MISP3 monoclonal antibody as well as preparation method and application of MISP3 monoclonal antibody kit

Abstract

The invention provides an MISP3 monoclonal antibody and a preparation method and application of a kit of the MISP3 monoclonal antibody. The monoclonal antibody hybridoma cell strain MISP3 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.46119, and the prepared monoclonal antibody can effectively recognize the MISP3.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a MISP3 monoclonal antibody and a preparation method and use thereof. Background Art

[0002] It is predicted to affect the expression of related genes in some way, potentially associated with developmental abnormalities. It is predicted to indirectly participate in cell cycle regulation by affecting the expression or function of other MISP family members. It is predicted to influence gene expression during cell division through the form of non-coding RNA. Transcriptionally produced long non-coding RNA is predicted to participate in the regulation of gene expression. Its subcellular localization is predicted to be nuclear. The protein has a predicted molecular weight of 24 kD.

[0003] There is currently no report on how to prepare MISP3 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 MISP3 monoclonal antibody and a preparation method and use thereof, so as to fill the gaps in the prior art.

[0005] Another aspect of the present invention provides a monoclonal antibody hybridoma cell line MISP3, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No.46119.

[0006] Another aspect of the present invention provides the MISP3 monoclonal antibody secreted and produced by the above-mentioned monoclonal antibody hybridoma cell line MISP3.

[0007] Furthermore, the MISP3 monoclonal antibody is IgG1.

[0008] Another aspect of the present invention provides use of a MISP3 monoclonal antibody in preparing a sample for detecting MISP3.

[0009] Another aspect of the present invention provides a kit for detecting MISP3 in a sample, wherein the kit contains the above-mentioned MISP3 monoclonal antibody.

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

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

[0012] The above detection reagents for immunofluorescence 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, cells, plasma, etc.

[0014] As described above, the detection and use of the protein MISP3 of the present invention have the following beneficial effects: The monoclonal antibody prepared in the present application and the monoclonal antibody prepared by the hybridoma cell line MISP3 can effectively recognize MISP3.

[0015] Cell name: Hybridoma cell Storage time: September 25, 2024 Depository: General Microbiology Center of China Culture Collection Administration Deposit number: CGMCC No.46119 Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a schematic diagram of the method of cell immunofluorescence in Example 1 to verify the subcellular localization of MISP3 antibody in HepG2 cells. DETAILED DESCRIPTION

[0017] Example 1 Preparation of Monoclonal Antibody Hybridoma Cell Lines and Monoclonal Antibodies MISP3, or missing division-associated protein 3, is a member of the missing division protein (MISP) family. MISP family proteins typically play a role in cell division and cell cycle regulation, particularly during mitosis. However, MISP3 itself may not encode a functional protein. Instead, it may be a pseudogene or noncoding RNA that indirectly participates in cell cycle regulation by regulating the expression or function of other MISP family members. Because MISP3 does not encode a protein, its direct role in disease is unclear. However, other members of the MISP family may be involved in diseases associated with cell cycle disruption, such as cancer.

[0018] The molecular mechanism of MISP3 may involve the regulation of its transcripts in cell cycle regulation. As a pseudogene, MISP3 may influence gene expression by producing long noncoding RNAs, potentially involving epigenetic regulation or interactions with other RNA molecules. The gene function of MISP3 may be related to its role in transcriptional regulation, although this has not yet been fully elucidated. Furthermore, MISP3 may have unstudied functions, such as roles in cellular responses to environmental changes or in intercellular communication. With further research into the roles of pseudogenes and noncoding RNAs in cell biology, MISP3 may be revealed as a novel player in disease pathogenesis and cellular physiology.

[0019] 1) Peptide sequence analysis and design The amino acid sequence of the MISP3 protein was analyzed for antigenic epitopes using Abdesigner software, primarily evaluating hydrophilicity, antigenicity, surface potential, and flexible regions. Combined with past experience in antibody preparation, the complexity of the amino acid structure, susceptibility to oxidation, synthesis difficulty, and amino acid type and distribution were considered. Ultimately, the 15 amino acids at positions 204 to 218 of the MISP3 protein were identified as the amino acid sequence for the synthetic peptide, designated RRKVSENGLEQEERK (SEQ ID NO. 1).

[0020] 2) Peptide synthesis and cross-linking 4. Synthesis: Use the ACT396 fully automatic multi-channel peptide synthesizer to automatically synthesize the target peptide through the programmed program. The target peptide is dissolved in 50% acetonitrile solution, and its structure is identified by mass spectrometry to confirm that it is the target protein.

