A method for preparing a specific phenylacetylated lysine pan antibody

Abstract

This invention provides a method for preparing specific phenylacetylated lysine pan-antibodies, relating to the fields of biomedicine and antibody engineering technology. The method for preparing specific phenylacetylated lysine pan-antibodies includes a target recognition body containing a phenylacetylated modification group specifically recognizing the lysine-amino group, which is covalently bound to the lysine-amino group via an amide bond. This group is independent of the protein primary sequence and can bind to phenylacetylated lysine sites in histones and various non-histone substrates. A specific distinguisher precisely distinguishes between phenylacetylated and structurally similar acylation modifications, with a specific binding rate to phenylacetylated modifications >90%. By synthesizing specific phenylacetylated antigens, optimizing recognition sites, and refining purification processes, highly specific and sensitive pan-antibodies are prepared, filling detection gaps and solving many problems in existing technologies.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and antibody engineering technology, specifically to a method for preparing specific phenylacetylated lysine pan-antibodies. Background Technology

[0002] Protein or polypeptide modification, a ubiquitous post-translational regulatory mechanism in organisms, refers to the process by which specific functional groups are covalently linked to amino acid residues of proteins after synthesis, through enzymatic reactions or chemical modifications. This modification process does not alter the primary structure of the protein, but it can precisely regulate a series of core life activities within the cell, such as gene expression, signal transduction, metabolic homeostasis, protein degradation, and subcellular localization, by changing the protein's charge distribution, stereoconformity, or intermolecular interaction interfaces. Currently, over 400 types of protein modifications have been identified. Among them, the acylation family has attracted much attention due to its involvement in numerous physiological and pathological processes; common types include acetylation, lactylation, phosphorylation, succinylation, crotonylation, and benzoylation.

[0003] Different modification types possess unique biological characteristics: acetylation regulates the open state of chromatin through the dynamic balance of histone acetyltransferases and deacetylases, and its dysregulation is closely related to neurodegenerative diseases such as Alzheimer's and Parkinson's; lactylation, as a metabolite-derived modification, uses lactate as a substrate and regulates cellular metabolic reprogramming by linking lactyl groups, playing a key role in diseases such as tumors and osteoporosis; phosphorylation acts as a "molecular switch" for signal transduction through the interaction of kinases and phosphatases, and abnormal phosphorylation of epidermal growth factor receptor is one of the important mechanisms driving tumor proliferation. Benzoylation, as a specific acylation modification on lysine residues, has a unique benzene ring structure, and its regulatory effects on protein structure and function are significantly different from traditional acylation modifications, especially in histone epigenetic regulation, where it is closely related to gene transcription activation. Based on the principle of structural similarity, phenylacetyl groups, which have a high homology to the chemical structure of benzoyl groups, can also covalently induce phenylacetylation of lysine residues. The discovery of this modification type further expands the research scope of the acylation modification family. As a novel post-translational modification, the in vivo presence and dynamic changes of phenylacetylation have not been fully elucidated, and establishing efficient and sensitive detection techniques is of great significance for revealing its biological functions.

[0004] Typically, the detection of protein modifications requires antibodies that specifically recognize the modification. For example, lysine acetylation modification can be recognized by pan-alcoholic antibodies against lysine acetylation. Currently, while antibodies against lysine benzoylation have been successfully developed for the detection of lysine benzoylation, antibodies against lysine phenylacetylation have not been successfully developed. Establishing an in vivo detection method for phenylacetylation modification would not only provide technical support for elucidating the physiological function of this modification but also hold promise as a novel molecular marker for early disease diagnosis, opening new avenues for developing precision treatment strategies targeting modification regulation. Therefore, it is necessary to develop a novel antibody for the detection of lysine phenylacetylation modification. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing specific pan-antibodies against phenylacetylated lysine. This method solves the problems of existing technologies lacking specific detection antibodies for lysine phenylacetylation modification, lacking efficient and sensitive detection methods, and being unable to elucidate the in vivo presence and dynamic changes of this modification, which seriously restricts its functional research and clinical translation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a specific phenylacetylated lysine pan-antibody, comprising: The target recognizer contains a substance that specifically recognizes lysine. -amino group, which specifically recognizes lysine - A phenylacetylated modification group covalently linked to the amino group via an amide bond, independent of the protein primary sequence, can bind to phenylacetylated lysine sites in various histone and non-histone substrates; A specific distinguisher that precisely distinguishes phenylacetylation from structurally similar acylation modifications, exhibiting a specific binding rate of >90% to phenylacetylation modifications and a cross-reactivity rate of <5% with acetylated, propionylated, crotonylated, benzoylated, phosphorylated, and unmodified lysine residues; The low-abundance detector is adapted to the low abundance of phenylacetylation modification in vivo, with a detection limit as low as ng, meeting the detection needs of low-expression phenylacetylated proteins under physiological conditions.

