An esophageal cancer biomarker, antigen, antibody, and preparation method and application thereof

Abstract

The application discloses an esophageal cancer biomarker, an antigen, an antibody and a preparation method and application thereof, the biomarker is a human SERPINB3 acetylated polypeptide, and the polypeptide sequence is DAIK-K(acetyl)-FYQTSVESVDC.The antigen is a conjugate of the human SERPINB3 acetylated polypeptide and a carrier protein.The antibody can be used for detecting the acetylation level of the human SERPINB3 acetylated polypeptide K125 site in a biological sample.A detection kit of the application can be used for detecting the difference in the acetylation level of the human SERPINB3 protein K125 site between different samples, the application explores the influence of the acetylation of the SERPINB3 protein K125 site on the tumor cell proliferation, migration and invasion process, and provides a new tool for tumor diagnosis and prognosis judgment, and research on the tumor cell proliferation and metastasis mechanism.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and in particular to an esophageal cancer biomarker, antigen, antibody, preparation method, and application thereof. Background Technology

[0002] The serine proteinase inhibitor (Serpin) superfamily is the largest family of protease inhibitors discovered to date. This family participates in regulating many physiological and pathological processes in the body, such as blood coagulation, fibrinolysis, apoptosis, inflammation, complement activation, and cell migration. The Serpin B family is the largest member of the human Serpin family. Many members of the Serpin B family are closely related to tumorigenesis and development. Currently, Serpin B1, Serpin B3, Serpin B4, and Serpin B5 are the most studied in digestive system tumors. Serpin B3, in particular, can inhibit the activity of cysteine ​​proteases such as cathepsins K, L, S, and V, exhibiting anti-apoptotic, cell proliferation, and migration effects. Serpin B3 plays an important role in tumorigenesis and development. Studies have shown that Serpin B3 is closely related to the occurrence, invasion, and migration of squamous cell carcinomas such as cervical cancer, lung cancer, esophageal cancer, and head and neck tumors.

[0003] Protein acetylation refers to the addition of acetylated groups to proteins, a type of post-translational modification. It 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 specialized functional complexes. 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 novel protein acetylation sites and acetylation modification omics can provide important guidance for elucidating the causes of more diseases and offer new insights for disease treatment.

[0004] Esophageal cancer (also known as esophageal squamous cell carcinoma) is one of the ten most common malignant tumors worldwide. my country has a high incidence of esophageal cancer, with squamous cell carcinoma being the primary histological type. Esophageal cancer often presents with few or no symptoms, and most patients are already in an advanced stage when they seek medical attention. The overall 5-year survival rate for patients in the middle and late stages is only about 10%, while the 5-year survival rate can reach 90% if surgery is performed in the early stages of esophageal cancer. Therefore, finding key technologies for early detection of esophageal cancer is crucial for reducing its incidence and mortality. Currently, gastroscopy is the most effective clinical examination for esophageal cancer; however, gastrointestinal endoscopy is invasive, inconvenient, and causes patient discomfort, with risks of infection and bleeding, and is also expensive. Therefore, there is an urgent need for an esophageal cancer biomarker with high specificity and sensitivity for predicting the risk of developing esophageal cancer, enabling early screening to guide clinical intervention and treatment as early as possible. Summary of the Invention

[0005] To address the deficiencies in existing technologies, this invention provides an esophageal cancer biomarker, an antigenic peptide, and an antibody that can specifically bind to it, for detecting the acetylation level of the SERPINB3 peptide at the K125 site in esophageal cancer cells.

[0006] This invention provides an esophageal cancer biomarker, wherein the biomarker is a human SERPINB3 acetylated polypeptide, the sequence of which is DAIK-K(acetyl)-FYQTSVESVDC, where acetyl represents acetylation.

[0007] Furthermore, the esophageal cancer biomarker is any one of the biomarkers used for the diagnosis, prognosis, and treatment efficacy detection of esophageal cancer.

[0008] The present invention also provides an antigen, wherein the antigen is a conjugate of the human SERPINB3 acetylated polypeptide and a carrier protein; the carrier protein is selected from KLH, OVA, THY or BSA.

[0009] The present invention also provides an antibody that can specifically bind to the human SERPINB3 acetylated polypeptide or the antigen.

[0010] Furthermore, the polyclonal antibody is obtained by immunizing non-human animals with a conjugate of DAIK-K(acetyl)-FYQTSVESVDC and a carrier protein, followed by purification and extraction of the serum from the non-human animals.

