SPRY4 monoclonal antibody and preparation method and application of kit of SPRY4 monoclonal antibody

Abstract

The invention provides an SPRY4 monoclonal antibody and a preparation method and application of a kit of the SPRY4 monoclonal antibody. The monoclonal antibody hybridoma cell strain SPRY4 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.46101, and the prepared monoclonal antibody and the kit thereof can effectively recognize the SPRY4.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a SPRY4 monoclonal antibody and a preparation method and use thereof. Background Art

[0002] Predicted to regulate cell growth, differentiation and migration. Predicted to predict angiogenesis, tumorigenesis and immune response. Predicted to be subcellular localized in the nucleus. Predicted protein molecular weight 23kD.

[0003] There is no report on how to prepare SPRY4 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 SPRY4 monoclonal antibody and a preparation method and use of 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 SPRY4, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No.46101.

[0006] Another aspect of the present invention provides a SPRY4 monoclonal antibody secreted and produced by the above-mentioned monoclonal antibody hybridoma cell line SPRY4.

[0007] Furthermore, the SPRY4 monoclonal antibody is IgG1.

[0008] Another aspect of the present invention provides use of a SPRY4 monoclonal antibody in preparing a sample for detecting SPRY4.

[0009] Another aspect of the present invention provides a kit for detecting SPRY4 in a sample, wherein the kit contains the above-mentioned SPRY4 monoclonal antibody.

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

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

[0012] The above detection reagents for western blot 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 SPRY4 of the present invention have the following beneficial effects: The monoclonal antibody prepared by the monoclonal antibody hybridoma cell line SPRY4 prepared in the present application can effectively recognize SPRY4.

[0015] Cell name: Hybridoma cell Storage time: September 25, 2024 Depository: China National Microbiological Culture Collection Administration General Microbiology Center Deposit number: CGMCC No.46101 Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram showing the method of western blotting in Example 1 for verifying the expression of SPRY4 antibody in Hela cells. DETAILED DESCRIPTION

[0017] Example 1 Preparation of monoclonal antibody hybridoma cell lines and monoclonal antibodies SPRY4 protein, a member of the SPRY family, is increasingly important in disease-related research. The protein participates in intracellular signal transduction through its SPRY domain, especially the regulation of the RAS / MAPK pathway, affecting cell growth, differentiation and migration. In oncology, the expression level of SPRY4 is closely related to the development of various cancers, and its abnormal expression may promote the proliferation and invasion of tumor cells. In addition, the role of SPRY4 in cardiovascular disease has also attracted the attention of researchers, especially in angiogenesis and cardiac development. In terms of gene function, SPRY4 may act as a negative regulator to prevent excessive cell proliferation by inhibiting certain signaling pathways. However, the potential functions of SPRY4 in fields such as immunology, neurobiology, metabolic regulation and cell aging have not been fully explored, and these unstudied areas may reveal its new roles in health and disease.

[0018] The molecular mechanism of SPRY4 protein involves its interaction with multiple proteins, and these interactions are essential for the balance of intracellular signaling. SPRY4 binds to other proteins through its SPRY domain, which may affect cell cycle control, cell proliferation and apoptosis. Although some biological functions of SPRY4 have been confirmed, its role in cell aging and metabolic regulation is still unclear, and these areas may provide new perspectives and therapeutic targets for future research. With a better understanding of the molecular mechanism of SPRY4, we may find new applications in disease prevention and treatment.

[0019] 1) Analysis and design of peptide sequences Abdesigner software was used to perform antigen epitope analysis on the amino acid sequence of SPRY4 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 188 to 202 of SPRY4 protein were determined as the amino acid sequence of the synthetic polypeptide, and the sequence was FALWDGELLTHSGLE (SEQ ID NO.1).

[0020] 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 procedure and dissolved in 50% acetonitrile. The structure was characterized using mass spectrometry to confirm that it was the target peptide.

[0021] Cross-linking: Use Sulfo-SMCC as a cross-linking agent to cross-link KLH with the synthetic peptide. Dissolve 3 mg of sulfo-SMCC in 0.6 ml of ultrapure water, take 18 mg of KLH and dissolve it in 1.2 ml of ultrapure water, and adjust the pH to about 7 with 3 M sodium hydroxide. After mixing, slowly add sulfo-SMCC solution dropwise to the KLH solution and stir it 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 20 minutes to obtain an eluent, namely the activated Sulfo-SMCC / KLH solution. Dissolve 4 mg of cross-linked peptide in 400 ul PBS (pH 7.4), add 0.4 volume of Sulfo-SMCC / KLH complex solution to the peptide solution, adjust the pH to 7.3, shake at room temperature for 9 h, freeze at -70°C, place in a freeze dryer for 24 h, and set aside. The peptide cross-linking efficiency was determined by measuring the peptide thiol groups before and after cross-linking using the Ellman method, and the coupling rate was 98.7%.