[0007] Preferably, the phenylacetylated lysine recognized by the antibody is -phenylacetyl- -methyl- -Lysine, the molecular weight of lysine residues increases by 118.0419 Da after phenylacetylation modification.

[0008] Preferably, the specificity of the antibody is verified by both competitive ELISA and dot blot experiments, which can effectively eliminate interference from other acylation modifications and ensure that there are no false positive results in subsequent protein immunoblotting and immunoprecipitation experiments.

[0009] Preferably, the antibody can detect 1-10 in a Western blot assay. The modified protein showed a positive antibody dilution greater than 54,000 in the enzyme-linked immunosorbent assay (ELISA), and the signal intensity was more than 10 times higher than that for the unmodified peptide.

[0010] Preferably, the antibody can be prepared by immunizing animals, or by screening phage display libraries, yeast display libraries, bacterial display libraries, and ribosome display libraries; The antibody can be used for the detection and enrichment of phenylacetylated peptides and proteins in dot blot, enzyme-linked immunosorbent assay, protein immunoblotting and immunoprecipitation experiments.

[0011] A method for preparing a specific pan-antibody containing phenylacetylated lysine includes the following steps: S1. Synthesize modified phenylacetylated lysine to obtain -phenylacetyl- -methyl- - Pure lysine; S2. Modified phenylacetylated lysine and its derived polypeptide are coupled to a carrier protein to prepare a complete antigen for immunization. S3. Immunize experimental animals with the whole antigen and collect and screen positive serum; S4. The positive serum was subjected to multi-stage affinity purification to obtain a highly specific phenylacetylated lysine pan-antibody.

[0012] Preferably, the specific steps for synthesizing the modified phenylacetylated lysine in step S1 are as follows: S11, with -tert-Butyloxycarbonyl- -fluorenemethyloxycarbonyl- -methyl- Using lysine as a raw material, the solution was fully dissolved in dichloromethane, and then freshly prepared HCl gas was continuously bubbled through it at room temperature for 1.5 hours. The reaction was then continued at room temperature for 5 hours under a strong acidic HCl environment. After confirming the complete reaction by thin-layer chromatography, the solution was evaporated to dryness and then purified by silica gel column chromatography to obtain... -fluorenemethyloxycarbonyl- -methyl- -Lysine; S12. Dissolve the above product in acetone, add 1.1M NaHCO3 aqueous solution and stir at room temperature for 2 hours to remove the bound HCl. After cooling to 0-5°C in an ice-water bath, slowly add an acetone solution of phenylacetyl chloride, maintaining the reaction in an ice-water bath for 1 hour. After extraction, drying, and rotary evaporation, obtain the product by silica gel column chromatography. -phenylacetyl- -fluorenemethyloxycarbonyl- -methyl- -Lysine; S13. Add piperidine to the above product and stir overnight at room temperature to remove the Fmoc protecting group. Obtain the esterified product by vacuum distillation and column chromatography. Then add sodium hydroxide and hydrolyze in an ice-water bath for 2 hours in a system of water and methanol with a volume ratio of 1:1. Adjust the pH to 3.0 with 2NHCl and evaporate to dryness. Dissolve in ethanol, filter, and evaporate to dryness to obtain the final product.

[0013] Preferably, the specific steps for preparing the complete antigen in step S2 are as follows: S21. Dissolve 10 mg of hemocyanin in 1 ml of LME buffer, and add 0.4 mg of [unclear text - possibly a typo, should be "S21"] to each solution. With 1.1mg - After mixing thoroughly, react at room temperature for 15 minutes; S22, add 1.4μ 10 minutes of reaction with β-mercaptoethanol, using Adjust the pH of the solution to 7.4; S23, Dissolve 2 mg of modified phenylacetylated lysine and its derived polypeptide in... Then add the above-mentioned activated After thoroughly mixing the solution, react at room temperature for 2 hours. S24, add 50 mM Tris to terminate the reaction. Dialyze overnight at 4°C in the solution; The sequence of the modified phenylacetylated lysine-derived polypeptide is as follows: , and , It is phenylacetylated lysine.

[0014] Preferably, the specific steps of animal immunization and serum screening in step S3 are as follows: S31. Eight-week-old New Zealand white rabbits were used. Two subcutaneous injections were administered on both shoulders and two intramuscular injections were administered on both hind legs. Each rabbit was immunized a total of four times, on day 1, day 21, day 28, and day 35, respectively. S32. On the 45th day after immunization, 30 mL of whole blood was collected for the first time. After centrifugation, the supernatant was collected and serum was screened by enzyme-linked immunosorbent assay and dot blot analysis. On days 33, 50, 65, and 70, 20 mL of whole blood was collected each time. After centrifugation, the supernatant was collected and screened repeatedly. Serum with positive test results was collected.

