A phosphorylation site-specific antibody for transcription factor HOMEZ, its preparation method and applications

Abstract

This application relates to the field of biomedical technologies, and specifically discloses a transcription factor HOMEZ phosphorylation site-specific antibody, its preparation method and applications. The specific antibody provided in this application can specifically recognize the phosphorylation of threonine at position 451 of HOMEZ in tumor specimens, thereby judging the prognosis of tumors and providing potential therapeutic targets for the diagnosis and treatment of clinical tumors. Moreover, the specific antibody of this application can be used in Western blot experiments to detect the expression differences between normal cells and tumor cells, which helps to study the role of phosphorylation modification of HOMEZ protein in the occurrence and development of tumor diseases. It can also be used in experiments such as immunofluorescence and immunohistochemical staining to detect the phosphorylation level of HOMEZ protein in clinical specimens, and has broad clinical application prospects in aspects such as disease diagnosis, treatment and prognosis determination.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to a transcription factor HOMEZ phosphorylation site-specific antibody, its preparation method and applications. Background Art

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

[0003] Currently, there are very few reports on the HOMEZ gene. Some studies have shown that the Homez gene plays a role in the neuronal development downstream of the Xenopus anterior neural / neural progenitor genes; there are two missense mutations in the HOMEZ gene in Chinese patients with isolated ventricular septal defect, which are directly related to the etiology; the Homez gene is highly expressed in the testes of mice, but the Homez gene knockout mice are fertile and it is not important for the development of germ cells. However, there is no report on the phosphorylation modification of HOMEZ and its function in tumors. Summary of the Invention

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

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

[0006] This application provides a HOMEZ protein Thr451 site phosphorylation antigen peptide, and the active amino acid sequence of the antigen peptide is LNERAE(pT)PPLPC, as shown in SEQ ID NO:1, where T is phosphorylated.

[0007] The inventors of this application have first discovered that there is phosphorylation modification of threonine at the 451st site of the HOMEZ protein, and it is closely related to the metastasis and prognosis of tumors. 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 (sequence: LNERAE(pT)PPLPC) near the Thr451 site of the HOMEZ protein as an immunogen to immunize animals, and the specific antibody obtained after purification of the animal serum can specifically recognize the Thr451 phosphorylated HOMEZ protein, and this specific antibody can be applied to the detection of Thr451 phosphorylation of the HOMEZ protein and functional research.

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

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

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

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

[0013] S2. Design the phosphorylated antigen peptide and non-phosphorylated antigen peptide of the Thr451 site of the HOMEZ protein according to the HOMEZ target sequence and the phosphorylation site of the Thr451 of the HOMEZ protein determined in step S1;

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

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

[0016] As a preferred embodiment of the preparation method of the specific antibody against phosphorylation at the Thr451 site of the HOMEZ protein described in the present application, in step S1, determining the phosphorylation site of the Thr451 of the HOMEZ protein includes the following steps:

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

[0018] 2) Construct a HOMEZ point mutation plasmid, replace the potential phosphorylation site with alanine that cannot be phosphorylated, and perform second-generation sequencing to verify that the amino acid sequence of the potential phosphorylation site is replaced with alanine, which is regarded as successful construction of the HOMEZ point mutation plasmid;

[0019] 3) Transfect the wild-type plasmid of HOMEZ and the point mutant plasmid of HOMEZ into 293T cells, then collect the cell precipitate and detect the phosphorylation modification level of the HOMEZ protein through co-immunoprecipitation (co-IP) experiment to determine the phosphorylation site of Thr451 of the anti-human HOMEZ protein.

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

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

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

[0023] Among them, when replacing the potential phosphorylation site with alanine that cannot be phosphorylated, there may be a situation of failed replacement. Only when the amino acid sequence of the T site is verified to be replaced by A through next-generation sequencing is the construction successful. For example, T451A indicates that the construction of the HOMEZ point mutant plasmid corresponding to the T451 site is successful.

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

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

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

[0027] After the primary immunization of the animal, 2 to 3 booster immunizations are carried out respectively. Each immunization uses the phosphorylation antigen peptide or non-phosphorylated antigen peptide of the Thr451 site of the HOMEZ protein, mixes it with adjuvant, injects it subcutaneously in the back, and collects the antiserum.

