CN116444677A - FoxM1 protein Y575 phosphorylated polyclonal antibody, preparation method and application thereof - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
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- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C07K—PEPTIDES
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
The application relates to the technical field of antibodies, and particularly discloses a FoxM1 protein Y575 phosphorylated polyclonal antibody, a preparation method and application thereof. The antigen synthesized peptide has an amino acid sequence at least 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO. 1, and the phosphorylation site is tyrosine at position 575 of FoxM1 protein. The antigen synthesized peptide can specifically recognize the phosphorylated polyclonal antibody of the FoxM1 protein Y575; the method for preparing the FoxM1 protein Y575 phosphorylated polyclonal antibody by using the antigen synthetic peptide is simple, low in cost and strong in operability; the polyclonal antibody for the phosphorylation of the FoxM1 protein Y575 can be applied to the specific detection of the Y575 phosphorylated FoxM1 protein in an immunoblotting experiment, and has important significance in the research and application of the FoxM1 protein.
Description
Technical Field
The application relates to the technical field of antibodies, in particular to a FoxM1 protein Y575 phosphorylated polyclonal antibody, a preparation method and application thereof.
Background
FoxM1 belongs to the family of FOX transcription factors, and is a tumor-associated protein with a winged helical structure in a DNA binding domain. FoxM1 is widely expressed in various tissue cells and participates in physiological functions such as proliferation and differentiation of cells, apoptosis, formation of tumors and the like. In normal tissue cells, foxM1 protein exhibits a low expression state. However, high expression of FoxM1 is detected in various malignant tumor tissue cells, and the higher the expression level of FoxM1, the stronger the cell proliferation and metastasis capacity and the higher the malignancy of tumor, it has become a marker protein for prognosis of tumor patients. Therefore, detection of FoxM1 protein expression level in tissue cells has important reference significance for diagnosis and treatment of tumors.
The expression level of FoxM1 protein in tissue cells is closely related to its degree of phosphorylation. Wherein tyrosine 575 at position 575 of FoxM1 (Y575) can be modified by non-receptor tyrosine kinase c-Abl phosphorylation, and the phosphorylation modification of FoxM1Y575 is critical for the maintenance of protein stability and high expression level. Therefore, it is important to develop antibodies capable of specifically recognizing the Y575 phosphorylated FoxM1 protein.
Antibodies recognizing FoxM1 proteins are mostly obtained by immunizing animals with an amino acid sequence of FoxM1 protein as an immunogen, such as Anti-FoXM1 antibody (ab 264210) from Abcam, inc., polyclonal antibodies obtained by immunizing animals with 450-500 aa of FoXM1 protein as an immunogen, recognizing total FoxM1 protein (including phosphorylated and non-phosphorylated) in tissue cells. There are also antibodies that specifically recognize the FoxM1 phosphorylation site, such as the FoxM1Ser35 phosphorylated antibody from cell signaling technology, but recognize the serine/threonine phosphorylation site, and there are currently no polyclonal antibodies that specifically recognize FoxM1 (Y575 phosphorylated).
Disclosure of Invention
The purpose of the present application is to overcome the shortcomings of the prior art and provide a FoxM1 protein Y575 phosphorylated polyclonal antibody, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides an antigen-synthesized peptide of a FoxM1 protein phosphorylated antibody, said antigen-synthesized peptide having an amino acid sequence at least 98%, 99%, 99.5% or 99.9% identical to SEQ ID No. 1, the phosphorylation site being tyrosine at position 575 of the FoxM1 protein.
The inventor of the application obtains the antigen synthetic peptide of the application through a great deal of creative work, and can specifically identify the polyclonal antibody phosphorylated by the FoxM1 protein Y575, and then the polyclonal antibody can specifically identify the Y575 phosphorylated FoxM1 protein.
The antigen synthesized peptide has the characteristics of short amino acid sequence and strong immunogenicity.