[0011] Furthermore, the antibody is used to detect the acetylation level of the K125 acetylation site of the SERPINB3 protein in biological samples.

[0012] This invention also provides a method for preparing an antibody, comprising the following steps:

[0013] S1: The artificially synthesized human SERPINB3 acetylated polypeptide;

[0014] S2: Human SERPINB3 acetylated polypeptide is coupled to a carrier protein;

[0015] S3: Animal immunization and blood collection;

[0016] S4: Antibody titer detection;

[0017] S5: Antibody affinity purification;

[0018] S6: The purified antibody is then identified.

[0019] Furthermore, the use of the biomarker, antigen, or antibody in the preparation of reagents or kits for esophageal cancer detection.

[0020] The present invention also provides an esophageal cancer screening kit, comprising the antibody.

[0021] Furthermore, the screening kit also 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.

[0022] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0023] (1) This invention provides a biomarker for detecting esophageal cancer, filling the gap in biomarkers for acetylated modified proteins in esophageal cancer and providing a new approach for biomarker identification.

[0024] (2) The present invention uses artificial synthesis to prepare an antigenic polypeptide of the K125 acetylation site of SERPINB3 protein, and to prepare the corresponding polyclonal antibody.

[0025] (3) The polyclonal antibody of the present invention can specifically recognize the K125 acetylation site of SERPINB3 protein and detect the acetylation level of this site.

[0026] (4) The present invention provides a kit for esophageal cancer screening. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the process of the present invention;

[0029] Figure 2 The heatmap shows the top ten differentially expressed acetylated modified proteins in normal esophageal tissue and esophageal cancer tissue of Example 1 of the present invention (arranged from top to bottom according to the degree of difference);

[0030] Figure 3 The results are from the ELISA experiment in Example 4 of this invention.

[0031] Figure 4 This is an immunohistochemical section image of Example 5 of the present invention;

[0032] Figure 5 This is a box plot showing the differential expression in Embodiment 5 of the present invention;

[0033] Figure 6 This is a survival curve diagram of Embodiment 5 of the present invention;

[0034] Figure 7 This is the ROC curve of Embodiment 5 of the present invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0036] The operation process of this invention is as follows: Figure 1 As shown.

[0037] Example 1: Synthesis of SERPINB3 acetylated polypeptide

[0038] I. Obtaining SERPINB3 acetylated peptide

[0039] 1. The amino acid sequence of the human SERPINB3 protein obtained from GenBank is shown below:

[0040] >NP_008850.1

[0041] 1MNSLSEANTKFMFDLFQQFRKSKENNIFYSPISITSALGMVLLGAKDNTAQQIKKVLHFD

[0042] 61QVTENTTGKAATYHVDRSGNVHHQFQKLLTEFNKSTDAYELKIANKLFGEKTYLFLQEYL

[0043] 121DAIKKFYQTSVESVDFANAPEESRKKINSWVESQTNEKIKNLIPEGNIGSNTTLVLVNAI

[0044] 181YFKGQWEKKFNKEDTKEEKFWPNKNTYKSIQMMRQYTSFHFASLEDVQAKVLEIPYKGKD

[0045] 241LSMIVLLPNE IDGLQKLEEKLTAEKLMEWTSLQNMRETRVDLHLPRFKVEESYDLKDTLR

[0046] 301TMGMVDIFNGDADLSGMTGSRGLVLSGVLHKAFVEVTEEGAEAAAATAVVGFGSSPTSTN

[0047] 361EEFHCNHPFL FFIRQNKTNS ILFYGRFSSP

[0048] 2. The characteristics of human SERPIB3 protein were analyzed using ProtParam software. The results are shown in Table 1:

[0049] Table 1. Characteristics of SERPIB3 protein

[0050] Number of amino acids 390 Molecular weight 27900 Theoretical pI 6.52

[0051] The results showed that the human SERPINB3 protein contains 390 amino acids, has a molecular weight of 27,900 Daltons, an isoelectric point of 6.52, and is a neutral protein.

[0052] 3. Differentially expressed SERPINB3 acetylation in normal esophageal tissue and esophageal cancer tissue was obtained by mass spectrometry analysis, revealing an acetylation mutation at lysine position 125, such as... Figure 2 As shown, the K125 acetylation level of SERPINB3 protein is significantly downregulated in esophageal cancer compared to normal tissue.