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

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

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

[0031] Preferably, the purified antiserum was identified by enzyme-linked immunosorbent assay (ELISA), and the specific process 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 enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4°C. The next day, the coating solution was discarded, and each well was washed once with 150 μl of 1×PBST washing buffer. 150 μl of 2% BSA was added to each well for blocking and incubated at 37°C for 1 hour, and then the blocking solution was discarded. The purified antiserum was serially diluted with 2% BSA, 100 μl of which was added to the above-mentioned blocked reaction wells and incubated at 37°C for 1 hour. The reaction solution was discarded, and each well was washed 3 times with 150 μl of 1×PBST washing buffer. The horseradish peroxidase-conjugated secondary antibody was diluted at a ratio of 1:5000, 100 μl of which was added to the above-mentioned reaction wells and incubated at 37°C for 1 hour. The reaction solution was discarded, and each well was washed 3 times with 150 μl of 1×PBST washing buffer. 100 μl of 3,3’,5,5’-tetramethylbenzidine substrate solution was added to each well and incubated at 37°C for 5 minutes for color development. Then 100 μl of 1 M sulfuric acid was added to each well to terminate the reaction. The ELISA plate was placed in a preheated microplate reader for reading, and the data was saved and analyzed. The excitation wavelength of the microplate reader was 450 nm.

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

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

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

[0036] Preferably, the tumor includes breast cancer.

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

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

[0039] The present application provides a phosphorylation site-specific antibody for transcription factor HOMEZ, its preparation method and application. The specific antibody provided by the present application can specifically recognize the phosphorylation of threonine at position 451 of HOMEZ in tumor specimens, thereby judging the prognosis of tumors and providing potential action targets for the diagnosis and treatment of clinical tumors. Moreover, the specific antibody of the present application can be used in Western blot experiments to detect the expression differences between normal cells and tumor cells, which helps to study the role of phosphorylation modification of HOMEZ protein in the occurrence and development of tumor diseases. It can also be used in experiments such as immunofluorescence and immunohistochemical staining to detect the phosphorylation level of HOMEZ protein in clinical specimens, and has broad clinical application prospects in aspects such as disease diagnosis, treatment and prognosis determination. Brief Description of the Drawings

[0040] Figure 1 It is a result diagram for detecting the phosphorylation modification of HOMEZ after transfection with the wild-type plasmid of HOMEZ using a pan-phosphorylation antibody (p-Ser and p-Thr);

[0041] Figure 2 It is the potential phosphorylation sites of human HOMEZ protein shown by phosphorylation mass spectrometry data;

[0042] Figure 3 It is the phosphorylation level of HOMEZ after transfection with the wild-type or mutant plasmid of HOMEZ using a pan-phosphorylation antibody (p-Thr);

[0043] Figure 4 It is the expression level of HOMEZ pT451 after transfection with the wild-type or T451-site mutant plasmid of HOMEZ in HOMEZ stably knocked-out cells using a specific antibody against the phosphorylation of Thr451 site of HOMEZ protein;

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

[0045] Figure 6 It is a statistical chart of the expression levels of HOMEZ pT451 in 65 pairs of breast cancer tissues and adjacent normal tissues. Detailed Embodiments

[0046] To better illustrate the purpose, technical solution and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific embodiments.

[0047] In the following examples, unless otherwise specified, the experimental methods used are conventional methods. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified, and the component raw materials used in each parallel experiment are of the same kind.

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

[0049] The present application first provides an antigenic peptide for the phosphorylation of the Thr451 site of the HOMEZ protein. The active amino acid sequence of the antigenic peptide is LNERAE(pT)PPLPC, as shown in SEQ ID NO:1, where T is phosphorylated. Without phosphorylation modification, it is a non-phosphorylated antigenic peptide (sequence: LNERAETPPLPC).

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

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

[0052] This example provides a preparation method for a specific antibody against the phosphorylation of the Thr451 site of the HOMEZ protein, including the following steps:

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

[0054] (1.1) Transfect 293T cells with the HOMEZ wild-type plasmid and the corresponding empty vector plasmid. After 48 hours, collect the cell pellet and detect the phosphorylation modification level on the HOMEZ protein through an immunoprecipitation (co-IP) experiment to determine that the HOMEZ protein indeed has phosphorylation modification.

[0055] Specifically, transfect 293T cells with the HOMEZ wild-type plasmid and the corresponding empty vector plasmid. After 48 hours, collect the cell pellet. Through the co-IP experiment, use the pan-threonine phosphorylation antibody and the pan-serine phosphorylation antibody to detect the phosphorylation modification level on the HOMEZ protein respectively. The results are as Figure 1 shown.

[0056] The results show that compared with the control group, phosphorylation modification of threonine was detected on the HOMEZ protein, while phosphorylation modification of serine was not detected, indicating that the HOMEZ protein has threonine phosphorylation modification.

