A2ml1 protein acetylation polypeptide, antigen, antibody and application thereof

Abstract

The application discloses a human A2ML1 protein acetylation polypeptide, an antigen, an antibody and application thereof, and belongs to the technical field of antibodies. The sequence of the human A2ML1 protein acetylation polypeptide is YVLPFK(acetyl)VEVVEPKEC, wherein acetyl represents acetylation. The antigen is a conjugate of the human A2ML1 protein acetylation polypeptide and a carrier protein. The antibody can specifically bind to the human A2ML1 protein acetylation polypeptide. The antibody is a polyclonal antibody or an antibody fragment obtained after chemical treatment or enzyme digestion. The polyclonal antibody is obtained by immunizing a non-human animal with the conjugate of the human A2ML1 protein acetylation polypeptide and the carrier protein, and then purifying and extracting serum of the non-human animal. The human A2ML1 protein acetylation polypeptide, the antigen and the antibody provided by the application can specifically recognize an A2ML1 protein K220 acetylation site, are used for detecting expression differences of normal cells, tumor cells and tumor cells after drug treatment, provide a tool for exploring a tumor cell proliferation and metastasis mechanism, and provide help for tumor diagnosis and prognosis.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more particularly to a human A2ML1 protein acetylated polypeptide, antigen, antibody, and their applications. Background Technology

[0002] Malignant tumors, commonly known as cancer, are among the most difficult diseases to treat worldwide. Big data shows that the global mortality rate from malignant tumors remains consistently high, and even after treatment, the prognosis for some cancers is not ideal. Therefore, many scholars are researching the pathogenesis and mechanisms of various cancers, hoping to find effective treatments. Furthermore, early cancer monitoring and prognostic analysis are crucial. They help doctors identify a patient's risk of developing cancer early, serving as an early warning system and enabling patients to receive treatment sooner, thus improving their survival rate. Prognostic analysis is also a scientific method for assessing a patient's survival status after treatment. However, both early cancer monitoring and prognostic analysis rely heavily on various tumor markers. Tumor markers (TMs) are antigens and bioactive substances produced by tumor tissues and cells due to the abnormal expression of proto-oncogenes, tumor suppressor genes, and other tumor-related genes and their products. While these markers are generally expressed in normal human tissues and cells, their expression levels are higher in the tissues and cells of patients with various cancers.

[0003] Protein acetylation is a protein modification process in which acetyl groups are transferred to the protein molecule chain under the action of acetyltransferases. It belongs to post-translational modifications of proteins and includes histone acetylation and non-histone acetylation, primarily occurring at lysine residues. Protein acetylation affects protein function, such as enzyme activation and inactivation, protein stability, subcellular structural localization, and the formation of specific functional complexes. Acetylation of histone H3 and H4 residues can activate gene transcription. Besides activating the transcription of specific genes, histone acetylation is also a reversible, dynamic regulatory process; maintaining this reversible acetylation process is crucial for stabilizing chromatin structure and regulating gene expression. Non-histone acetylation also participates in key cellular processes related to physiology and disease, such as gene transcription, DNA damage repair, cell division, signal transduction, protein folding, autophagy, and metabolism. In conclusion, research on non-histone acetylation and tumorigenesis has deepened our understanding of tumorigenesis mechanisms and provided new ideas and targets for tumor treatment.

[0004] A2ML1 is a newly discovered member of the macroglobulin inhibitor family. Similar to α2-macroglobulin, the A2ML1 gene encodes a 1454-amino acid sequence located on chromosome 12p13.31 and is a 180 kDa protein monomer distributed in human epithelial cells. The functional inhibitory unit of A2ML1 is a monomer that relies on covalent binding to a protease (mediated by A2ML1 thioesters) for inhibition. In contrast to the A2M tetramer, which traps the protease in two lumens formed by four subunits, in the protease-cleaved monomeric A2ML1, a disordered region surrounds the trapped protease and may prevent substrate entry. In native A2ML1, the decoy region traverses a hydrophobic channel, suggesting that decoy region cleavage disrupts this arrangement, triggering extensive conformational changes that lead to protease inhibition. Structural comparison with complement C3 / C4 indicates that the A2M protein superfamily shares a common mechanism that triggers conformational changes during proteolytic activation. A2ML1 protein inhibits extracellular proteases through its unique "trapping" mechanism, thereby significantly promoting epithelial cell shedding.

