Transcription factor HOMEZ phosphorylation site specific antibody as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a transcription factor HOMEZ phosphorylation site specific antibody as well as a preparation method and application thereof. The specific antibody provided by the invention can specifically recognize phosphorylation of the 451st site threonine of HOMEZ in a tumor specimen, so that the prognosis condition of the tumor is judged, and a potential action target is provided for clinical diagnosis and treatment of the tumor; moreover, the specific antibody disclosed by the invention can be used for detecting the expression difference between normal cells and tumor cells in a Western blot experiment, and is beneficial to research on the effect of phosphorylation modification of HOMEZ protein in the occurrence and development process of tumor diseases; and the kit can also be used for detecting the phosphorylation level of the HOMEZ protein in a clinical specimen through experiments such as immunofluorescence and immunohistochemical staining, and has a wide clinical application prospect in the aspects of disease diagnosis, treatment, prognosis judgment and the like.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a transcription factor HOMEZ phosphorylation site-specific antibody and a preparation method and application thereof. Background Art

[0002] The HOMEZ gene encodes 550 amino acids containing three atypical homeodomains, two leucine zipper-like motifs and an acidic domain, and is ubiquitously expressed in tissues of humans and mice.

[0003] At present, there are very few reports on the HOMEZ gene. Studies have shown that the Homez gene plays a role in the development of neurons downstream of the proneural / neurogenic genes in Xenopus laevis; there are two missense mutations in the HOMEZ gene in Chinese patients with isolated ventricular septal defect, which are directly related to the cause of the disease; the Homez gene is highly expressed in the testis of mice, but Homez gene knockout mice are fertile and are not important for the development of germ cells. However, there are no reports on the phosphorylation modification of HOMEZ and its function in tumors. Summary of the invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a transcription factor HOMEZ phosphorylation site specific antibody and its preparation method and application. The specific antibody of the present application can specifically recognize the phosphorylation of amino acid 451 of HOMEZ gene in tumor tissue specimens, thereby judging the prognosis of tumors.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] The present application provides a phosphorylated antigenic peptide at Thr451 site of HOMEZ protein, wherein the active amino acid sequence of the antigenic peptide is LNERAE(pT)PPLPC, as shown in SEQ ID NO: 1, wherein T is phosphorylated.

[0007] The inventors of this application first discovered that the 451st site of the HOMEZ protein has phosphorylation modification of threonine, and is closely related to tumor metastasis and prognosis. These studies will provide a theoretical basis for the diagnosis and treatment of tumors and have broad clinical significance.

[0008] This application uses a phosphorylated peptide segment near the Thr451 site of the HOMEZ protein (sequence: LNERAE(pT)PPLPC) as an immunogen to immunize animals. The specific antibody obtained after purification of the animal serum has the ability to specifically recognize Thr451 phosphorylated HOMEZ protein. The specific antibody can be used for Thr451 phosphorylation detection and functional research of the HOMEZ protein.

[0009] The present application also provides a specific antibody against phosphorylation of Thr451 site of HOMEZ protein, wherein the specific antibody is prepared by immunizing an animal with the phosphorylated antigen peptide.

[0010] The specific antibody against phosphorylation of Thr451 site of HOMEZ protein provided in the present application can detect the post-translational phosphorylation modification of human HOMEZ protein and can specifically identify phosphorylation of amino acid 451 of HOMEZ in tumor specimens.

[0011] The present application also provides a method for preparing the specific antibody against phosphorylation of Thr451 site of HOMEZ protein, comprising the following steps:

[0012] S1. Determine the phosphorylation site of Thr451 of anti-HOMEZ protein;

[0013] S2, designing the phosphorylated antigenic peptide and non-phosphorylated antigenic peptide at Thr451 site of HOMEZ protein according to the HOMEZ target sequence and the anti-HOMEZ protein Thr451 phosphorylation site determined in step S1;

[0014] S3, coupling the phosphorylated antigen peptide at Thr451 site of HOMEZ protein obtained in step S2 and the non-phosphorylated antigen peptide with a carrier protein, respectively, to obtain an antigen;

[0015] S4. Immunize the animal with the antigen obtained in step S3 and collect antiserum; purify and identify the antiserum to obtain a specific antibody against the phosphorylation of Thr451 site of HOMEZ protein.