[0022] 3) Peptide immunization and antiserum preparation 90 µg of cross-linked KLH-peptide was dissolved in 90 µl 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 25 g were used for immunization and injected intradermally on the back, with at least 3 injections. Two weeks after the first immunization, 90 µg of the peptide was dissolved in 90 µl 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 was required to inject intradermally on the back, with at least 3 injections. Two weeks after the second immunization, the second booster immunization was performed, and the specific operation steps were the same as the first booster immunization. One week later, 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. Antibody and titer detection were performed multiple times until the antibody titer was greater than 1:10000. 90 μg of the polypeptide was dissolved in 0.45 ml of phosphate buffer (0.01 M PBS) and injected intraperitoneally for boosting immunization.

[0023] 4) Hybridoma cell preparation (1) Cell fusion: Three days after the booster immunization, blood was collected from the eye sockets of mice. The mice were killed by cervical dislocation and then immersed in 75% alcohol for disinfection. The spleen of the mice was removed from the clean bench and gently ground on a 200-mesh steel sieve. Serum-free DMEM was added to make the volume 8 ml. The ground spleen was blown with a Pasteur pipette to disperse it into single cells. 8 ul of the mixed solution was counted to calculate the total number of spleen cells. The corresponding number of myeloma cells was added according to the ratio of myeloma cells: spleen cells of 1:8, mixed evenly, centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The cells were gently shaken to disperse and placed in a water bath at 37°C. 800 ul of PEG1500 was added with a dropper, shaking while adding, and the reaction was completed within 50 seconds. After standing for 90 seconds, 1.1 ml of serum-free DMEM was added dropwise, shaking while adding, and the reaction was slowly terminated. 35 ml of serum-free DMEM was added, shaking while adding, centrifuged at 800 rpm / min for 5 minutes, and the supernatant was discarded.

[0024] (2) Cell culture screening: Use 1 ml of cell culture medium to gently blow away the fused cells at the bottom, and use HAT culture medium (HAT dissolved in DMEM containing 20% ​​fetal bovine serum) to make up the volume to 50 ml. Evenly spread it into a 96-well plate with feeder cells, 150 μl / well. After 3 days, replace the supernatant in the 96-well plate with newly prepared HAT culture medium (HAT dissolved in DMEM containing 20% ​​fetal bovine serum). After 4 days of changing the medium, take 80 ul 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. Replace the wells with higher positive values ​​with HAT culture medium and test again one day later. After retesting, take the well with the highest positive value and use HT culture medium (HT dissolved in DMEM containing 20% ​​fetal bovine serum) for limiting dilution, and evenly spread it into a 96-well plate with feeder cells, 150 μl / well. After seven days, the positive value was tested, and the monoclonal clone with high positive value was subcloned with DMEM containing 20% ​​fetal bovine serum. After the positive monoclonal clone was subcloned, 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 expanded and cultured, 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 Seven days before cell injection, five healthy 8-week-old BalB / C female mice were intraperitoneally injected with 1.1 ml paraffin oil per mouse. When the cultured cells were in the logarithmic phase, the cells in the culture dish were blown off and counted, and 3*100 cells were taken and placed in a centrifuge tube and centrifuged at 800 r / min for 5 min. The supernatant was discarded, 3 ml of sterilized phosphate buffer (0.01M PBS) was added, and the cell clusters were gently blown away with a pipette, and the phosphate buffer (0.01M PBS) was added to the volume to 30 ml, and centrifuged at 800 r / min for 5 min. 2.5 ml of phosphate buffer (0.01M PBS) was added to the centrifuge tube, mixed evenly, and 0.5 ml of the mixture was intraperitoneally injected into each mouse. Seven days after the injection, the mice were killed by cervical dislocation, placed in 75% alcohol for disinfection, the abdominal epithelium was cut open with surgical scissors, a 10 ml syringe was inserted to pick up the peritoneal membrane, and the ascites was extracted into a centrifuge tube, and centrifuged at 4000 r / min for 5 min. 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: Use a pipette to take 8 ml of 50% Protein-A Sepharose suspension and add it to a 25 ml chromatography column. Remove the top and bottom caps. The column bed volume after the liquid flows out is 4 ml. Then rinse the chromatography column with 4 times the column volume of phosphate buffer. Take out 8 ml of the corresponding serum, mix it with 2 ml of phosphate buffer and add it to a 25 ml chromatography column. Rotate it on a rotating mixer at room temperature (20-25°C) for 3 hours to allow the serum sample to flow out. Then wash the chromatography column with 3 times the column volume of phosphate buffer and add 8 ml of eluent for elution.