[0021] Cross-linking: Use Sulfo-SMCC as a cross-linker to cross-link the carrier protein KLH to the synthetic peptide. Dissolve 12 mg of KLH in 0.8 ml of ultrapure water; dissolve 2 mg of Sulfo-SMCC in 0.4 ml of ultrapure water and adjust the pH to approximately 7 with 3 M sodium hydroxide. After mixing, add the Sulfo-SMCC solution dropwise to the KLH and stir at room temperature for 45 minutes. Next, load the prepared Sulfo-SMCC / KLH mixture onto a Sephadex G25 column pre-equilibrated with equilibration buffer (0.05 M PB, pH 6.0) for 25 minutes. Collect the light gray eluate, which is the activated Sulfo-SMCC / KLH solution. Dissolve 2.5 mg of cross-linked peptide in 250 μl of PBS (pH 7.4). Add 0.25 volumes of sulfo-SMCC / KLH complex solution to the peptide solution, adjust the pH to 7.3, shake at room temperature for 9.5 hours, freeze at -70°C, and lyophilize for 24 hours before use. The peptide cross-linking efficiency was determined by measuring the thiol groups on the peptides before and after cross-linking using the Ellman method, resulting in a coupling efficiency of 98.9%.

[0022] 3) Peptide immunization and antiserum preparation 110 μg of cross-linked KLH peptide was dissolved in 110 μl of phosphate buffered saline (0.01M PBS) and thoroughly emulsified with an equal volume of Freund's complete adjuvant (until it does not diffuse in water). Eight-week-old healthy BalB / C mice weighing 20-25 g were immunized by intradermal injection into the back, with injections administered at least three times. Two weeks after the initial immunization, 110 μg of peptide was dissolved in 110 μl of phosphate buffered saline (0.01M PBS) and thoroughly emulsified with an equal volume of Freund's incomplete adjuvant. This was then administered intradermally as the first booster immunization, with injections administered at least three times. Two weeks after the second immunization, a second booster immunization was administered using the same procedures and requirements as the first booster. One week later, tail blood was collected, and the titer of the immune serum was determined by indirect ELISA using an ELISA plate coated with uncross-linked synthetic peptide. Repeat the booster immunization and titer determination until the serum titer reaches 1:10000 or above. After the titer reaches the required level, take 110ug of the polypeptide and dissolve it in 0.55ml of phosphate buffer (0.01M PBS) and inject it intraperitoneally as a booster immunization.

[0023] 4) Hybridoma cell preparation (1) Cell fusion: Three days after the stimulation and boosting of the immunized mice, blood was collected from the eye sockets. The mice were killed by dislocating the neck and disinfected with 75% alcohol. The spleen of the mice was removed in a clean bench and gently ground on a 200-mesh steel sieve. Serum-free DMEM was added to make the volume 9 ml. The ground spleen was blown up with a pipette to disperse it in the solution. The number of cells in 9 ul of the mixed solution was counted to calculate the total number of spleen cells. According to the ratio of myeloma cells to spleen cells, the corresponding number of myeloma cells was added to the spleen cells at a ratio of 1:8.5, mixed evenly, centrifuged at 900 r / min for 5 minutes, and then the supernatant was discarded. The cells were gently shaken to disperse them and placed in a 37°C water bath. 900 ul of PEG1500 was added with a pipette, shaking while adding, and the reaction was completed within 45 seconds. After standing for 90 seconds, 1.3 ml of serum-free DMEM was added dropwise, shaking while adding, and the reaction was slowly stopped. Serum-free DMEM was added to make up the volume to 25 ml, and the mixture was shaken while adding. Centrifuge at 900 rpm / min for 5 minutes and discard the supernatant.

[0024] (2) Cell culture screening: Gently disperse the fused cells at the bottom with 1.5 ml of cell culture medium and dilute to 50 ml with HAT medium (HAT dissolved in DMEM containing 10% calf serum). Evenly spread 170 μl / well on a 96-well plate with feeder cells. After 2 days, replace the supernatant in the 96-well plate with freshly prepared HAT medium (HAT dissolved in DMEM containing 10% calf serum). Three days after the solution replacement, remove 70 μl of supernatant from each well and add it to the ELISA plate coated with the antigen. Detect the positive value of the supernatant by indirect ELISA. Replace the wells with high positive values ​​with HAT medium and retest one day later. After retesting, take the well with the highest positive value and dilute it with HT medium (HT dissolved in DMEM containing 10% calf serum) for limiting dilution. Evenly spread it on a 96-well plate with feeder cells at 170 μl / well. After 9 days, the positive values ​​were tested and monoclones with high positive values ​​were subcloned using DMEM containing 10% calf serum. After subcloning the positive monoclones, the positive rate of the supernatant in the 96-well plate was 100%. Cell lines with high positive values ​​and large cell clusters were selected for amplification culture, and the obtained monoclonal antibody hybridoma cell lines were sent to the General Microbiology Center of the China Culture Collection Administration for Microorganisms for preservation. 5) Preparation of monoclonal antibodies Five days before cell injection, five healthy 8-week-old BalB / C female mice were injected intraperitoneally with 0.8 ml of paraffin oil per mouse. When the cultured cells were in the logarithmic growth phase, the cells were collected and counted. 3*100 cells were taken and placed in a centrifuge tube and centrifuged at 900 r / min for 5 minutes. The supernatant was poured out, 6 ml of sterile phosphate buffer (0.01 M PBS) was added, the cell clumps were gently dispersed with a pipette, and the volume was made up to 30 ml with phosphate buffer (0.01 M PBS), and centrifuged at 900 r / min for 5 minutes. 2 ml of phosphate buffer (0.01 M PBS) was added to the centrifuge tube, mixed thoroughly, and 0.4 ml of the mixture was injected into the abdominal cavity of each mouse. 10 days after injection, the mice were killed by cervical dislocation and disinfected with 75% alcohol. The abdominal epithelium was incised with surgical scissors, and a 10 ml syringe was inserted to lift the abdominal membrane. The ascites was extracted into a centrifuge tube and centrifuged at 3800 r / min for 5 minutes. The supernatant was collected, mixed with binding buffer at a ratio of 1:1, and filtered using a 0.45 μm filter.