[0053] The results showed that the K125 site in the SERPINB3 amino acid sequence is located in the intracellular segment of SERPINB3 and is a site for acetylation modification of the SERPINB3 protein. The level of acetylation at the K125 site of the SERPINB3 protein may serve as a biomarker for the diagnosis of esophageal cancer.

[0054] Based on the above analysis, the selected polypeptide sequence is DAIK-K(AC)-FYQTSVESVDC.

[0055] II. Synthesis of Polypeptides

[0056] 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 125 was acetylated: DAIK-K(AC)-FYQTSVESVDC. A control peptide sequence containing a non-acetylated lysine residue at position 125 was also synthesized: DAIKKFYQTSVESVDC. The peptides were synthesized by Suzhou Baiyuan Biotechnology Co., Ltd.

[0057] 1. The peptide is synthesized using a solid-phase synthesis method. 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, the peptide chain is lengthened by reacting with an excess of activated carboxyl components. 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 polypeptide.

[0058] 2. Purification: RP-HPLC purification

[0059] (1) HPLC conditions:

[0060] Mobile phase: A) 0.1% TFA aqueous solution; B) 0.1% TFA acetonitrile solution;

[0061] Gradient: A / B(90 / 40) to A / B(40 / 90) for 30 minutes;

[0062] Flow rate: 1 mL / min;

[0063] Temperature: Room temperature (23℃);

[0064] Detection: 214nm ultraviolet light;

[0065] Sample: freeze-dried crude product.

[0066] (2) Steps:

[0067] a. Dissolve the crude product in the mobile phase;

[0068] b. Inject 20-30 mg (2-2.5 mL) of sample;

[0069] c. Collect the main peak into a 50 mL tube;

[0070] d. Freeze-drying.

[0071] (3) Identification: LC / MS

[0072] Conditions: Mobile phase: A) 0.05% TFA aqueous solution; B) 0.1% TFA acetonitrile solution;

[0073] Gradient: A / B(90 / 10) to A / B(40 / 60) 15 min;

[0074] Flow rate: 1 mL / min;

[0075] Temperature: Room temperature (23℃);

[0076] Detection: 214nm ultraviolet light;

[0077] Atmospheric pressure ionization mass spectrometry (MS API): electrospray ionization source (ESI).

[0078] The results of polypeptide synthesis are shown in Table 2.

[0079] Table 2 Results of peptide synthesis

[0080] polypeptide synthesis number polypeptide sequence Peptide purity (%) 20072402 DAIK-K(AC)-FYQTSVESVDC 96.81％ 20072412 DAIKKFYQTSVESVDC 96.32％

[0081] Example 2: Coupling of SERPINB3 acetylated peptide with carrier protein

[0082] 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 limpet hemacyanin (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.

[0083] Peptide conjugation process:

[0084] 1. Solution preparation: The coupling buffer (AH solution) includes Na2HPO4, NaH2PO4, NaCl, and EDTA, and the pH is adjusted to 7.2.

[0085] 2. Experimental steps:

[0086] (1) Preparation of the post bed:

[0087] Wash the column bed with pure water and coupling buffer.

[0088] (2) Preparation:

[0089] Dissolve the peptide in a small amount of dimethylformamide (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.

[0090] (3) KLH, Sulfo-SMCC preparation:

[0091] Based on the mass ratio of total conjugated peptide to pure KLH = 1:1, the amount of pure KLH is calculated; based on the mass ratio of pure KLH to Sulfo-SMCC = 10:1, the amount of Sulfo-SMCC is calculated.

[0092] (4) KLH and Sulfo-SMCC reactions and reactant collection:

[0093] Dissolve the weighed KLH in an appropriate amount of AH solution to prepare a final concentration of 10 mg / mL. 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.

[0094] (5) Coupling of KLH and Sulfo-SMCC reactants with peptides:

[0095] Add the appropriate amount of KLH and Sulfo-SMCC reactant to each tube of peptide to be coupled, react at room temperature for 2 hours or overnight at room temperature, mix thoroughly with a vertical mixer, and store the coupled peptide at -20°C; Note: The peptide is coupled using KLH carrier protein and the resulting coupled peptide is used as an immunogenic antigen.

