Hybridoma cell strain and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and relates to a hybridoma cell strain C8-3-7 and a preparation method thereof. The preservation number of the cell strain C8-3-7 is CCTCC (China Center For Type Culture Collection) No: C202573. The hybridoma cell strain C8-3-7 efficiently expresses the DHX33 antibody and has an infinite proliferation capability. The DHX33 antibody can be specifically combined with DHX33 protein, and can specifically recognize DHX33 protein antigen in ELISA (enzyme-linked immuno sorbent assay) and immunohistochemistry or immunocytochemistry analysis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a hybridoma cell strain expressing and secreting DHX33 protein antigen antibody and a preparation method thereof. BACKGROUND

[0002] Monoclonal antibody is different from conventional serum antibody, which is a mixture of antibodies against many antigenic determinants and is produced by polyclonal cells. Monoclonal antibody is an antibody secreted by a hybridoma cell line formed by the fusion of a myeloma cell and a B cell with the ability to secrete antibodies. Monoclonal antibody has better application in the industry due to its higher specificity and stability of the culture production system. Monoclonal antibody has the best utilization value and product stability in disease diagnosis. The basic principle of preparing monoclonal antibody by hybridoma technology is as follows: B cells can proliferate and differentiate into plasma cells under the stimulation of specific foreign antigens to secrete antibodies specific to a single determinant of the antigen. Such antibodies have specificity. The foreign antigen is immunized in animals one or more times to obtain a large number of B cells that specifically react. The hybridoma cells produced by cell fusion technology can maintain the characteristics of both parent cells. The hybridoma cells are screened by metabolic deficiency rescue mechanism, cloned, and then cultured and proliferated to prepare the required monoclonal antibody. PEG is the most widely used fusion agent in lymphocyte hybridoma technology. PEG can make cells aggregate together. PEG can combine with water near the cell membrane, so that the water between cells is replaced, thereby reducing the polarity of the cell surface and causing cell membrane fusion.

[0003] The screening of hybridoma cells is achieved by rescuing the DNA metabolic pathway. DNA synthesis has a main biosynthetic pathway and a rescue pathway. The main biosynthetic pathway is to synthesize DNA using glutamine (Gln) or monophosphate uridine acid (UMP) under the catalysis of dihydrofolate reductase; the rescue pathway is to rescue DNA synthesis using hypoxanthine or thymidine deoxyriboside under the catalysis of hypoxanthine guanine phosphoribosyl transferase (HGPRT) or thymidine deoxyriboside kinase (TK). HAT culture solution includes hypoxanthine + aminopterin + thymidine deoxyriboside. Aminopterin is an inhibitor of dihydrofolate reductase, so it can effectively block the main synthesis pathway of DNA synthesis. After the main biosynthetic pathway of DNA of general cells is blocked by aminopterin, DNA synthesis can still occur through the rescue pathway. If the cells are provided with thymidine deoxyriboside and hypoxanthine in the presence of TK and HGPRT enzymes, DNA synthesis can still occur, but if one of the enzymes is missing, DNA synthesis cannot occur.

[0004] The myeloma cell lines used for cell fusion are drug-induced metabolically deficient cells, lacking TK or HGPRT enzymes. When aminopterin is present, it blocks the main pathway of DNA synthesis, thus requiring a compensatory DNA synthesis pathway. However, because the myeloma cells and their fusion products lack HGPRT or TK enzymes, they die rapidly in HAT culture medium. While immune spleen cells and their fusion products possess HGPRT and TK enzymes, they are short-lived cells that cannot grow and reproduce in culture medium, generally dying within 5-7 days. Only hybridoma cells fused with immune spleen cells acquire both the immortalization and highly proliferative characteristics of myeloma cells, while simultaneously obtaining the gene products of functional HGPRT and TK enzymes from immune spleen cells. Therefore, these hybridoma cells can grow and reproduce in HAT selective culture medium.

[0005] DHX33 protein is a member of the RNA helicase family containing a DEAH box. It is highly expressed in various cancer tissues. Cancer cells lacking DHX33 protein exhibit severely inhibited growth or death. DHX33 has potential therapeutic value. Currently, there are few commercially available DHX33 antibodies, and monoclonal DHX33 antibodies suitable for immunohistochemical detection are even scarcer. There is an urgent need for existing technologies to provide hybridoma cells that efficiently express DHX33 antibodies and are easy to isolate, culture, and purify for subsequent immunohistochemical and ELISA detection and analysis of DHX33 protein. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a hybridoma cell line that efficiently expresses and secretes DHX33 antibody, as well as methods for its preparation, isolation and purification, and the use of DHX33 antibody in ELISA and immunohistochemical detection, so as to make up for the deficiencies of the prior art and achieve the goal of stable production of antibodies for detection.

