Phosphorylation-modified cuedc1 polypeptides, antigens, vaccines, antibodies, and methods of making and uses thereof

Abstract

The present application belongs to the technical field of protein engineering, and particularly relates to a phosphorylation-modified CUEDC1 polypeptide, antigen, vaccine, antibody and a preparation method and use thereof. The present application provides a CUEDC1 protein after phosphorylation modification and a kit prepared by the same. The present application provides an antigen of human CUEDC1 protein S123 site phosphorylation. The antigen is used for immunizing animals, and high-sensitivity and high-specificity antiserum and purified antibodies are obtained, which can distinguish the phosphorylation state of CUEDC1, and are expected to be used for detecting the phosphorylation level of CUEDC1 protein, and have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology, specifically relating to phosphorylated CUEDC1 peptides, antigens, vaccines, antibodies, their preparation methods and uses. Background Technology

[0002] Ubiquitination is a crucial mode of post-translational modification (PTM). Mechanistically, ubiquitin binds sequentially to lysine residues of target proteins through a cascade of enzyme reactions induced by the ubiquitination proteasome system (UPS). Different ubiquitin chains formed on the target protein lead to varying biological effects of the substrate protein. For example, K48-linked ubiquitinated proteins are ultimately degraded by the 26S proteasome; while proteins modified with K63-linked ubiquitin chains participate in DNA repair and activation of nuclear factor κβ (NFκB). The UPS is a complex and tightly controlled system responsible for 80-90% of protein degradation, enabling proteins to perform their normal biological functions and maintain their expression stability within cells. It regulates the turnover of various key proteins involved in cell cycle progression and tumorigenesis. Therefore, ubiquitination not only regulates the levels of specific proteins but also participates in signal transduction, protein complex assembly, and enzyme activity; disruption of these functions can lead to disease. It is worth noting that increasing evidence suggests that the progression of liver cancer cells is often accompanied by disruption of protein ubiquitination and deubiquitination processes.

[0003] CUE domain-containing protein 1 (CUEDC1) is a member of the ubiquitin-coupled estrogen receptor degradation (CUE) domain-containing protein family, consisting of 386 amino acids with a theoretical molecular weight of approximately 42.3 kDa. It possesses a moderately conserved CUE structure, with the CUE domain being approximately 40 amino acids long and found in proteins with various functions, including the degradation of misfolded proteins in the endoplasmic reticulum and protein sorting. Studies have confirmed that the CUE domain can bind both monoubiquitin and polyubiquitin. Some proteins containing the CUE domain undergo ubiquitination in a CUE domain-dependent manner, but their affinities for ubiquitin vary. Furthermore, some CUE domain-containing proteins from higher eukaryotes cannot effectively bind monoubiquitin. Therefore, whether the CUE domain in CUEDC1 can bind ubiquitin and participate in the ubiquitination process requires further investigation, which poses challenges to the diagnosis and treatment of diseases related to CUEDC1.

[0004] After a protein is phosphorylated, its phosphorylation site and surrounding sequence may form a degradation signal (degron), which is recognized by ubiquitin ligases, thereby promoting ubiquitination modification of the protein. Moreover, the detection of phosphorylated CUEDC1 has significant value in both basic research and clinical applications. First, the phosphorylation state of CUEDC1 is closely related to the regulatory mechanisms of tumorigenesis and development, and is involved in regulating tumor proliferation, migration, and invasion, as well as inflammatory signaling pathways. Second, CUEDC1 at specific phosphorylation sites can serve as potential biomarkers to reflect the active state, malignant state, or specific subtype of tumors, aiding in early tumor screening and prognostic assessment. Phosphorylated CUEDC1 can also serve as a therapeutic target, potentially providing new strategies for tumor treatment. However, the mechanism between the phosphorylation state of CUEDC1 and its ubiquitination remains unclear, and existing detection methods struggle to distinguish between different phosphorylation modification states of CUEDC1, which greatly limits its application value in disease diagnosis, mechanism research, and drug efficacy evaluation.

[0005] Therefore, developing detection reagents that can distinguish the phosphorylation modification state of CUEDC1 is an urgent problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides phosphorylated CUEDC1 peptides, antigens, vaccines, antibodies, their preparation methods, and uses.

[0007] The present invention provides a phosphorylated CUEDC1 polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1, wherein the phosphorylation site is located at the 11th amino acid of the sequence shown in SEQ ID NO. 1.