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

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

[0059] (1.3) The HOMEZ point mutation plasmid was constructed. Specifically, the potential phosphorylation sites were replaced with alanine (A) that could not be phosphorylated, and second-generation sequencing was performed to verify that the amino acid sequence of the potential phosphorylation site was successfully replaced with alanine.

[0060] (1.4) The 293T cells were transfected with the wild-type HOMEZ plasmid or the successfully constructed HOMEZ point mutation plasmid above. After 48 h, the cell pellet was collected and the phosphorylation modification level on the HOMEZ protein was detected by co-IP experiment to determine the Thr451 phosphorylation site of the HOMEZ protein.

[0061] Specifically, the 293T cells were transfected with the wild-type HOMEZ plasmid or the successfully constructed HOMEZ point mutation plasmid above. After 48 h, the cell pellet was collected. Through co-IP experiment, the phosphorylation modification level on the HOMEZ protein was detected using a pan-threonine phosphorylation antibody. The results were as 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 there was no significant difference in T437A. According to this result, it could be determined that the site of threonine phosphorylation modification of the HOMEZ protein was T451.

[0063] (2) According to 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) The corresponding antigen peptides were synthesized according to the phosphorylated polypeptide sequence and the non-phosphorylated polypeptide sequence in step (2), and the antigen peptide was conjugated with a carrier protein to obtain an antigen synthesis peptide for use as an antigen.

[0065] Specifically, the corresponding antigen peptides were synthesized according to the phosphorylated polypeptide sequence and the non-phosphorylated polypeptide sequence in step (2), which specifically included: using solid-phase synthesis method to synthesize the corresponding antigen peptides according to the phosphorylated polypeptide sequence and the non-phosphorylated polypeptide sequence in step (2), and purifying them by high performance liquid chromatography (HPLC) method until their purity was greater than 95%.

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

[0067] Specifically, select healthy New Zealand white rabbits as host animals, conduct adaptive feeding for about 2 weeks, and collect blood before immunization as a negative control. After mixing the antigen synthetic peptide with an adjuvant, conduct primary immunization by subcutaneous multi-point injection, with 0.2 ml at each point. Conduct a second immunization 14 days after the first immunization, and a third immunization 7 days later. On the 7th day after the three immunizations, collect a small sample of serum from the auricular artery for testing. If the test is qualified, boost immunize 7 days later. Whole blood can be collected 7 days after the boost immunization, and the antiserum is collected.

[0068] (5) Purify and identify the antiserum collected in step (4) to obtain an anti-human HOMEZ protein Thr451 site phosphorylation antibody.

[0069] Specifically, use the HPLC method to purify the antiserum collected in step (4); use ELISA to identify the purified antiserum.

[0070] Furthermore, use ELISA to identify the purified antiserum, and its process specifically includes:

[0071] (5.1) Dilute the antigen obtained in step (3) to 1 μg / ml with carbonate buffer, take 100 μl of the diluted antigen and add it to an enzyme-linked immunosorbent assay (ELISA) plate, and coat it overnight at 4 °C; the next day, discard the coating solution, and add 150 μl of 1×PBST washing buffer to each well to wash 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) Gradiently dilute the purified antiserum with 2% BSA (1:250, 1:1000, 1:4000, 1:16000, 1:64000, 1:256000, and 1:1024000), take 100 μl and add it to the above-mentioned blocked reaction wells. At the same time, add PBS to set up a negative control well, incubate at 37 °C for 1 hour, discard the reaction solution, and add 150 μl of 1×PBST washing buffer to wash 3 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-mentioned reaction wells, incubate at 37 °C for 1 hour, discard the reaction solution, and add 150 μl of 1×PBST washing buffer to wash 3 times.

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

[0076] (5.6) Place the microplate in a preheated microplate reader for reading, save the data and analyze it. The excitation wavelength of the microplate reader is 450 nm.

[0077] When analyzing the data, it is necessary to compare the readings of the wells coated with modified polypeptide and unmodified polypeptide. If the reading of the well coated with modified polypeptide is significantly higher than that of the well coated with unmodified polypeptide, and the reading of the well coated with unmodified polypeptide is close to 0, it is regarded as successful immunization. At this time, the anti-human HOMEZ protein Thr451 site phosphorylated antibody obtained is the phosphorylated antibody of this application.