[0007] On the one hand, the present invention provides a hybridoma cell line C8-3-7, which was deposited at the China Center for Type Culture Collection (CCTCC) on October 28, 2025, with accession number CCTCC No: C202573.

[0008] In an embodiment of the present invention, the hybridoma cell line C8-3-7 expresses and secretes DHX33 antibody.

[0009] In an embodiment of the present invention, the hybridoma cell line C8-3-7 has unlimited proliferative capacity and a short doubling time.

[0010] In a second aspect, the present invention provides a DHX33 antibody expressed and secreted by the hybridoma cell line C8-3-7.

[0011] In an embodiment of the present invention, the DHX33 antibody can specifically recognize the DHX33 protein.

[0012] In a third aspect, the present invention provides a kit for detecting DHX33 protein, the kit comprising an antibody against DHX33 expressed and secreted by hybridoma cell line C8-3-7.

[0013] In a fourth aspect, the present invention provides the use of DHX33 antibodies expressed and secreted by hybridoma cell line C8-3-7 in the specific recognition of DHX33 protein antigens in ELISA and immunohistochemical assays.

[0014] In a fifth aspect, the present invention provides the use of DHX33 antibodies expressed and secreted by hybridoma cell line C8-3-7 in detecting DHX33 protein in tissue and cell samples from various sources.

[0015] In a sixth aspect, the present invention provides a method for preparing the hybridoma cell line C8-3-7, comprising the following steps: S1. Inject the DHX33 protein antigen into mice for 2-5 immunizations; S2. Isolate mouse spleen cells and hybridize them with mouse myeloma cells in vitro; S3. Isolate and culture monoclonal hybridoma cell lines; and S4. Identify and isolate the hybridoma cell line C8-3-7 that specifically recognizes the DHX33 protein antigen.

[0016] In an embodiment of the method of the present invention, in step S1, the dose of DHX33 protein antigen is 100-250 μg per mouse per dose.

[0017] In an embodiment of the present invention, mice can be immunized 2, 3, 4 or 5 times.

[0018] In an embodiment of the method of the present invention, the cell fusion agent used in step S2 is polyethylene glycol. In a specific embodiment, the polyethylene glycol has a molecular weight of 4000, a concentration of 50%, and a pH of 8.0-8.2.

[0019] In an embodiment of the method of the present invention, in step S2, the ratio of mouse spleen cells to mouse myeloma cells is 3-10:1. In a specific embodiment, this ratio can be, for example, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0020] In an embodiment of the method of the present invention, the method may further include step S5 after step S4, namely, culturing and amplifying the isolated hybridoma cell line C8-3-7.

[0021] The hybridoma cell line C8-3-7 provided by this invention is easy to isolate, culture, and purify, has unlimited proliferation capacity, and efficiently expresses DHX33 antibody that specifically recognizes DHX33 protein antigen. Therefore, DHX33 antibody can be used to recognize DHX33 protein antigen in ELISA and immunohistochemical analysis, serving as an experimental tool for subsequent pathological testing or other related scientific research. Attached Figure Description

[0022] Figure 1 The results of Western blot analysis of mouse pre-immune serum against H1299 cancer cell lysate are shown.

[0023] Figure 2 The results of Western blot analysis of mouse serum against H1299 cancer cell lysate following immunization are shown.

[0024] Figure 3 The growth morphology of isolated mouse spleen cells is shown.

[0025] Figure 4 The growth morphology of C8-3-7 hybridoma cells after isolation is shown.

[0026] Figure 5 The growth and proliferation curves of hybridoma cells C8-3-7 are shown.

[0027] Figure 6 shows the specific recognition of DHX33 protein by hybridoma cells C8-3-7 in ELISA detection. 6A shows the ELISA data of C8-3-7 on the initial ELISA selection plate, with highlighted areas indicating C8-3-7 cell clone locations; 6B shows the ELISA data of the antibody purified from 10 mL of C8-3-7 cell supernatant using a Protein A affinity column (1:3 dilution).

[0028] Figure 7 shows the specific recognition of DHX33 protein by hybridoma cells C8-3-7 in immunohistochemical tissue. Among them, Figure 7A DHX33 antibody produced by C8-3-7 -- Analysis of U251-MG cells; Figure 7B DHX33 antibody produced by C8-3-7 was analyzed in normal mouse lung tissue.

