CASC2 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention provides a CASC2 monoclonal antibody as well as a preparation method and application thereof. An applicant is preserved in the China General Microbiological Culture Collection Center (CGMCC) through a hybridoma cell strain CASC2, and the preservation number is CGMCC No.45959. The prepared monoclonal antibody can effectively recognize the CASC2.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a CASC2 monoclonal antibody and a preparation method and use thereof. Background Art

[0002] Prediction may play an important role in tumorigenesis and progression. Prediction can inhibit the Wnt / β-catenin signaling pathway, thereby reducing tumor cell proliferation and invasion. Prediction can serve as a potential diagnostic marker and therapeutic target. Prediction is active in the cell nucleus.

[0003] There is no report on how to prepare CASC2 monoclonal antibodies. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a CASC2 monoclonal antibody and a preparation method and use thereof, so as to fill the gap in the prior art.

[0005] Another aspect of the present invention provides a monoclonal antibody hybridoma cell line CASC2, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.45959.

[0006] Another aspect of the present invention provides CASC2 monoclonal antibodies secreted and produced by the above-mentioned monoclonal antibody hybridoma cell line CASC2.

[0007] Furthermore, the CASC2 monoclonal antibody is IgG1.

[0008] Another aspect of the present invention provides use of a CASC2 monoclonal antibody in preparing a sample for detecting CASC2.

[0009] Furthermore, the sample is a biological sample, which may be tissue, cell, etc.

[0010] As described above, the detection and use of the protein CASC2 of the present invention have the following beneficial effects:

[0011] The monoclonal antibody prepared in the present application and the monoclonal antibody prepared by the hybridoma cell line CASC2 can effectively recognize CASC2. Cell name: Monoclonal antibody hybridoma cell line Storage time: June 14, 2024 Depository: China National Microbiological Culture Collection Administration General Microbiology Center Deposit number: CGMCC No.45959 Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure shows a schematic diagram of an immunohistochemical method for verifying the subcellular localization of CASC2 antibody in mouse kidney tissue in Example 1. DETAILED DESCRIPTION

[0013] Example 1 Preparation of hybridoma cell lines and monoclonal antibodies Hepatocellular carcinoma (HCC) is the most common hepatocellular malignancy. Researchers have reported that cancer susceptibility candidate 2 (cas2), a long noncoding RNA, is downregulated in a variety of cancers including HCC. Our study aimed to explore the molecular mechanism of CASC2 in HCC. Real-time quantitative PCR (RT-qPCR) was used to analyze the expression of CASC2 and miR-183 in HCC tissues and cells. The MTT method was used to detect the viability of liver cancer SMMC-7721 and Huh-7 cells. The colony formation ability of HCC cells was evaluated by colony formation assay. The migration and invasion ability of liver cancer cells were evaluated by Transwell method. Western blot was used to detect the levels of key Wnt / β-catenin signaling pathway factors, C-myc, cyclinD, survivin and β-catenin. The interaction between CASC2 and miR-183 was confirmed by bioinformatics analysis and luciferase reporter gene assay. CASC2 was downregulated and miR-183 was upregulated in HCC tissues and cell lines. The expression of miR-183 was negatively correlated with that of CASC2 in HCC tissues. In HCC cells, overexpression of CASC2 inhibited cell viability, colony formation, migration and invasion, and the activity of the Wnt / β-catenin signaling pathway. miR-183 is a downstream target of CASC2 and is negatively regulated by CASC2. The introduction of miR-183 rescued the inhibitory effects of CASC2 on HCC cell viability, colony formation, migration and invasion, and Wnt / β-catenin signaling induced by CASC2. CASC2 directly downregulated miR-183 by inactivating the Wnt / β-catenin signaling pathway, thereby inhibiting cell viability and the colony formation, migration and invasion of liver cancer cells. CASC2 plays an important role in the pathogenesis of liver cancer and may serve as a potential target for cancer diagnosis and treatment.