In a second aspect, the present application provides a method for preparing a polyclonal antibody phosphorylated by FoxM1 protein Y575, comprising the steps of:
1) Coupling the antigen synthetic peptide with carrier protein to obtain antigen;
2) Immunizing an animal with the antigen of step 1), and collecting antisera;
3) And (3) performing affinity column separation and purification on the antiserum obtained in the step (2) by using the synthesized modified peptide to obtain the FoxM1 protein Y575 phosphorylated polyclonal antibody.
The method for preparing the FoxM1 protein Y575 phosphorylated polyclonal antibody by using the antigen synthetic peptide is simple, low in cost and high in operability; the prepared FoxM1 protein Y575 phosphorylated polyclonal antibody has strong immunity and high specificity, and the FoxM1 protein Y575 phosphorylated polyclonal antibody can be applied to specific detection of Y575 phosphorylated FoxM1 protein in immunoblotting experiments, and has important significance in research and application of the FoxM1 protein.
As a preferred embodiment of the method for preparing a polyclonal antibody phosphorylated by FoxM1 protein Y575 described herein, in the step 2), the animal is immunized four times with the antigen described in the step 1), the antigen is emulsified with Freund's incomplete adjuvant, and antisera are collected and assayed for serum titer.
As a preferred embodiment of the method for preparing a polyclonal antibody phosphorylated by FoxM1 protein Y575 described herein, the carrier protein comprises carrier protein KLH.
In the step 3), the antigen synthetic peptide and the FoxM1 polypeptide are taken and respectively added into a chromatographic column for reaction, then the reaction is carried out at room temperature, a coupling solution is added for cleaning a filler, then a sealing solution is added for reaction at room temperature, and after the reaction is finished, the sealing solution is removed, so that an antigen synthetic peptide affinity column and a FoxM1 polypeptide affinity column are obtained; loading the antiserum obtained in the step 2) into an antigen synthetic peptide affinity column for loading, collecting the flow-through liquid, eluting, and collecting antibodies; and (3) dialyzing an eluent containing the antibody, loading the eluent into a FoxM1 polypeptide affinity column for loading, collecting the flow-through liquid, and eluting to obtain the FoxM1 protein Y575 phosphorylated polyclonal antibody.
As a preferred embodiment of the method for preparing a polyclonal antibody phosphorylated by FoxM1 protein Y575 described herein, the detection of antiserum titers was detected by an indirect ELISA method.
In a third aspect, the present application provides a FoxM1 protein Y575 phosphorylated polyclonal antibody prepared by the above preparation method.
In a fourth aspect, the present application provides the use of the polyclonal antibody phosphorylated by FoxM1 protein Y575 as described above in the preparation of a reagent for immunoblotting detection.
In a fifth aspect, the present application provides the use of the above FoxM1 protein Y575 phosphorylated polyclonal antibody in the preparation of a reagent for recognizing FoxM1 protein tyrosine phosphorylated at position 575.
The polyclonal antibody of the FoxM1 protein Y575 phosphorylation obtained by immunizing animals with the antigen synthetic peptide can be applied to an immunoblotting experiment and is used for specifically detecting the FoxM1 protein phosphorylated by the Y575. The FoxM1 protein Y575 phosphorylated polyclonal antibody can be applied to detection and research of Y575 phosphorylation of FoxM 1.
Compared with the prior art, the application has the following beneficial effects:
the application provides a polyclonal antibody phosphorylated by FoxM1 protein Y575, a preparation method and application thereof, wherein the antigen synthetic peptide can specifically identify the polyclonal antibody phosphorylated by FoxM1 protein Y575, and the polyclonal antibody can specifically identify the Y575 phosphorylated FoxM1 protein. The method for preparing the FoxM1 protein Y575 phosphorylated polyclonal antibody by using the antigen synthetic peptide is simple, low in cost and high in operability; the prepared FoxM1 protein Y575 phosphorylated polyclonal antibody has strong immunity and high specificity, and the FoxM1 protein Y575 phosphorylated polyclonal antibody can be applied to specific detection of Y575 phosphorylated FoxM1 protein in immunoblotting experiments, and has important significance in research and application of the FoxM1 protein.
