Antigen peptide targeting PGK1 protein Thr255 O-GlcNAc glycosylation site, antibody and application thereof

Abstract

The invention provides an antigen peptide of a Thr255 O-GlcNAc glycosylation site of a targeted PGK1 protein, an antibody and application of the antigen peptide and the antibody. The antigen peptide has immunogenicity of a Thr255 O-GlcNAc glycosylation site of a targeted PGK1 protein, and the amino acid sequence of the antigen peptide is as shown in any one of SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4. The antigen peptide provided by the invention is helpful for researching the important effect of PGK1 in cell energy balance, and also provides a potential action target for the key effect possibly played in the biological processes of tissue repair, stem cell differentiation, damage repair and the like; and how to regulate and control glycosylation modification of PGK1 is further explored, so that proliferation, differentiation and tissue repair capacities of stem cells are enhanced, and a potential application prospect is provided for the field of regenerative medicine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antigenic peptide, an antibody and applications thereof targeting the Thr255 O-GlcNAc glycosylation site of a PGK1 protein. Background Art

[0002] O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important reversible post-translational modification (PTM) of proteins, primarily occurring on serine (Ser) and threonine (Thr) residues. Sugar addition is catalyzed by O-GlcNAc transferase (OGT), while O-GlcNAc hydrolase (OGA) removes the sugar moiety. O-GlcNAc glycosylation regulates diverse biological processes, such as cell cycle control, gene transcription, cell motility, and endocrine function. Due to its dynamic regulatory nature, O-GlcNAc glycosylation plays a key role in numerous physiological and pathological processes. Recent studies have demonstrated that O-GlcNAc glycosylation is closely linked to cell repair, tissue regeneration, and organ function recovery in regenerative medicine. In core areas of regenerative medicine, such as stem cell differentiation, tissue engineering, and wound healing, O-GlcNAc glycosylation influences cell regenerative capacity by regulating metabolic pathways, enhancing cell survival, and promoting proliferation and differentiation. With advances in glycomics, hundreds of O-GlcNAc modification sites can now be identified in a single study, enabling quantitative analysis of O-GlcNAc site-specific changes under different conditions. Using O-GlcNAc site-specific antibodies, researchers can conduct site-specific studies similar to those of phosphorylation, providing a new research direction for further exploring and optimizing the efficacy of regenerative medicine.

[0003] Phosphoglycerate kinase 1 (PGK1) is a key enzyme in the glycolysis pathway, catalyzing the conversion of 3-phosphoglycerate to 2-phosphoglycerate. PGK1 is not only a key regulator of cellular energy metabolism, but also regulates its activity and function through multiple post-translational modifications (such as phosphorylation, acetylation, and glycosylation). It is also closely associated with biological processes such as cell proliferation, stress response, and differentiation. Glycosylation of PGK1 may promote tissue repair and cell regeneration by regulating cellular metabolic status, enhancing cell proliferation, and modulating cellular stress responses. Because O-GlcNAc glycosylation of PGK1 mediates glucose metabolism reprogramming, affecting stem cell survival and function, further investigation of PGK1 O-GlcNAc glycosylation is of great significance.

[0004] Current antibodies targeting O-GlcNAc glycosylation mainly focus on the detection of whole proteins or non-specific sites. Because O-GlcNAc glycosylation is dynamic, reversible, and can occur at multiple sites, the development of highly specific antibodies that can recognize O-GlcNAc modifications at specific sites is crucial for revealing the role of glycosylation in disease and its physiological functions. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention conducted an in-depth study of the PGK1 protein structure and discovered that its threonine (Thr) at position 255 may undergo O-GlcNAc glycosylation. Therefore, developing relevant technologies and methods for effectively detecting O-GlcNAc glycosylation of threonine (Thr) at position 255 of the PGK1 protein has important application value for the screening of early disease markers, the study of disease mechanisms, and drug development.

[0006] The primary purpose of the present invention is to provide an antigenic peptide specifically targeting the O-GlcNAc glycosylation modification of the Thr255 site of the PGK1 protein. The antigenic peptide has immunogenicity targeting the Thr255 O-GlcNAc glycosylation site of the PGK1 protein, wherein the amino acid sequence of the antigenic peptide is shown in any one of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, which can indirectly explore the biological processes of cell metabolism and diseases.

