ZB10P monoclonal antibody and preparation method and application of kit of ZB10P monoclonal antibody

Abstract

The invention provides a ZB10P monoclonal antibody and a preparation method and application of a kit of the ZB10P monoclonal antibody. The monoclonal antibody hybridoma cell strain ZB10P is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.46118, and the prepared monoclonal antibody can effectively recognize the ZB10P.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a ZB10P monoclonal antibody and a preparation method and use thereof. Background Art

[0002] Predicted to be involved in transcriptional regulatory network. Predicted to be related to dysfunction of zinc finger protein family in some genetic diseases. Predicted to play a role in regulating the expression of some genes, and its abnormality may be related to related diseases. Predicted to be subcellular localized in cytoplasm and nucleus. Predicted protein molecular weight 26kD.

[0003] There is no report on how to prepare ZB10P 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 ZB10P 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 strain, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.46118.

[0006] Another aspect of the present invention provides the ZB10P monoclonal antibody secreted and produced by the above monoclonal antibody hybridoma cell line.

[0007] Furthermore, the ZB10P monoclonal antibody is IgG1.

[0008] Another aspect of the present invention provides use of a ZB10P monoclonal antibody in preparing a sample for detecting ZB10P.

[0009] Another aspect of the present invention provides a kit for detecting ZB10P in a sample, wherein the kit contains the above-mentioned ZB10P 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 the art.

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

[0014] As described above, the detection and use of protein ZB10P of the present invention have the following beneficial effects:

[0015] The monoclonal antibody prepared by the monoclonal antibody hybridoma cell line prepared in the present application can effectively recognize ZB10P.

[0016] Cell name: Monoclonal antibody hybridoma cell line

[0017] Storage time: September 25, 2024

[0018] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0019] Deposit number: CGMCC No.46118

[0020] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The schematic diagram of Example 1 is a cell immunofluorescence method to verify the subcellular localization of ZB10P antibody in HepG2 cells. DETAILED DESCRIPTION

[0022] Example 1 Preparation of monoclonal antibody hybridoma cell lines and monoclonal antibodies

[0023] ZB10P, short for Zinc Finger Protein 10 Pseudogene, is a pseudogene, which means that it is derived from the gene sequence of zinc finger protein 10 (ZNF10) but has lost the ability to encode protein. Pseudogenes may have regulatory functions in the genome, such as participating in the regulation of gene expression as long non-coding RNA. Although ZB10P itself does not encode protein, it may indirectly participate in cell function and the development of disease by affecting the transcription or post-transcriptional regulatory process of genes.

[0024] The relevance of ZB10P to disease may not be direct because it does not encode a functional protein. However, pseudogenes sometimes exert their effects by affecting the expression or function of homologous genes and may be associated with certain disease states. For example, if the transcript of ZB10P interacts with the mRNA of ZNF10 or other zinc finger protein family members, it may affect the expression levels of these genes, thereby affecting processes such as cell signaling, proliferation, differentiation, or apoptosis. In addition, abnormal expression or regulation of pseudogenes may be associated with certain pathological conditions, especially at the level of epigenetic regulation. Although the exact molecular mechanism and gene function of ZB10P have not yet been fully elucidated, with in-depth research on the role of pseudogenes in the genome, new roles in disease occurrence and cell physiology may be revealed in the future.

[0025] 1) Analysis and design of peptide sequences

[0026] Abdesigner software was used to perform antigen epitope analysis on the amino acid sequence of ZB10P protein, mainly evaluating indexes such as hydrophilicity, antigenicity, surface possibility, and flexible area. Combined with past practical experience in preparing antibodies, 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 209 to 223 of the ZB10P protein were determined as the amino acid sequence of the synthetic polypeptide, and the sequence was QDDPLAYWEKKREAW (SEQ ID NO.1).

[0027] 2) Peptide synthesis and cross-linking

[0028] 4. Synthesis: Use ACT396 fully automatic multi-channel peptide synthesizer to automatically synthesize the target peptide through the programmed program, dissolve the target peptide in 50% acetonitrile solution, identify its structure by mass spectrometry, and confirm it is the target protein.

[0029] Cross-linking: Use Sulfo-SMCC as a cross-linking agent to cross-link the carrier protein KLH with 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 about 7 with 3M sodium hydroxide. After mixing, add the Sulfo-SMCC solution dropwise to KLH and stir at room temperature for 45 minutes. Then, load the prepared 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 25 minutes, and collect the light gray eluate, which is the activated Sulfo-SMCC / KLH solution. Dissolve 2.5 mg of cross-linked peptide in 250 ul PBS (pH 7.4), add 0.25 volume 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 freeze-dry for 24 hours before 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 98.9%.

[0030] 3) Peptide immunization and antiserum preparation

[0031] 110 μg of cross-linked KLH-peptide was dissolved in 110 μl 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, 110 μg of polypeptide was dissolved in 110 μl 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 uncross-linked synthetic polypeptides, and the immune serum titer was detected by indirect ELISA. Repeat the booster immunization and titer determination until the serum titer reached 1:10000 or more. After the titer reaches the requirement, 110 ug of the polypeptide is dissolved in 0.55 ml of phosphate buffer (0.01 M PBS) and injected intraperitoneally as a booster immunization.

[0032] 4) Hybridoma cell preparation

[0033] (1) Cell fusion: Three days after the stimulation of the boosted immunized mice, blood was collected from the eye sockets. The mice were killed by dislocation of the neck and disinfected with 75% alcohol. The spleen of the mouse was removed from the 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 9ul 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 900r / 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. 900ul of PEG1500 was added with a pipette, and the mixture was shaken while dripping. The reaction was completed within 45 seconds. After standing for 90 seconds, 1.3ml of serum-free DMEM was added dropwise, and the reaction was slowly stopped while adding and shaking. Serum-free DMEM was added to make up to 25 ml, and the volume was shaken while adding. Centrifuge at 900 rpm / min for 5 minutes and the supernatant was discarded.