[0008] The present invention provides an antigen, which is a conjugate of phosphorylated CUEDC1 polypeptide and carrier protein as described above.

[0009] Furthermore, the carrier protein is keyhole hemocyanin.

[0010] Further, the conjugate is prepared by the following steps: phosphorylated CUEDC1 polypeptide and carrier protein are linked by a Schiff base reaction with glutaraldehyde to obtain the conjugate; the weight ratio of phosphorylated CUEDC1 polypeptide to carrier protein is 0.05-0.1:0.5-1.

[0011] Furthermore, the weight ratio of phosphorylated CUEDC1 peptide to carrier protein was 0.05:1.

[0012] The present invention provides the use of the phosphorylated CUEDC1 peptide as described above, or the antigen as described in any of the preceding claims, in the preparation of vaccines and / or antibodies.

[0013] The present invention provides a vaccine comprising the antigen as described in any of the preceding claims.

[0014] Furthermore, the vaccine also includes an adjuvant.

[0015] Furthermore, the adjuvant is selected from at least one of Freund's complete adjuvant and Freund's incomplete adjuvant.

[0016] The present invention provides an antibody against phosphorylated CUEDC1, characterized in that it is prepared by immunizing animals with an antigen as described in any of the preceding claims or a vaccine as described in any of the preceding claims.

[0017] Furthermore, it is prepared according to the following steps: The antigen is mixed with an adjuvant to form a water-in-oil emulsion. After emulsification, it is injected subcutaneously into the neck and back of the animal. After 3-6 immunizations, venous blood is collected.

[0018] Furthermore, in the 3-6 immunizations, the adjuvant used for the first immunization is Freund's complete adjuvant, while the adjuvant used for the other immunizations is Freund's incomplete adjuvant. And / or, the volume ratio of antigen to adjuvant is 1:1; And / or, each immunization takes place every 10-14 days; And / or, the animal is a rabbit; And / or, the antibody is a polyclonal antibody.

[0019] The present invention provides a method for preparing an antibody against phosphorylated CUEDC1 as described in any of the preceding claims, characterized in that it comprises: preparing the antibody after immunizing an animal with the antigen or the vaccine.

[0020] The present invention provides the use of the phosphorylated CUEDC1 peptide as described above, or the antiphosphorylated CUEDC1 antibody as described in any of the preceding claims, in the preparation of a kit for detecting the phosphorylation level of CUEDC1 protein.

[0021] Furthermore, the detection of CUEDC1 protein phosphorylation level refers to the detection of serine phosphorylation level at position 123 in the CUEDC1 protein sequence.

[0022] This invention provides a phosphorylated CUEDC1 protein and a kit for its preparation. This invention also provides an antigen phosphorylated at site S123 of the human CUEDC1 protein. Using this antigen to immunize animals yields highly sensitive and specific antiserum and purified antibodies, which can distinguish the phosphorylation state of CUEDC1 and show promise for detecting the phosphorylation level of CUEDC1 protein.

[0023] This invention provides phosphorylated CUEDC1 protein and a kit for its preparation, which has the following advantages compared to existing technologies targeting unmodified / mixed modified CUEDC1 protein: (1) The target status is clear and the batch-to-batch stability is good. By preparing a phosphorylation modification antibody specific to CUEDC1, the phosphorylation state and modification site of CUEDC1 protein can be detected, thus solving the problem of fluctuation and insufficient comparability of results caused by CUEDC1 being in a mixed modification state in the existing technology.

[0024] (2) It is beneficial to study the function and mechanism of CUEDC1 under specific modification states. Using phosphorylated CUEDC1 protein as a research subject can significantly improve the interpretability of studies on downstream interactions and ubiquitination, and reduce the risk of false negatives / false positives due to inconsistent CUEDC1 modification status.

[0025] (3) Improved antibody detection specificity, enabling differentiation of modified states. By using phosphorylated CUEDC1 antibodies, the "phosphorylation status of CUEDC1" can be identified, solving the problem that existing detection systems have difficulty distinguishing whether CUEDC1 is phosphorylated, thereby improving detection accuracy.