[0014] 1) Analysis and design of peptide sequences Abdesigner software was used to perform antigen epitope analysis on the amino acid sequence of CASC2 protein, mainly evaluating the hydrophilicity, antigenicity, surface possibility, flexible area and other indexes. Combined with the actual experience of preparing antibodies in the past, the complexity of the amino acid structure, the degree of oxidation, the difficulty of synthesis, the type and distribution of amino acids were considered, and finally the 14 amino acids at positions 2-15 of CASC2 protein were determined as the amino acid sequence of the synthetic polypeptide, and the sequence was TQEKMYPHLFSERC (SEQ ID NO.1).

[0015] 2) Peptide synthesis and cross-linking Synthesis: The target peptide was automatically synthesized using an ACT396 fully automatic multi-channel peptide synthesizer through a pre-programmed program. The target peptide was dissolved in 50% acetonitrile solution, and its structure was identified by mass spectrometry to confirm that it was the target protein.

[0016] Cross-linking: Use Sulfo-SMCC as a cross-linking agent to cross-link KLH with the synthetic peptide. Dissolve 30 mg of KLH in 2.0 ml of ultrapure water; dissolve 2.5 mg of Sulfo-SMCC in 0.5 ml of ultrapure water, and adjust the pH to about 7 with 3 M sodium hydroxide. After mixing, slowly add the Sulfo-SMCC solution to the KLH solution, and stir the reactants evenly at room temperature for 45 minutes. On this basis, load the reacted Sulfo-SMCC / KLH mixture onto a Sephadex G25 column that has been pre-equilibrated with an equilibrium buffer (0.05M PB, pH 6.0) for 30 minutes, and collect the light gray eluate, which is the activated sulfo-SMCC / KLH solution. Dissolve 3 mg of cross-linked peptide in 300 ul PBS (pH 7.4), add 0.3 volume of sulfo-SMCC / KLH complex solution to the peptide solution, adjust the pH to 7.3, shake at room temperature for 10 hours, freeze at -70°C and place in a freeze dryer for 24 hours for later use. The peptide cross-linking efficiency was determined by detecting the peptide thiol groups before and after cross-linking using the Ellman method, and the coupling rate was 98.4%.

[0017] 3) Peptide immunization and antiserum preparation 85µ g of cross-linked KLH-peptide was dissolved in 85µ l of phosphate buffer (0.01 M PBS) and an equal volume of Freund's complete adjuvant was added (until water does not diffuse) for full emulsification. Healthy BalB / C mice aged 8 weeks and weighing about 20 grams were used for immunization and injected intradermally on the back, with at least 3 injections. Two weeks after the first immunization, 90µ g of peptide was dissolved in 85µ l of phosphate buffer (0.01 M PBS) and fully emulsified with an equal amount of Freund's incomplete adjuvant for intradermal immunization. As the first booster immunization, it is required to inject intradermally on the back, with at least 3 injections. Two weeks after the second immunization, the second booster immunization was carried out, and the specific operation steps were the same as the first booster immunization. One week later, the tail blood was collected, and the ELISA plate was coated with non-cross-linked synthetic peptides, and the immune serum titer was detected by indirect ELISA. The antibody and titer detection were repeated until the antibody titer was greater than 1:10000. 85 µg of peptide was dissolved in 0.425 ml of phosphate buffer (0.01 M PBS) and injected intraperitoneally for boosting immunization.