Drawings
FIG. 1 is a graph showing the purification results of FoxM1 Protein Y575 phosphorylated polyclonal antibody (lane M: protein Marker; lane 1: serum (diluted 10-fold); lane 2: flow-through (diluted 10-fold); lane 3: eluate obtained in step b of the antibody purification of example 1; lane 4: eluate obtained in step c of the antibody purification of example 1);
FIG. 2 is a graph showing the results of ELISA titers for detection of FoxM1 protein Y575 phosphorylated polyclonal antibodies;
FIG. 3 is a graph showing the results of ELISA titers for FoxM1 non-phosphorylated antibodies;
FIG. 4 is a graph showing the results of FoxM1 protein Y575 phosphorylated polyclonal antibody applied to immunoblotting experiments (in vivo experiments);
FIG. 5 is a graph showing the results of FoxM1 protein Y575 phosphorylated polyclonal antibody applied to immunoblotting experiments (in vitro kinase assay).
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
The application finds that an antigen synthesized peptide of a phosphorylated peptide (the amino acid sequence of which is CPASQLS (pY) SQEVGG, and SEQ ID NO: 1) is used as a FoxM1 protein phosphorylated antibody to immunize animals, the antibody obtained by purifying animal serum can specifically identify Y575 phosphorylated FoxM1 protein, and the antibody can be applied to immunoblotting experiments and is used for specifically detecting Y575 phosphorylated FoxM1 protein. The antibody can be applied to detection and research of Y575 phosphorylation of FoxM 1.
In some embodiments, the antigen-synthesizing peptides described herein have an amino acid sequence that is at least 98%, 99%, 99.5%, or 99.9% identical to SEQ ID NO. 1, with the phosphorylation site being tyrosine at position 575 of the FoxM1 protein.
Specific information on the antigen-synthesized peptide (FoxM 1 (pY 575)) and the FoxM1 polypeptide are shown in Table 1.
TABLE 1
Example 1, foxM1 protein Y575 phosphorylated polyclonal antibody and method for preparing the same
The embodiment provides a preparation method of a FoxM1 protein Y575 phosphorylated polyclonal antibody, which comprises the following steps:
1) Coupling 4mg of the obtained antigen synthetic peptide with 4mg of carrier protein KLH to obtain an antigen;
2) Immunizing an animal with the antigen of step 1), wherein the specific steps include the following steps:
a. primary immunization: 2 New Zealand white rabbits (Ming's agriculture and animal husbandry science and technology, rabbit No. 23, 09) were taken, the rabbit was aged for 4 months and weighing about 2.5kg, 1mg of antigen was immunized (calculated by total amount of polypeptide and carrier protein KLH) per rabbit, and Freund's complete adjuvant (SIGMA, F5881) was used to emulsify the antigen.
b. Secondary immunization: two weeks later, a second immunization was performed, emulsifying the antigen with Freund's incomplete adjuvant (SIGMA, F5506).
c. Three immunizations: after one week, three immunizations were performed, and antigens were emulsified with Freund's incomplete adjuvant.
d. Four immunizations: four immunizations were performed after one week, and the antigen was emulsified with Freund's incomplete adjuvant.
e. Blood collection: serum samples were collected one week after four immunizations and assayed for serum titers by ELISA.
f. And finally, blood collection: after four immunizations for one week, final blood collection was performed to collect antisera.