[0015] Preferably, the specific steps for antibody purification in step S4 are as follows: S41. After filtering the positive serum, load it into the pre-equilibrated solution. Affinity chromatography column, using After washing, elute with 150 mM glycine buffer, collect the eluent and neutralize the pH to 7.0 to obtain crude purity. ; S42, the crude pure The sample is loaded onto an antigen-peptide affinity chromatography column to specifically enrich the target antibody; S43. Load the enriched antibody onto an unmodified peptide affinity chromatography column and collect the eluent directly to remove non-specific antibodies. S44. Determine the antibody protein content, add 10% glycerol, dispense into smaller containers, and store at -20℃.

[0016] This invention provides a method for preparing specific pan-antibodies against phenylacetylated lysine. It has the following beneficial effects: 1. This invention utilizes artificially synthesized modified phenylacetylated lysine and specifically derived polypeptides as immunoantigens, and combines animal immunization and library screening as dual pathways to prepare the world's first pan-antibody for phenylacetylated lysine. This fills the gap in the lack of specific detection antibodies for this novel modification and provides core tool support for in vivo functional studies of phenylacetylated modifications.

[0017] 2. This invention designs a target lysine... The system identifies the phenylacetylation modification site and uses a dual-specificity verification system to specifically identify phenylacetylation modifications, accurately distinguishing various structurally similar acylation modifications and controlling the cross-reactivity rate to within 5%, thus avoiding false positives at the source.

[0018] 3. This invention optimizes the antibody multi-level affinity purification process and combines it with high-sensitivity performance design to achieve efficient detection of endogenous low-abundance phenylacetylated proteins, with the lowest detection limit reaching the ng level and ELISA titer exceeding 54,000, solving the core problem that existing technologies cannot detect low-abundance modifications at the physiological level.

[0019] 4. This invention establishes a standardized process for antigen synthesis, conjugation, immunization, and purification, and combines it with specific screening and non-specific antibody removal steps to comprehensively optimize the antibody preparation process, significantly improving the success rate and batch-to-batch stability, and solving the industry pain points of difficult antigen synthesis and large batch-to-batch antibody differences in existing technologies. Attached Figure Description

[0020] Figure 1 This is an experimental detection diagram of the present invention; Figure 2 This is a diagram illustrating the antibody specificity of the present invention; Figure 3 This is a diagram of the ELISA experimental detection method of the present invention; Figure 4 This is an animal experiment testing diagram of the present invention; Figure 5 This is a data comparison chart for the present invention; Figure 6 This is a comparison chart of the reaction efficiency of the present invention; Figure 7 The reagents and materials of this invention Figure 1 ; Figure 8 The reagents and materials of this invention Figure 2 . Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0022] Example: like Figure 1-8 As shown, this embodiment of the invention provides a method for preparing a specific pan-antibody containing phenylacetylated lysine, comprising: The target receptor contains a specific receptor for lysine. -Amino group, specifically recognizes lysine. The phenylacetylated modification group on the -amino group, covalently linked by an amide bond, is independent of the protein primary sequence and can bind to phenylacetylated lysine residues in various substrates, including histones and non-histones. The phenylacetylated lysine residues recognized by the antibody are... -phenylacetyl- -methyl- -Lysine, the molecular weight of lysine residues increases by 118.0419 Da after phenylacetylation modification; This specific distinguisher precisely differentiates phenylacetylation from structurally similar acylation modifications, exhibiting a specific binding rate >90% to phenylacetylation and a cross-reactivity rate <5% with acetylated, propionylated, crotonylated, benzoylated, phosphorylated, and unmodified lysine residues. The antibody's specificity is validated through both competitive ELISA and dot blot assays, effectively eliminating interference from other acylation modifications and ensuring false-positive results in subsequent Western blotting and immunoprecipitation experiments. The antibody can detect 1-10 [types of acylation modifications] in Western blotting experiments. The modified protein showed a positive antibody dilution greater than 54,000 in the enzyme-linked immunosorbent assay (ELISA), and the signal intensity was more than 10 times higher than that for the unmodified peptide. The low-abundance detector is adapted to the low abundance of phenylacetylated modification in vivo, with a detection limit as low as ng, meeting the detection requirements of low-expression phenylacetylated proteins under physiological conditions. The antibody can be prepared by immunizing animals or by screening phage display libraries, yeast display libraries, bacterial display libraries, and ribosome display libraries. The antibody can be used for the detection and enrichment of phenylacetylated modified peptides and proteins in dot blot, enzyme-linked immunosorbent assay, protein immunoblotting, and immunoprecipitation experiments.