[0005] Non-histone acetylation modification plays a crucial role in the regulation of A2ML1 protein as a post-translational regulatory mechanism, thus requiring the elucidation of the effects of specific acetylation sites on A2ML1. Furthermore, A2ML1 can regulate epithelial cell homeostasis through interactions with growth factors and cytokines. The prognostic correlation between A2ML1 and cancers, including pancreatic cancer, esophageal cancer, and breast cancer, has also been confirmed. Therefore, investigating the impact of A2ML1 on the development and progression of ESCC and its potential mechanisms is of great significance for identifying new targets for the diagnosis and treatment of ESCC. Based on previous work, lysine 220 (K220) of the A2ML1 protein is predicted to be a potential acetylation site, possibly related to the protein's stability and activation functions. However, antibodies for detecting the K220 acetylation site status in the lysine sequence of human A2ML1 protein are currently lacking. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide a human A2ML1 protein acetylated polypeptide, antigen, antibody, and their applications, which can specifically identify the K220 acetylation site of the A2ML1 protein expressed in human tumor cells. Moreover, compared to adjacent normal tissues, it is highly expressed in various cancer tissue cells and can be used to detect the acetylation level at this site in tumor cells.

[0007] This invention provides a human A2ML1 protein acetylated polypeptide, the sequence of which is YVLPFK(acetyl)VEVVEPKEC, where acetyl represents acetylation.

[0008] Furthermore, the present invention also provides an antigen, which is a conjugate of a human A2ML1 protein acetylated polypeptide and a carrier protein as described above.

[0009] Specifically, the carrier protein includes any one of KLH, OVA, THY, or BSA.

[0010] Furthermore, the present invention also provides an antibody that can specifically bind to the human A2ML1 protein acetylated polypeptide as described above.

[0011] Specifically, the antibody is a polyclonal antibody or an antibody fragment processed by chemical methods or enzymatic digestion.

[0012] More specifically, the method for preparing the polyclonal antibody includes the following steps:

[0013] S1: Synthetic human A2ML1 protein acetylated peptide;

[0014] S2: The acetylated peptide of human A2ML1 protein was conjugated with a carrier protein, and the conjugate was purified by desalting column and then immunized with non-human animals.

[0015] S3: Detect antibody titers in immunized non-human animal serum, collect immunized non-human animal serum, and purify antibodies;

[0016] S4: Detect the purified antibody.

[0017] Furthermore, the present invention also provides the application of the aforementioned human A2ML1 protein acetylated peptide, antigen, or antibody in the detection of acetylation levels at the K220 acetylation site of human A2ML1 protein.

[0018] Furthermore, the present invention also provides the use of the aforementioned human A2ML1 protein acetylated peptide, antigen, or antibody in the preparation of reagents or kits for the diagnosis, treatment, and prognosis of tumors.

[0019] Furthermore, the present invention also provides a detection kit comprising the antibody as described above, and further comprising antigen retrieval solution, PBS buffer solution, enzyme blocking agent, horseradish enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB chromogenic agent, hematoxylin staining solution, ethanol, environmentally friendly clearing agent, 0.5% ammonia water and ultrapure water.

[0020] The beneficial effects of this invention are as follows: Compared with the prior art, the improvements of this invention are:

[0021] 1. This invention synthesizes a human A2ML1 acetylated polypeptide using an artificial design method and prepares the corresponding polyclonal antibody. This polyclonal antibody, as identified by ELISA and other methods, specifically recognizes the K220 acetylation site of the A2ML1 protein, and exhibits high expression in various cancer cells compared to adjacent normal tissues, with statistically significant differences.