The indirect ELISA method initially detects serum titers (as shown in table 2):
a. antigen coating and sealing: diluting the antigen to 1 mug/ml by using CBS coating buffer, coating the antigen into a 96-well plate according to 100 mu l of each well, and coating the antigen at 4 ℃ overnight; the next day PBST plates were washed 5 times and blocked with 5% milk powder-CBS at room temperature for 1h; the plates were washed 5 times again with PBST for use.
b. Incubating primary antibodies: serum was diluted in a double ratio of 1:2000 with PBS, and finally diluted in a ratio of 1:128000, and PBS was used as a negative control, and added to a 96-well plate for incubation at 37℃for 1h.
c. Incubating a secondary antibody: plates were washed 5 times with PBST, the secondary antibody (goat anti-rabbit IgG-HRP) was diluted 1:10000 with 5% milk powder-PBS, 100 μl of diluted secondary antibody was added to each well, and incubated at 37deg.C for 40min.
d. Color development and reading: PBST plate was washed 5 times, 100. Mu.l TMB working solution was added to each well, incubated at 37℃for 5min, 50. Mu.l stop solution was added to each well, and OD was measured by an ELISA reader 450 Absorbance values.
Table 2ELISA preliminary detection of serum titers
3) Antibody purification:
a. polypeptide affinity column preparation: taking 3mg of FoxM1 (pY 575) polypeptide and 6mg of FoxM1 polypeptide, respectively adding into a 2ml balanced SulfoLink Coupling Resin chromatographic empty column, carrying out oscillation reaction at room temperature for 1 hour, standing at room temperature for 15 minutes, and cleaning a filler by using a coupling solution (the coupling solution obtained by coupling 4mg of the obtained antigen synthetic peptide with 4mg of carrier protein KLH); adding 2 times of column volume of blocking solution (5% milk powder prepared by PBST), oscillating at room temperature for 30 minutes, standing and incubating for 30 minutes; after the sealing liquid is discharged, cleaning liquid with the volume of 10 times of the column volume is added, and the mixture is stored in sealing liquid with the volume of 2 times of the column volume and is stored at the temperature of 4 ℃.
b. Affinity purification of FoxM1 phosphorylated antibody (FoxM 1 protein Y575 phosphorylated polyclonal antibody of the present application): taking 20mL of rabbit serum, centrifugally filtering, loading an antigen synthetic peptide of FoxM1 protein phosphorylated antibody on a column, collecting a running-through liquid, repeatedly loading the solution on the column once, flushing the solution with an existing balancing buffer (such as HBSS, shanghai Beacon Biotechnology Co., ltd.) with 5 times of bed volume, adding an existing eluent (such as Simer fly), and collecting the eluted antibody; this step was repeated until no more antibody was eluted from the eluate, giving an eluted sample.
c. Affinity purification of FoxM1 non-phosphorylated antibodies: b, after dialyzing the elution sample in the step b, carrying out affinity column loading on the FoxM1 polypeptide antigen, collecting the flow-through liquid, repeatedly carrying out column loading once, washing with an equilibrium buffer solution with the volume of 5 times of the column bed, adding an eluent, and collecting the eluted antibody; this step was repeated until no more antibody was eluted from the eluate, designated as FoxM1 phosphorylated rabbit polyclonal antibody (FoxM 1 phosphorylated antibody), and designated as FoxM1 non-phosphorylated rabbit polyclonal antibody.
d. The eluted antibody was dialyzed into PBS, and the dialyzed antibody was concentrated by ultrafiltration in an ultrafiltration tube.
Example 2 detection of FoxM1 protein Y575 phosphorylated polyclonal antibody
1. And (3) purity detection: and (3) carrying out SDS-PAGE electrophoresis on the prepared FoxM1 protein Y575 phosphorylated polyclonal antibody, and then, dyeing with a protein fast dye solution, and carrying out antibody purity detection.
The purification results of the phosphorylated polyclonal antibody of FoxM1 protein Y575 are shown in FIG. 1 (FIG. 1A shows the purification result of 09. Mu.l. Rabbit serum; FIG. 1B shows the purification result of 23. Mu.l. Rabbit serum).