[0029] Figure 8 shows the detection activity of antibodies secreted by hybridoma cells C8-3-7 against cancer cell proteins in DHX33-deficient cells. Figure 8A This is an H1299 cell control (DHX33 is present); Figure 8B These are H1299 cells that have had DHX33 knocked out (DHX33 protein deficiency).

[0030] Figure 9 This shows the knockout efficiency of DHX33 protein in cancer cells as analyzed by quantitative PCR.

[0031] Figure 10 Statistical analysis of immunocytochemical staining results of cancer cells secreted by C8-3-7 with purified antibody during DHX33 knockout, compared with the control group (DHX33 not knocked out).

[0032] Hybridoma cell line C8-3-7 has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China, with accession number CCTCC No: C202573 and deposit date of October 28, 2025. The scientific description of this cell line is hybridoma cell line C8-3-7. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments described below are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] 1. Mouse immunization method DHX33 protein (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was used. 1 mg of protein was weighed, dissolved in 0.5 mL of 4 M urea, and aliquoted into four EP tubes (100 μL / tube), stored at -20°C. On the day of use, the thawed DHX33 protein solution was dissolved in 100 μL of water, followed by 200 μL of Freund's complete adjuvant (F850325, Shanghai Maclean's Biochemical Technology Co., Ltd.). The solution was vortexed at high speed for 30 min to achieve complete emulsification. To verify the emulsification effect, 10 μL of DHX33 protein solution was pipetted and added to the aqueous solution on a petri dish. If the DHX33 protein solution did not disperse within 5 min, the emulsification was considered complete. Then, 0.2 mL of the completely emulsified DHX33 protein solution (corresponding to 100-250 μg DHX33 protein) was inoculated into one Balb / c mouse (Beijing Vital River Laboratory Animal Technology Co., Ltd.). Specifically, mice were immunized by subcutaneous injection at five points, with 40-50 μL of the mixed antigen injected at each point. Another mouse was immunized with the same dose. Subsequent booster immunizations were administered via single intramuscular injection, using Freund's incomplete adjuvant (F850326, Shanghai Maclean's Biochemical Technology Co., Ltd.) in a 1:1 emulsification ratio, vortexed for 30 min. The antigen dosage per mouse was 100-250 μg per immunization. 2-4 booster immunizations were administered.

[0035] 2. Isolation of B cells from mouse spleen 1) Material preparation Consumables and instruments: medical ophthalmic scissors, forceps, tray (or petri dish), mortar and pestle (or petri dish and syringe plunger), 200-400 mesh filter, 50 mL centrifuge tubes, 1000 μL pipettes, 100 μL pipettes.

[0036] Reagents: Red blood cell lysis buffer (for specific preparation method, please refer to: Carcinogenesis. 2017 38(6):649-660 Role of DHX33 in c-Myc-induced cancers, Jijun Fu, Yuchu Liu, Xingshun Wang, Baolei Yuan, Yandong Zhang), PBS solution, RMPI-1640 complete culture medium, 75% alcohol.

[0037] 2) Preparation of single-cell suspension For the preparation method of single-cell suspension, please refer to: Li Wenjuan, Zou Jiaqi, Han Xinxin, et al. Establishment of purification and culture method of mouse spleen B lymphocytes [J]. Chinese Journal of Tissue Engineering Research, 2015, 19 (02): 207-212. Specifically, mice were euthanized by cervical dislocation and immersed in 75% alcohol for 5 min. A clean sterilization tray was prepared and placed in the biosafety cabinet in advance. The sterilized mice were placed in the tray. The mice were placed on their sides with their left abdomen facing up. The skin of the mice was cut open in the middle of the abdomen with medical ophthalmic scissors to expose their peritoneum. The red strips seen were the spleen. The peritoneum was lifted with forceps, cut open, and the spleen was removed and placed in PBS phosphate buffer (a culture dish or mortar can be used). A small amount of PBS buffer solution (about 5 mL) was added to the mortar (or culture dish), and the mixture was gently ground until there were no obvious red lumps. Then, PBS buffer solution (to about 10 mL) was drawn into the mortar (or culture dish) and blown into it. Filter the cell suspension through a 200-400 mesh filter and collect it in a 50 mL centrifuge tube. Centrifuge at 300 g for 5 min at room temperature and discard the supernatant. Add 2 mL of erythrocyte lysis buffer and resuspend the cells, mix thoroughly by pipetting, and lyse at room temperature for 2-3 min. Immediately add 10 mL of PBS and centrifuge at 300 g for 5 min at room temperature. Discard the supernatant and resuspend the cells in RMPI-1640 complete culture medium. Observe the cell concentration under a microscope.