[0026] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0027] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0028] Figure 1 The image shows the results of a dot blot hybridization experiment to determine antibody sensitivity. Lane 1 is CUEDC1S123ph peptide A, lane 2 is CUEDC1S123ph peptide B, lane 3 is CUEDC1S122ph control peptide C, and lane 4 is CUEDC1 unmodified peptide. The primary antibody was diluted 1:2000 and incubated for 1 h, while the secondary antibody was diluted 1:10000 and incubated for 45 min with an exposure time of 30 s. Ab1, Ab3, and Ab4 represent antibodies purified from the serum of rabbits R1, R3, and R4, respectively.

[0029] Figure 2The image shows the results of Western Blot (WB) assays to determine the binding of antibodies to target proteins in lysates of four human cell lines. Lane 1 is for MCF-7 cell lysates, lane 2 is for HeLa cell lysates, lane 3 is for HepG2 cell lysates, and lane 4 is for A549 cell lysates. The primary antibody was diluted 1:500 and incubated for 2 hours, while the secondary antibody was diluted 1:10000 and incubated for 45 minutes. The exposure times for the three antibody binding results to the target proteins were 60 s+, 60 s, and 60 s, respectively. Detailed Implementation

[0030] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0031] Example 1: Preparation of phosphorylated CUEDC1 protein antibody 1. Synthesis of immune antigens (1) This embodiment provides a phosphorylated CUEDC1 protein with the amino acid sequence CLERTLEPDS-(phospho)S-DEE (SEQ ID NO. 1), wherein the phosphorylation site is located at the 11th amino acid of the sequence shown in SEQ ID NO. 1. The antigenic short peptide CUEDC1 S123ph polypeptide A is synthesized according to the protein sequence shown in SEQ ID NO. 1.

[0032] (2) Linkage between antigenic short peptides and carrier proteins 10 mg of keyhole hemocyanin (KLH) was dissolved in 1 mL of PBS buffer (0.01 mol / L, pH 7.4) to prepare a mixture. 0.5 mg of the aforementioned antigenic peptide (CUEDC1 S123ph polypeptide A) was dissolved in this mixture, and 1 mL of 0.25% glutaraldehyde solution was added. The mixture was stirred at room temperature for 4 h. The reaction was terminated by adding 0.5 mL of 1 mol / L glycine. At 4 °C, the supernatant was transferred to a PBS buffer (0.01 mol / L, pH 7.4) dialysis bag and dialyzed at 4 °C for 72 h, changing the dialysate every 3–4 h. After 72 h, the solution was aliquoted to prepare CUEDC1 S123ph-KLH. Half of the dialysate was stored at 4 °C for detection, and the other half was stored at -20 °C for immunization.

[0033] 2. Animal immunization Four New Zealand White rabbits were immunized with the prepared CUEDC1 S123ph-KLH, for a total of three immunizations. Immunization was performed via subcutaneous injection at multiple sites on the nape of the neck. For the first immunization, CUEDC1 S123ph-KLH (1 mg / mL) was mixed with Freund's complete adjuvant at a 1:1 volume ratio, and the mixture was repeatedly aspirated using a connector to fully emulsify it into a water-in-oil emulsion. One mL of the emulsion was then injected into each rabbit. Two weeks later, a second immunization was performed. CUEDC1 S123ph-KLH (1 mg / mL) was emulsified with Freund's incomplete adjuvant at a 1:1 volume ratio, and one mL of the emulsion was injected into each rabbit. Two weeks later, a third immunization was performed using the same method (same as the second immunization). Ten days later, blood was collected from the ear vein to determine the antiserum titer.

[0034] The specific ELISA detection process for antiserum titer is as follows: (1) Antigen coating Dilute the CUEDC1 S123ph polypeptide A antigen with coating buffer, add 100 μL / well to each well of the polystyrene ELISA plate, coat at 37°C for 2 h, wash twice for 5 min each time, and pat dry.

[0035] (2) Closed Add 200 μL of blocking solution per well, block at 37°C for 3 h, wash 3 times, and pat dry.

[0036] (3) Add the antiserum to be tested 100 μL of antiserum per well, with normal rabbit negative serum as a control, incubated at 37°C for 30 min, washed 3 times, and patted dry.

[0037] (4) Add secondary antibody Dilute the enzyme-labeled goat anti-rabbit enzyme with blocking buffer at 100 μL / well, incubate at 37°C for 30 min, wash 3 times, and pat dry.

[0038] (5) Color development and detection Add 80 μL each of chromogenic solution A and B to each well, and incubate at 37℃ for 15 min. Add 80 μL of stop solution to each well. Measure the OD value of each well at a single wavelength of 450 nm. The ratio of the OD value of the well to the negative control well (P / N) greater than 2.1 is used as the cutoff point for judging potency.