[0016] As a preferred embodiment of the method for preparing a specific antibody against HOMEZ protein Thr451 phosphorylation in the present application, in step S1, determining the anti-HOMEZ protein Thr451 phosphorylation site comprises the following steps:

[0017] 1) Screening out potential phosphorylation sites of HOMEZ protein by phosphorylation modification mass spectrometry analysis; wherein the potential phosphorylation sites include Thr437 and Thr451 sites;

[0018] 2) Constructing a HOMEZ point mutation plasmid, replacing the potential phosphorylation site with alanine that cannot be phosphorylated, and performing next-generation sequencing to verify that the amino acid sequence of the potential phosphorylation site is replaced with alanine, which is considered to be the successful construction of the HOMEZ point mutation plasmid;

[0019] 3) HOMEZ wild-type plasmid and HOMEZ point mutation plasmid were transfected into 293T cells, and then the cell pellets were collected and the phosphorylation modification level of HOMEZ protein was detected by co-immunoprecipitation (co-IP) experiment to determine the Thr451 phosphorylation site of anti-human HOMEZ protein.

[0020] Among them, 293T cells are used to detect phosphorylation-modified vector cells.

[0021] Among them, co-IP experiment is a commonly used experimental method, through which the phosphorylation modification level of HOMEZ protein can be detected.

[0022] Among them, phosphorylation modification mass spectrometry analysis is a commonly used detection method, through which mass spectrometry analysis can be used to determine the phosphorylation modification sites on the HOMEZ protein.

[0023] Among them, when the potential phosphorylation site is replaced with alanine that cannot be phosphorylated, the replacement may fail. The construction is successful only when the amino acid sequence of the T site is replaced with A as verified by the second-generation sequencing, such as T451A, indicating that the HOMEZ point mutation plasmid corresponding to the T451 site is successfully constructed.

[0024] As a preferred embodiment of the method for preparing a specific antibody against HOMEZ protein Thr451 phosphorylation described in the present application, in step S2, the sequence of the non-phosphorylated antigen peptide is LNERAETPPLPC, and the non-phosphorylated antigen peptide does not have a phosphorylation group added at the threonine site at position 451.

[0025] Preferably, the carrier protein is bovine serum albumin (BSA).

[0026] As a preferred embodiment of the method for preparing the specific antibody against the phosphorylation of Thr451 site of HOMEZ protein described in the present application, the immunization method in step S4 specifically includes:

[0027] After the initial immunization of the animal, 2 to 3 booster immunizations were performed respectively. Each immunization used the phosphorylated antigen peptide or non-phosphorylated antigen peptide of the HOMEZ protein Thr451 site, mixed with an adjuvant, and injected subcutaneously on the back to collect antiserum.

[0028] As a preferred embodiment of the method for preparing the specific antibody against the phosphorylation of Thr451 site of HOMEZ protein described in the present application, the purification and identification of the antiserum in step S4 specifically includes:

[0029] Purifying the collected antiserum by high performance liquid chromatography;

[0030] The purified antiserum was identified by enzyme-linked immunosorbent assay.

[0031] Preferably, the purified antiserum is identified by enzyme-linked immunosorbent assay (ELISA), and the process specifically includes:

[0032] The obtained antigen peptide was diluted to 1 μg / ml with carbonate buffer, 100 μl of the diluted antigen peptide was added to the ELISA plate, and coated overnight at 4°C; the next day, the coating solution was discarded, 150 μl of 1× PBST washing buffer was added to each well for washing once; 150 μl of 2% The plate was blocked with BSA, incubated at 37°C for 1 hour, and the blocking solution was discarded; the purified antiserum was graded diluted with 2% BSA, 100 μl was added to the blocked reaction wells, incubated at 37°C for 1 hour, the reaction solution was discarded, and 150 μl of 1×PBST washing buffer was added for washing 3 times; the horseradish peroxidase-coupled secondary antibody was diluted at a ratio of 1:5000, 100 μl was added to the reaction wells, incubated at 37°C for 1 hour, the reaction solution was discarded, and 150 μl of 1×PBST washing buffer was added for washing 3 times; 100 μl of 3,3',5,5'-tetramethylbenzidine substrate solution was added to each well, incubated at 37°C for 5 minutes for color development, and 100 μl of 1M sulfuric acid was added to each well to terminate the reaction; the ELISA plate was placed in a preheated ELISA reader for reading, and the data was saved and analyzed. The excitation wavelength of the ELISA reader was 450 nm.