[0096] Example 3: Preparation of Rabbit Polyclonal Antibody Against SERPINB3 Peptide

[0097] 1. The immunization and blood collection process is shown in Table 3:

[0098] Table 3 Immunization and Blood Collection Procedures

[0099]

[0100]

[0101] 2. Antibody titer ELISA detection method

[0102] ① Serum ELISA test:

[0103] (1) Solution preparation:

[0104] Coating solutions: 50 mM Na2CO3 (pH 9.6), 20 mM Tris HCl (pH 8.5) or 10 mM PBS (pH 7.4);

[0105] Blocking solutions: Commonly used sealing agents include BSA, skim milk powder, casein, and gelatin.

[0106] Washing solution: PBST or pure water.

[0107] (2) Experimental steps:

[0108] a. Dissolve the antigen in the coating solution at an appropriate concentration;

[0109] b. Add 100 μL of antigen to the corresponding well and incubate overnight at 4°C;

[0110] c. Empty the liquid and pat dry any remaining liquid, then rinse 3 times with detergent;

[0111] d. Add 200 μL of blocking solution to each well and incubate at 37°C for 1 hour;

[0112] e. Empty the liquid and pat dry any remaining liquid, then rinse 3 times with detergent;

[0113] f. Add 100 μL of primary antibody to each well and incubate at 37°C for 1 hour;

[0114] g. Empty the liquid and pat dry any remaining liquid, then rinse 3 times with detergent;

[0115] h. Add 100 μL of secondary antibody to each well and incubate at 37°C for 1 hour;

[0116] i. Empty the liquid and pat dry any remaining liquid, then rinse 5 times with detergent;

[0117] j. Pat dry any remaining liquid in the wells, add 100 μL of colorimetric solution to each well, and develop the color at 7°C in the dark for 10 min;

[0118] k. Add 50 μL of 2M H2SO4 to each well to stop the color development and immediately read the OD value at 450 nm.

[0119] (3) Serum ELISA test results are shown in Table 4:

[0120] Table 4. Serum ELISA Detection Results

[0121]

[0122] Note: The above mixed serum from rabbits showed positive results in ELISA (1:27000, OD value>1.0). Antigen affinity purification and ELISA antibody identification were performed.

[0123] ②The antibody affinity purification results are shown in Table 5:

[0124] Table 5. Antibody affinity purification results

[0125]

[0126] ③ The antibody ELISA results are shown in Table 6:

[0127] Table 6. Antibody ELISA Results

[0128]

[0129]

[0130] The R01751 serum and antibody (SA201124X02) ELISA test (reaction with acetylated antigen) of this project were positive, and the antibody reaction with acetylated antigen (20072402) at a concentration of 0.125 μg / mL was much greater than the reaction with non-acetylated antigen (20072412); the R01752 serum and antibody (SA201125X02) ELISA test was positive, and the antibody reaction with acetylated antigen (20072402) at a concentration of 0.125 μg / mL was much greater than the reaction with non-acetylated antigen (20072412); the antibody was purified by a two-step affinity method, and a total of 9.05 mg of acetylated antibody was obtained.

[0131] The above results indicate that the antibody prepared in this embodiment has good efficacy and was successfully prepared.

[0132] Example 4: Identification of Rabbit Polyclonal Antibody Against SERPINB3 Peptide

[0133] I. ELISA Identification of Anti-SERPINB3 (acK125) Polyclonal Antibody

[0134] Since there are no reports on the acetylation of acSERPINB3-Lys125, this invention commissioned a company to customize the acSERPINB3-Lys125 antibody.

[0135] (1) Dissolve the peptides SERPINB3-Lys125 (Lys125 unacetylated control group) and acSERPINB3-Lys125 (Lys125 acetylated experimental group) in 1×CBS coating buffer to a concentration of 0.2 μg / 100 μL, spread 100 μL into each well of the microplate, and coat overnight at 4°C.

[0136] (2) On the second day, take out the coated microplate, shake off the coating solution, and tap the plate.

[0137] (3) Add blocking solution (3% BSA), 200 μL per well, and incubate at 37°C for 2 h.

[0138] (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 minutes and then shake it dry (to avoid contamination between wells). Place the plate on a clean gauze and pat the liquid in the well dry.

[0139] (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.

[0140] (6) Add 100 μL of SERPINB3 primary antibody to each well at a dilution of 1:4000 and incubate at 37°C for 1 h.

[0141] (7) After 1 hour, remove the microplate, wash it three times, add HRP secondary antibody, and incubate at 37°C for 30 minutes.

[0142] (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.

[0143] (9) After 15 minutes, remove the microplate (which turns blue), immediately add the stop solution, shake the microplate for 30 seconds, take a reading at 450 nm, and analyze the results.