[0023] A method for preparing a specific pan-antibody containing phenylacetylated lysine includes the following steps: S1. Synthesize modified phenylacetylated lysine to obtain -phenylacetyl- -methyl- - Pure lysine; S11, with -tert-Butyloxycarbonyl- -fluorenemethyloxycarbonyl- -methyl- Using lysine as a raw material, the solution was fully dissolved in dichloromethane, and then freshly prepared HCl gas was continuously bubbled through it at room temperature for 1.5 hours. The reaction was then continued at room temperature for 5 hours under a strong acidic HCl environment. After confirming the complete reaction by thin-layer chromatography, the solution was evaporated to dryness and then purified by silica gel column chromatography to obtain... -fluorenemethyloxycarbonyl- -methyl- -Lysine; S12. Dissolve the above product in acetone, add 1.1M NaHCO3 aqueous solution and stir at room temperature for 2 hours to remove the bound HCl. After cooling to 0-5°C in an ice-water bath, slowly add an acetone solution of phenylacetyl chloride, maintaining the reaction in an ice-water bath for 1 hour. After extraction, drying, and rotary evaporation, obtain the product by silica gel column chromatography. -phenylacetyl- -fluorenemethyloxycarbonyl- -methyl- -Lysine; S13. Add piperidine to the above product and stir at room temperature overnight to remove the Fmoc protecting group. The esterified product is obtained by vacuum distillation and column chromatography. Then, sodium hydroxide is added and hydrolyzed in an ice-water bath for 2 hours in a system of water and methanol with a volume ratio of 1:1. The pH is adjusted to 3.0 with 2NHCl and then evaporated to dryness. The final product is obtained by dissolving in ethanol, filtering, and evaporating to dryness. S2. Modified phenylacetylated lysine and its derived polypeptide are coupled to a carrier protein to prepare a complete antigen for immunization. S21. Dissolve 10 mg of hemocyanin in 1 ml of LME buffer, and add 0.4 mg of [unclear text - possibly a typo, should be "S21"] to each solution. With 1.1mg - After mixing thoroughly, react at room temperature for 15 minutes; S22, add 1.4μ 10 minutes of reaction with β-mercaptoethanol, using Adjust the pH of the solution to 7.4; S23, Dissolve 2 mg of modified phenylacetylated lysine and its derived polypeptide in... Then add the above-mentioned activated After thoroughly mixing the solution, react at room temperature for 2 hours. S24, add 50 mM Tris to terminate the reaction. Dialyze overnight at 4°C in the solution; The sequence of the modified phenylacetylated lysine-derived polypeptide is as follows: , and , It is phenylacetylated lysine; S3. Immunize experimental animals with the whole antigen and collect and screen positive serum; S31. Eight-week-old New Zealand white rabbits were used. Two subcutaneous injections were administered on both shoulders and two intramuscular injections were administered on both hind legs. Each rabbit was immunized a total of four times, on day 1, day 21, day 28, and day 35, respectively. S32. On the 45th day after immunization, 30 mL of whole blood was collected for the first time. After centrifugation, the supernatant was collected and serum was screened by enzyme-linked immunosorbent assay and dot blot analysis. S33, day 50, day 65, and day 70, 20 mL of whole blood was collected each time. After centrifugation, the supernatant was collected and screened repeatedly. Serum with positive test results was collected. S4. The positive serum was subjected to multi-stage affinity purification to obtain a highly specific pan-antibody of phenylacetylated lysine. S41. After filtering the positive serum, load it into the pre-equilibrated solution. Affinity chromatography column, using After washing, elute with 150 mM glycine buffer, collect the eluent and neutralize the pH to 7.0 to obtain crude purity. ; S42, the crude pure The sample is loaded onto an antigen-peptide affinity chromatography column to specifically enrich the target antibody; S43. Load the enriched antibody onto an unmodified peptide affinity chromatography column and collect the eluent directly to remove non-specific antibodies. S44. Determine the antibody protein content, add 10% glycerol, dispense into smaller containers, and store at -20℃.

[0024] The specific embodiments described in this invention are for illustrative purposes only and do not constitute a limitation on the scope of protection of this invention. The chemical reagents, biological materials, and experimental consumables used in these embodiments are all commercially available products; the cell lines involved are all deposited in patent-approved depository institutions, specifically: HeLa cells (ATCC® CCL-2™), HB-8065 cells (ATCC® HB-8065™), and CCL-243 cells (ATCC® CCL-243™).