[0034] (2) Cell culture screening: Use 1.5 ml of cell culture medium to gently disperse the fused cells at the bottom, and dilute to 50 ml with HAT medium (HAT dissolved in DMEM containing 10% calf serum). Spread evenly on a 96-well plate with feeder cells at 170 μl / well. After 2 days, replace the supernatant in the 96-well plate with freshly prepared HAT medium (HAT dissolved in DMEM containing 10% calf serum). After 3 days of changing the solution, take 70 ul of supernatant from each well and add it to the ELISA plate coated with the antigen. Detect the positive value of the supernatant using the indirect ELISA method. 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. Spread evenly on a 96-well plate with feeder cells at 170 μl / well. After 9 days, the positive value was detected, and the monoclonal clone with high positive value was subcloned using DMEM containing 10% calf serum. After subcloning the positive monoclonal clone, the positive rate of the supernatant of the 96-well plate reached 100%. The cell line with high positive value and large cell cluster was selected for amplification culture, and the obtained monoclonal antibody hybridoma cell line was sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation.

[0035] 5) Preparation of monoclonal antibodies

[0036] Five days before cell injection, five healthy 8-week-old BalB / C female mice were injected intraperitoneally with 0.8 ml / mouse paraffin oil. 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 900r / min for 5 minutes. The supernatant was poured out, 6 ml of sterilized phosphate buffer (0.01M PBS) was added, the cell mass was gently dispersed with a pipette, and the phosphate buffer (0.01M PBS) was added to the volume to 30 ml, and centrifuged at 900r / min for 5 minutes. 2 ml of phosphate buffer (0.01M PBS) was added to the centrifuge tube, mixed thoroughly, and 0.4 ml of the mixture was injected into the peritoneal cavity of each mouse. Ten days after injection, the mice were killed by cervical dislocation and disinfected with 75% alcohol. The abdominal epithelium was cut open 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 3800r / min for 5 minutes. The supernatant was collected, mixed with binding buffer at a 1:1 ratio, and filtered using a 0.45 μm filter.

[0037] 6) Antibody affinity purification

[0038] (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 with 2 ml of PBS, and then add to the 28 ml chromatography column, and rotate the rotary mixer at room temperature (20-25°C) for 1.5 hours to discharge the serum sample. In addition, wash the chromatography column three times with 18 ml of purification washing solution, and add 9 ml of eluent for elution.

[0039] (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 ZB10P 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. Dialyze overnight, then centrifuge at 4600 rpm × 42 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 1.077 mg / ml.

[0040] 7) Cell immunofluorescence analysis:

[0041] Confirm that the ZB10P antibody information is correct, inoculate the cells in a 24-well plate with a slide, and wait until the cells grow to a suitable density before processing the sample. The specific steps are as follows:

[0042] 7.1. Fixation

[0043] Use 4% PFA to fix 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 used. Currently, 4% formaldehyde is more commonly used and is suitable for studying cell membrane proteins.

[0044] 7.2 Transparency

[0045] After fixation, wash three times with 1×PBS, add 0.1% TritonX-100 100μL / slice, and permeabilize for 10 minutes at room temperature. The purpose of permeabilization is to make holes in the cell membrane to allow antibodies to enter the cell and bind to the antigen.

[0046] 7.3. Closure

[0047] After permeabilization, wash three times with 1×PBS and add 10% FBS PBS 100 μL / slice for blocking. The purpose of blocking is to prevent endogenous nonspecific protein antigen binding.

[0048] 7.4. Primary Antibody Incubation

[0049] Dilute the corresponding primary antibody with 10% FBS PBS at a dilution ratio of 1:500, add 100 μL of the diluted primary antibody per slide, and incubate at room temperature for 1 hour.

[0050] 7.5. Secondary Antibody Incubation

[0051] After 1 hour, wash three times with 1×PBS, dilute the secondary antibody of the same species as the primary antibody with 10% FBS, PBS at a dilution ratio of 1:500 or 1:1000, add 100 μL of the diluted secondary antibody per piece, and incubate at room temperature for 1 hour.

[0052] 7.6. DAPI nuclear staining

[0053] After the secondary antibody incubation, the slides were washed three times with 1× PBS, and DAPI 100 μL / slide was added to stain the nuclei for 10 min at room temperature.

[0054] 7.7. Sealing

[0055] After the nuclear staining is completed, wash it three times with 1×PBS, then wash it three times with ultrapure water, add anti-quenching mounting medium on the slide, discard the water from the slide, gently invert it on the slide, dry it at room temperature away from light, and store it at -20℃. Observe under a microscope, the results are as follows Figure 1 .

[0056] The results showed that the prepared ZB10P monoclonal antibody could effectively recognize ZB10P.

[0057] 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 ZB10P, characterized in that: The monoclonal antibody hybridoma cell line ZB10P is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.46118.

2. The ZB10P monoclonal antibody secreted and produced by the monoclonal antibody hybridoma cell line ZB10P as claimed in claim 1.

3. The ZB10P monoclonal antibody according to claim 2, characterized in that The ZB10P monoclonal antibody is IgG1.

4. Use of the ZB10P monoclonal antibody according to claim 2 in the preparation of a preparation for detecting ZB10P in a sample.

5. A kit for detecting ZB10P in a sample, characterized in that: The kit contains the ZB10P 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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