[0038] 3. Fusion of mouse myeloma cells Polyethylene glycol (PEG) used as a cell fusion agent typically has a molecular weight of 4000, a commonly used concentration of 50%, and a pH of 8.0–8.2 (adjusted with 10% NaHCO3). Lower molecular weight PEG exhibits poor fusion effects and is toxic; higher molecular weight results in excessive viscosity and difficulty in handling. The fusion effect is highest at a 50% PEG concentration and a slightly alkaline pH. Different batches of PEG, even with the same molecular weight, can show significant differences in fusion rates, requiring careful selection. Each batch must undergo cytotoxicity testing before application. High-purity PEG for gas chromatography is usually chosen.

[0039] There are many methods for cell fusion, the most common being rotation and centrifugation. During fusion, the ratio of spleen cells to myeloma cells is 3-10:1. The reagents and materials used for fusion are as follows: (1) Spleen cells and myeloma cells for fusion. (2) 100 mL of RPMI-1640 culture medium. (3) 100 mL of RMPI-1640 complete culture medium. (4) 50 mL of 2.5% FCS-1640 solution. (5) 100 mL of HAT culture medium. (6) 50% PEG: Take 10 g of high-purity PEG (SERVAS) with a molecular weight of 4000 and autoclave it in a 25 mL bottle. Before use, mix it with 10 mL of RMPI-1640 culture medium preheated to 40 ℃ (w / v) and check the pH with phenol red. Generally, it is not necessary to adjust the pH. If the pH changes, it can be adjusted with HCl or NaHCO3. (7) 10 mL and 50 mL sterile precipitation tubes or bottles. (8) 40-well plastic culture dish. The specific cell fusion steps are as follows: 1) Prepare mouse spleen cells, 2) Mix 10... 8 10 mouse spleen cells and 10 71) Collect mouse myeloma cells and add 50 mL of 2.5% FCS-1640 solution. 2) Centrifuge at 400 g for 3 min at room temperature to pellet the cells. 3) Remove the supernatant. 4) Gently tap the bottom of the tube to allow the pellet to flow. Place the pellet tube in a 40 ℃ water bath to allow it to reach the fusion temperature. 5) Add 0.8 mL of 50% PEG preheated to 40 ℃. Add slowly dropwise using a 1 mL pipette while shaking the pellet tube. Particles should be visible to the naked eye. The dropwise addition process should last for 2 min. 6) Add 1 mL of RMPI-1640 culture medium while shaking for 1 min. 7) Repeat step 7) once. 8) Add 1 mL of RMPI-1640 culture medium while shaking for 0.5 min. 9) Repeat step 9) once. 10) Add 15 mL of RMPI-1640 culture medium. 11) Centrifuge at 400 xg for 1 min at room temperature to pellet the cells. 12) 14) Remove the supernatant, gently tap the bottom of the tube, and add 25 mL of RMPI-1640 complete culture medium containing HAT. 15) Add one drop (approximately 0.05 mL) of fusion cell solution to each 40-well plastic culture dish inoculated with feeder cells the previous day. 16) Gently shake and incubate at 37 ℃ with 5% CO2 saturated humidity. 17) On days 3, 6, 9, and 10, replace the supernatant with RMPI-1640 complete culture medium containing HAT. Carefully aspirate the supernatant, being careful not to remove cells fixed at the bottom of the wells. Add an appropriate amount of feeder cells as needed. 18) On days 12 and 15, add RMPI-1640 complete culture medium containing HAT. Observe with an inverted microscope before each medium change. Hybridoma cells should appear in approximately 10 days. Most hybridoma cells appear within 10-20 days, but some may take up to a month. After hybridoma cells appear, aspirate the supernatant and check for antibodies. 18) Propagate and passage the continuing-growing hybridoma cells. During passage, HAT medium and RMPI-1640 complete medium may be discontinued and replaced with 10% FCS-1640 medium, depending on the situation. The cells are simultaneously stored in liquid nitrogen and cloned. Antibodies are checked at each generation to prevent mutations and loss of antibody-producing cells.