[0039] The serum was diluted 1:64,000. The immunization effect was continuously monitored, and the antibody titer in the serum was reflected by ELISA results. Western blotting was also performed on the serum. When the serum was diluted 1:10,000 and the OD450 was >1.0 (after the 3rd or 4th immunization), it was considered to have met the standard and could proceed with subsequent purification.

[0040] 3. Purification and preservation of antiserum (1) Magnetic beads capture short antigen peptides Take 400 μL of streptavidin magnetic beads, wash three times with PBS buffer, and redisperse in 5 mL of PBS buffer. Accurately transfer 1 mL of biotin-labeled CUEDC1 S123ph peptide to the washed Dynabeads™ magnetic beads and incubate in PBS suspension for 30 minutes. The biotin-labeled CUEDC1 S123ph peptide specifically binds to streptavidin, thereby being captured by the magnetic beads.

[0041] (2) Antibody purification Take 1 mL of serum obtained from CUEDC1 S123ph-KLH immunization and bind it to 2.5 mL of magnetic beads (2 mg, 10 mg / mL) containing CUEDC1 S123ph peptide. Incubate at 4°C with gentle shaking for 2 h. Separate the antibody-coated beads with a magnet for 2–3 minutes. Bind the supernatant to magnetic beads (2 mg, 10 mg / mL) containing CUEDC1 S123ph peptide again and incubate with gentle shaking for 2 h. Wash the coated beads 4–5 times in PBS elution buffer containing 0.1% BSA and collect the eluent to obtain CUEDC1 phosphorylated antibody, which is a polyclonal antibody.

[0042] The amino acid sequence of the CUEDC1 protein is as follows: MTSLFRRSSSGSGGGGTAGARGGGGGTAAPQELNNSRPARQVRRLEFNQAMDDFKTMFPNMDYDIIECVLRANSGAVDATIDQLLQMNLEGGGSSSGGVYEDSSDSEDSIPPEILERTLEPDSSDEEPPPVYSPPAYHMHVFDRPYPLAPPTPPPRIDALGSGAPTSQRRYRNWNPPLLGNLPDDFLRILPQQLDS IQGNAGGPKPGSGEGCPPAMAGPGPGDQESRWKQYLEDERIALFLQNEEFMKELQRNRDFLLALERDRLKYESQKSKSSSVAVGNDFGFSSPVPGTGDANPAVSEDALFRDKLKHMGKSTRRKLFELARAFSEKTKMRKSKRKHLLKHQSLGAAASTANLLDDVEGHACDEDFRGRRQEAPKVEEGLREGQ (SEQ ID NO. 2).

[0043] Example 2: CUEDC1 phosphorylation antibody used to detect CUEDC1 phosphorylation status This embodiment provides a kit for detecting the phosphorylation status of CUEDC1 based on the CUEDC1 phosphorylation antibody prepared in Example 1, specifically detecting the phosphorylation status of serine at position 123 of the CUEDC1 protein. This kit can detect phosphorylation using a double-antibody sandwich ELISA method or a Western blot method. This embodiment uses a kit for the ELISA method as an example.

[0044] I. Reagent Kit The kit consists of: 1. Pre-coated ELISA plate: 96-well microplate, pre-coated with capture antibody against CUEDC1 protein (non-phosphorylation dependent) and blocked with blocking buffer; several sealing films are also provided.

[0045] 2. Standard products Phosphorylated CUEDC1 protein standards (lyophilized powder or stock solution with calibrated concentrations), containing known concentrations of phosphorylated CUEDC1 recombinant protein, are used for gradient dilution to plot standard curves.

[0046] 3. Antibody detection The CUEDC1 phosphorylated antibody was prepared according to the method in Example 1. It was used after dilution to a working concentration, typically prepared with antibody dilution buffer to a concentration of 1–5 µg / mL.

[0047] 4. Enzyme-labeled secondary antibody Horseradish peroxidase (HRP)-labeled anti-rabbit IgG.

[0048] 5. Total protein detection antibody (optional, for dual-channel detection) Biotin- or directly HRP-labeled anti-CUEDC1 total protein antibodies (recognizing non-phosphorylation-dependent epitopes) are used to simultaneously determine the total CUEDC1 content.