[0026] (2) Peptide affinity purification: Add 1.5 ml of Sulfo-link gel suspension (0.75 ml gel) to the column, drain the column liquid, and rinse the column with 6 ml coupling buffer. Dissolve the prepared SPRY4 peptide in 1.5 ml coupling buffer and add it to the column. Add 1.5 ml coupling buffer to the column and mix at room temperature for 1 hour. Rinse the column with 9 ml coupling buffer, add 4.5 ml blocking agent, and mix at room temperature for 1 hour. Rinse the column 3 times, then add 9 ml IgG and 4.5 ml PBS to the column, and mix at room temperature for 1 hour. Rinse the column 3 times with PBS, and then elute with 3 ml elution buffer. Place the purified antibody in a dialysis bag and dialyze at 4°C. Dialyze overnight, then centrifuge at 4500 rpm × 32 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 1.078 mg / ml.

[0027] 7) Western Blot Analysis: Confirm that the SPRY4 antibody information is correct and perform the following operations after protein extraction: 7.1. Glue making After the glue filling device is assembled, water can be poured into the glass plate to check for leaks, and then inverted to dry for about 10 minutes. If glue leakage occurs: pay attention to check whether the device is clamped and whether the glue strip is aged; if the glass plate is not clean, it may cause uneven glue / strip tailing. The long plate is inside and the short plate is outside. After adding the separation gel, add 1ml of water or anhydrous ethanol to seal. After 30 minutes, pour out the liquid seal. After the glue is dry, pour it out and drain it. Add concentrated gel and wait for 30 minutes. After the gel is completely solidified, it can be used for subsequent electrophoresis.

[0028] 7.2 Electrophoresis The cooked protein sample was taken out of the refrigerator and placed in an ice box to melt. The melted protein was heated again at 100°C for 5-10 minutes.

[0029] Add the protein maker and protein samples to the SDS-PAGE gel. The concentration gel is usually 80V, and the separation gel is 100V.

[0030] 7.3. Transfer First, remove the gel and cut it. Assemble the transfer device according to the filter paper-gel-membrane-filter paper sandwich structure. Add pre-cooled electrotransfer solution and ice packs and place it in an ice box. Turn on the power and run the gel.

[0031] 7.4. Milk sealing Take out the PVDF membrane. Place it in an incubation box with 5% skim milk in advance and block it by slowly shaking it on a shaker at room temperature for 1 hour. Rinse the hybridization membrane with TBST buffer to wash away the residual blocking solution.

[0032] 7.5 Antibody hybridization Primary antibody hybridization: Incubate at room temperature for 2 hours, or overnight at 4°C. Pour off the primary antibody or recover the primary antibody solution, add TBST to wash the membrane, 10 minutes each time, three times. Secondary antibody hybridization: Place the secondary antibody in the incubation box and incubate at room temperature for 1 hour.

[0033] 7.6. Development Discard the secondary antibody solution, wash the membrane three times with TBST, 10 min each time. Drain the water, place the membrane in a chemiluminescent imager, and evenly add ECL luminescent solution. Operate the instrument for luminescent development.

[0034] The results showed that the prepared SPRY4 monoclonal antibody could effectively recognize SPRY4.

[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 SPRY4, characterized in that: The monoclonal antibody hybridoma cell line SPRY4 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number: CGMCC No.46101.

2. The SPRY4 monoclonal antibody secreted and produced by the monoclonal antibody hybridoma cell line SPRY4 according to claim 1.

3. The SPRY4 monoclonal antibody according to claim 2, characterized in that The SPRY4 monoclonal antibody is IgG1.

4. Use of the SPRY4 monoclonal antibody according to claim 2 in preparing a preparation for detecting SPRY4 in a sample.

5. A kit for detecting SPRY4 in a sample, characterized in that: The kit contains the SPRY4 monoclonal antibody as claimed in claim 2.

6. The kit according to claim 5, characterized in that: The kit also includes detection reagents for Western blotting.

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

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