[0040] 4. Immunohistochemical methods Pathological tissue sections were deparaffinized in xylene and hydrated in a series of solutions with gradually decreasing ethanol concentrations. The antigen was presented in a vaporizer with Tris buffer (pH 9.0). The tissues were then incubated in a methanol solution containing 1% H2O2 to inactivate endogenous peroxidase. After blocking with phosphate-buffered saline (PBS) containing 5% fetal bovine serum for 1 h at room temperature, the tissues were incubated with the primary antibody at 25°C for 2 h. Subsequent immunohistochemical analysis was performed according to the manufacturer's instructions using the DAKO kit (DAKO, K5007). The antibodies used were as follows: primary antibody: DHX33 antibody; secondary antibody: ready-to-use rapid immunohistochemistry kit MaxVision™ HRP (mouse / rabbit), purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.

[0041] 5. Western blot Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher). After incubation on ice for 10 min, cell lysates were further disrupted by sonication. Whole-cell extracts were then subjected to SDS-PAGE gel electrophoresis, and protein content was determined using the BCA method. Protein analysis was performed on equal volumes of each sample (50 μg total protein per sample). Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% skim milk and incubated in 1×TBST buffer at room temperature for 1 h. The primary antibody was diluted in 5% FBS (with 1×TBST) and incubated with the membrane overnight at 4°C. The membranes were then washed several times with 1×TBST buffer and incubated at room temperature for 2 h with HRP (horseradish peroxidase)-labeled secondary antibody (Thermo Fisher) in 5% FBS (diluted in 1×TBST). The blot was visualized using an ECL kit (Thermo Fisher).

[0042] 6. Real-time quantitative PCR Primers were designed using the online "Realtime PCR Tool" of IDT (http: / / sg.idtdna.com / site) and purchased from Guangzhou Aiji Biotechnology Co., Ltd. Total RNA was extracted using an RNA isolation kit (Shanghai Yisheng Biotechnology Co., Ltd.) and then transcribed into cDNA using a PrimeScript mixing kit (Takara). Real-time PCR was performed using Bio-Rad CFX fluorescence quantitative PCR, managed with the corresponding software. To analyze mRNA levels, standard quantitative PCR reactions were performed using SYBR greenSupermix (Bio-Rad), and the target gene transcript content was calculated using ΔΔCT values ​​after normalization to GAPDH values. Melting curves were used to confirm the amplification of single products. Primer sequences for DHX33 and GAPDH can be found in the paper Identification of DHX33 as a mediator in rRNA synthesis and cellgrowth. Zhang, Y., Forys, JT., Miceli, A., Gwinn, A., Weber, JD. MolecularCellular Biology 2011, 31 (23), 4676-4691.

[0043] 7. Cell Culture The isolated hybridoma cells were cultured in F12K medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, streptomycin, and penicillin. Human primary HFF (human foreskin fibroblast) were cultured in DMEM medium containing 10% FBS, 2 mM L-glutamine, streptomycin, and penicillin, and incubated in a humidified environment containing 5% carbon dioxide.

[0044] 8. Cell growth curve analysis Cells were digested with trypsin and resuspended in complete cell culture medium to produce a single-cell suspension. Cells were counted and then seeded in 6-well plates at 50,000 cells per well. The number of cells was counted daily from the start of plate formation.

[0045] 9. ELISA Experiment The reagents used in the ELISA experiment were prepared as follows: Coating buffer: 1.59 g Na₂CO₃ + 2.93 g NaHCO₃ + 900 mL pure water, mix thoroughly, adjust the pH to 9.6, and bring the volume to 1 L to obtain carbonate buffer. Washing buffer: 0.2 g KH₂PO₄ + 1.356 g Na₂HPO₄ + 0.2 g KCl + 8.0 g NaCl + 900 mL pure water, mix thoroughly, and bring the volume to 1 L to obtain 0.15 mol / L PBS. Add 0.5 mL Tween-20 to obtain PBS-T. BSA blocking solution: 1 g bovine serum albumin (BSA) + 100 mL washing buffer to obtain 1% BSA blocking solution. Diluent: 0.1 g bovine serum albumin + 100 mL washing buffer to obtain 0.1% BSA diluent.