[0007] The present invention also provides an antigen construct, which comprises the antigen peptide and a carrier for loading or coupling the antigen peptide.

[0008] In some embodiments, the carrier is selected from any one of keyhole limpet hemocyanin, bovine serum albumin, chicken ovalbumin, and bovine thyroglobulin.

[0009] The present invention also provides an application of the antigenic peptide or the antigenic construct, wherein the application is any one of the following: (1) Application in the preparation of antibodies specific for Thr255 O-GlcNAc glycosylation of PGK1 protein; (2) Application in the preparation of vaccines for diseases related to Thr255 O-GlcNAc glycosylation of PGK1 protein.

[0010] The present invention also provides an antibody, which specifically binds to the antigen peptide or the antigen construct.

[0011] The present invention also provides a method for preparing the antibody, comprising the steps of immunizing an animal with the antigen peptide or the antigen construct, collecting antiserum and purifying the antiserum.

[0012] In one embodiment of the present invention, the animal is preferably a rabbit or a mouse.

[0013] The present invention also provides an ELISA kit, which comprises the antibody or the antibody obtained by the preparation method.

[0014] The present invention also provides a pharmaceutical composition comprising the antibody or the antibody obtained by the preparation method, and a pharmaceutically acceptable excipient or carrier.

[0015] In summary, compared with the prior art, the present invention achieves the following technical effects: The present application provides an antigenic peptide targeting the Thr255 O-GlcNAc glycosylation site of the PGK1 protein and an antibody prepared therefrom, which helps to study the important role of PGK1 in cellular energy balance and provides a potential target for its key role in biological processes such as tissue repair, stem cell differentiation and injury repair. Further, by exploring how to regulate the glycosylation modification of PGK1, the proliferation, differentiation and tissue repair ability of stem cells are enhanced, providing potential application prospects in the field of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a flow chart for the preparation of the human PGK1 protein Thr255 site O-GlcNAc glycosylated antibody of the present invention; Figure 2 This is a three-dimensional structure diagram of the PGK1 protein in Example 1 of the present invention; Figure 3 This is a schematic diagram of the PGK1 domain structure of Example 1 of the present invention; Figure 4 This is a hydrophilicity analysis diagram of PGK1 protein in Example 1 of the present invention; Figure 5 This is the antigenicity score of PGK1 protein in Example 1 of the present invention; Figure 6 This is the antiserum titer ratio determination of Example 1 of the present invention; Figure 7 This is the titer ratio determination of the purified antibody in Example 2 of the present invention; Figure 8 This is the specific identification of the O-GlcNAc-PGK1-Thr255 antibody in Example 2 of the present invention. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0019] Previous studies of this application have found that the Thr255 site in the amino acid sequence of PGK1 is one of the sites of protein O-GlcNAc glycosylation modification, and affects the enzymatic activity of PGK1. To this end, the embodiments of this application disclose an antigenic peptide, which has the immunogenicity of targeting the Thr255 O-GlcNAc glycosylation site of the PGK1 protein, wherein the amino acid sequence of the antigenic peptide is shown in any one of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The Thr255 site of PGK1 is a protein O-GlcNAc glycosylation that may play a key role in tissue repair, stem cell differentiation, and damage repair by regulating cellular energy balance. By developing antibodies that specifically recognize the O-GlcNAc glycosylation modification of the Thr255 site of PGK1, it is helpful to explore how to enhance the proliferation, differentiation and tissue repair ability of stem cells by regulating the glycosylation modification of PGK1, providing a new perspective on the role of PGK1 in stem cell biology, and also providing a theoretical basis for its potential application in regenerative medicine. Among them, the antibody preparation process is as follows Figure 1 shown.

[0020] Example 1 Preparation of Antibodies Against O-GlcNAc Glycosylation of Thr255 of Human PGK1 Protein The three-dimensional structure of human PGK1 protein was obtained using the Protein Data Bank (PDB) protein three-dimensional structure database (https: / / www.rcsb.org / ) and the molecular visualization software PyMOL, as shown in Figure 2. Figure 2 As shown, multiple α-helices are staggered, connecting adjacent amino acid residues through various hydrogen bonds, thereby maintaining their stability. Furthermore, the molecule contains multiple reverse-folded regions that form β-pleated sheets, with Thr255 located near the active center of the PGK1 enzyme.