[0018] 4) Hybridoma cell preparation (1) Cell fusion: 4 days after boosting the immunization of positive mice, blood samples were collected from the mouse eyeballs, and then the mice were killed by cervical dislocation. The mice were immersed in 75% alcohol, the spleen was removed, ground using a 200-mesh steel sieve, and 6 ml of DMEM solution was added. The ground spleen cells were gently blown away using a pipette to disperse them into single cells in the solution. 7 μl of the mixed solution was taken for counting and the total number of spleen cells was obtained. The corresponding number of myeloma cells was supplemented according to the myeloma cell / spleen cell ratio of 1:8.5, and the cells were mixed evenly in the solution. Centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were shaken to mix and make them loose and flowable, and then placed in a water bath with the water temperature adjusted to 37°C. 980 μl of PEG1500 was slowly added using a pipette, stirring slowly during the dripping process and completed within 48 seconds. After adding the droplet, let it stand for 90 seconds. 0.98 ml of serum-free DMEM was added again, stirring and slowly diluting the PEG to stop the reaction. Add 47 ml of serum-free DMEM, stir, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0019] (2) Cell culture screening: 3.7 ml of fusion cell culture solution was extracted, and the fusion cells were slowly blown off from the bottom. HAT culture medium (HAT dissolved in DMEM containing 8% calf serum) was added to calibrate it to 48 ml. 178 μl / well was evenly distributed in a 96-well plate and the cells were cultured. After 2 days, the supernatant of the 96-well plate was discarded and replaced with a newly prepared HAT culture medium (HAT dissolved in DMEM containing 8% calf serum). After 3 days of replacement, 95 ul of supernatant was taken from each well and added to an ELISA plate that had been coated with an antigen, and the positive value of the supernatant was detected by the indirect ELISA method. The wells with higher positive values ​​were selected, and the HAT culture medium was replaced again, and then checked again after 24 hours. The wells with high positive results were limitedly diluted with HT culture solution (HT dissolved in DMEM containing 8% calf serum), and 178 ul / well was distributed in a 96-well plate that already contained cultured cells. After 8 days, the positive value was tested, and the highly positive monoclonal clone was selected for subcloning with DMEM containing 8% calf serum. When the positive monoclonal clone completed subcloning, the positive rate of the supernatant in the 96-well plate could reach 100%. Cell lines with large cell surface area and high positive value were selected for proliferation culture, and the obtained hybridoma cell strains were sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation. 5) Preparation of monoclonal antibodies Two days before cell injection, five healthy female BALB / c mice aged 8 weeks were selected and injected with 0.9 ml paraffin oil into the mouse peritoneum. When the cells were in the logarithmic growth phase, the cells were blown down and counted, 3*100 cells were taken, placed in a centrifuge tube, and centrifuged at 1000 rpm for 5 minutes. The supernatant was poured out, 3.6 ml sterile phosphate buffer (0.01M PBS) was added, the cells were dispersed using a pipette, 30 ml phosphate buffer (0.01M PBS) was added, and centrifuged at 1000 rpm for 5 minutes. 3.5 ml phosphate buffer (0.01M PBS) was added to the centrifuge tube, mixed thoroughly and homogenized, and 0.7 ml of the mixture was injected into the peritoneum of each mouse. Eight days after the injection, the mice were killed by cervical dislocation, placed in 75% alcohol, the abdominal epithelium was cut, a syringe was inserted into the peritoneal cavity, the ascites was slowly taken out and placed in a container and centrifuged at 4100 rpm for 5 minutes. All supernatants were collected and filtered using a 0.45 μm filter tip and mixed with binding buffer at a 1:1 ratio.

[0020] 6) Antibody affinity purification (1) IgG purification: Use a liquid pipette to add 11 ml of 50% Protein-A Sepharose suspension to a 35 ml chromatography column, remove the top and bottom caps, and the column bed volume after the liquid flows out is 5.5 ml. Then rinse with 27.5 ml of deionized water three times. Take out 12 ml of the corresponding serum, mix it with 2 ml of PBS, and add it to a 40 ml chromatography column. Rotate it on a rotating mixer at room temperature (20-25°C) for 2.5 hours to allow the serum sample to flow out. Then wash the chromatography column three times with 22 ml of purification washing solution, and add 11 ml of eluent for elution.

[0021] (2) Peptide affinity purification: Add 1.6 ml of Sulfo-link gel suspension (0.8 ml gel) to the column, drain the column liquid, and rinse the column with 6.4 ml coupling buffer. Dissolve the prepared CASC2 peptide in 1.6 ml coupling buffer and add it to the column, then add 1.6 ml coupling buffer to the column, and mix at room temperature for 1 hour. Rinse the column with 9.6 ml coupling buffer, then add 4.8 ml blocking agent, and mix at room temperature for 1 hour. Rinse the column 3 times, then add 9.6 ml IgG and 4.8 ml PBS to the column, and mix at room temperature for 1 hour. Rinse the column 3 times with PBS, and then elute with 2 ml eluent. Put the purified antibody into a dialysis bag and dialyze at 4 ° C. Dialyze overnight, then centrifuge at 4000 rpm × 40 minutes to separate the precipitate and collect the supernatant. The antibody titer was determined by indirect ELISA method and was 1:32000, and the protein content was determined by Bradford method and was 1.076 mg / ml.

