R3HD4 monoclonal antibody as well as preparation method and application of kit of R3HD4 monoclonal antibody

Abstract

The invention provides an R3HD4 monoclonal antibody and a preparation method and application of a kit of the R3HD4 monoclonal antibody. The monoclonal antibody hybridoma cell strain R3HD4 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.46114, and the prepared monoclonal antibody can effectively recognize R3HD4. The invention further discloses a preparation method of the monoclonal antibody.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation method and application of an R3HD4 monoclonal antibody and a kit thereof. Background Art

[0002] Predicted to be involved in DNA maintenance mechanisms, and its dysfunction may be associated with genetic diseases. Predicted to play a key role in the cell cycle and DNA replication, and abnormalities in R3HD4 may be associated with tumor development. Predicted to be involved in DNA repair pathways such as nucleotide excision repair (NER), mismatch repair (MMR), or homologous recombination repair (HRR). Predicted to play a role in the S and / or M phases of the cell cycle. Predicted subcellular localization is cytoplasmic and nucleus. Predicted protein molecular weight 30kD.

[0003] There is no report on how to prepare R3HD4 monoclonal antibody. 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 method for preparing and using an R3HD4 monoclonal antibody and a kit 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 R3HD4, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No.46114.

[0006] Another aspect of the present invention provides the R3HD4 monoclonal antibody secreted and produced by the monoclonal antibody hybridoma cell line R3HD4.

[0007] Furthermore, the R3HD4 monoclonal antibody is IgG1.

[0008] Another aspect of the present invention provides use of R3HD4 monoclonal antibody in preparing a sample for detecting R3HD4.

[0009] Another aspect of the present invention provides a kit for detecting R3HD4 in a sample, wherein the kit contains the above-mentioned R3HD4 monoclonal antibody.

[0010] Furthermore, the kit also includes a detection reagent for immunohistochemistry.

[0011] Furthermore, the kit also includes a detection reagent for enzyme-linked immunosorbent assay (ELISA).

[0012] The above detection reagents for immunohistochemistry and detection reagents for enzyme-linked immunosorbent assay (ELISA) are commonly used reagents in this field.

[0013] Furthermore, the sample is a biological sample, which may be tissue, cell, plasma, etc.

[0014] As described above, the detection and use of the protein R3HD4 of the present invention have the following beneficial effects: The monoclonal antibody prepared by the monoclonal antibody hybridoma cell line R3HD4 prepared in the present application can effectively recognize R3HD4.

[0015] Cell name: Hybridoma cell Storage time: September 25, 2024 Depository: China National Microbiological Culture Collection Administration General Microbiology Center Deposit number: CGMCC No.46114 Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows a schematic diagram of Example 1, which shows the use of an immunohistochemical method to verify the expression of R3HD4 antibody in rat myocardial tissue. DETAILED DESCRIPTION

[0017] Example 1 Preparation of monoclonal antibody hybridoma cell lines and monoclonal antibodies The R3HD4 protein, or RAD3809-like DNA helicase 4, is a helicase involved in DNA metabolism. This protein plays an important role in maintaining genome stability as it is involved in key processes such as DNA replication, repair, and recombination. The function of R3HD4 is essential to prevent the occurrence of mutations and genetic diseases. In the context of disease-related research, abnormal expression or dysfunction of R3HD4 may be associated with a variety of diseases, especially those involving DNA damage response and cell cycle control, such as certain types of cancer and hereditary progeria.

[0018] The molecular mechanism of R3HD4 involves its activity in the process of DNA unwinding, which may affect the replication and repair of genes. As part of the function of the gene, R3HD4 may be involved in regulating specific stages of the cell cycle to ensure that genetic information is accurately transmitted to daughter cells. In addition, R3HD4 may play a role in the cell's stress response to DNA damage, affecting cell survival or apoptosis. Although many of R3HD4's functions have not been fully understood, its potential role in cell physiology may include participation in intercellular communication, tissue-specific functions, and regulation during development. With further research on the R3HD4 protein, we may find new applications for it in disease diagnosis, treatment, and prevention.

[0019] 1) Analysis and design of peptide sequences Abdesigner software was used to perform antigen epitope analysis on the amino acid sequence of R3HD4 protein, mainly evaluating indexes such as hydrophilicity, antigenicity, surface possibility, and flexible area. 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 15 amino acids at positions 243 to 257 of the R3HD4 protein were determined as the amino acid sequence of the synthetic polypeptide, and the sequence was QMKVSNRHLDFLPPG (SEQ ID NO.1).

[0020] 2) Peptide synthesis and cross-linking Synthesis: The target peptide was automatically synthesized by ACT396 fully automatic multi-channel peptide synthesizer according to the programmed program and dissolved in 50% acetonitrile. The structure was characterized by mass spectrometry to confirm that it was the target peptide.