[0021] Step 1: Design an antigen peptide containing Thr255 O-GlcNAc glycosylation.

[0022] First, the full-length amino acid sequence of human PGK1 protein (SEQ ID NO. 1) was obtained from the UniProt website (https: / / www.uniprot.org / ). The homology of the human PGK1 protein sequence with other species was analyzed using the bioinformatics software DNAMAN. The sequence near the Thr255 site was highly conserved in multiple species. The secondary structure of the human PGK1 protein was predicted and the hydrophilicity and hydrophobicity were analyzed. The schematic diagram of the PGK1 domain is shown in the figure. Figure 3 As shown, it includes three main domains (N domain, core domain and C domain) and their functional regions. N domain (amino terminus, positions 1-150): contains the 3-phosphoglycerate binding region, which is responsible for binding to the substrate. Core domain (positions 150-280): contains the catalytic site, which is the core region of the PGK1 enzyme for catalytic reactions. C domain (amino terminus, positions 280-415): mainly the coenzyme binding region, involved in the interaction and regulation with other molecules. Among them, the region at amino acids 80-160 has functions related to ATP binding and hydrolysis, so this region is closely related to energy metabolism. Positions 200-280 represent the transfer of responsible phosphate groups. The specific functions of each region are crucial for the catalytic activity and substrate binding of the enzyme. The hydrophilicity of PGK1 protein is as follows Figure 4 As shown in the figure, due to the folding state of human PGK1 protein and the structure of its functional region, its hydrophilicity shows a periodic fluctuation, among which the Thr255 site shows strong hydrophilicity, indicating that it is in an important functional region and is related to substrate binding or catalysis. Sites in the area near the strongly hydrophilic Thr255 site also show strong hydrophilicity or weak hydrophobicity. Therefore, the region sequence with the Thr255 site as the core is a recommended antigen sequence. At the same time, the antigenic epitope prediction online tool IEDB (Immune Epitope Database, https: / / www.iedb.org / bcell / ) was used to predict antigenic epitopes, such as Figure 5 The figure below shows the distribution of antigenicity scores for human PGK1 protein. A higher score (>0.5) indicates that the amino acid at that site has high antigenicity. Therefore, the fragments in the yellow region are potentially antigenic fragments, with the sequence at sites 252-265 exhibiting high antigenicity. Sequences near Thr255 with high homology, surface exposure, random coils or junctions, high hydrophilicity, and high antigenicity scores were selected as antigenic peptides.

[0023] The following three amino acid antigenic peptides containing O-GlcNAc glycosylation at Thr255 were designed and obtained: antigenic peptide 1 sequence: EIGT(O-GlcNAc)SLFDEEGAKI (252-265, SEQ ID NO.2), antigenic peptide 2 sequence: NNMEIGT(O-GlcNAc)SLFDEEG (249-262, SEQ ID NO.3), and antigenic peptide 3 sequence: NMEIGT(O-GlcNAc)SLFDE (250-260, SEQ ID NO.4), where (O-GlcNAc) indicates that the amino acid at this site is O-GlcNAc glycosylated.

[0024] Step 2: Peptide synthesis technology was used to synthesize the three amino acid antigen peptides designed above, each containing O-GlcNAc glycosylation at Thr255. An O-GlcNAc glycosylation group was added to the amino acid Thr255 and coupled to the carrier protein hemocyanin (KLH) to prepare O-GlcNAc glycosylated antibodies as immunogens; the peptides were coupled to the carrier protein bovine serum albumin (BSA) and used as affinity purification fillers for affinity purification. Correspondingly, non-O-GlcNAc glycosylated synthetic peptides were synthesized and coupled to the carrier protein bovine serum albumin (BSA) to serve as affinity separation fillers. All synthetic peptides were purified by HPLC with a purity greater than 95%.

[0025] Step 3: Immunize animals with all antigens and collect antiserum.