[0033] The present application also provides the use of the HOMEZ protein Thr451 phosphorylated antigen peptide or the specific antibody against HOMEZ protein Thr451 phosphorylated site in the preparation of a preparation for detecting HOMEZ protein Thr451 phosphorylated site.

[0034] The present application also provides a pharmaceutical preparation for detecting HOMEZ protein with phosphorylation modification at Thr451 site, comprising the specific antibody against HOMEZ protein phosphorylation at Thr451 site.

[0035] The present application also provides a diagnostic kit for tumor prognosis, which comprises the specific antibody against phosphorylation of Thr451 site of HOMEZ protein.

[0036] Preferably, the tumor comprises breast cancer.

[0037] The specific antibody obtained by the above-mentioned preparation method in the present application can specifically recognize the phosphorylation of threonine at position 451 of HOMEZ protein in tumor tissue specimens, thereby judging the prognosis of breast cancer.

[0038] Compared with the prior art, this application has the following beneficial effects:

[0039] The present application provides a transcription factor HOMEZ phosphorylation site-specific antibody and a preparation method and application thereof. The specific antibody provided in the present application can specifically recognize the phosphorylation of TH451 of HOMEZ in tumor specimens, thereby judging the prognosis of the tumor and providing a potential target for the diagnosis and treatment of clinical tumors. Moreover, the specific antibody of the present application can be used for Western blot experiments to detect the expression difference between normal cells and tumor cells, which is helpful to study the role of phosphorylation modification of HOMEZ protein in the occurrence and development of tumor diseases. It can also be used for immunofluorescence, immunohistochemical staining and other experiments to detect the phosphorylation level of HOMEZ protein in clinical specimens, and has broad clinical application prospects in disease diagnosis, treatment and prognosis determination. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the result of using pan-phosphorylation antibodies (p-Ser and p-Thr) to detect the phosphorylation modification of HOMEZ after transfection of HOMEZ wild-type plasmid;

[0041] Figure 2 Phosphorylation mass spectrometry data show potential phosphorylation sites of human HOMEZ protein;

[0042] Figure 3 To detect the phosphorylation level of HOMEZ after transfection of HOMEZ wild-type or mutant plasmids using a pan-phospho-antibody (p-Thr);

[0043] Figure 4 To use a specific antibody against the phosphorylation site of Thr451 of HOMEZ protein to detect the expression level of HOMEZpT451 in HOMEZ stable knockout cells after transfection with HOMEZ wild-type or T451 site mutant plasmids;

[0044] Figure 5 This is an example of immunohistochemical staining of breast cancer tissue and normal breast tissue using a specific antibody against the phosphorylation site of HOMEZ protein Thr451;

[0045] Figure 6 The statistical graph of HOMEZpT451 expression levels in 65 paired breast cancer tissues and adjacent normal tissues. DETAILED DESCRIPTION

[0046] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0047] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0048] In the following examples and figures, HOMEZpT451 refers to the phosphorylation of HOMEZ protein at Thr451.

[0049] The present application first provides a phosphorylated antigenic peptide at Thr451 of HOMEZ protein, the active amino acid sequence of which is LNERAE(pT)PPLPC, as shown in SEQ ID NO: 1, wherein T is phosphorylated. The one that is not phosphorylated is a non-phosphorylated antigenic peptide (sequence is LNERAETPPLPC).

[0050] The present application also provides a specific antibody against phosphorylation of Thr451 site of HOMEZ protein and a preparation method thereof. The specific antibody is prepared by immunizing an animal with the phosphorylated antigen peptide.

[0051] Example 1: A specific antibody against phosphorylation of Thr451 site of HOMEZ protein and its preparation method

[0052] This embodiment provides a method for preparing a specific antibody against phosphorylation of Thr451 site of HOMEZ protein, comprising the following steps:

[0053] (1) Determine the Thr451 phosphorylation site of human HOMEZ protein:

[0054] (1.1) 293T cells were transfected with HOMEZ wild-type plasmid and the corresponding empty vector plasmid. After 48 h, the cell pellets were collected and the phosphorylation level of HOMEZ protein was detected by co-immunoprecipitation (co-IP) experiment to confirm that HOMEZ protein was indeed phosphorylated.

