by adding serum-free 1640 medium preheated at 37°C. After centrifugation and discarding the supernatant, the cells were resuspended in RPMI 1640 complete medium containing HAT. The fused cells were plated into 96-well cell culture plates at 200 μL / well and cultured in an incubator at 37°C and 5% CO2.

[0054] Ten days after the fusion culture, hybridoma cell clones were selected and the cell line numbers were marked. The culture supernatants of the hybridoma cells were detected by indirect ELISA method. The culture supernatants of the positive wells were discarded and replaced with RPMI 1640 complete medium containing HA. Two days later, the culture supernatants were detected by indirect ELISA again. Hybridoma cell clones showing strong positive reactions were selected, and the positive clones were cloned by the limited dilution method to obtain hybridoma cell lines with a positive rate of 100% and stably secreting 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 in the China General Microbiological Culture Collection Center on April 9, 2025, with the deposit number CGMCC No. 46343.

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

[0056] The hybridoma cell line (2F8) was amplified to the logarithmic growth phase and inoculated at 5.0×10 5Cells / mL were inoculated into a shake flask containing CDM4 medium (Hyclone, USA) supplemented with L-glutamine and cultured in a shaker at 37°C, 5% CO2, and 100 rpm. Starting from the next day, Cell Boost (Hyclone, USA) was replenished daily. 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 normal pressure purifier. The chromatography column was equilibrated with 5 column volumes of equilibration buffer; the cell supernatant to be purified was slowly loaded onto the chromatography column, and washed with 10 column volumes of equilibration buffer until the baseline; 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 the hybridoma cell line 2F8 was named MAb-2F8. After sampling the purified antibody, protein gel electrophoresis was performed, and the purity and size of the monoclonal antibody were identified after Coomassie brilliant blue staining. The results are shown in Figure 2 , the purity of the purified MAb-2F8 monoclonal antibody of the present invention reached over 90%. The molecular weights of the heavy chain and light chain of this monoclonal antibody were approximately 50 kDa and 28 kDa, respectively.

[0057] The hybridoma cell line 2F8 was sent to Nanjing Mingyan Biotechnology Co., Ltd. for analysis of the variable region genes and amino acid sequences of the monoclonal antibody. Total RNA was extracted from the hybridoma cells, and after reverse transcription to obtain cDNA, the variable region genes of the heavy chain and light chain were amplified using specific primers respectively, ligated to a cloning vector, transformed and plated, and positive clone strains were picked for gene sequencing. The nucleotide sequences of the heavy chain variable region (shown in SEQ ID NO: 3) and light chain variable region (shown in SEQ ID NO: 4) of the monoclonal antibody prepared by the present invention were obtained, and the amino acid sequences of the heavy chain variable region (shown in SEQ ID NO: 5) and light chain variable region (shown in SEQ ID NO: 6) were deduced.

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

[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 full-length heavy chain variable region contains 120 amino acids. The numbers of amino acids in the 4 domains of FR are 25, 17, 38, and 11 respectively. The numbers of amino acids in the 3 domains of HCDR are 8, 10, and 11 respectively. The amino acid sequences of the regions of HCDR1, HCDR2, and HCDR3 are: GFTFNTNS, IRSKSDNYAT, VRDRTGGGFAY respectively.

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

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

[0067] The full-length light chain variable region contains 111 amino acids. The numbers of amino acids in the 4 domains of FR are 26, 17, 36, and 10 respectively. The numbers of amino acids in the 3 domains of LCDR are 10, 3, and 9 respectively. The amino acid sequences of the regions of LCDR1, LCDR2, and LCDR3 are: KSVSTSGYSY, LAS, QHSRELPLT respectively.