[0026] SPF New Zealand white rabbits were immunized with the full antigen conjugated to the Thr255 O-GlcNAc-glycosylated peptide-KLH from Step 2 and adjuvant. The synthetic peptide powders of the Thr255 O-GlcNAc-glycosylated peptide-KLH were dissolved in sterile 0.1× PBS. The antigen solution was drawn up into one sterile syringe, while an equal amount of complete Freund's adjuvant (CFA) was drawn up into another syringe. The Thr255 O-GlcNAc-glycosylated peptide-KLH and CFA were mixed thoroughly by repeated pipetting through the plastic tubing connecting the two syringes until a complete emulsion formed. The emulsion was tested for non-spreading upon dripping into water. For the first immunization, the antigen emulsion was injected subcutaneously into the thin, loose skin of the neck and back, into the gluteal and thigh muscles, intradermally on both sides of the waist, and into the paw pads. The total dose of both antigens for the first immunization was approximately 0.61 mg. Twenty days after the first immunization, the first booster immunization was administered. An antigen emulsion was prepared using incomplete Freund's adjuvant (IFA) instead of CFA and injected as in the initial immunization. The total amount of antigen in this booster immunization was approximately 0.9 mg. A second booster immunization was performed 12 days after the previous booster immunization, using incomplete Freund's adjuvant (IFA) instead of CFA to prepare the antigen emulsion and following the initial injection procedure. The total amount of antigen in this booster immunization was also 0.9 mg. Three days after the final booster immunization, blood was collected from the rabbits' abdominal aorta to collect a large amount of serum. The blood was placed in a sealed beaker and allowed to stand at room temperature overnight to allow the clot to shrink. The next day, the serum was aseptically extracted and aliquoted into 50 mL centrifuge tubes. The supernatant was collected and aliquoted into 1 mL tubes (approximately 50 mL total), labeled as post-immunization antiserum, and stored at -20°C. The titer of the antiserum against the target synthetic peptide was determined by ELISA. Dissolve the Thr255 O-GlcNAc-glycosylated antigen peptide-BSA and the Thr255 non-O-GlcNAc-glycosylated antigen peptide-BSA in 0.05 M carbonate buffer (pH 9.6) to prepare a 10 μg / mL working solution. Coat a 96-well microtiter plate with 100 μL of the antigen solution per well, setting up triplicate wells. Incubate the plate overnight at 4°C, then discard the solution and add 200 μL / well of 5% skim milk blocking solution. Incubate at room temperature for 1 hour. After blocking, discard the solution and wash the plate three times with PBS-T (0.05% Tween-20), letting it sit for 3 minutes each time and patting dry.Antiserum was prepared at a dilution gradient of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000. 100 μL of the corresponding dilution of antiserum was added to each well. For blank control wells, an equal volume of PBS was added and the cells were incubated at room temperature for 1 hour. After incubation, the wells were discarded and washed five times with PBS-T, each time for 3 minutes, and then patted dry. Then, 100 μL of HRP-conjugated anti-rabbit IgG diluted solution was added to each well and incubated at room temperature for 1 hour. After washing five times with PBS-T, each time for 3 minutes, 100 μL of TMB substrate solution was added to develop color. The cells were reacted for 15 minutes in the dark and the color development was observed. After the reaction, 50 μL of 2 M sulfuric acid was added to each well to terminate the reaction. The color changed from blue to yellow, and the OD value was read at 450 nm using a microplate reader. A titer curve was drawn based on the antiserum dilution factor (1:1000 to 1:512000). The binding ability of the Thr255 O-GlcNAc-glycosylated antigen peptide-BSA and the non-Thr255 O-GlcNAc-glycosylated antigen peptide-BSA was compared to evaluate the antiserum titer.

[0027] The results are as follows Figure 6 As shown, the titers of antisera generated from specific antibodies prepared against different antigenic peptide sequences are compared. The OD450 values ​​of all antibodies gradually decrease with increasing antiserum dilution. Furthermore, antisera with different antigenic designs exhibit varying binding capacities after dilution, reflecting differences in antibody titer. The titers of antisera generated by all O-GlcNAc-modified antigenic peptides were significantly higher than those generated by the corresponding unmodified antigenic peptides. Among all antigenic peptide sequences glycosylated at Thr255 by O-GlcNAc, antigenic peptide 1 (252-265, SEQ ID NO. 2) generated the highest antiserum titer, indicating that it is the optimal antigenic peptide design.

