CR018 monoclonal antibody and preparation method and application of CR018 monoclonal antibody kit

Abstract

The invention provides a CR018 monoclonal antibody and a preparation method and application of a kit of the CR018 monoclonal antibody. The monoclonal antibody hybridoma cell strain CR018 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.46121. The prepared monoclonal antibody can effectively recognize CR018. The invention further discloses a method for preparing the monoclonal antibody.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to CR018 monoclonal antibody and a preparation method and use thereof. Background Art

[0002] Predicted to play a role in regulating cell proliferation, migration and apoptosis. Predicted to be involved in angiogenesis or cardiac function regulation, and its abnormality may be associated with certain cardiovascular diseases. Predicted to affect cell proliferation and differentiation by regulating the expression of cell cycle-related genes. Predicted to be a regulatory factor of signal transduction molecules and participate in intracellular signal transduction pathways. Predicted to be subcellularly localized in the cytoplasm and nucleus. Predicted protein molecular weight 10kD.

[0003] There is currently no report on how to prepare the CR018 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 CR018 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 CR018, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.46121.

[0006] Another aspect of the present invention provides the CR018 monoclonal antibody secreted and produced by the above-mentioned monoclonal antibody hybridoma cell line CR018.

[0007] Furthermore, the CR018 monoclonal antibody is IgG1.

[0008] Another aspect of the present invention provides use of the CR018 monoclonal antibody in preparing CR018 for detection samples.

[0009] Another aspect of the present invention provides a kit for detecting CR018 in a sample, wherein the kit contains the above-mentioned CR018 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, cell, plasma, etc.

[0014] As described above, the detection and use of the protein CR018 of the present invention have the following beneficial effects: The monoclonal antibody prepared by the monoclonal antibody hybridoma cell line CR018 prepared in the present application can effectively recognize CR018.

[0015] Cell name: Hybridoma cell Storage time: September 25, 2024 Depository: China National Microbiological Culture Collection Administration General Microbiology Center Deposit number: CGMCC No.46121 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 CR018 antibody in HepG2 cells. DETAILED DESCRIPTION

[0017] Example 1 Preparation of monoclonal antibody hybridoma cell lines and monoclonal antibodies CR018, also known as LINC00526, is a long noncoding RNA (lncRNA) that may encode an understudied protein, which is biologically known as a putative uncharacterized protein. Since the function of LINC00526 and the protein it encodes have not been described in detail, we can only speculate on its possible biological roles and disease associations. This putative protein may be involved in a variety of physiological processes in cells, including cell signaling, cell cycle control, gene expression regulation, etc. Considering the role of lncRNA in regulating gene expression, LINC00526 may be involved in the development of diseases such as cancer, neurodegenerative diseases, or other diseases caused by imbalanced gene regulation by affecting the expression of specific genes.

[0018] The molecular mechanism of LINC00526 may involve the transcriptional regulation of its encoded protein or the regulatory function of its RNA molecule itself. This lncRNA may affect intracellular signaling pathways by interacting with DNA, RNA or proteins, thereby regulating cell behavior and response. In terms of gene function, LINC00526 may be involved in regulating genes related to cell proliferation, differentiation and apoptosis. In addition, LINC00526 may play a role in the response of cells to external stimuli, such as oxidative stress or cell damage. Although the exact biological role and pathological effects of LINC00526 require further study, its potential role in intercellular communication, tissue-specific functions and developmental processes may reveal its broader impact in human health and disease. With further research on the LINC00526 protein, we may find new applications 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 CR018 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 15 amino acids at positions 2 to 16 of the CR018 protein were determined as the amino acid sequence of the synthetic polypeptide, and the sequence was SHSYKKAISDEALRP (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, dissolve the target peptide in 50% acetonitrile solution, identify its structure by mass spectrometry, and confirm that it is the target protein.

[0021] 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 3 M sodium hydroxide. After mixing, add the Sulfo-SMCC solution dropwise to KLH and stir at room temperature for 45 min. 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 min, and collect the light gray eluate, which is the activated Sulfo-SMCC / KLH solution. Dissolve 2.5 mg of cross-linked peptide in 250ul PBS (pH7.4), 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℃ and freeze-dry in a freeze dryer 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%.

[0022] 3) Peptide immunization and antiserum preparation 110μg of cross-linked KLH-peptide was dissolved in 110μl 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 110μl 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 110ug of polypeptide and dissolve it in 0.55ml phosphate buffer (0.01M PBS) and inject it intraperitoneally as booster immunization.

[0023] 4) Hybridoma cell preparation (1) Cell fusion: Three days after the stimulation of the booster 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 dropwise within 45 seconds. After standing for 90 seconds, 1.3ml of serum-free DMEM was added dropwise, and the reaction was slowly stopped by shaking. Serum-free DMEM was added to make up to 25 ml, and the volume was shaken while adding. Centrifugation was performed at 900 rpm / min for 5 minutes, and the supernatant was discarded.

[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, take 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 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. 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 / 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.

[0025] 6) Antibody affinity purification (1) IgG purification: Add 9 ml of 50% Protein-A Sepharose suspension to a 28 ml 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 column. Rotate the mixer at room temperature (20-25°C) for 1.5 hours to discharge the serum sample. In addition, wash the 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 CR018 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 × 42 min, and the supernatant was collected. The antibody titer was determined to be 1:32000 by indirect ELISA, and the protein content was 1.077 mg / ml by Bradford method.

[0027] 7) Cell immunofluorescence analysis: Confirm that the CR018 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 to process the sample. The specific operations are as follows: 7.1. Fixation Use 4% PFA to fix for 30 minutes at room temperature. 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.

[0028] 7.2 Transparency 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 antigens.

[0029] 7.3. Closure 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.

[0030] 7.4. Primary Antibody Incubation 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.

[0031] 7.5. Secondary Antibody Incubation 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 / plate of the diluted secondary antibody and 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 DAPI 100 μL / slide was added to stain the nuclei for 10 min at room temperature.

[0033] 7.7. Sealing 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 .

[0034] The results showed that the prepared CR018 monoclonal antibody could effectively recognize CR018.

[0035] 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 CR018, characterized in that: The monoclonal antibody hybridoma cell line CR018 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.46121.

2. The CR018 monoclonal antibody secreted and produced by the monoclonal antibody hybridoma cell line CR018 as claimed in claim 1.

3. The CR018 monoclonal antibody according to claim 2, characterized in that The CR018 monoclonal antibody is IgG1.

4. Use of the CR018 monoclonal antibody as claimed in claim 2 in the preparation of a preparation for detecting CR018 in a sample.

5. A kit for detecting CR018 in a sample, characterized in that: The kit contains the CR018 monoclonal antibody as described 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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