[0046] Take an appropriate concentration of DHX33 protein, dilute it to 10 μg / mL with coating buffer, and add 100 μL to each well using a pipette. Incubate overnight at 2-8°C. Discard the coating buffer, wash the plate with 270 μL of washing buffer for 5 min, repeat three times, and pat dry on a paper towel. Block: Add 200 μL of 1% BSA blocking buffer to each well and block at room temperature for 60 min. Wash: Discard the blocking buffer, wash the plate with 270 μL of washing buffer for 5 min, repeat three times, and pat dry on a paper towel. Add antibody: Dilute the antibody to an appropriate concentration using antibody diluent, add 100 μL to each well, and incubate at 37°C for 120 min. Discard the antibody / peptide antigen mixture, wash the plate with 270 μL of washing buffer for 5 min, repeat three times, and pat dry on a paper towel. Add 100 μL of HRP-labeled secondary antibody (Thermo Fisher Scientific) to each well and incubate at 37°C for 120 min. Discard the enzyme conjugate buffer, wash the plate with 270 μL of washing buffer for 5 min. Repeat the reaction three times, patting dry on a paper towel as much as possible; add 100 μL of TMB chromogenic solution to each well, wrap with aluminum foil to protect from light, and react at room temperature for 30 min; add 100 μL of TMB chromogenic stop solution to each well; read the absorbance at 450 nm using a multi-mode microplate reader.

[0047] 10. Antibody purification Collect all cell suspensions and centrifuge at 2500 xg for 5 min at 4°C. Collect the supernatant and filter it through a 0.22 μm filter membrane. Then, dilute the supernatant two-fold with binding buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0) to prepare the sample. Incubate the diluted sample at room temperature for 15–20 min to ensure protein solubility. Equilibrate a 1 mL HiTRap Protein G HP chromatography column with 10 mL of pre-chilled PBS buffer. Load the sample at a flow rate of 1 mL / min or 1.5 mL / min, with a column pressure not exceeding 0.3 MPa. After loading, wash away unbound material with 10 or 20 column volumes of binding buffer. Gradient elution: Remove non-specific contaminants with 5 column volumes of 70% elution buffer (100 mM glycine-HCl, pH 2.7), then elute the target protein with 10 column volumes of 100% elution buffer (100 mM glycine-HCl, pH 2.7) (or elute the antibody with 5 mL of 100 mM glycine (pH 2.5; protein ligands may hydrolyze if the elution buffer pH is below 2). Collect the eluent fractions into centrifuge tubes containing 25 μL of 1M TRIS (pH 9.5) and gently neutralize (neutralization steps may vary depending on the specific situation). Combine all fractions, set aside a small amount for protein concentration assays, and assess the purity and specificity of the purified monoclonal antibody by SDS-polyacrylamide gel electrophoresis (using a 10% or 12.5% ​​polyacrylamide gel) and immunoassay. Wash the column with 20 mL of PBS, and finally seal the purified column in 20% ethanol and store at 2–8°C. The amount of opsin determines the specific antibody preparation steps that follow.

[0048] 11. Preparation and infection of lentiviruses with DHX33 knockout To investigate whether the generated hybridoma cells secrete specific antibodies against the DHX33 protein, this invention uses lentivirus-mediated shRNA to silence the messenger RNA of the DHX33 protein. This invention uses plasmid pLKO.1 to construct a lentiviral vector, and the gene of the shRNA sequence is cloned into the pLKO.1 vector via the restriction enzyme sites AgeI / EcoRI. Restriction enzyme sites AgeI / EcoRI are added to both ends of the sh-DHX33 oligoDNA. DNA sequence: sh-DHX33-prooligonucleotide: 5'-CCGGGCTATCGCAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGCTTTTTG-3', sh-DHX33-post-oligonucleotide: 5'-AATTCAAAAAGCTATCGCAAAGTGATCATTTCTCGAGAAATGATCACTTTGCGATAGC-3'.

[0049] Then, the RNA sequence of the above sequence was cloned into the restriction enzyme site AgeI / EcoR of the pLKO.1 vector to obtain a lentivirus that knocks down the expression level of DHX33 protein. This lentivirus contains the above-mentioned lentiviral plasmid. Control group: The control small RNA (shScrambled) sequence is: 5'-CCTAAGGTTAAGTCGCCCTCG-3'. The present invention also provides a method for preparing the lentivirus, including the following steps: transfecting the plasmid mixture into 293T cells (Chinese Academy of Sciences Cell Bank) using the PEI introduction method: Specifically, transfection is performed using cell culture dishes with a diameter of 10 cm. When the 293T cells grow to 90% confluence, the following plasmids are mixed: pLKO.1-shRNA (i.e., the above-mentioned lentiviral plasmid), pCMV-VSV-G, and pCMV-dR8.2 dvpr, using an appropriate plasmid mixing ratio to make the total DNA amount 12 µg per culture dish. Vectors pLKO.1, pCMV-VSV-G, and pCMV-dR8.2 dvpr were all purchased from Biovector Inc. After 16-18 hours, the medium was replaced with RPMI-1640 complete culture medium containing antibiotics (penicillin and streptomycin). After another 24 or 48 hours, the cell culture medium was collected using sterile pipettes, centrifuged at 1000 rpm for two minutes, and the virus in the supernatant was aliquoted into 5 mL sterile centrifuge tubes and stored at -80°C. Lentiviral infection generally uses a 1:10 dilution of the virus, incubated overnight, and then replaced with fresh culture medium.