Measuring the protein concentration by a spectrophotometer, wherein the concentration of 23 # rabbit phosphorylated polyclonal antibody (FoxM 1 protein Y575 phosphorylated polyclonal antibody) is 1.2mg/ml, and the volume is 2ml; the concentration of the 09 # phosphorylated polyclonal antibody (FoxM 1 protein Y575 phosphorylated polyclonal antibody) is 0.9mg/ml, and the volume is 2ml; the concentration of the No. 23 non-phosphorylated polyclonal antibody (FoxM 1 non-phosphorylated rabbit polyclonal antibody) is 1.3mg/ml, and the volume is 3ml; the concentration of the No. 09 non-phosphorylated polyclonal antibody (FoxM 1 non-phosphorylated rabbit polyclonal antibody) is 0.9mg/ml, and the volume is 3ml.
2. Potency detection: polyclonal antibodies to FoxM1 protein Y575 were phosphorylated and ELISA titers were measured (as shown in tables 3-4).
1) Antigen coating and sealing: the CBS coating buffer was diluted to 1. Mu.g/ml of FoxM1 (pY 575), coated in 100. Mu.l per well of FoxM1 (pY 575) into 96 well plates, coated overnight at 4 ℃; the next day PBST plates were washed 5 times and blocked with 5% milk powder-CBS at room temperature for 1h; the plates were washed 5 times again with PBST for use.
2) Incubating primary antibodies: serum was diluted in a double ratio of 1:2000 with PBS, and finally diluted in a ratio of 1:128000, and PBS was used as a negative control, and added to a 96-well plate for incubation at 37℃for 1h.
3) Incubating a secondary antibody: plates were washed 5 times with PBST, the secondary antibody (goat anti-rabbit IgG-HRP) was diluted 1:10000 with 5% milk powder-PBS, 100 μl of diluted secondary antibody was added to each well, and incubated at 37deg.C for 40min.
4) Color development and reading: PBST plate was washed 5 times, 100. Mu.l TMB working solution was added to each well, incubated at 37℃for 5min, 50. Mu.l stop solution was added to each well, and OD was measured by an ELISA reader 450 Absorbance values.
TABLE 3 Table 3
TABLE 4 Table 4
As shown in FIGS. 2-3, new Zealand white rabbits were subjected to subcutaneous multipoint immunization with FoxM1 (pY 575) to generate antibodies, the purity of the antibodies was measured by using a Coomassie Brilliant blue method after purification of the antibodies, and the antibody titer was detected by ELISA, so that the obtained FoxM1 protein Y575 phosphorylated polyclonal antibody (rabbit) was capable of specifically recognizing the phosphorylated FoxM1 (pY 575) coating protein, whereas the prepared FoxM1 non-phosphorylated antibody (control) was incapable of specifically recognizing the FoxM1 (pY 575) coating protein, and thus the FoxM1 protein Y575 phosphorylated polyclonal antibody (rabbit) was successfully prepared.
EXAMPLE 3 application of FoxM1 protein Y575 phosphorylated polyclonal antibody to immunoblotting experiments
1. In vivo experiments:
the 293 cells were co-transfected with wild-type plasmid Flag-FoxM1WT or Flag-FoxM1Y575F mutant plasmid (tyrosine at position 575 was mutated to phenylalanine so that it could not be phosphorylated by c-Abl) and Myc-c-Abl, after 48 hours cells were collected for lysis, lysates were immunoprecipitated with Flag or IgG (negative control) agarose beads, flag-tagged Flag-FoxM1WT and Flag-FoxM1Y575F proteins were precipitated, proteins were subjected to SDS-PAGE electrophoresis, and immunoblotted with Flag antibodies and the FoxM1Y575 phosphorylated polyclonal antibodies of the present application after transfer.
As shown in FIG. 4, in vivo experiments confirm that the polyclonal antibody phosphorylated by FoxM1 protein Y575 prepared above specifically recognizes phosphorylated FoxM1Y575, and hardly recognizes FoxM1Y575F in which Y575 is mutated (tyrosine mutation to phenylalanine).