[0025] Example 1: Synthesis of phenylacetylated lysine small molecules and preparation of rabbit polyclonal antibodies 1.1 Synthesis of modified phenylacetylated lysine 1.1.1 Intermediates -fluorenemethyloxycarbonyl- -methyl- Preparation of lysine Pick -tert-Butyloxycarbonyl- -fluorenemethyloxycarbonyl- -methyl- -Lysine (Molecular weight 482.57 g / mol) was placed in a reaction flask and dissolved completely in dichloromethane; freshly prepared hydrogen chloride gas was continuously introduced at room temperature for 1.5 h, and the reaction was continued at room temperature for 5 h in a strong acidic environment of hydrogen chloride. The reaction was monitored for completeness by thin-layer chromatography; the reaction solution was placed at 50 °C and 0.03 MPa and rotary evaporated to dryness to obtain a viscous solid crude product; it was purified by silica gel column chromatography with an eluent gradient of dichloromethane → dichloromethane:methanol = 50:1 → dichloromethane:methanol = 20:1. The target component was collected and rotary evaporated to dryness to obtain N-fluorenylmethoxycarbonyl-N'-methyl-L-lysine ( (Molecular weight 382.45 g / mol), yield 98%.

[0026] 1.1.2 Preparation of intermediate N'-phenylacetyl-N-fluorenylmethoxycarbonyl-N'-methyl-L-lysine The above intermediate was placed in a reaction flask, acetone was added and stirred to dissolve it, then 1.1M sodium bicarbonate aqueous solution was added and stirred at room temperature for 2 hours to remove the bound hydrogen chloride; the reaction system was placed in an ice-water bath to cool to 0-5℃, and phenylacetyl chloride solution was slowly added dropwise. An acetone solution (molecular weight 154.59 g / mol) was added dropwise, and the reaction was maintained in an ice-water bath for 1 hour. The reaction endpoint was monitored by thin-layer chromatography. The pH of the system was adjusted to 3.0 with 2N hydrochloric acid, and the mixture was extracted twice with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then rotary evaporated to dryness at 50 °C and 0.03 MPa to obtain a viscous solid crude product. The crude product was purified by silica gel column chromatography with an eluent gradient of dichloromethane → dichloromethane:methanol = 50:1 → dichloromethane:methanol = 30:1. The target fraction was collected and rotary evaporated to dryness to obtain... -phenylacetyl- -fluorenemethyloxycarbonyl- -methyl- -Lysine, yield 74%.

[0027] 1.1.3 Final Products -phenylacetyl- -methyl- Preparation of lysine Place the above intermediate in a reaction flask and add... , Dimethylformamide was dissolved in a mixture of dimethylformamide and tetrahydrofuran by stirring, then piperidine was added, and the mixture was stirred overnight at room temperature until the Fmoc protecting group was completely removed. The solvent was removed by vacuum distillation at 0.88 kPa to obtain the mixed product. The esterified product was purified by silica gel column chromatography. A mixed solvent of sodium hydroxide, water, and methanol (volume ratio 1:1) was added to the esterified product, and the mixture was reacted in an ice-water bath for 2 hours until complete hydrolysis. The pH of the system was adjusted to 3.0 with 2N hydrochloric acid, and the mixture was rotary evaporated to dryness. The residual solid was dissolved in ethanol, and the insoluble impurities were removed by filtration. The filtrate was rotary evaporated to dryness to obtain a white solid. -phenylacetyl- -methyl- -Lysine ( (Molecular weight 266.34 g / mol), yield 78%.

[0028] 1.2 Preparation of phenylacetylated lysine complete antigen 1) Dissolve 10 mg of hemocyanin in 1 mL In buffer solution; 2) Add 0.4mg With 1.1mg - Mix thoroughly and react at room temperature for 15 minutes; 3) Add 1.4 1-Mercaptoethanol, react for 10 min, then adjust the pH of the system to 7.4 with phosphate buffer; 4) Take 2mg -phenylacetyl- -methyl- - Lysine was dissolved in phosphate buffer and added to the activated hemocyanin solution. The mixture was thoroughly mixed and reacted at room temperature for 2 hours. 5) Add 50 mM Tris and react for 15 min. Dialyze overnight in phosphate buffer at 4°C to obtain the complete antigen for immunization.

[0029] 1.3 Animal Immunization Eight-week-old New Zealand white rabbits were selected. The whole antigen was diluted with physiological saline and emulsified with an equal volume of adjuvant. Immunogen was injected at two subcutaneous points on both shoulders and two intramuscular points on both hind legs. An equal volume of immunogen was injected at each site. Each rabbit was immunized a total of 4 times, on day 1, day 21, day 28 and day 35.

[0030] 1.4 Serum Collection and Screening On day 45, 30 mL of whole blood was collected for the first time. On days 50, 65, and 70, 20 mL of whole blood was collected each time. Serum was separated by centrifugation. Serum was specifically screened by enzyme-linked immunosorbent assay (ELISA) and dot blot assay to obtain positive serum.