[0050] 12. Statistical Analysis

[0051] Data are expressed as mean ± SD. Using Stokes... t The Student's t-test is used to determine statistical significance. P A value <0.05 indicates a significant difference.

[0052] Example The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0053] Unless otherwise specified, all examples were conducted under standard experimental conditions, as described in Sam-brook et al., Molecular Cloning: a Laboratory Manual (Sam-brook J & Russell DW, 2012), or as recommended by the manufacturer's instructions. All materials and reagents used, unless otherwise specified, were commercially available.

[0054] 1. Immunizing mice to acquire resistance specifically recognizing the DHX33 protein. Initial immunization in mice: Two 6-week-old Balb / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were vaccinated. Pre-immunization serum was obtained one day prior to immunization via orbital blood sampling. The following day, the DHX33 protein antigen was administered to the mice subcutaneously at multiple sites using Freund's complete adjuvant. Booster immunization: Every 10-14 days, the same antigen, mixed with Freund's incomplete adjuvant, was administered to both mice via intramuscular injection. Antiserum from each initial or subsequent immunization was obtained one day before the next immunization via orbital blood sampling for subsequent serum antibody analysis.

[0055] Western blot analysis of whole-cell lysates of H1299 cancer cells (Chinese Academy of Sciences Cell Bank) was performed using serum from mice before and after a second immunization. The results showed a strong protein detection signal at the DHX33 protein site in the post-immunization serum. Figure 1 and Figure 2 ).

[0056] 2. Spleen isolation and cell extraction from immunized mice On day 14 after four antigen immunizations, mice were removed from the animal room and euthanized by cervical dislocation. The abdominal skin was then thoroughly disinfected with 75% ethanol. The abdominal skin and peritoneum were cut open with sterile scissors, and the spleen was removed with sterile forceps. The spleen tissue was then placed in a sterile mortar and 2 mL of sterile phosphate buffer was added. The spleen tissue was ground. Once all tissue fragments were broken up, the cell suspension was filtered through a sterile 70 mm filter to remove the tissue fragments. The filtered cell suspension was centrifuged at 1500 rpm. The cell pellet was incubated with 2 mL of erythrocyte lysis buffer at room temperature for 2 min (see Role of DHX33 in c-Myc-induced cancers. Fu J, Liu Y, Wang X, Yuan B, Zhang Y. Carcinogenesis. 2017 Jun 1;38(6):649-660) to lyse the erythrocytes. The lysed cell suspension was diluted with 20 mL of sterile PBS and then centrifuged at 1500 rpm for 3 min at room temperature. Red blood cell-free mouse spleen cells could be directly cryopreserved using cell cryopreservation medium at a cell density of 1 x 10⁻⁶ cells / mL. 7 / mL. Cell morphology as Figure 3 As shown.

[0057] 3. Preparation and screening of hybridoma cell lines SP20 cells (Wuhan Pronosei Biotechnology Co., Ltd.) were fused in vitro with spleen B cells from immunized mice. Specific cell fusion methods can be found in the previously described experimental procedures. To enhance the proliferation capacity of the fused cells, the diluted cells were first seeded into 96-well plates, with 1 x 10⁻⁶ cells added to each well. 5 Normal mouse spleen cells (isolated using the same method as described above) were used as supporting cells to provide sufficient growth conditions to stimulate hybridoma cell proliferation. To adaptively improve the survival rate of initial hybridoma cells, they were cultured in 50% theoretical HAT medium during early hybridoma cell screening, and then the HAT concentration was gradually increased to 75% and 100%, respectively. The morphology of the hybridoma cell lines obtained after screening is shown below. Figure 4 As shown in the figure. The clone name of this cell line is C8-3-7 cells. It has a relatively rapid growth and proliferation capacity, and its in vitro proliferation curve is shown in the figure. Figure 5 As shown.