[0025] 6) Antibody affinity purification (1) IgG purification: Add 9 ml of 50% Protein-A Sepharose suspension to a 28 ml chromatography column, remove the top and bottom caps, and the column bed volume after the liquid flows out is 4.5 ml. Then rinse with 22.5 ml of deionized water three times. Take 9 ml of the corresponding serum, mix it with 2 ml of PBS, and then add it to the 28 ml chromatography column. Rotate it on a rotary mixer at room temperature (20-25°C) for 1.5 hours to drain the serum sample. In addition, wash the chromatography column three times with 18 ml of purification wash solution and add 9 ml of eluent for elution.

[0026] (2) Peptide affinity purification: Add 1.8 ml of Sulfo-link gel suspension (0.9 ml of gel) to the chromatography column, drain the column liquid, and rinse the column with 7.2 ml of coupling buffer. Dissolve the prepared MISP3 peptide in 1.8 ml of coupling buffer and add it to the chromatography column. Then add 1.8 ml of coupling buffer to the chromatography column and mix it by inversion at room temperature for 1 hour. Rinse the chromatography column with 10.8 ml of coupling buffer, add 5.4 ml of blocking agent, and mix it by inversion at room temperature for 1 hour. Rinse the chromatography column 3 times, then add 10.8 ml of IgG and 5.4 ml of PBS to the chromatography column, and mix it by inversion at room temperature for 1 hour. Rinse the chromatography column 3 times with PBS, and then elute with 3.6 ml of eluent. Place the purified antibody in a dialysis bag and dialyze it at 4°C. After overnight dialysis, the precipitate was separated by centrifugation at 4600 rpm for 42 minutes, and the supernatant was collected. The antibody titer was determined to be 1:32,000 by indirect ELISA, and the protein content was 1.077 mg / ml by Bradford assay.

[0027] 7) Cell immunofluorescence analysis: After confirming that the MISP3 antibody information is correct, the cells were seeded in a 24-well plate with a slide. After the cells grew to an appropriate density, the samples were processed as follows: 7.1. Fixation Fix with 4% PFA at room temperature for 30 minutes. The choice of fixative depends on the nature of the antigen being studied and the characteristics of the antibody being used. Currently, 4% formaldehyde is commonly used and is suitable for studying cell membrane proteins.

[0028] 7.2 Transparency After fixation, wash three times with 1× PBS, add 0.1% TritonX-100 (100 μL / slice), and permeabilize for 10 minutes at room temperature. The purpose of permeabilization is to create pores in the cell membrane, allowing the antibody to enter the cell and bind to the antigen.

[0029] 7.3. Closure After permeabilization, wash three times with 1× PBS and add 10% FBS PBS (100 μL / slide) for blocking. This is to prevent endogenous nonspecific protein antigen binding.

[0030] 7.4 Primary Antibody Incubation Dilute the corresponding primary antibody in 10% FBS PBS at a dilution ratio of 1:500, add 100 μL of diluted primary antibody per slide, and incubate at room temperature for 1 hour.

[0031] 7.5. Secondary Antibody Incubation After 1 hour, wash three times with 1x PBS. Dilute the secondary antibody of the same species as the primary antibody in 10% FBS / PBS at a dilution ratio of 1:500 or 1:1000. Add 100 μL of the diluted secondary antibody per slide. Incubate at room temperature for 1 hour.

[0032] 7.6. DAPI nuclear staining After the secondary antibody incubation, the slides were washed three times with 1× PBS, and 100 μL / slide of DAPI was added and the nuclei were stained at room temperature for 10 min.

[0033] 7.7. Sealing After the nuclear staining is completed, wash the slides with 1×PBS three times, then wash them with ultrapure water three times, add anti-quenching mounting medium on the slide, discard the water from the slide, gently invert it and place it on the slide, dry it in the dark at room temperature, and store it at -20℃. Figure 1 .

[0034] The results showed that the prepared MISP3 monoclonal antibody could effectively recognize MISP3.

[0035] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods and compositions in the invention will be apparent 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 apparent 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 MISP3, characterized in that: The monoclonal antibody hybridoma cell line MISP3 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.46119.

2. The MISP3 monoclonal antibody secreted and produced by the monoclonal antibody hybridoma cell line MISP3 as claimed in claim 1.

3. The MISP3 monoclonal antibody according to claim 2, characterized in that The MISP3 monoclonal antibody is IgG1.

4. Use of the MISP3 monoclonal antibody according to claim 2 in the preparation of a preparation for detecting MISP3 in a sample.

5. A kit for detecting MISP3 in a sample, characterized in that: The kit contains the MISP3 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 immunofluorescence.

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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