[0028] In some embodiments, the fully antigen-immunized animal of the present application may also be a mouse, and the preparation scheme is as follows: Mice were immunized four times, two weeks apart, with either the full antigen conjugated to the Thr255 O-GlcNAc-glycosylated peptide-KLH or the full antigen conjugated to the non-O-GlcNAc-glycosylated peptide-KLH, respectively, in combination with an adjuvant. Three days after the final immunization, mice were sacrificed and spleens were collected under sterile conditions. Splenic B lymphocyte suspensions were prepared by trituration. Sensitized B lymphocytes were mixed with myeloma cells from the same mouse strain at a specified ratio, washed twice with serum-free incomplete medium, and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the remaining liquid was aspirated. The bottom of the centrifuge tube was flicked and preheated polyethylene glycol (PEG) fusogen was added with gentle mixing. Preheated incomplete culture medium was then added to terminate the PEG reaction. After standing at room temperature for 10 minutes, the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in 5 mL of HAT selection medium. The volume was then filled with HAT medium containing peritoneal macrophages to a volume of 80–100 mL. 1 mL was added to each well of a 24-well plate and incubated at 37°C in a 5% CO2 incubator. After 5 days, half of the medium was replaced with HAT medium, and after 10 days, the HAT medium was replaced with HT medium. Hybridoma cell growth was regularly monitored, and clones were screened, expanded, and cultured to enrich for secreted antibodies. Antibody titers against synthetic peptides of the glycosylated antigen were assessed by ELISA. Antibodies were purified using affinity separation and affinity purification cycles. Western blotting confirmed that the antibodies specifically recognized PGK1 glycosylated at Thr255 with O-GlcNAc.

[0029] Example 2 Purification and identification of antibodies glycosylated with O-GlcNAc at Thr255 of human PGK1 protein.

[0030] The antibody affinity separation-affinity purification cycle purification technology was performed using affinity chromatography using synthetic peptides coupled to bovine serum albumin and agarose chromatography columns. First, a non-O-GlcNAc glycosylated synthetic peptide corresponding to the Thr255 site was coupled to bovine serum albumin (BSA) and agarose as the filler of the chromatography column to perform affinity separation to remove non-O-GlcNAc glycosylated antibodies in the antiserum, and obtain the effluent containing O-GlcNAc glycosylated antibodies; the second time, an O-GlcNAc glycosylated antigen peptide was coupled to bovine serum albumin (BSA) and agarose as the filler of the chromatography column to perform affinity purification to remove low-affinity, low-sequence complexity epitope antibodies in the O-GlcNAc glycosylated antibodies, and Western blotting was used to separate the antibodies. Blot analysis confirmed that the antiserum specifically recognized the O-GlcNAc-glycosylated antigenic peptide. Purified anti-O-GlcNAc-Thr255 glycosylated antibodies, designated O-GlcNAc-PGK1-Thr255 antibodies, were obtained and stored in a mixture of 2.5% (wt / vol) BSA, 0.01% (vol / vol) Tween-20, and 25% (vol / vol) glycerol. ELISA was used to test the titer of the purified antibodies and their recognition of both O-GlcNAc-glycosylated and non-O-GlcNAc-glycosylated antigenic peptides. Synthetic Thr255 O-GlcNAc-glycosylated antigenic peptides-BSA and non-O-GlcNAc-glycosylated Thr255 antigenic peptides-BSA were coated onto 96-well microtiter plates. Then, the purified antiserum was added in a gradient dilution of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 and 1:512000, incubated and washed. Then, HRP-labeled goat anti-rabbit IgG dilution was added, incubated again and washed. After TMB substrate color development, the stop solution was added to stop the reaction and the OD450 value of each well was measured. The results are shown in Figure 2. Figure 7As shown, a comparison of the titers of specific antibodies after purification from antisera prepared from different antigenic peptide sequences revealed that the titer of specific antibodies prepared from PGK1 protein antigenic peptide 1 (252-265, SEQ ID NO. 2) was higher than that of antibodies prepared from antigenic peptide 2 and 3. Western blot analysis was performed to characterize the antibodies. Human 293T cells were transfected with either the wild-type FLAG-tagged plasmid pCMV-FLAG-Thr255WT or the plasmid pCMV-FLAG-Thr255Ala, in which the Thr255 position was mutated to alanine. After 48 hours of transfection, the cells were harvested and total protein was extracted using lysis buffer. FLAG-tagged proteins were enriched by immunoprecipitation using anti-FLAG antibodies coupled to protein A / G magnetic beads. After washing the precipitate, SDS-PAGE loading buffer was added and boiled. The sample was separated by SDS-PAGE and transferred to a membrane. Western blot analysis was performed using an antibody prepared against an O-GlcNAc-glycosylated antigen peptide at the Thr255 site. The specificity of the antibody was evaluated by comparing the signal intensity of histones transfected with pCMV-FLAG-Thr255WT and pCMV-FLAG-Thr255Ala.