[0055] Specifically, 293T cells were transfected with HOMEZ wild-type plasmid and the corresponding empty vector plasmid. Cell pellets were collected 48 hours later. Co-IP experiments were performed to detect the phosphorylation modification level of HOMEZ protein using pan-threonine phosphorylation antibody and pan-serine phosphorylation antibody, respectively. The results are shown in Figure 2. Figure 1 shown.

[0056] The results showed that compared with the control group, threonine phosphorylation modification was detected on the HOMEZ protein, but serine phosphorylation modification was not detected, indicating that threonine phosphorylation modification existed on the HOMEZ protein.

[0057] (1.2) 293T cells were transfected with HOMEZ wild-type plasmid, and cell pellets were collected 48 hours later. After co-IP experiment, phosphorylation modification mass spectrometry analysis was performed to determine the phosphorylation modification sites of HOMEZ.

[0058] The results are as follows Figure 2 As shown, the results showed that there were two potential phosphorylation sites, T437 and T451, on the HOMEZ protein.

[0059] (1.3) Constructing a HOMEZ point mutation plasmid, specifically replacing the potential phosphorylation site with alanine (A) that cannot be phosphorylated, and performing next-generation sequencing to verify that the construction is successful when the amino acid sequence of the potential phosphorylation site is replaced with alanine.

[0060] (1.4) 293T cells were transfected with HOMEZ wild-type plasmid or the HOMEZ point mutation plasmid constructed as described above. Cell pellets were collected 48 h later and the phosphorylation level of HOMEZ protein was detected by co-IP assay to determine the phosphorylation site of HOMEZ protein Thr451.

[0061] Specifically, 293T cells were transfected with HOMEZ wild-type plasmid or the above-constructed HOMEZ point mutation plasmid, and cell pellets were collected 48 hours later. The phosphorylation modification level of HOMEZ protein was detected by co-IP experiment using pan-threonine phosphorylation antibody. The results are as follows: Figure 3 shown.

[0062] The results showed that compared with the wild type, T451A could significantly inhibit the phosphorylation modification level on the HOMEZ protein, while T437A had no significant difference. Based on this result, it can be determined that the site of threonine phosphorylation modification of the HOMEZ protein is T451.

[0063] (2) Based on the HOMEZ target sequence and the Thr451 phosphorylation site of the human HOMEZ protein determined in step (1), the phosphorylated polypeptide sequence (LNERAE(pT)PPLPC) and the non-phosphorylated polypeptide sequence (LNERAETPPLPC) to be synthesized were designed.

[0064] (3) Synthesizing corresponding antigenic peptides according to the phosphorylated polypeptide sequences and non-phosphorylated polypeptide sequences in step (2), and coupling the antigenic peptides with carrier proteins to obtain antigenic synthetic peptides for use as antigens.

[0065] Specifically, the corresponding antigenic peptide is synthesized according to the phosphorylated polypeptide sequence and the non-phosphorylated polypeptide sequence in step (2), which specifically includes: synthesizing the corresponding antigenic peptide according to the phosphorylated polypeptide sequence and the non-phosphorylated polypeptide sequence in step (2) by solid phase synthesis, and purifying it by high performance liquid chromatography (HPLC) method until its purity is greater than 95%.

[0066] (4) Immunize animals with the antigen synthetic peptide obtained in step (3) and collect antiserum.

[0067] Specifically, healthy New Zealand white rabbits were selected as host animals, and adaptive breeding was performed for about 2 weeks, and blood was collected before immunization as a negative control. After the antigen synthetic peptide was mixed with the adjuvant, the primary immunization was performed by subcutaneous multi-point injection, with 0.2 ml per point. The secondary immunization was performed on the 14th day after the first immunization, and the third immunization was performed 7 days later. On the 7th day after the third immunization, a small sample of serum was collected from the middle auricular artery for testing. If the test was qualified, an additional immunization was performed 7 days later. After the additional immunization, whole blood could be collected 7 days later to collect antiserum.

[0068] (5) Purifying and identifying the antiserum collected in step (4) to obtain an antibody against the phosphorylated Thr451 site of human HOMEZ protein.

[0069] Specifically, the antiserum collected in step (4) is purified by HPLC; and the purified antiserum is identified by ELISA.