[0058] 4. Detection of antibody activity in hybridoma cell line C8-3-7 by ELISA. Hybridoma cell line C8-3-7 was cultured in 96-well plates. When the cell density reached approximately 10%, the supernatant was collected and added to an ELISA plate coated with DHX33 protein antigen for ELISA. Specific experimental procedures can be found in the previously described experimental methods. As a positive control, a commercially available DHX33 antibody (B4, Santa Cruz Biotechnology, Inc.) was diluted 1:200. RPMI-1640 medium was used as the negative control. The final experimental results are shown below. Figure 6A As shown. Experimental results showed that the ELISA signal value of the bright yellow cell line in one well was higher than that of the surrounding cells. After isolating this cell line, it was further cultured in 10 cm plates, and the cell supernatant was further purified for antibody purification. Multiple elutions were separated by protein A affinity chromatography, eluents E1-E9, among which E5 and E6 showed significantly higher ELISA activity than the other elution fractions (E5 and E6). Figure 6B C8-3-7 cells showed a stronger ELISA positive characteristic, significantly higher than the control.

[0059] 5. Activity analysis of antibodies produced by hybridoma cell line C8-3-7 To further analyze the specificity of the antibody produced by C8-3-7 against DHX33 protein in immunohistochemistry (IHC), immunohistochemical analysis was performed using cancer tissue from a U251-MG cell-bearing mouse xenograft tumor model (Shenzhen Kaiyue Life Science Co., Ltd.). The specific experimental methods are as described above. The results showed that the DHX33 antibody produced by the C8-3-7 cell line exhibited superior activity compared to the commercially available DHX33 antibody (B4). Experimental data showed that most of the staining occurred in the nucleus region. Figure 7A The antibodies produced by this cell line were mostly negative for staining normal mouse tissues. Figure 7B ).

[0060] 6. The antibody produced by C8-3-7 does not recognize other non-specific proteins. To further analyze the specific recognition activity of antibodies produced by the hybridoma cell line C8-3-7 against the DHX33 protein antigen, DHX33 gene knockout cancer cells were used for analysis. To achieve precise knockout of the DHX33 gene in cancer cells, shRNA inhibiting the DHX33 gene was expressed in the lung cancer H1299 cell line via lentivirus-mediated gene silencing of the DHX33 messenger RNA. Three days after lentivirus infection, immunocytochemical experiments were performed on control H1299 cells and DHX33 gene knockout H1299 cells. The experimental results are shown in Figures 8-10. The DHX33 antibody produced by C8-3-7 did not recognize other proteins in the DHX33 gene knockout cancer cells because the recognition signal of the antibody produced by C8-3-7 was significantly reduced after DHX33 gene knockout. Real-time quantitative PCR and Western blotting showed that the DHX33 gene had been effectively knocked out.

Claims

1. The hybridoma cell line C8-3-7, characterized in that, The cell line has the accession number CCTCC No: C202573.

2. The hybridoma cell line C8-3-7 according to claim 1, characterized in that, The cell line has a short doubling time and the ability to proliferate indefinitely.

3. DHX33 antibody, characterized in that, The DHX33 antibody is expressed and secreted by the hybridoma cell line C8-3-7 as described in claim 1 or 2.

4. The DHX33 antibody according to claim 3, characterized in that, The DHX33 antibody specifically recognizes the DHX33 protein.

5. A kit for detecting DHX33 protein, characterized in that, The kit contains the DHX33 antibody as described in claim 3 or 4.

6. Use of the DHX33 antibody according to claim 3 or 4 in detecting DHX33 protein in tissue and cell samples from various sources.

7. A method for preparing the hybridoma cell line C8-3-7 according to claim 1 or 2, characterized in that, Includes the following steps: S1. Inject the DHX33 protein antigen into mice for 2-5 immunizations; S2. Isolate mouse spleen cells and hybridize them with mouse myeloma cells in vitro; S3. Isolate and culture monoclonal hybridoma cell lines; and S4. Identify and isolate the hybridoma cell line C8-3-7 that specifically recognizes the DHX33 protein antigen.

8. The method according to claim 7, characterized in that, In step S1, the dose of the DHX33 protein antigen is 100-250 μg per mouse per dose.

9. The method according to claim 7, characterized in that, In step S2, the cell fusion agent used is polyethylene glycol, preferably with a molecular weight of 4000, a concentration of 50%, and a pH of 8.0-8.

2.

10. The method according to claim 7, characterized in that, In step S2, the ratio of mouse spleen cells to small myeloma cells is 3-10:1.

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