[0031] Figure 8 The results show the specificity of the antibody prepared using the PGK1 protein antigen peptide 1 sequence (252-265, SEQ ID NO. 2). In the T255 group, a significant O-GlcNAc-PGK1-T255 signal was detected, indicating that the antibody specifically recognizes the O-GlcNAc-modified form at Thr255 of human PGK1 protein. No O-GlcNAc signal was detected in the T255A mutant group, indicating that the antibody does not nonspecifically bind to unmodified (or mutated) PGK1 protein. These results demonstrate that the antibody prepared using the PGK1 protein antigen peptide 1 sequence (252-265, SEQ ID NO. 2) specifically recognizes the O-GlcNAc-modified form at Thr255 of human PGK1 protein, but not the mutant form (T255A), demonstrating excellent specificity.

[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0033] Sequence Listing SEQ ID No.1 MSLSNKLTLDKLDVKGKRVVMRVDFNVPMKNNQITNNQRIKAAVPSIKFCLDNGAKSVVLMSHLGRPDGVPMPDKYSLEPVAVELKSLLGKDVLFLKDCVGPEVEKACANPAAGSVILLENLRFHVEEEGKGKDASGNKVKAEPAKIEAFRASLSKLGDVYVNDAFGTAHRAHSSMVGVNLPQKAGGFLMKKELNYFAKALESPERPFLAILGGAKVADKIQLINNMLDKVNEMIIGGGMAFTFLKVLNNMEIGTSLFDEEGAKIVKDLMSKAEKNGVKITLPVDFVTADKFDENAKTGQATVASGIPAGWMGLDCGPESSKKYAEAVTRAKQIVWNGPVGVFEWEAFARGTKALMDEVVKATSRGCITIIGGGDTATCCAKWNTEDKVSHVSTGGGASLELLEGKVLPGVDALSNI SEQ ID No.2 EIGT(O-GlcNAc)SLFDEEGAKI SEQ ID No.3 NNMEIGT(O-GlcNAc)SLFDEEG SEQ ID No.4 NMEIGT(O-GlcNAc)SLFDE

Claims

1. An isolated antigenic peptide, characterized in that: The antigenic peptide has the immunogenicity of targeting the Thr255 O-GlcNAc glycosylation site of the PGK1 protein, wherein the amino acid sequence of the antigenic peptide is shown in any one of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.

4.

2. An antigen construct, characterized in that The antigen construct comprises the antigen peptide according to claim 1 and a carrier for loading or coupling the antigen peptide according to claim 1.

3. The antigen construct according to claim 2, characterized in that The carrier is selected from any one of keyhole limpet hemocyanin, bovine serum albumin, chicken ovalbumin and bovine thyroglobulin.

4. Use of the antigen peptide according to claim 1 or the antigen construct according to any one of claims 2 to 3, characterized in that: The application is any of the following: (1) Application in the preparation of Thr255 O-GlcNAc glycosylation-specific antibodies of PGK1 protein; (2) Application in the preparation of vaccines for diseases related to Thr255 O-GlcNAc glycosylation of PGK1 protein.

5. An antibody, characterized in that The antibody specifically binds to the antigen peptide according to claim 1 or the antigen construct according to any one of claims 2 to 3.

6. The method for preparing the antibody according to claim 5, characterized in that: The method comprises the steps of immunizing an animal with the antigen peptide according to claim 1 or the antigen construct according to any one of claims 2 to 3, collecting antiserum and purifying the antiserum.

7. An ELISA kit, characterized in that: The ELISA kit comprises the antibody according to claim 5 or the antibody obtained by the preparation method according to claim 6.

8. A pharmaceutical composition, characterized in that The invention comprises the antibody according to claim 5 or the antibody obtained by the preparation method according to claim 6, and a pharmaceutically acceptable auxiliary material or carrier.

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