[0031] 1.5 Antibody Purification 1) Protein A affinity chromatography: After filtering the positive serum, the sample was loaded onto a pre-equilibrated Protein A affinity chromatography column. After washing the column with phosphate buffer, it was eluted with 150 mM glycine buffer. The pH of the elution buffer was adjusted to 7.0 to obtain crude purified immunoglobulin G. 2) Antigen-peptide affinity chromatography: Crude purified immunoglobulin G is loaded onto an antigen-peptide affinity chromatography column to specifically enrich the target antibody; 3) Removal of non-specific antibodies: The enriched product is loaded onto an unmodified peptide affinity chromatography column, the eluent is collected, and non-specific binding components are removed. 4) Antibody preservation: Determine the antibody protein concentration, add 10% glycerol (v / v), and aliquot and store at -20℃.

[0032] Example 2: Preparation of phenylacetylated lysine-derived polypeptide antibodies 2.1 Derivatized Peptide Synthesis Synthesize the following modified phenylacetylated lysine-derived polypeptides, where K* represents phenylacetylated lysine: Peptide 1: CHAVSEGTK*AVTK (preferred sequence); Polypeptide 2: CLLLPGELAK*HAVSEGTK; Polypeptide 3: CPEPAK*SAPAPK.

[0033] 2.2 Preparation of complete polypeptide antigen Using the conjugation method described in Example 1.2, the above-derived polypeptides were conjugated with hemocyanin to prepare polypeptide-type full antigens.

[0034] 2.3 Antibody Preparation Following the animal immunization, serum screening, and antibody purification steps described in Examples 1.3-1.5, a pan-antibody of phenylacetylated lysine-derived polypeptide was prepared.

[0035] Example 3 demonstrates the preparation of pan-antibodies against phenylacetylated lysine from a library screening database. by -phenylacetyl- -methyl- Using lysine and CHAVSEGTK*AVTK peptides as screening targets, three rounds of affinity enrichment screening were performed from phage display libraries, yeast display libraries, bacterial display libraries, and ribosome display libraries, respectively. Antibody genes that specifically bind to phenylacetylated modifications were obtained, expression vectors were constructed and induced to express, and phenylacetylated lysine pan-antibodies were obtained after purification.

[0036] Experimental example: Experiment Example 1: Dot blot assay for antibody specificity 2.1.1 Experimental Samples Test samples: Lysine phenylacetylated peptides (1ng, 4ng, 16ng, 64ng, 100ng gradient). Control samples: Lysine acetylated peptide library, serine phosphorylated peptide library, threonine phosphorylated peptide library, lysine propionylated peptide library, lysine crotonylated peptide library, tyrosine nitrated peptide library, lysine benzoylated peptide library, and unmodified lysine peptide library. The loading amount of each control sample was 100 ng.

[0037] 2.1.2 Experimental Methods Spot each polypeptide onto Membrane, air dry; add blocking solution and seal at room temperature for 60 min; 1× After washing, the antibody prepared in Example 1 was added and incubated at room temperature for 2 hours; after washing, the secondary antibody was added and incubated at room temperature for 45 minutes to 1 hour; and the chromogenic substrate was added for exposure and imaging.

[0038] 2.1.3 Experimental Results The antibody specifically binds only to phenylacetylated modified peptides and has no significant cross-binding with acetylated, phosphorylated, propionylated, crotonylated, benzoylated, or unmodified lysine residues, with a cross-reactivity rate of <5%.

[0039] Experiment Example 2: Enzyme-linked immunosorbent assay (ELISA) to detect antibody titer 2.2.1 Experimental Methods Benzyl acetylated polypeptide antigen was coated onto an ELISA plate at 50 μg / well and incubated overnight at 4°C; 1× After washing, block with 1% bovine serum albumin blocking solution at 37°C for 1 hour; antibody is serially diluted 3-fold starting from 1:1000 and incubated at 37°C for 1.5 hours; secondary antibody is diluted 1:10000 and incubated at 37°C for 45 minutes. The reaction was terminated with 1M sulfuric acid after color development, and the OD450 value was measured.

[0040] 2.2.2 Experimental Results The antibody positive dilution > 54000 (OD450 > 1.0) showed that the recognition signal intensity of the phenylacetylated peptide was more than 10 times that of the unmodified peptide.

[0041] Experiment Example 3: Western blot assay for detecting endogenous phenylacetylated proteins 2.3.1 Experimental Samples Total protein from Hela cells (ATCC®CCL-2™) and HepG2 cells (ATCC®HB-8065™).

[0042] 2.3.2 Experimental Methods The procedure was performed using standard Western blotting. The primary antibody was the phenylacetylated lysine pan-antibody prepared in Example 1, and the secondary antibody was used for incubation followed by color development and exposure.