[0021] Cross-linking: Sulfo-SMCC is used as a cross-linking agent to cross-link the carrier protein KLH with the synthetic peptide: 9 mg KLH is dissolved in 0.6 ml ultrapure water; 1.5 mg Sulfo-SMCC is dissolved in 0.3 ml ultrapure water, and the pH value is adjusted to about 7 with 3M NaOH. While mixing, slowly add the Sulfo-SMCC solution dropwise to the KLH solution, and rotate and mix the reactants at room temperature for 50 minutes. Load the reacted Sulfo-SMCC / KLH mixture onto a Sephadex G25 column that has been pre-equilibrated with an equilibrium buffer (0.05MPB, pH 6.0) for 20 minutes, and collect the light gray eluate, which is the activated Sulfo-SMCC / KLH solution. Dissolve 3mg of cross-linked peptide in 300ul PBS (pH7.4), add 0.3 volume of Sulfo-SMCC / KLH complex solution to the peptide solution, adjust pH to 7.3, shake at room temperature for 10 hours, freeze at -70℃, and freeze-dry for 24 hours. Use Ellman method to detect peptide thiol groups before and after cross-linking to determine the peptide cross-linking efficiency, and the coupling rate is 98.2%.

[0022] 3) Peptide immunization and antiserum preparation 105μg of cross-linked KLH-peptide was dissolved in 105μl phosphate buffer (0.01M PBS), and an equal volume of Freund's complete adjuvant was added to fully emulsify (until it does not diffuse in water). Healthy BalB / C mice aged 8 weeks and weighing 20-25g were used for immunization, and intradermal injection was performed on the back, and at least 3 points were injected. Two weeks after the first immunization, 110μg of polypeptide was dissolved in 105μl phosphate buffer (0.01M PBS), and after being fully emulsified with an equal amount of Freund's incomplete adjuvant, intradermal injection was performed as the first booster immunization, requiring intradermal injection on the back, and at least 3 points were injected. Two weeks after the second immunization, the second booster immunization was performed, and the method and requirements were the same as the first booster immunization. One week later, tail blood was collected, and the enzyme-labeled plate was coated with non-cross-linked synthetic polypeptides, and the titer of the immune serum was detected by indirect ELISA. Repeat booster immunization and titer determination until the serum titer reaches 1:10000 or above. After the titer reaches the requirement, take 105ug of polypeptide and dissolve it in 0.525ml phosphate buffer (0.01M PBS) and inject it intraperitoneally as booster immunization.

[0023] 4) Hybridoma cell preparation (1) Cell fusion: Two days after boosting immunization of positive mice, blood samples were collected from the mouse eyeballs, and then the mice were killed by dislocating the neck. The mice were soaked in 75% alcohol, and their spleens were obtained in a clean bench. They were ground using a 200-mesh steel sieve, and DMEM solution was added to 12 ml. The ground spleen was gently blown with a pipette to disperse it in the solution. 12 ul of the mixed solution was taken and counted to obtain the total number of spleen cells. According to the ratio of myeloma cells to spleen cells of 1:7.5, the corresponding number of myeloma cells was added, mixed thoroughly, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The mixed cells were shaken to make them loose and fluid, and then placed in a water bath set at 37°C. 880 ul of PEG1500 was slowly added using a pipette, and the addition was completed within 50 seconds. After the addition, it was allowed to stand for 90 seconds. 1.2 ml of serum-free DMEM was added dropwise again, and the mixture was shaken while adding to slowly dilute the PEG to terminate the reaction. 30 ml of serum-free DMEM was added, and the mixture was shaken while adding, and the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.

[0024] (2) Cell culture screening: Pipette 4 ml of fusion cell culture medium, slowly blow up the fusion cells at the bottom, and add HAT culture medium (HAT dissolved in DMEM containing 10% calf serum) to make it fixed to 50 ml. 145 μl / well is evenly spread on a 96-well plate with feeder cells. After 4 days, discard the supernatant in the 96-well plate and replace it with newly prepared HAT culture medium (HAT dissolved in DMEM containing 10% calf serum). Three days after the replacement, extract 75 μl of supernatant from each well and add it to the ELISA plate coated with antigen, and use the indirect ELISA method to detect the positive value of the supernatant. Select the wells with higher positive values, replace the HAT culture medium again, and re-test after 24 hours. Use HT culture medium (HT dissolved in DMEM containing 10% calf serum) to perform limiting dilution on the wells with higher re-test positive values, and distribute 145 μl / well evenly on the 96-well plate containing feeder cells. After 6 days, the positive value of the supernatant was detected, and the monoclonal clones with high positive values ​​were selected for subcloning with DMEM containing 10% calf serum. When the positive monoclonal clones were subcloned, the positive rate of the supernatant on the plate could reach 100%. Cell lines with large cell areas and high positive values ​​were selected for reproduction and culture. The obtained monoclonal antibody hybridoma cell lines were transferred to the General Microbiology Center of the China Microbiological Culture Collection Administration for preservation. 5) Preparation of monoclonal antibodies Four days before the cell injection, four healthy 8-week-old BalB / C female mice were selected and 0.8 ml paraffin oil was injected into the peritoneal cavity of each mouse. When the cells were in the logarithmic growth phase, the cells were blown off and counted, and 3*100 cells were obtained. They were collected and placed in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was poured out, 4 ml of sterile phosphate buffer (0.01M PBS) was added, the cells were blown off with a pipette, and 26 ml of phosphate buffer (0.01M PBS) was added, and centrifuged at 1000 rpm for 5 minutes. 4 ml of phosphate buffer (0.01M PBS) was added to the centrifuge tube, mixed well, and 0.8 ml of the mixture was injected into the peritoneal cavity of each mouse. Nine days after the injection, the mice were killed by cervical dislocation, soaked in 75% alcohol, the abdominal epithelium was cut open, the peritoneal membrane was lifted with a syringe, and the ascites was slowly drawn into a container and centrifuged at 3900 rpm for 5 minutes. All the supernatant was collected and filtered with a 0.45 μm filter to mix it with the binding buffer solution in a 1:1 ratio.