[0144] like Figure 3 As shown, the OD value of acSERPINB3-Lys125 antibody binding to acSERPINB3-Lys125 antigen is significantly greater than the OD value of acSERPINB3-Lys125 antibody binding to non-acetylated SERPINB3-Lys125 antigen. Furthermore, the OD value of acSERPINB3-K125 antibody binding to acSERPINB3-K125 antigen decreases with increasing antibody concentration. These results suggest that the acSERPINB3-K125 antibody is an antibody with a certain specificity to the acSERPINB3-K125 site.

[0145] The above experimental results demonstrate that the acetylated polyclonal antibody of the present invention can specifically recognize the acK125 acetylation site of SERPINB3 protein.

[0146] Example 5: Immunohistochemical identification of anti-SERPINB3 (acK125) polyclonal antibody

[0147] Immunohistochemistry was performed using esophageal cancer tissue microarrays to verify the differential expression of ac-SERPINB3(K125) antibody in esophageal cancer and adjacent tissues.

[0148] 1. Experimental steps:

[0149] (1) Bake the slices in a 60℃ oven for 2 hours.

[0150] (2) Sectioning dewaxing and hydration procedure:

[0151] a. Dewaxing the environmentally friendly transparent agent on a shaker for 10 minutes, 3 times;

[0152] b. Soak in 100%-95%-85%-75%-50% ethanol for 5 minutes each.

[0153] (3) Rinse with ultrapure water for 3 minutes. The washing must be thorough.

[0154] (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.

[0155] (5) Stop heating and let it cool to room temperature for 20 to 30 minutes.

[0156] (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.

[0157] (7) Place the sample in 3% H2O2 endogenous peroxidase inhibitor and incubate at room temperature in the dark for 15 minutes. Wash with PBS buffer three times for 5 minutes each time.

[0158] (8) Take out the slide, add 60 μL of primary antibody, and place it in a special incubation box at 4°C overnight.

[0159] (9) On the second day, take out the slides and warm them for 30 minutes. Then wash them in PBS buffer three times for 5 minutes each time to ensure thorough washing and prevent non-specific staining caused by incomplete washing (discard the PBS from the first two washes).

[0160] (10) After wiping the fluid around the tissue dry, add 60 μL of horseradish enzyme-labeled goat anti-rabbit IgG polymer (cover the sample as required by the actual situation), incubate at room temperature for 20 minutes, wash with PBS buffer for 3 minutes, and repeat 3 times.

[0161] (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.

[0162] (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.

[0163] (13) Place the slices in 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol for 3 minutes each.

[0164] (14) After removing the slices, place them in the clearing agent for 5 minutes each time, repeating 3 times.

[0165] (15) Mount the slide with neutral resin and observe it under an optical microscope.

[0166] 2. Appraisal Results

[0167] Esophageal cancer sections after immunohistochemical experiments were observed under a microscope. Figure 4 As shown, the stained portion in esophageal cancer tissue is relatively smaller compared to adjacent non-cancerous tissue. Immunohistochemical statistical results are as follows... Figure 5 As shown, the expression level of SERPINB3 (acK125) protein in adjacent normal tissues was higher than that in esophageal cancer. Statistical analysis was performed on 180 sites of esophageal cancer microarray data, using a Hamamatsu digital pathology scanner (Japan) for microarray scanning and Image Pro Plus software for staining intensity scoring. ROC analysis was performed on the expression level of SERPINB3-K125 acetylated protein in cancer tissues, and the sites with the highest specificity and sensitivity were selected as the dividing criteria for high and low expression groups. Kaplan-Meier survival analysis results showed... Figure 6 As shown, high expression levels of SERPINB3-K125 acetylated protein in cancerous tissues were associated with significantly prolonged patient survival (P<0.0001). This example statistically analyzed the expression levels in cancerous and adjacent tissues, and the results are as follows... Figure 7 As shown, the ROC curve has an area under the curve of 0.81, which is diagnostically significant.

[0168] The above results demonstrate that the SERPINB3 (acK125) protein of the present invention is beneficial to patient survival in cancer tissue and is closely related to tumor cells. The acetylated polyclonal antibody can specifically recognize the acetylated K125 site of SERPINB3. Detecting the acetylation level of this site can achieve the purpose of tumor diagnosis and clinical prognosis assessment.