[0043] 2.3.3 Experimental Results The antibody can specifically recognize endogenous phenylacetylated modified proteins in cells, with a detection limit as low as 1-10 ng, which can meet the detection requirements of low-abundance post-translational modifications.

[0044] Experiment Example 4: Quantitative Verification of Antibody Specificity Using acetylated, propionylated, crotonylated, benzoylated, and phosphorylated modified peptides as coating antigens, the antibody binding rate was quantitatively detected by enzyme-linked immunosorbent assay (ELISA). The results showed that the antibody had a specific binding rate of >90% to phenylacetylated modifications and no significant cross-binding with other acylation modifications.

[0045] Comparative example: Comparative Example 1: Preparation of unmodified lysine antigen-antibody The antigen was prepared by replacing phenylacetylated lysine with unmodified lysine, and the animals were immunized and the antibody was purified according to the method described in Example 1. The results showed that the obtained antibody had no phenylacetylation modification specificity, could not distinguish between modified and unmodified peptides, and had no detection application value.

[0046] Comparative Example 2: Omit antigen affinity purification steps The antibody was purified using only Protein A affinity chromatography, omitting the antigen peptide affinity enrichment and unmodified peptide removal steps, and prepared according to Example 1. The results showed that the obtained antibody had high non-specific background and large batch-to-batch variation, and a large number of non-specific bands appeared in the Western blot experiment, making it unsuitable for accurate detection of phenylacetylation modification.

[0047] Comparative Example 3: Traditional acetylation antibody preparation process The antibody was prepared using a conventional process for lysine acetylation antibody preparation, without using phenylacetylation-modified antigen, according to Example 1. The results showed that the obtained antibody had a binding rate of <30% with phenylacetylation modification and a cross-reactivity rate of >60% with acetylation modification, and did not have phenylacetylation specificity.

[0048] Comparative Example 4: Preparation of Antigen-Antibody Without Phenylacetyl Chloride Modification Phenylacetyl chloride was not used in the antigen synthesis process; instead, acetyl chloride and benzoyl chloride were used. The antigen and antibody were prepared according to Example 1. The results showed that the obtained antigen had no phenylacetylation modification site, and the prepared antibody did not recognize the phenylacetylation modification at all, making the experiment invalid.

[0049] Comparative Example 5: Preparation of Non-specific Peptide Sequence Antigen-Antibody The antigen and antibody were prepared according to Example 2 by replacing the preferred phenylacetylated derivative peptide with a random polypeptide sequence. The results showed that the specificity of the obtained antibody was significantly reduced, and the cross-reactivity with various acylation modifications was >20%, which could not meet the requirements for pan-antibody detection.

[0050] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a specific pan-antibody containing phenylacetylated lysine, characterized in that, include: The target recognizer contains a substance that specifically recognizes lysine. -amino group, which specifically recognizes lysine - A phenylacetylated modification group covalently linked to the amino group via an amide bond, independent of the protein primary sequence, can bind to phenylacetylated lysine sites in various histone and non-histone substrates; A specific distinguisher that precisely distinguishes phenylacetylation from structurally similar acylation modifications, exhibiting a specific binding rate of >90% to phenylacetylation modifications and a cross-reactivity rate of <5% with acetylated, propionylated, crotonylated, benzoylated, phosphorylated, and unmodified lysine residues; The low-abundance detector is adapted to the low abundance of phenylacetylation modification in vivo, with a detection limit as low as ng, meeting the detection needs of low-expression phenylacetylated proteins under physiological conditions.

2. The method for preparing a specific phenylacetylated lysine pan-antibody according to claim 1, characterized in that: The antibody recognizes phenylacetylated lysine as... -phenylacetyl- -methyl- -Lysine, the molecular weight of lysine residues increases by 118.0419 Da after phenylacetylation modification.

3. The method for preparing a specific phenylacetylated lysine pan-antibody according to claim 1, characterized in that: The specificity of the antibody was verified by both competitive ELISA and dot blot assays, which can effectively eliminate interference from other acylation modifications and ensure that there are no false positive results in subsequent protein immunoblotting and immunoprecipitation experiments.

4. The method for preparing a specific phenylacetylated lysine pan-antibody according to claim 1, characterized in that: The antibody can detect 1-10 in a Western blot assay. The modified protein showed a positive antibody dilution greater than 54,000 in the enzyme-linked immunosorbent assay (ELISA), and the signal intensity was more than 10 times higher than that for the unmodified peptide.

5. The method for preparing a specific phenylacetylated lysine pan-antibody according to claim 1, characterized in that: The antibodies can be prepared by immunizing animals, or by screening phage display libraries, yeast display libraries, bacterial display libraries, and ribosome display libraries. The antibody can be used for the detection and enrichment of phenylacetylated peptides and proteins in dot blot, enzyme-linked immunosorbent assay, protein immunoblotting and immunoprecipitation experiments.