2. In vitro kinase assay:
wild type FoxM1 or FoxM1Y575F protein and c-Abl kinase protein are respectively incubated for 40 minutes under the conditions of ATPase and 37 ℃, and after the mixture is subjected to SDS-PAGE electrophoresis, commercial FoxM1 antibodies and polyclonal antibodies phosphorylated by the FoxM1 protein Y575 in the application are used for immunoblotting detection.
As shown in FIG. 5, in vitro kinase assay experiments confirm that the antibody specifically recognizes phosphorylated FoxM1Y575, and cannot recognize FoxM1Y575F (tyrosine to phenylalanine) where Y575 is mutated.
There is no antibody in the prior art that specifically recognizes the Y575 phosphorylated FoxM1 protein. The antigen synthetic peptide has the characteristics of short amino acid sequence and strong immunogenicity, and the method for preparing the FoxM1 protein Y575 phosphorylated polyclonal antibody by using the antigen synthetic peptide is simple, low in cost and strong in operability; the prepared polyclonal antibody for the phosphorylation of the FoxM1 protein Y575 has strong immunity and high specificity, can be applied to the specific detection of the Y575 phosphorylated FoxM1 protein in an immunoblotting experiment, and has important significance in the research and application of the FoxM1 protein.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present application and not for limiting the scope of protection of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.
Claims (9)
1. An antigen-synthesized peptide of a FoxM1 protein phosphorylated antibody, wherein the antigen-synthesized peptide has an amino acid sequence at least 98%, 99%, 99.5% or 99.9% identical to SEQ ID No. 1, and the phosphorylation site is tyrosine at position 575 of the FoxM1 protein.
2. A method for preparing a polyclonal antibody phosphorylated by FoxM1 protein Y575, comprising the steps of:
1) Coupling the antigen-synthesizing peptide according to claim 1 with a carrier protein to obtain an antigen;
2) Immunizing an animal with the antigen of step 1), and collecting antisera;
3) And (3) performing affinity column separation and purification on the antiserum obtained in the step (2) by using the synthesized modified peptide to obtain the FoxM1 protein Y575 phosphorylated polyclonal antibody.
3. The method of claim 2, wherein in step 2), the animal is immunized four times with the antigen of step 1), the antigen is emulsified with Freund's incomplete adjuvant, and antisera are collected and assayed for serum titer.
4. The method of claim 2, wherein the carrier protein comprises carrier protein KLH.
5. The preparation method of claim 2, wherein in the step 3), the antigen synthetic peptide and the FoxM1 polypeptide of claim 1 are taken and respectively added into a chromatographic column for reaction, then the chromatographic column is kept still at room temperature, a coupling solution is added for cleaning a filler, then a sealing solution is added for room temperature reaction, and after the reaction is finished, the sealing solution is removed to obtain an antigen synthetic peptide affinity column and a FoxM1 polypeptide affinity column; loading the antiserum obtained in the step 2) into an antigen synthetic peptide affinity column for loading, collecting the flow-through liquid, eluting, and collecting antibodies; and (3) dialyzing an eluent containing the antibody, loading the eluent into a FoxM1 polypeptide affinity column for loading, collecting the flow-through liquid, and eluting to obtain the FoxM1 protein Y575 phosphorylated polyclonal antibody.
6. The method of claim 3, wherein the detection of antisera titers is by an indirect ELISA method.
7. The FoxM1 protein Y575 phosphorylated polyclonal antibody prepared by the preparation method according to any one of claims 2 to 4.
8. Use of the FoxM1 protein Y575 phosphorylated polyclonal antibody of claim 7 in the preparation of a reagent for immunoblotting detection.
9. Use of a FoxM1 protein Y575 phosphorylated polyclonal antibody according to claim 7 in the preparation of a reagent for recognizing the FoxM1 protein tyrosine phosphorylated at position 575.
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