[0022] 2. This invention discloses a polyclonal antibody targeting the acetylation of the human A2ML1 protein at the K220 site, which helps to study the kinase activity that mediates its acetylation and explore the various biological functions of the human A2ML1 protein.

[0023] 3. The polyclonal antibody targeting the K220 acetylation site of human A2ML1 protein in this invention can recognize the K220 acetylation site of A2ML1 protein and can be used to detect the acetylation level of this site in tumor cells. This provides a tool for exploring the mechanisms of tumor cell proliferation and metastasis, helps in tumor diagnosis, and can guide the clinical prognosis. Attached Figure Description

[0024] Figure 1 This is the result of the ELISA experiment in Example 4 of the present invention to detect whether the acA2ML1-K220 antibody is a specific acetylation site antibody.

[0025] Figure 2 This is a microscopic image of the esophageal cancer tissue and the adjacent tissue after immunohistochemical experiments in Example 5 of the present invention.

[0026] Figure 3 The results of Kaplan-Meier survival analysis of tissue sections in Example 5 of this invention are shown.

[0027] Figure 4 The results are the expression analysis of A2ML1 protein and acetylated protein in Example 5 of this invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0029] Example 1:

[0030] Example 1 provides a human A2ML1 protein acetylated polypeptide, the sequence of which is YVLPFK(acetyl)VEVVEPKEC, where acetyl represents acetylation. This human A2ML1 protein acetylated polypeptide is synthesized through artificial design, and the specific synthesis method includes the following steps:

[0031] (1) The amino acid sequence of human A2ML1 protein (NP_001269353.2) obtained from GenBank is as follows:

[0032] TLPNVPGMYTLEASGQGCVYVQTVLRYNILPPTNMKTFSLSVEIGKARCEQPTSPRSLTLTIHTSYVGSR

[0033] SSSNMAIVEVKMLSGFSPMEGTNQLLLQQPLVKKVEFGTDTLNIYLDELIKNTQTYTFTISQSVLVTNLK

[0034] PATIKVYDYYLPDEQATIQYSDPCE

[0035] The results showed that the human A2ML1 protein contains 165 amino acids.

[0036] (2) The characteristics of human A2ML1 protein were analyzed using ProtParam software. The results are shown in Table 1 below.

[0037] Table 1 Characteristics of human A2ML1 protein

[0038] Molecular weight 29 Theoretical pI 5.246

[0039] The results showed that the human A2ML1 protein has a molecular weight of 29 Daltons and an isoelectric point of 5.246, making it an acidic protein.

[0040] (3) The immunogenicity, hydrophilicity and surface accessibility of human A2ML1 protein were analyzed using DNAstar software.

[0041] (4) Design of human A2ML1 protein acetylated peptide sequence: The K220 site in the A2ML1 amino acid sequence is located in the intracellular segment of A2ML1 and is a site for A2ML1 protein acetylation modification, which may play an important role in the dynamic regulation of A2ML1 protein degradation and inactivation. Based on the above analysis, the selected peptide sequence is YVLPFK(acetyl)VEVVEPKEC.

[0042] Example 2:

[0043] Example 2 provides a method for synthesizing an antigen by coupling the human A2ML1 protein acetylated polypeptide designed in Example 1 with a carrier protein.

[0044] To facilitate coupling with the carrier protein, a cysteine ​​residue was added to the C-terminus of the synthesized peptide, and the lysine residue at position 220 was acetylated: YVLPFK(acetyl)VEVVEPKEC. The peptide was synthesized by Suzhou Baiyuan Biotechnology Co., Ltd.

[0045] (1) Peptide synthesis process

[0046] A: Peptide synthesis: Solid-phase synthesis is used. First, the hydroxyl groups of the hydroxyl-terminal amino acids of the desired peptide chain are covalently linked to an insoluble polymer resin. Then, the amino acid bound to the solid support is used as the amino component. After removing the amino protecting group, it reacts with an excess of activated carboxyl components to lengthen the peptide chain. This process is repeated until the desired peptide chain length is reached. Finally, the peptide chain is cleaved from the resin and purified to obtain the desired peptide.