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

[0079] Table 1 ELISA detection of the purified specific antibody against phosphorylation of HOMEZ protein at Thr451 site

[0080]

[0081] Example 2: Application detection of a specific antibody against phosphorylation of Thr451 site of HOMEZ protein in cells and tissues Detection

[0082] 1. Verification of the recognition of HOMEZ phosphorylation by the specific antibody against phosphorylation of HOMEZ protein at Thr451 site in in vitro cell experiments:

[0083] Use the CRISPR / Cas9 technology to construct a ZR75-1 cell line with HOMEZ knockout, and transfect the empty vector, HOMEZ wild-type plasmid and HOMEZ-T451A point mutant plasmid respectively. After 48 h, collect the cells and use Western blot technology to detect the expression levels of HOMEZ protein and HOMEZpT451 (phosphorylation of HOMEZ protein at Thr451 site), with β-actin as the internal reference.

[0084] The results showed that, compared with the control group transfected with empty vector, the specific antibody against phosphorylation of HOMEZ protein 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 antibody could no longer recognize HOMEZ after the mutation at T451 site, so as to verify the specificity and effectiveness of the specific antibody against phosphorylation of HOMEZ protein at Thr451 site, as Figure 4 shown.

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

[0086] Sections of breast cancer tissues and normal breast tissues were subjected to immunohistochemical staining with the specific antibody against phosphorylation of HOMEZ protein at Thr451 site, as Figure 5 and Figure 6 shown.

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

[0088] The specific antibody against phosphorylation of HOMEZ protein at Thr451 site prepared in the present invention can be used in experiments such as Western blot, immunofluorescence and immunohistochemical staining to detect the expression differences between normal cells and tumor cells, detect the phosphorylation level of HOMEZ protein in clinical samples, which helps 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 solutions of the present application rather than to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A HOMEZ protein Thr451 site phosphorylated antigen peptide, characterized in that, The active amino acid sequence of the antigenic peptide is LNERAE(pT)PPLPC, as shown in SEQ ID NO:1, where 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 antigenic peptide as described in claim 1, and the specific antibody is a polyclonal antibody.

3. A method for preparing a specific antibody against phosphorylation of the Thr451 site of the HOMEZ protein as described in claim 2, characterized in that, It includes the following steps: S1. Determine the phosphorylation site of Thr451 of the HOMEZ protein; S2. Design the phosphorylated antigenic peptide at the Thr451 site of the HOMEZ protein as described in claim 1 according to the HOMEZ target sequence and the phosphorylation site of Thr451 of the HOMEZ protein determined in step S1; S3. Couple the phosphorylated antigenic peptide at the Thr451 site of the HOMEZ protein obtained in step S2 with a carrier protein to obtain an antigen; S4. Immunize an animal with the antigen obtained in step S3 and collect the antiserum; Purify and identify the antiserum to obtain a specific antibody against phosphorylation at the Thr451 site of the HOMEZ protein.

4. The preparation method according to claim 3, characterized in that, In step S1, determining the phosphorylation site of Thr451 of the HOMEZ protein includes the following steps: 1) Screen out potential phosphorylation sites of the HOMEZ protein by phosphorylated modification mass spectrometry analysis; among them, the potential phosphorylation sites include Thr437 and Thr451 sites; 2) Construct a HOMEZ point mutation plasmid, replace the potential phosphorylation site with alanine that cannot be phosphorylated, and perform second-generation sequencing to verify that the amino acid sequence of the potential phosphorylation site is replaced with alanine, which is regarded as successful construction of the HOMEZ point mutation plasmid; 3) Transfect the HOMEZ wild-type plasmid and the HOMEZ point mutation plasmid into 293T cells, then collect the cell precipitate and detect the phosphorylation modification level of the HOMEZ protein by immunoprecipitation experiment to determine the phosphorylation site of Thr451 of the human HOMEZ protein.

5. The preparation method according to claim 3, characterized in that, In step S4, the specific immunization method includes: After the initial immunization of the animal, perform 2 to 3 booster immunizations. Each immunization uses the phosphorylated antigenic peptide at the Thr451 site of the HOMEZ protein, mix it with an adjuvant, inject it subcutaneously in the back, and collect the antiserum.

6. The preparation method according to claim 3, characterized in that, In step S4, purifying and identifying the antiserum specifically includes: Purify the collected antiserum by high performance liquid chromatography method; Identify the purified antiserum by enzyme-linked immunosorbent assay.

7. Use of the phosphorylated antigenic peptide at the Thr451 site of the HOMEZ protein as described in claim 1 or the specific antibody against phosphorylation at the Thr451 site of the HOMEZ protein as described in claim 2 in the preparation of a preparation for detecting phosphorylation at the Thr451 site of the HOMEZ protein.

8. A pharmaceutical preparation for detecting HOMEZ protein phosphorylated at Thr451 site, characterized in that, It includes the specific antibody against phosphorylation at the Thr451 site of the HOMEZ protein as described in claim 2.

Images