[0049] 6. Sample diluent Phosphate buffer (PBS, pH 7.4) containing 1% bovine serum albumin (BSA) or casein is used to dilute standards and test samples.

[0050] 7. Washing solution (10× or 20× concentrate) PBS containing 0.05% Tween-20 should be diluted to 1× with deionized water before use.

[0051] 8. Antibody diluent 0.1% BSA in PBS is used to dilute the detection antibody and enzyme-labeled secondary antibody buffer.

[0052] 9. Color developer TMB (3,3',5,5'-tetramethylbenzidine) single-component chromogenic substrate solution, or solution A (TMB) and solution B (hydrogen peroxide) are dispensed separately and mixed in equal volumes before use.

[0053] 10. Termination solution 2M sulfuric acid solution is used to terminate the colorimetric reaction.

[0054] 11. Total protein extraction / lysis buffer (optional) RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors is used for the processing of cell or tissue samples.

[0055] II. Detection Methods 1. Adding samples Discard the blocking solution from the pre-coated ELISA plate, wash 3 times, add the sample to be tested to the pre-coated ELISA plate pre-coated with capture antibody, and incubate overnight at 4°C or in an oven at 37°C for 2 hours.

[0056] 2. Primary antibody incubation: Discard the sample solution and wash 5 times. Perform 3-fold serial dilutions of the detection antibody, starting at a ratio of 1:1000, adjusting the dilution volume accordingly. Add the diluted antibody sequentially to the ELISA plate, incubate at 37°C for 1.5 h, and wash 1-3 times.

[0057] 3. Secondary antibody incubation: Dilute the secondary antibody to 1:10K with 1% BSA blocking buffer, incubate at room temperature or 37°C for 45 min, and then wash 1-3 times.

[0058] 6. Color rendering: After adding TMB chromogenic solution for 5-10 minutes, terminate the chromogenic reaction with 1M sulfuric acid, and read the data using an ELISA reader.

[0059] The technical solution of the present invention will be further explained through experiments below.

[0060] Experiment Example 1: Detection of Antiserum Titer I. Experimental Methods Antiserum was prepared according to steps 1-2 of Example 1, and its titer was detected. During the titer detection, CUEDC1 S123ph peptide A, CUEDC1 S123ph peptide B, CUEDC1 S122ph control peptide C, and CUEDC1 unmodified peptide were coated, respectively.

[0061] The amino acid sequences of CUEDC1 S123ph peptide A, CUEDC1 S123ph peptide B, CUEDC1 S122ph control peptide C, and CUEDC1 unmodified peptide are shown in the table below: Table 1 The specific methods for detecting antiserum titer are as follows: (1) Antigen coating CUEDC1 S123ph peptide A, CUEDC1 S123ph peptide B, CUEDC1 S122ph control peptide C, or CUEDC1 unmodified peptide antigen were diluted with coating buffer and added to each well of a polystyrene ELISA plate at 100 μL / well. The plates were coated at 37°C for 2 h, washed twice (5 min each time), and then patted dry.

[0062] (2) Closed Add 200 μL of blocking solution per well, block at 37°C for 3 h, wash 3 times, and pat dry.

[0063] (3) Add the antiserum to be tested 100 μL of antiserum per well, with normal rabbit negative serum as a control, incubated at 37°C for 30 min, washed 3 times, and patted dry.

[0064] (4) Add secondary antibody Dilute the enzyme-labeled goat anti-rabbit enzyme with blocking buffer at 100 μL / well, incubate at 37°C for 30 min, wash 3 times, and pat dry.

[0065] (5) Color development and detection Add 80 μL each of chromogenic solution A and B to each well, and incubate at 37℃ for 15 min. Add 80 μL of stop solution to each well. Measure the OD value of each well at a single wavelength of 450 nm. The ratio of the OD value of the well to the negative control well (P / N) greater than 2.1 is used as the cutoff point for judging potency.

[0066] II. Experimental Results The experimental results are shown in Table 2. A cross-sectional comparison at a 1:64K dilution showed that the antiserum from immunized rabbit R3 / R4 exhibited significant non-specific binding or cross-reactivity. The recognition strength of the control peptide C and the unmodified peptide was close to that of the immunogen, making them unsuitable for use as specific antibodies. The antibody produced by immunizing rabbit R1 showed good specificity against CUEDC1 S123ph peptide A / B, almost no cross-reactivity with the control peptide C, and very weak cross-reactivity with the unmodified peptide.