[0047] B: Purification: RP-HPLC purification

[0048] ①HPLC conditions:

[0049] Mobile phase: A) 0.1% TFA aqueous solution;

[0050] 0.1% TFA acetonitrile solution;

[0051] Gradient: A / B(90 / 40) to A / B(40 / 90) for 30 minutes;

[0052] Flow rate: 1 ml / min;

[0053] Temperature: Room temperature (23℃); Detection: 214nm ultraviolet light;

[0054] Sample: Freeze-dried crude product;

[0055] ②Steps:

[0056] a. Dissolve the crude product in the mobile phase;

[0057] b. Inject 20-30 mg (2-2.5 ml) of sample;

[0058] c. Collect the main peak into a 50 mL tube;

[0059] d. Freeze-drying;

[0060] ③ Identification: LC / MS

[0061] condition:

[0062] Mobile phase: A) 0.05% TFA aqueous solution; B) 0.1% TFA acetonitrile solution;

[0063] Gradient: A / B(90 / 10) to A / B(40 / 60) 15 min;

[0064] Flow rate: 1 ml / min; Temperature: room temperature (23℃);

[0065] Detection: 214nm ultraviolet light;

[0066] MS API: ESI.

[0067] (2) Coupling of peptides to carrier proteins

[0068] Chemically synthesized peptide antigens are small molecules and rarely possess strong antigenicity, inducing only weak immune responses in animals. Therefore, cross-linking with carrier proteins is crucial. Carrier proteins contain numerous antigenic determinants that stimulate T helper cells, thereby inducing B cell responses. Various carrier proteins are used for peptide cross-linking, with the most commonly used being keyhole limpethemacyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), and bovine thyroglobulin (THY). KLH exhibits higher antigenicity and is the most frequently used peptide cross-linking carrier. BSA is also commonly used as a peptide carrier, but its frequent use as an inhibitor in detection assays limits the application of antibodies produced by this method.

[0069] The process of conjugating peptides to carrier proteins includes the following steps:

[0070] a. Solution preparation: The coupling buffer includes Na2HPO4, NaH2PO4, NaCl, and EDTA, adjusted to pH 7.2;

[0071] b. Experimental steps:

[0072] 1) Column preparation: Wash the column with pure water and coupling buffer;

[0073] 2) Peptide preparation: Dissolve the peptide in a small amount of DMF, let it stand for half an hour until there are no particulate insoluble substances in the solution, add an appropriate amount of AH solution to prepare a 6 mg / ml peptide solution, and separate the amount to be coupled from the peptide solution.

[0074] 3) Preparation of KLH and Sulfo-SMCC: Based on the mass ratio of total amount of conjugated peptide to pure KLH = 1:1, calculate the amount of pure KLH. Based on the mass ratio of pure KLH to Sulfo-SMCC = 10:1, calculate the amount of Sulfo-SMCC.

[0075] 4) KLH and Sulfo-SMCC reaction and reactant collection: Dissolve the weighed KLH in an appropriate amount of AH solution to prepare a final concentration of 10 mg / ml, and dissolve Sulfo-SMCC in DMSO to prepare a solution of 100 mg / ml. Mix the two together and shake well. React at room temperature for 4 hours with intermittent shaking to ensure complete reaction. Separate the sample using a chromatography column.

[0076] 5) Coupling of KLH and Sulfo-SMCC reactants with peptides: Add the appropriate amount of KLH and Sulfo-SMCC reactants to each peptide to be coupled, react at room temperature for 2 hours or overnight at room temperature, mix well with a vertical mixer, and store the coupled peptides at -20°C.

[0077] The peptides are coupled with KLH carrier proteins, and the resulting coupled peptides can be used as immunoantigens.

[0078] Example 3:

[0079] Example 3 provides an antibody, specifically an anti-A2ML1 polypeptide rabbit polyclonal antibody, the synthesis of which includes the following steps:

[0080] (1) Artificially synthesized human A2ML1 protein acetylated peptide;

[0081] (2) The human A2ML1 protein acetylated peptide was conjugated with the carrier protein, and the conjugate was purified by desalting column and then immunized with non-human animals.