[0070] Furthermore, the purified antiserum was identified by ELISA, and the process specifically includes:

[0071] (5.1) Dilute the antigen obtained in step (3) to 1 μg / ml with carbonate buffer, add 100 μl of the diluted antigen to the ELISA plate, and coat overnight at 4°C. The next day, discard the coating solution and add 150 μl of 1×PBST washing buffer to each well for washing once.

[0072] (5.2) Add 150 μl of 2% BSA to each well for blocking, incubate at 37°C for 1 hour, and discard the blocking solution.

[0073] (5.3) The purified antiserum was serially diluted with 2% BSA (1:250, 1:1000, 1:4000, 1:16000, 1:64000, 1:256000 and 1:1024000), 100 μl was added to the blocked reaction wells, and PBS was added to set up negative control wells. The cells were incubated at 37°C for 1 hour, the reaction solution was discarded, and 150 μl of 1×PBST washing buffer was added to wash three times.

[0074] (5.4) Dilute the horseradish peroxidase-conjugated secondary antibody at a ratio of 1:5000, take 100 μl and add it to the above reaction wells, incubate at 37°C for 1 hour, discard the reaction solution, and add 150 μl of 1× PBST washing buffer to wash three times.

[0075] (5.5) Add 100 μl of 3,3',5,5'-tetramethylbenzidine substrate solution to each well and incubate at 37°C for 5 minutes for color development. Add 100 μl of 1 M sulfuric acid to each well to terminate the reaction.

[0076] (5.6) Place the ELISA plate in a preheated ELISA reader for reading, saving the data and analyzing it. The excitation wavelength of the ELISA reader is 450 nm.

[0077] When analyzing the data, it is necessary to compare the readings of the reaction wells coated with modified polypeptides and non-modified polypeptides. If the readings of the reaction wells coated with modified polypeptides are significantly higher than the readings of the reaction wells coated with non-modified polypeptides, and the readings of the reaction wells coated with non-modified polypeptides are close to 0, the immunization is considered to be successful. At this time, the anti-human HOMEZ protein Thr451 site phosphorylated antibody obtained is the phosphorylated antibody of the present application.

[0078] The results are shown in Table 1. The purified specific antibody against the phosphorylation of Thr451 site of HOMEZ protein can detect phosphorylated peptides, but is negative for non-phosphorylated peptides.

[0079] Table 1 ELISA detection of purified specific antibodies against HOMEZ protein Thr451 phosphorylation

[0080]

[0081] Example 2: Application of specific antibodies against HOMEZ protein Thr451 phosphorylation in cells and tissues Test

[0082] 1. In vitro cell experiments verify the recognition of HOMEZ phosphorylation by specific antibodies against HOMEZ protein Thr451 site phosphorylation:

[0083] The HOMEZ knockout ZR75-1 cell line was constructed using CRISPR / Cas9 technology. Empty vector, HOMEZ wild-type plasmid and HOMEZ-T451A point mutation plasmid were transfected, respectively. The cells were collected 48 hours later, and the expression levels of HOMEZ protein and HOMEZpT451 (phosphorylated Thr451 site of HOMEZ protein) were detected by Western blot technology, with β-actin as the internal reference.

[0084] The results showed that compared with the empty vector transfection control group, the specific antibody against HOMEZ protein phosphorylation at Thr451 site could detect the expression of HOMEZpT451 in ZR75-1 cells transfected with HOMEZ wild-type plasmid, while the expression of HOMEZpT451 could not be detected in ZR75-1 cells transfected with HOMEZ-T451A point mutation plasmid, indicating that the T451 site of HOMEZ was no longer recognized by the antibody after mutation, so as to verify the specificity and effectiveness of the specific antibody against HOMEZ protein phosphorylation at Thr451 site. Figure 4 shown.

[0085] 2. Application of specific antibody against HOMEZ protein phosphorylation at Thr451 site in immunohistochemical staining of breast cancer tissue (65 cases) and normal breast tissue (65 cases).

[0086] Immunohistochemical staining of sections of breast cancer tissue and normal breast tissue with specific antibodies against HOMEZ protein phosphorylation at Thr451 site, such as Figure 5 and Figure 6 shown.

[0087] The results showed that HOMEZpT451 was mainly localized in the cell nucleus, and its expression level in breast cancer tissue was significantly higher than that in normal breast tissue, ***p<0.001.