6. A method for preparing a specific phenylacetylated lysine pan-antibody, using a method for preparing a specific phenylacetylated lysine pan-antibody as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Synthesize modified phenylacetylated lysine to obtain -phenylacetyl- -methyl- - Pure lysine; S2. Modified phenylacetylated lysine and its derived polypeptide are coupled to a carrier protein to prepare a complete antigen for immunization. S3. Immunize experimental animals with the whole antigen and collect and screen positive serum; S4. The positive serum was subjected to multi-stage affinity purification to obtain a highly specific phenylacetylated lysine pan-antibody.

7. The method for preparing a specific phenylacetylated lysine pan-antibody according to claim 6, characterized in that: The specific steps for synthesizing modified phenylacetylated lysine in step S1 are as follows: S11, with -tert-Butyloxycarbonyl- -fluorenemethyloxycarbonyl- -methyl- Using lysine as a raw material, the solution was fully dissolved in dichloromethane, and then freshly prepared HCl gas was continuously bubbled through it at room temperature for 1.5 hours. The reaction was then continued at room temperature for 5 hours under a strong acidic HCl environment. After confirming the complete reaction by thin-layer chromatography, the solution was evaporated to dryness and then purified by silica gel column chromatography to obtain... -fluorenemethyloxycarbonyl- -methyl- -Lysine; S12. Dissolve the above product in acetone, add 1.1M NaHCO3 aqueous solution and stir at room temperature for 2 hours to remove the bound HCl. After cooling to 0-5°C in an ice-water bath, slowly add an acetone solution of phenylacetyl chloride, maintaining the reaction in an ice-water bath for 1 hour. After extraction, drying, and rotary evaporation, obtain the product by silica gel column chromatography. -phenylacetyl- -fluorenemethyloxycarbonyl- -methyl- -Lysine; S13. Add piperidine to the above product and stir overnight at room temperature to remove the Fmoc protecting group. Obtain the esterified product by vacuum distillation and column chromatography. Then add sodium hydroxide and hydrolyze in an ice-water bath for 2 hours in a system of water and methanol with a volume ratio of 1:

1. Adjust the pH to 3.0 with 2NHCl and evaporate to dryness. Dissolve in ethanol, filter, and evaporate to dryness to obtain the final product.

8. A method for preparing a specific phenylacetylated lysine pan-antibody according to claim 6, characterized in that: The specific steps for preparing the complete antigen in step S2 are as follows: S21. Dissolve 10 mg of hemocyanin in 1 ml of LME buffer, and add 0.4 mg of [unclear text - possibly a typo, should be "S21"] to each solution. With 1.1mg - After mixing thoroughly, react at room temperature for 15 minutes; S22, add 1.4μ 10 minutes of reaction with β-mercaptoethanol, using Adjust the pH of the solution to 7.4; S23, Dissolve 2 mg of modified phenylacetylated lysine and its derived polypeptide in... Then add the above-mentioned activated After thoroughly mixing the solution, react at room temperature for 2 hours. S24, add 50 mM Tris to terminate the reaction. Dialyze overnight at 4°C in the solution; The sequence of the modified phenylacetylated lysine-derived polypeptide is as follows: , and , It is phenylacetylated lysine.

9. A method for preparing a specific phenylacetylated lysine pan-antibody according to claim 6, characterized in that: The specific steps for animal immunization and serum screening in step S3 are as follows: S31. Eight-week-old New Zealand white rabbits were used. Two subcutaneous injections were administered on both shoulders and two intramuscular injections were administered on both hind legs. Each rabbit was immunized a total of four times, on day 1, day 21, day 28, and day 35, respectively. S32. On the 45th day after immunization, 30 mL of whole blood was collected for the first time. After centrifugation, the supernatant was collected and serum was screened by enzyme-linked immunosorbent assay and dot blot analysis. On days 33, 50, 65, and 70, 20 mL of whole blood was collected each time. After centrifugation, the supernatant was collected and screened repeatedly. Serum with positive test results was collected.

10. A method for preparing a specific phenylacetylated lysine pan-antibody according to claim 6, characterized in that: The specific steps for antibody purification in step S4 are as follows: S41. After filtering the positive serum, load it into the pre-equilibrated solution. Affinity chromatography column, using After washing, elute with 150 mM glycine buffer, collect the eluent and neutralize the pH to 7.0 to obtain crude purity. ; S42, the crude pure The sample is loaded onto an antigen-peptide affinity chromatography column to specifically enrich the target antibody; S43. Load the enriched antibody onto an unmodified peptide affinity chromatography column and collect the eluent directly to remove non-specific antibodies. S44. Determine the antibody protein content, add 10% glycerol, dispense into smaller containers, and store at -20℃.

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