[0082] It should be noted that the coupling operations in steps (1) and (2) both adopt the method in Example 2. The specific procedures for animal immunization and blood collection are shown in Table 2 below.

[0083] Table 2 Animal Immunization Blood Collection Procedure

[0084]

[0085] (3): Antibody titer was tested on the serum of immunized non-human animals, the serum of immunized non-human animals was collected, and the antibodies were purified;

[0086] The antibody titer ELISA detection method includes the following steps:

[0087] a. Solution preparation:

[0088] Coating solutions: 50 mM Na2CO3 (pH 9.6), 20 mM Tris-HCl (pH 8.5) or 10 mM PBS (pH 7.4);

[0089] Blocking solutions: Commonly used sealing agents include BSA, skim milk powder, casein, and gelatin.

[0090] Washing solution: PBST or pure water;

[0091] b. Experimental steps:

[0092] 1) Dissolve the antigen in the coating solution at an appropriate concentration;

[0093] 2) Add 100 μl of antigen to the corresponding well and incubate overnight at 4°C;

[0094] 3) Empty the liquid and pat dry any remaining liquid, then rinse 3 times with detergent;

[0095] 4) Add 200 μl of blocking solution to each well and incubate at 37°C for 1 hour;

[0096] 5) Empty the liquid and pat dry any remaining liquid, then rinse 3 times with detergent;

[0097] 6) Add 100 μl of primary antibody to each well and incubate at 37°C for 1 hour;

[0098] 7) Empty the liquid and pat dry any remaining liquid, then rinse 3 times with detergent;

[0099] 8) Add 100 μl of secondary antibody to each well and incubate at 37°C for 1 hour;

[0100] 9) Empty the liquid and pat dry any remaining liquid, then rinse 5 times with detergent;

[0101] 10) Pat dry any remaining liquid in the wells, add 100 μl of colorimetric solution to each well, and develop the color at 37°C in the dark for 10 min;

[0102] 11) Add 50 μl of 2M H2SO4 to each well to stop the color development and immediately read the OD value at 450 nm.

[0103] c. Serum ELISA test results are shown in Table 3 below.

[0104] Table 3. Serum ELISA Detection Results

[0105]

[0106] Both R02927 and R02928 mixed serum ELISA tests were positive (1:16000, OD value>1). Two-step affinity purification and ELISA antibody identification will be arranged. The antibody affinity purification results are shown in Table 4 below.

[0107] Table 4. Antibody affinity purification results

[0108] R02927NP Sp200724BF SA210810X02 1.20 28.00 33.60 R02927P Sp200724AF SA210810X01 0.52 6.50 3.38 R02928NP Sp200724BF SA210810X02 1.10 25.00 27.50 R02928P Sp200724AF SA210810X01 0.51 9.10 4.64

[0109] S4: The purified antibody was tested, and the results are shown in Table 5 below.

[0110] Table 5. Results of antibody detection after purification

[0111]

[0112] In summary, the ELISA test of R02927P and R02928P serum immunized in this invention was positive, indicating that the antibodies prepared in this embodiment have good effects and were successfully prepared.

[0113] Example 4:

[0114] Example 4 identifies the anti-A2ML1 (acK220) polyclonal antibody synthesized in Example 3 of the present invention, and simultaneously synthesizes a polypeptide sequence containing a non-acetylated lysine at position 220: YVLPFKVEVVEPKEC as a control.

[0115] I. ELISA Identification of Anti-A2ML1 (acK220) Polyclonal Antibody

[0116] (1) Dissolve peptide A2ML1-K220 (K220 unacetylated control group) and acA2ML1-K220 (K220 acetylated experimental group) in 1×CBS coating buffer to 0.2 μg / 100 μl, spread 100 μl into each well of the microplate, and coat overnight at 4°C.

[0117] (2) On the second day, take out the coated microplate, shake off the coating solution, and tap the plate.