[0169] Example 6: A test kit and its usage method

[0170] I. The test kit specifically includes the following components:

[0171] Polyclonal antibody (the antibody prepared in Example 3).

[0172] It may also contain 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.

[0173] Antigen retrieval solution can be EDTA(1X) antigen retrieval solution;

[0174] The pH of the PBS buffer solution is 7.4;

[0175] The inhibitor is an endogenous peroxidase inhibitor, such as 3% H2O2;

[0176] The environmentally friendly transparent agent is Van-Clear.

[0177] II. This embodiment also provides a method for using the above-mentioned test kit, including the following steps:

[0178] (1) The tissue to be tested was routinely dewaxed in paraffin, and then dewaxed in a shaker with an environmentally friendly clearing agent and ethanol, and then rinsed with ultrapure water.

[0179] (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, and then wash with PBS 3 times.

[0180] (3) Place the sample in 3% H2O2, an endogenous peroxidase inhibitor, and incubate at room temperature in the dark. Wash with PBS buffer.

[0181] (4) Take out the slice, add diluted acetylated antibody against the K125 site of human SERPINB3 protein (antibody prepared in Example 3), put it in an incubator, freeze overnight at 4°C, and wash thoroughly in PBS buffer.

[0182] (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.

[0183] (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.

[0184] This invention selected 14 peptides near the 125th threonine residue (K125) of the SERPINB3 protein as candidate peptides and synthesized the peptide containing acK125 using artificial methods, and prepared a complete antigen. Mass spectrometry analysis revealed differentially expressed SERPINB3 acetylation in normal esophageal tissue and esophageal cancer tissue, showing an acetylation mutation at lysine 125, and a suitable peptide sequence was determined for artificial synthesis. The synthesized peptide was conjugated with a maleimide-activated vector mcKLH, and the conjugated product was purified using a desalting column and used to immunize New Zealand rabbits. After five immunizations, the rabbit serum was analyzed for antibody titer using ELISA. Once the titer reached the desired 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 immunohistochemistry. The results showed that the polyclonal antibody specifically recognizes the acK125 site of the SERPINB3 protein. Survival curve and ROC curve analysis show that detecting the acetylation level at this site in tumor cells can help in tumor diagnosis and guide clinical prognosis, providing a tool for exploring the mechanisms of tumor cell proliferation and metastasis.

[0185] 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 within the protection scope of the present invention.

Claims

1. An esophageal cancer biomarker, characterized by, The esophageal cancer biomarker is a human SERPINB3 acetylated polypeptide, the sequence of which is DAIK-K(acetyl)-FYQTSVESVDC, where acetyl represents acetylation.

2. The esophageal cancer biomarker of claim 1, characterized in that, The esophageal cancer biomarker is any one of the biomarkers used for the diagnosis, prognosis, and treatment efficacy of esophageal cancer.

3. An antigen, characterized in that, The antigen is a conjugate of the human SERPINB3 acetylated polypeptide and the carrier protein as described in claim 1; the carrier protein is selected from KLH, OVA, bovine thyroglobulin THY, or BSA.

4. An antibody, characterized in that, The antibody is a polyclonal antibody obtained by immunizing non-human animals with a conjugate of DAIK-K(acetyl)-FYQTSVESVDC and a carrier protein, followed by purification and extraction of the serum from the non-human animals. The carrier protein is keyhole hemocyanin; the non-human animal is a rabbit.

5. The antibody of any one of claim 4, characterized in that, The antibody is used to detect the acetylation level of the K125 acetylation site of the SERPINB3 protein in biological samples.

6. A method for the production of an antibody according to claim 4 or 5, characterized in that, Includes the following steps: S1: Artificially synthesize the human SERPINB3 acetylated polypeptide as described in claim 1; S2: Human SERPINB3 acetylated polypeptide is coupled to a carrier protein; S3: Animal immunization and blood collection; S4: Antibody titer detection; S5: Antibody affinity purification; S6: The purified antibody is then identified.

7. The use of the biomarker of claim 1, the antigen of claim 3, or the antibody of any one of claims 4-5 in the preparation of reagents or kits for esophageal cancer detection.

8. An esophageal cancer screening kit, characterized by, Includes the antibody as described in any one of claims 4 to 5.

9. The screening kit of claim 8, wherein, The screening kit also includes antigen retrieval solution, PBS buffer solution, enzyme blocking agent, horseradish enzyme-labeled goat anti-mouse IgG polymer or horseradish enzyme-labeled goat anti-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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