[0022] 7) Immunohistochemical analysis: After confirming that the CASC2 antibody information is correct and the required samples are prepared, proceed with the following steps: 7.1. Dehydration and transparency Dehydrate and transparentize the samples using gradient ethanol and xylene. After trimming the tissue, place it in a dehydration box and place it in the following solutions in sequence: 75% ethanol (1h) - 85% ethanol (1h) - 95% ethanol (1h) - 100% ethanol (50min) - 100% ethanol (50min) - 100% ethanol (50min) - xylene (40min) - xylene (40min) - xylene (40min) - 63℃ paraffin (50min) - 63℃ paraffin (50min) - 63℃ paraffin (50min) 7.2 Wax Embedding After dehydration, place the samples in the preheated embedding machine, determine the embedding direction as required, and place them on a freezing table to cool.

[0023] 7.3 Slicing Place the trimmed wax block into a microtome for slicing. The slice thickness is 3-5um. Float the slices on 40℃ warm water to flatten the tissue. Pick up the tissue with an adhesive slide and place it in a 60℃ oven for baking for 1-2h.

[0024] 7.4 Dewaxing and rehydration Use xylene and gradient ethanol for dewaxing and rehydration. Place the slices in a slice rack and place them in the following solutions in sequence: xylene (5 min) - xylene (5 min) - xylene (5 min) - 100% ethanol (5 min) - 100% ethanol (5 min) - 95% ethanol (3 min) - 85% ethanol (3 min) - 75% ethanol (3 min) - deionized water (5 min) 7.5 High-pressure antigen retrieval Heat the pressure cooker to 100°C and place the staining rack in it. (Note: To prevent a large amount of water vapor from entering the beaker during the heating of the buffer solution, seal the beaker with plastic wrap) 7.6. Closure The tissue on the section was blocked with 10% non-immune goat serum and placed in a humidified box at room temperature for 30 min.

[0025] 7.7. Primary Antibody Incubation The blocking solution was shaken off, and the primary antibody diluted with 1% casein was added at a dilution ratio of 1:200, and incubated overnight at 4°C. The sections were placed in a staining rack and washed four times in PBST.

[0026] 7.8. Secondary Antibody Incubation Secondary antibodies diluted with casein were added to the sections and incubated in a humidified chamber at 37°C for 1 h. The sections were placed in a staining rack and washed four times in PBST.

[0027] 7.9. Triple Antibody Incubation Add casein-diluted tertiary antibodies to the sliced ​​tissue and incubate in a humidified box at 37°C for 1 hour. Place the slice in a staining rack and wash three times in PBST. Then wash twice in PBS.

[0028] 7.10 DAB staining Add DAB staining solution to the sliced ​​tissue and place it in a humidified box at room temperature for 10-15 minutes. (Pay attention to the staining of the slices under the microscope. If the staining is dark yellow and there is no obvious background coloring, you can proceed to the next step.) Place the slices in a staining rack and wash them twice in PBS, 5 minutes each time.

[0029] 7.11. Hematoxylin staining Hematoxylin staining solution was added to the sliced ​​tissue and placed in a humidified box at room temperature for 30 min.

[0030] 7.12 Dehydration and sealing Wash and soak the staining rack in clean water for 30 minutes, and then put it in an oven to dry. Add neutral gum to the sliced ​​tissue, cover it with a cover glass, and observe it under a microscope. The results are as follows: Figure 1 .

[0031] The results showed that the prepared CASC2 monoclonal antibody could effectively recognize CASC2.

[0032] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A hybridoma cell line CASC2, characterized in that: The hybridoma cell line CASC2 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.45959.

2. The CASC2 monoclonal antibody secreted and produced by the hybridoma cell line CASC2 as claimed in claim 1.

3. The CASC2 monoclonal antibody according to claim 2, characterized in that The CASC2 monoclonal antibody is IgG1.

4. Use of the CASC2 monoclonal antibody according to claim 2 in preparing a preparation for detecting CASC2 in a sample.

5. The use according to claim 4, characterized in that: The sample is a biological sample.

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