[0088] The specific antibody against phosphorylation of Thr451 site of HOMEZ protein prepared by the present invention can be used in experiments such as Western blot, immunofluorescence and immunohistochemical staining to detect the expression difference between normal cells and tumor cells, and to detect the phosphorylation level of HOMEZ protein in clinical samples, which is helpful to study the role of phosphorylation modification of HOMEZ protein in the occurrence and development of tumor diseases, and has broad clinical application prospects in disease diagnosis, treatment and prognosis determination.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A HOMEZ protein Thr451 site phosphorylated antigen peptide, characterized in that: The active amino acid sequence of the antigen peptide is LNERAE(pT)PPLPC, as shown in SEQ ID NO: 1, wherein T is phosphorylated.

2. A specific antibody against phosphorylation of Thr451 site of HOMEZ protein, characterized in that: The specific antibody is prepared by immunizing an animal with the phosphorylated antigen peptide as claimed in claim 1.

3. A method for preparing a specific antibody against phosphorylation of Thr451 site of HOMEZ protein as claimed in claim 2, characterized in that: The following steps are involved: S1. Determine the phosphorylation site of Thr451 of the anti-HOMEZ protein; S2, designing the phosphorylated antigenic peptide and non-phosphorylated antigenic peptide at Thr451 site of HOMEZ protein as claimed in claim 1 according to the HOMEZ target sequence and the anti-HOMEZ protein Thr451 phosphorylation site determined in step S1; S3, coupling the phosphorylated antigen peptide at Thr451 site of HOMEZ protein obtained in step S2 and the non-phosphorylated antigen peptide with a carrier protein, respectively, to obtain an antigen; S4, immunizing the animal with the antigen obtained in step S3, and collecting antiserum; The antiserum was purified and identified to obtain a specific antibody against the phosphorylation site Thr451 of HOMEZ protein.

4. The preparation method according to claim 3, characterized in that: In step S1, determining the phosphorylation site of Thr451 of the anti-HOMEZ protein comprises the following steps: 1) Screening out potential phosphorylation sites of HOMEZ protein by phosphorylation modification mass spectrometry analysis; wherein the potential phosphorylation sites include Thr437 and Thr451 sites; 2) Constructing a HOMEZ point mutation plasmid, replacing the potential phosphorylation site with alanine that cannot be phosphorylated, and performing next-generation sequencing to verify that the amino acid sequence of the potential phosphorylation site is replaced with alanine, which is considered to be the successful construction of the HOMEZ point mutation plasmid; 3) HOMEZ wild-type plasmid and HOMEZ point mutation plasmid were transfected into 293T cells, and then the cell pellets were collected and the phosphorylation modification level of HOMEZ protein was detected by co-immunoprecipitation experiment to determine the Thr451 phosphorylation site of anti-human HOMEZ protein.

5. The preparation method according to claim 3, characterized in that: In the step S2, the sequence of the non-phosphorylated antigen peptide is LNERAETPPLPC, and the non-phosphorylated antigen peptide does not have a phosphorylation group added at the 451st threonine site.

6. The preparation method according to claim 3, characterized in that: The immunization method in step S4 specifically includes: After the initial immunization of the animal, 2 to 3 booster immunizations were performed respectively. Each immunization used the phosphorylated antigen peptide or non-phosphorylated antigen peptide at Thr451 site of the HOMEZ protein, mixed with an adjuvant, and injected subcutaneously on the back to collect antiserum.

7. The preparation method according to claim 3, characterized in that: Purifying and identifying the antiserum in step S4 specifically includes: Purifying the collected antiserum by high performance liquid chromatography; The purified antiserum was identified by enzyme-linked immunosorbent assay.

8. Use of the HOMEZ protein Thr451 phosphorylated antigenic peptide according to claim 1 or the anti-HOMEZ protein Thr451 phosphorylated specific antibody according to claim 2 in the preparation of a preparation for detecting HOMEZ protein Thr451 phosphorylated.

9. A pharmaceutical preparation for detecting HOMEZ protein phosphorylated at Thr451, characterized in that: It comprises the specific antibody against phosphorylation of Thr451 site of HOMEZ protein as claimed in claim 2.

10. A diagnostic kit for tumor prognosis, characterized in that: The diagnostic kit comprises the specific antibody against phosphorylation of Thr451 site of HOMEZ protein as claimed in claim 2.

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