[0118] (3) Add blocking solution (3% BSA), 200 μl per well, and incubate at 37°C for 2 h.

[0119] (4) After sealing, take out the microplate, wash the plate three times, add 300 μl of PBST to each well, let it stand for 2 min and then shake it dry (to avoid contamination between wells), and pat the plate on a clean gauze to dry the liquid in the well.

[0120] (5) It can be directly used for the next experiment, or it can be packaged in a self-sealing bag and stored at 4℃ for later use.

[0121] (6) Add 100 μl of A2ML1 primary antibody to each well at a dilution of 1:4000 and incubate at 37°C for 1 h.

[0122] (7) After 1 hour, remove the microplate, wash it three times, add HRP secondary antibody, and incubate at 37°C for 30 minutes.

[0123] (8) After 30 minutes, remove the microplate, wash it three times, add TMB chromogenic reagent and incubate at 37°C in the dark for 15 minutes.

[0124] (9) After 15 minutes, remove the ELISA plate (which turns blue), immediately add the stop solution, shake the ELISA reader for 30 seconds, take a reading at 450 nm, and analyze the results.

[0125] II. ELISA assay detects acA2ML1-K220 antibody as a specific acetylation site antibody.

[0126] Since there are no reports on acetylation at the A2ML1-K220 site, this invention also commissioned a company to customize an acA2ML1-K220 antibody. The acA2ML1-K220 antibody was tested using ELISA to determine whether it was a specific acetylation site antibody; the results are attached. Figure 1 As shown. From the appendix Figure 1 As can be seen, the OD value of the acA2ML1-K220 antibody binding to the acA2ML1-K220 antigen is significantly greater than the OD value of the acA2ML1-K220 antibody binding to the non-acetylated A2ML1-K220 antigen. Furthermore, the OD value of the acA2ML1-K220 antibody binding to the acA2ML1-K220 antigen decreases with the dilution of the antibody concentration. These results suggest that the acA2ML1-K220 antibody is a specific antibody targeting the acA2ML1-K220 site.

[0127] Example 5:

[0128] Example 5 describes the immunohistochemical identification of the anti-A2ML1 (acK220) polyclonal antibody synthesized in Example 3 of this invention. Esophageal cancer tissue sections were used for immunohistochemical detection to verify the differential expression of acA2ML1-K220 antibody in cancerous and adjacent tissues. The specific experimental procedure includes the following steps:

[0129] (1) Place the tissue slices in a 60℃ oven and bake for 2 hours.

[0130] (2) Sectioning dewaxing and hydration procedure;

[0131] Specifically, first try dewaxing on an environmentally friendly transparent agent for 10 minutes three times, then soak in 100%-95%-85%-75%-50% ethanol for 5 minutes each in sequence.

[0132] (3) Rinse with ultrapure water for 3 minutes. The washing must be thorough.

[0133] (4) Antigen thermal retrieval: Heat the EDTA(1X) antigen retrieval solution in a microwave oven to boiling, then place the paraffin section into the boiling antigen retrieval solution and microwave on medium speed for 20-30 minutes.

[0134] (5) Stop heating and let it cool to room temperature for 20 to 30 minutes.

[0135] (6) After antigen retrieval, the slices were placed in ultrapure water and soaked twice for 3 minutes each time. Then they were washed three times with PBS for 3 minutes each time.

[0136] (7) Place the sample in 3% H2O2 endogenous peroxidase inhibitor and incubate at room temperature in the dark for 15 minutes. Then wash with PBS buffer three times for 5 minutes each time.

[0137] (8) Take out the slice, add 60 μl of primary antibody, and place it in a special incubation box at 4°C overnight.

[0138] (9) On the second day, remove the slides and warm them for 30 minutes. Then wash them three times in PBS buffer for 5 minutes each time to ensure thorough washing and prevent non-specific staining caused by incomplete washing. Discard the PBS used for the first two washes.