[0067] The endpoint of the antiserum immunized against R1 rabbits against CUEDC1 S123ph peptide A / B was 1:64K (OD>0.5), which is a high-titer antiserum.

[0068] Table 2 Serum ELISA Detection Results Note: R1, R2, R3, and R4 represent the numbers of the immunized rabbits.

[0069] Experiment Example 2: Antibody ELISA Detection Experiment I. Experimental Methods Antibodies were prepared according to the method in Example 1. Serum from rabbits R1, R3, and R4 was purified, and the resulting antibodies were designated Ab1, Ab3, and Ab4, respectively. In this experiment, the titers of Ab1, Ab3, and Ab4 were determined using an ELISA assay. The specific steps of the ELISA assay are as follows: (1) Antigen coating: Dilute the antigen (CUEDC1 S123ph peptide A, CUEDC1 S123ph peptide B, CUEDC1 S122ph control peptide C or CUEDC1 unmodified peptide) with coating buffer, and add it sequentially to the microplate at a rate of 50 μg / well. Incubate overnight at 4°C or for 2 hours in a 37°C oven.

[0070] (2) Washing the plate: Take out the ELISA plate coated the day before and wash it three times with 1×TBST.

[0071] (3) Closed: Add 1% BSA blocking solution to the cleaned ELISA plate, incubate at 37°C for 1 hour, and then wash 1-3 times.

[0072] (4) Primary antibody incubation: Antibodies Ab1, Ab3, and Ab4 were serially diluted 3-fold, starting at a ratio of 1:1000, and the dilution volume was adjusted accordingly. The solutions were then added sequentially to the ELISA plate, incubated at 37°C for 1.5 h, and washed 1-3 times.

[0073] (5) Secondary antibody incubation: Dilute the secondary antibody to 1:10K with 1% BSA blocking buffer, incubate at room temperature or 37°C for 45 min, and then wash 1-3 times.

[0074] (6) Color development: After adding TMB chromogenic solution for 5-10 minutes, terminate the chromogenic reaction with 1M sulfuric acid, and read the data using an ELISA reader.

[0075] II. Experimental Results The experimental results are shown in Table 3. The results indicate that the endpoint titer of Ab1 against CUEDC1 S123ph peptide A is approximately 1:1,458,000, which is considered a high titer. At a dilution of 1:486K, the OD of Ab1 against CUEDC1 S123ph peptide A / B is... 450 The OD value was 0.22, while the OD values ​​for the control peptide C and the unmodified peptide were significantly lower. 450 All values ​​were below 0.03, and the P / N ratio was greater than 7, indicating that the antibody has excellent specificity in distinguishing different phosphate modification states.

[0076] Table 3. Antibody ELISA Detection Results Experiment Example 3: Antibody Sensitivity Detection I. Experimental Methods Dot blot hybridization: After activating the PVDF membrane with methanol, 2 μL of different doses of CUEDC1 S123ph peptide A, CUEDC1 S123ph peptide B, CUEDC1 S122ph control peptide C, and CUEDC1 unmodified peptide were added, with antigen peptide masses of 1 ng, 4 ng, 16 ng, and 64 ng, respectively. After drying at room temperature, the membrane was blocked with 5% skim milk for 1 h. The membrane was then immersed in 1:2000 diluted Ab1, Ab3, and Ab4 antibodies and incubated at room temperature for 1 h. The membrane was washed three times with PBST. 1:10000 diluted enzyme-labeled goat anti-rabbit antibody was added, and the membrane was incubated with shaking at room temperature for 45 min. The membrane was washed three times with PBST. The membrane was then immersed in a fluorescent dye and developed later.

[0077] II. Experimental Results The results are as follows Figure 1 As shown, when the antigen peptide content is within 64 ng, Ab1, Ab3, and Ab4 antibodies only specifically recognize CUEDC1 S123ph peptide A / B, but cannot recognize CUEDC1 S122ph control peptide C or CUEDC1 unmodified peptide. Therefore, the prepared CUEDC1 S123ph antibody has high efficiency, sensitivity, and significant specificity.

[0078] Experiment Example 4: Western Blot (WB) Experiment I. Experimental Methods Western blot (WB) experiments were used to evaluate the binding of Ab1, Ab3, and Ab4 to target proteins in four human cell lines (MCF-7, HeLa, HepG2, and A549). The specific steps are as follows: (1) Cell lysis: MCF-7, HeLa, HepG2, and A549 cells were lysed to obtain total protein samples, and protein concentration was determined by the BCA method.