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

[0069] 1. Subtype identification

[0070] The immunoglobulin subtype of the purified monoclonal antibody was identified using a mouse monoclonal antibody subtype identification kit (Proteintech). The monoclonal antibody sample was diluted to 200 ng / mL with 1×PBST and added to the reaction wells coated with anti-heavy chain IgG1, IgG2a, IgG2b, IgG3, IgA, IgM and light chain Kappa, Lambda antibodies at 50 μL / well. Then, the diluted goat anti-mouse IgA+IgM+IgG-HRP was added to the reaction wells at 50 μL / well, gently mixed on a mixer, covered with a sealing film, and incubated at room temperature for 1 h; the liquid in the wells was discarded, and the plate was washed 3 times with 1×PBST and patted dry on absorbent paper; the prepared chromogenic solution was added to the reaction wells at 100 μL / well, and the color was developed at room temperature in the dark for 15 min; finally, the stop solution was added to the reaction wells at 100 μL / well, and the OD value at 450 nm was read by an enzyme-linked immunosorbent assay reader. The results showed ( Figure 3 ), that the monoclonal antibody of the present invention is an immunoglobulin of the IgG1 class, and the light chain type is the Kappa chain.

[0071] 2. Antibody affinity determination

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

[0073] 3. Antibody specificity detection

[0074] Take the purified recombinant calmodulin 2 and human aortic smooth muscle cells in Example 1, make samples respectively, and detect the antigen-binding specificity of the monoclonal antibody of the present invention by immunoblotting. Perform electrophoresis using a 10% SDS-PAGE protein gel, and electrotransfer the gel proteins to a PVDF membrane using a Bio-Rad transfer device. Block the membrane with 5% skim milk at room temperature for 1 h, and wash the membrane 3 times with TBST. Take the purified monoclonal antibody in Example 2, add it at a dilution ratio of 1:2000, incubate overnight at 4 °C, and wash the membrane 3 times with TBST, 8 min each time. Add the HRP-labeled goat anti-mouse IgG secondary antibody (diluted at 1:5000), incubate at room temperature for 1 h, and wash the membrane 3 times with TBST, 8 min each time. Add the ECL hypersensitive chromogenic solution (Biosharp), and collect the chemiluminescence image data in a ChemiDoc MP all-in-one imaging system (BIO-RAD). The results are as Figure 4As shown, the monoclonal antibody secreted by the hybridoma cells prepared by the present invention can specifically recognize recombinant calponin 2 and calponin 2 in natural samples, and no other impurity bands are seen, indicating that the monoclonal antibody prepared by the present invention has good specificity and can be applied to immunoblotting experiments.

[0075] Example 4: Application of Monoclonal Antibody Against Human Calponin 2

[0076] 1. Immunofluorescence Detection

[0077] Human aortic smooth muscle cells (HASMC) and human umbilical vein endothelial cells (HUVEC) were respectively inoculated into 24-well cell culture plates with coverslips. When cultured to an appropriate density, they were washed 3 times with PBS. Fixed with 4% paraformaldehyde at room temperature for 30 min and washed 3 times with PBS. Permeabilized with 0.1% Triton X-100 at room temperature for 15 min and washed 3 times with PBS. Blocked with 5% BSA at room temperature for 30 min, and the blocking solution was discarded. The purified monoclonal antibody in Example 2 was taken and diluted with 5% BSA to a concentration of 2 μg / mL, and then dropped onto the coverslips respectively, incubated at room temperature for 2 h, and washed 3 times with PBS. The DyLight 594-labeled goat anti-mouse IgG fluorescent secondary antibody (Invitrogen) was diluted with 5% BSA at a ratio of 1:500 and dropped onto the coverslips respectively, incubated at room temperature for 1 h, and washed 3 times with PBS. A fluorescence anti-fading mounting medium (containing DAPI) was dropped, and the coverslips were inverted and mounted on glass slides, and observed and photographed through an inverted fluorescence microscope. The microscopic examination results showed ( Figure 5 ): The monoclonal antibody secreted by the hybridoma cell line 2F8 of the present invention can effectively recognize calponin 2 (red fluorescence) in cells. It can be seen that the red fluorescence is mainly distributed in the cytoplasm, and the staining result is clear and the background is clean, indicating that the monoclonal antibody prepared by the present invention can be used for cell immunofluorescence staining and has good specificity.