[0025] 6) Antibody affinity purification (1) IgG purification: Add 9 ml of 50% Protein-A Sepharose suspension to a 15 ml column, remove the top and bottom caps, and the column bed volume after the liquid flows out is 3.5 ml, then rinse with 12.25 ml of deionized water three times. Take 7 ml of the corresponding serum, mix it with 2 ml of PBS, and then add it to the 15 ml column. Rotate the mixer at room temperature (20-25°C) for 2 hours to discharge the serum sample. In addition, wash the column three times with 14 ml of purification wash solution, and add 7 ml of eluent for elution.

[0026] (2) Peptide affinity purification: Add 2 ml of Sulfo-link gel suspension (1 ml of gel) to the column, wait until the liquid in the column is drained, and rinse the column with 8 ml of coupling buffer. Dissolve the synthesized R3HD4 peptide with 2 ml of coupling buffer and add it to the column, then add 2 ml of coupling buffer to the column, and mix by inversion at room temperature for 1 hour. Rinse the column with 6 ml of coupling buffer, then add 6 ml of blocking buffer, and mix by inversion at room temperature for 1 hour. Rinse the column 3 times, then add 12 ml of IgG and 6 ml of PBS to the column, and mix by inversion at room temperature for 1 hour. Rinse the column 3 times with PBS, then elute with 4 ml of elution buffer. The purified antibody obtained is placed in a dialysis bag and dialyzed at 4°C. Dialyze overnight, then centrifuge at 3800 rpm × 40 min to remove the precipitate, and collect the supernatant. The antibody titer was determined by indirect ELISA and was 1:32000. The protein concentration was determined by Bradford method and was 1.075 mg / ml.

[0027] 7) Immunohistochemical analysis: Confirm that the R3HD4 antibody information is correct, prepare the required samples, and then perform the following operations in sequence: 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.

[0028] 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.

[0029] 7.4 Dewaxing and rehydration Take out the staining rack from the oven and put it into the staining box for dewaxing. (Note: The dewaxing time depends on the temperature and the number of times the dewaxing agent is used, 30 minutes to 1 hour) Take out the dewaxed staining rack, drain it, and put it into the staining box of anhydrous ethanol in turn. Put it in 3% H2O2 for 3 minutes to eliminate the activity of endogenous peroxidase in the tissue.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 7.10 DAB staining Add freshly prepared DAB for color development and stain for 5 minutes in the dark (3-10 minutes is recommended, or color development can be controlled under a light microscope. It is recommended to stain every 5 sheets and then put them in tap water to stop staining) 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.

[0035] 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 .

[0036] The results showed that the prepared R3HD4 monoclonal antibody could effectively recognize R3HD4.

[0037] 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 monoclonal antibody hybridoma cell line R3HD4, characterized in that: The monoclonal antibody hybridoma cell line R3HD4 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.46114.

2. The R3HD4 monoclonal antibody secreted and produced by the monoclonal antibody hybridoma cell line R3HD4 according to claim 1.

3. The R3HD4 monoclonal antibody according to claim 2, characterized in that The R3HD4 monoclonal antibody is IgG1.

4. Use of the R3HD4 monoclonal antibody according to claim 2 in the preparation of a preparation for detecting R3HD4 in a sample.

5. A kit for detecting R3HD4 in a sample, characterized in that: The kit contains the R3HD4 monoclonal antibody as claimed in claim 2.

6. The kit according to claim 5, characterized in that: The kit also includes a detection reagent for immunohistochemistry.

7. The kit according to claim 5, characterized in that: The kit also includes a detection reagent for enzyme-linked immunosorbent assay.

8. The kit according to claim 5, characterized in that: The sample is a biological sample.

9. The kit according to claim 8, characterized in that: The biological sample is tissue, cell, or plasma.

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