[0079] (2) Protein electrophoresis and transfer: Protein electrophoresis: Select a 10% separating gel according to the molecular weight of the target protein. When preparing the separating gel, after mixing all components, add 10% ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) last to initiate polymerization. After thorough mixing, pour the gel, with a volume of 4.7 mL per plate. After the gel solidifies, pour the stacking gel (stacking gel) and insert the comb. After boiling to denature the protein samples, load 20-40 μg of total protein per well. Electrophoresis is performed under constant voltage conditions: 80 V for the stacking gel stage and 120 V for the separating gel stage, until the bromophenol blue front migrates to the bottom of the gel.

[0080] Wet transfer: The transfer solution is pre-cooled before transfer. The gel, membrane, and filter paper are laid in the transfer solution in a sandwich structure to avoid air bubble formation. The transfer voltage is 80V-120V.

[0081] (3) Closed: Add blocking buffer to the transferred membrane and incubate at room temperature for 60 min. Wash with 1×TBST for 10 min after incubation.

[0082] (4) Primary antibody incubation: Dilute the antibody (Ab1, Ab3, or Ab4) with 2.5-5% skim milk powder, incubate at room temperature for 2 hours, and then wash three times with 1×TBST for 5-10 minutes each time.

[0083] (5) Secondary antibody incubation: Add the rabbit secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45 min, and then wash three times with 1×TBST for 5-10 min each time.

[0084] (6) Expose the washed film after adding a color developing substrate.

[0085] II. Experimental Results Experimental results are as follows Figure 2 As shown. The results indicate that the ELISA-positive antibodies Ab1, Ab3, and Ab4 were detected by Western blotting using MCF-7, HeLa, HepG2, and A549. Ab1, Ab3, and Ab4 showed bands of approximately 43 kDa in the MCF-7 and HepG2 lysis buffers, indicating a satisfactory result. In summary, this 43 kDa band represents the target protein CUEDC1 (S123ph), and the antibodies Ab1, Ab3, and Ab4 prepared in this invention can effectively detect CUEDC1 (S123ph).

[0086] As can be seen from the above embodiments and experimental examples, the present invention provides phosphorylated CUEDC1 peptides, antigens, vaccines, antibodies, and their preparation methods and uses. The present invention provides an antigen phosphorylated at the S123 site of the human CUEDC1 protein. Using this antigen to immunize animals yields highly sensitive and specific antiserum and purified antibodies, which can distinguish the phosphorylation state of CUEDC1 and are expected to be used for detecting the phosphorylation level of the CUEDC1 protein, showing promising application prospects.

Claims

1. A phosphorylated CUEDC1 polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO. 1, wherein the phosphorylation modification site is located at the 11th amino acid in the sequence shown in SEQ ID NO.

1.

2. An antigen, characterized in that: It is a conjugate of the phosphorylated CUEDC1 polypeptide described in claim 1 and a carrier protein.

3. The antigen according to claim 2, characterized in that: The carrier protein is keyhole hemocyanin.

4. Use of the phosphorylated CUEDC1 polypeptide of claim 1, or the antigen of claim 2 or 3, in the preparation of polyclonal antibodies against phosphorylated CUEDC1.

5. An antibody against phosphorylated CUEDC1, characterized in that: It is prepared by immunizing animals with an antigen including that described in claim 2 or 3, wherein the antibody is a polyclonal antibody.

6. The antibody against phosphorylated CUEDC1 according to claim 5, characterized in that, It is prepared according to the following steps: The antigen is mixed with an adjuvant to form a water-in-oil emulsion. After emulsification, it is injected subcutaneously into the neck and back of the animal. After 3-6 immunizations, venous blood is collected.

7. A method for preparing the antibody against phosphorylated CUEDC1 as described in claim 5 or 6, characterized in that, It includes: The antibody is prepared by immunizing an animal with the antigen described in claim 2 or 3, and the antibody is a polyclonal antibody.

8. Use of the antibody against phosphorylation-modified CUEDC1 as described in any one of claims 5-6 in the preparation of a kit for detecting the phosphorylation level of CUEDC1 protein.

9. The use according to claim 8, characterized in that: The detection of CUEDC1 protein phosphorylation level refers to the detection of serine phosphorylation level at position 123 in the CUEDC1 protein sequence.

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