[0139] (10) After wiping the fluid around the tissue dry, add 60 μl of horseradish enzyme-labeled goat anti-mouse / rabbit IgG polymer (cover the sample as required by the actual situation), incubate at room temperature for 20 minutes, and wash with PBS buffer for 3 minutes × 3 times.

[0140] (11) Color development: Add an appropriate amount of freshly prepared DAB color developer and develop the color at room temperature for 5-20 minutes. Stop the color development with tap water.

[0141] (12) Counterstaining: stain in hematoxylin solution for 5-10 minutes, wash with water, soak the sections in hydrochloric acid ethanol rapid differentiation solution for about 15 seconds, continue washing with water, put the sections in 0.5% ammonia water for 10 seconds, and wash with water.

[0142] (13) Place the slices in 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol for 3 minutes each.

[0143] (14) After removing the slices, soak them in the clearing agent for 5 minutes three times in sequence.

[0144] (15) Mount the slide with neutral resin and observe it under an optical microscope.

[0145] Esophageal cancer tissue sections after immunohistochemical experiments were observed under a microscope. The microscopic views of adjacent esophageal cancer tissue and esophageal cancer tissue are shown in the attached figure. Figure 2 As shown in the figure. Statistical analysis was performed on 180 sites of esophageal cancer tissue sections for cancer and adjacent tissue. Microarray scanning was performed using a Hamamatsu digital pathology scanner (Japan), and staining intensity was scored using Image Pro Plus software. ROC analysis was performed on the expression levels of total A2ML1 protein and A2ML1-K220 acetylated protein in cancer tissues. The sites with the highest specificity and sensitivity were selected as the dividing criteria, and high and low expression groups were formed. Kaplan-Meier survival analysis results are attached. Figure 3 As shown, from the appendix Figure 3 As can be seen, high expression levels of A2ML1 protein in cancer tissue were significantly associated with prolonged patient survival (P = 0.0346). Simultaneously, high expression of A2ML1-K220 acetylated protein in cancer tissue also tended to prolong patient survival (P = 0.112). Further analysis of A2ML1 protein and acetylated protein expression yielded the following results (see attached figure). Figure 4 As shown, from the appendix Figure 4The results show that when both total A2ML1 protein and acetylated protein are expressed at low levels, patient survival is significantly prolonged (P = 0.085). This suggests that total A2ML1 protein may be a protective factor against esophageal cancer, and its K220 acetylation mutation site exhibits a similar trend.

[0146] The above experimental results demonstrate that the acetylated polyclonal antibody of the present invention can specifically recognize the acK220 acetylation site of A2ML1 protein, and can be used to detect the acetylation level of this site in tumor cells such as esophageal cancer, providing a tool for exploring the mechanisms of tumor cell proliferation and metastasis, helping tumor diagnosis, and guiding its clinical prognosis.

[0147] Example 6:

[0148] Example 6 provides a detection kit containing the following components: antibody prepared in Example 3, antigen retrieval solution, PBS buffer solution, enzyme blocking agent, horseradish enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB chromogenic agent, hematoxylin staining solution, ethanol, environmentally friendly clearing agent, 0.5% ammonia water and ultrapure water.

[0149] The antigen retrieval solution may be EDTA(1X) antigen retrieval solution; the pH of the PBS buffer solution is 7.4; the blocking agent is an endogenous peroxidase blocking agent, such as 3% H2O2; and the environmentally friendly clearing agent is Van-Clear environmentally friendly clearing agent.

[0150] This embodiment also provides a method for using the above-mentioned test kit, including the following steps:

[0151] (1) The tissue to be tested was routinely paraffin-cut, dewaxed on a shaker with an environmentally friendly clearing agent and ethanol, and then rinsed with ultrapure water.

[0152] (2) Heat the antigen retrieval solution to boiling, put the paraffin section into the boiling antigen retrieval solution, microwave on medium-high speed, cool to room temperature and then soak in ultrapure water, then wash with PBS 3 times.

[0153] (3) Place the sample in 3% H2O2, an endogenous peroxidase inhibitor, and incubate at room temperature in the dark. Wash with PBS buffer.