[0078] 2. Immunohistochemical Analysis

[0079] The carotid plaque tissue sections were dewaxed twice in an environmentally friendly tissue clearing and dewaxing agent (Beyotime), 15 minutes each time. They were hydrated in a gradient manner in 100%, 100%, 95%, 85%, and 75% ethanol, 5 minutes each time, and finally rinsed with tap water for 5 minutes. Heat antigen retrieval was performed using citrate buffer. After natural cooling to room temperature, they were washed 3 times with PBS, 3 minutes each time. After draining the water, 3% hydrogen peroxide was used to inactivate endogenous peroxidase in the tissue, and it was incubated at room temperature in the dark for 15 minutes. They were washed 3 times with PBS, 3 minutes each time. 5% BSA was used for blocking, and it was incubated at room temperature for 30 minutes. After draining, a circle was drawn around the tissue with an immunohistochemistry pen, and monoclonal antibody secreted by the hybridoma cell line 2F8 of the present invention (1 μg / mL) and commercially available anti-human calmodulin 2 rabbit polyclonal antibody (Proteintech) were respectively dropped, and incubated overnight at 4°C. They were washed 5 times with PBS, 3 minutes each time. Enzyme-labeled goat anti-mouse / anti-rabbit IgG secondary antibody (Gene Tech) was added dropwise, and it was incubated at room temperature for 30 minutes. They were washed 5 times with PBS, 3 minutes each time. DAB chromogenic solution (Gene Tech) was added dropwise, and it was developed in the dark for 3 - 5 minutes (observed under a light microscope and the development time was controlled). The development was terminated by placing it in tap water, and it was rinsed for 10 minutes. Hematoxylin was used for counterstaining, differentiation, and bluing. The sections were dehydrated in a gradient of 75%, 85%, 95%, 100%, and 100% alcohol for 3 minutes each in turn, and finally cleared in an environmentally friendly tissue clearing and dewaxing agent for 3 minutes, sealed with neutral gum, and observed and photographed under a microscope. The microscopic examination results showed ( Figure 6 ), the monoclonal antibody secreted by the hybridoma cell 2F8 of the present invention could show calmodulin 2 in the tissue when detecting plaque tissue by IHC method, the staining was clear and there was no non-specific staining, the background was clean, and it was consistent with the detection results of the commercially available antibody on tissue sections. The staining localization was accurate and showed specific cytoplasmic expression in the tissue, indicating that the specificity of this antibody in the tissue was comparable to that of the commercially available antibody and it could be used for immunohistochemical detection.

[0080] 3. Immunohistochemical detection in different tumor tissues

[0081] Paraffin sections of human different tumors (prostate cancer, liver cancer, colorectal cancer) tissues were taken, and the monoclonal antibody MAb-2F8 of the present invention was used for immunohistochemical detection of the section tissues. The experimental steps were the same as those in Example 4.2. The experimental results were as Figure 7 shown. The immunohistochemical results showed that the staining localization of the monoclonal antibody of the present invention in each tumor tissue section was accurate, the staining was clear and the background was clean, and there was no non-specific staining, indicating that the monoclonal antibody of the present invention could be well used for immunohistochemical analysis to detect the expression level and expression position of calmodulin 2 in tumor tissues. Combining the results of immunoblotting and immunofluorescence, it was shown that the monoclonal antibody MAb-2F8 secreted by the hybridoma cell 2F8 had good effect and specificity in recognizing human calmodulin 2 antigen.

[0082] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A hybridoma cell line secreting calponin 2 monoclonal antibody, characterized in that Classified and named as 2F8, it was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 9, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46343.

2. A calmodulin 2 monoclonal antibody, characterized in that Named MAb-2F8, it is secreted by the hybridoma cell line 2F8 with the deposit number CGMCC No. 46343 as claimed in claim 1 or its subculture cell line.

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, wherein: The calmodulin 2 monoclonal antibody described above comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region has the amino acid sequence as shown in SEQ ID NO: 5, and the light chain variable region has the amino acid sequence as shown in SEQ ID NO:

6.

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

4.

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

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

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

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

10. Use of the hybridoma cell line as claimed in claim 1, the calmodulin 2 monoclonal antibody as claimed in any one of claims 2-4, or the polynucleotide as claimed in claim 5 in the preparation of an immunodetection reagent, a kit, a reagent for detecting calmodulin 2, a reagent for diagnosing cardiovascular diseases, or a kit for diagnosing cardiovascular diseases.

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