[0154] (4) Take out the slice, add diluted acetylated antibody against the K220 site of human A2ML1 protein (antibody prepared in Example 3), put it in an incubator, freeze overnight at 4°C, and wash thoroughly in PBS buffer.

[0155] (5) After wiping the fluid around the tissue dry, add horseradish enzyme-labeled goat anti-mouse / rabbit IgG polymer, incubate at room temperature, wash with PBS buffer, add an appropriate amount of freshly prepared DAB chromogenic agent, develop color at room temperature, stop the color development with tap water, stain in hematoxylin staining solution, wash with water and then soak the section in 0.5% ammonia water, and continue to wash with water.

[0156] (6) Place the sections in ethanol one by one, remove them and place them in a clearing agent, mount them with neutral resin, and observe them under an optical microscope.

[0157] This invention selected 14 peptides near the 220th threonine residue (K220) of the human A2ML1 protein as candidate peptides, and synthesized the peptide containing acK220 and prepared the complete antigen using artificial methods. The secondary structure, immunogenicity, hydrophilicity / hydrophobicity, and surface accessibility of the amino acid sequence near the 220th residue of the A2ML1 protein were analyzed to determine a suitable peptide sequence for artificial synthesis. The synthesized peptide was conjugated with a maleimide-activated vector mcKLH, and the conjugated product was purified by desalting column purification and then used to immunize New Zealand rabbits. The antibody titer of the rabbit serum after five immunizations was detected by ELISA. After the titer reached the ideal value, the immunized rabbit serum was collected, and the antibody was purified using a peptide-coated cyanogen bromide-activated sepharose (CNBr-activated sepharose) affinity purification column. The purified antibody was then identified by ELISA and other methods. The identification results show that the polyclonal antibody can specifically recognize the acK220 site of the A2ML1 protein, and can be used to detect the acetylation level of this site in tumor cells. This provides a tool for exploring the mechanisms of tumor cell proliferation and metastasis, helps in tumor diagnosis, and can guide the clinical prognosis.

[0158] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An acetylated polypeptide of human A2ML1 protein, characterized in that: The sequence of the human A2ML1 protein acetylated polypeptide is YVLPFK(acetyl)VEVVEPKEC, where acetyl represents acetylation.

2. An antigen, characterized in that: The antigen is a conjugate of the human A2ML1 protein acetylated polypeptide and the carrier protein as described in claim 1.

3. An antigen according to claim 2, characterized in that: The carrier protein includes any one of KLH, OVA, THY, or BSA.

4. An antibody, characterized in that: The antibody can specifically bind to the human A2ML1 protein acetylated polypeptide described in claim 1.

5. An antibody according to claim 4, characterized in that: The antibody is a polyclonal antibody or an antibody fragment that has undergone chemical or enzymatic digestion.

6. An antibody according to claim 5, characterized in that, The method for preparing the polyclonal antibody includes the following steps: S1: Synthetic human A2ML1 protein acetylated peptide; S2: The acetylated peptide of human A2ML1 protein was conjugated with a carrier protein, and the conjugate was purified by desalting column and then immunized with non-human animals. S3: Detect antibody titers in immunized non-human animal serum, collect immunized non-human animal serum, and purify antibodies; S4: Detect the purified antibody.

7. The use of the human A2ML1 protein acetylated polypeptide as described in claim 1, or the antigen as described in claim 2 or 3, or the antibody as described in any one of claims 4-6 in the detection of acetylation level at the K220 acetylation site of human A2ML1 protein.

8. The use of the human A2ML1 protein acetylated polypeptide as described in claim 1, or the antigen as described in claim 2 or 3, or the antibody as described in any one of claims 4-6 in the preparation of reagents or kits for the diagnosis, treatment and prognosis of tumors.

9. A detection kit, characterized in that: The antibody includes any one of claims 4-6, and further includes antigen retrieval solution, PBS buffer solution, enzyme blocking agent, horseradish enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB chromogenic agent, hematoxylin staining solution, ethanol, environmentally friendly clearing agent, 0.5% ammonia water and ultrapure water.

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