Rabbit polyclonal antibody for specific recognition of ANP32a K236 milk acylation modification and application of rabbit polyclonal antibody

Abstract

The invention discloses a rabbit polyclonal antibody for specific recognition of ANP32a K236 milk acylation modification and application of the rabbit polyclonal antibody. The rabbit polyclonal antibody is prepared by taking modified polypeptide as an immunogen and immunizing animals. The amino acid sequence of the adopted modified polypeptide is as shown in SEQ ID NO.1, and the 7th site in the amino acid sequence is modified by a lactic acid group and is CGSQKR-(lactyl) K-REPDD. The rabbit polyclonal antibody has high specificity and high sensitivity, can be used for detecting the milk acylation modification of the K236 site of the ANP32a protein, and fills the blank of detection tools in the field.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a rabbit polyclonal antibody specifically recognizing ANP32a K236 lactylated modification and an application thereof. Background Art

[0002] ANP32a (acidic nucleophosmin 32 family member A) is an important cellular regulatory protein. Lactylation at K236 plays a key role in epigenetic regulation, inflammatory responses, and tumorigenesis. Currently, there are no specific antibodies targeting ANP32a K236 lactylation, resulting in a lack of effective detection tools for related research.

[0003] Existing technologies for detecting protein lactoylation modifications primarily rely on pan-lactylation antibodies. However, these antibodies cannot distinguish modifications at specific sites, resulting in low specificity and high false-positive rates. Therefore, developing a highly specific and sensitive antibody for ANP32a K236 lactoylation is of great significance for advancing research on related disease mechanisms and drug development. Summary of the Invention

[0004] In order to overcome the problem of being unable to accurately detect the level of ANP32a K236 lactylation modification in tissues, the present invention provides a rabbit polyclonal antibody that specifically recognizes ANP32a K236 lactylation modification and its application, which has high specificity and high sensitivity, filling the gap in detection tools in this field.

[0005] In order to achieve the above objectives, this application adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an ANP32a K236 lactoylated modified polypeptide, the amino acid sequence of the modified polypeptide is shown in SEQ ID NO.1, and the 7th position in the amino acid sequence is modified with a lactic acid group, namely CGSQKR-(lactyl)K-REPDD.

[0007] In a second aspect, the present invention provides a rabbit polyclonal antibody that specifically recognizes the lactoylated modification of ANP32a K236. The rabbit polyclonal antibody is prepared by immunizing an animal using the modified polypeptide as an immunogen.

[0008] In the above technical solution, the animal is a New Zealand white rabbit.

[0009] In the above technical solution, the specific process of immunizing animals is as follows: the modified polypeptide is mixed with Freund's adjuvant, and subcutaneously injected into New Zealand white rabbits, with booster immunization once every two weeks for a total of 4 times, and serum is collected and purified to prepare the rabbit polyclonal antibody.

[0010] In the above technical solution, the purification adopts Protein A / G affinity chromatography purification.

[0011] In a third aspect, the present invention provides the use of the above rabbit polyclonal antibody in the preparation of a kit or reagent for detecting lactoylation modification at the K236 site of ANP32a protein.

[0012] The beneficial effects of the present invention are:

[0013] (1) Originality: The world's first specific antibody targeting ANP32a K236 lactylation modification fills the gap in detection tools in this field.

[0014] (2) High specificity: Through strict antigen design and screening, the antibody is ensured to recognize only the lactylated modification at the K236 site without cross-reaction.

[0015] (3) High sensitivity: ELISA detection sensitivity reaches 1:50,000 dilution, and Western Blot can detect modified proteins as low as microgram level.

[0016] (4) Wide applicability: Applicable to various detection methods such as ELISA, Dot blot, Western blot, etc., supporting basic research and clinical sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :Immunization and blood collection time axis diagram;

[0018] Figure 2 :Detection of the recognition ability of Ab3 antibody to modified peptide in Elisa experiment;

[0019] Figure 3 :Verification of the recognition ability of Ab3 antibody for modified peptides in Dot blot experiments;

[0020] Figure 4 : Western Blot detection results of Ab3 antibody in different tissue lysates. DETAILED DESCRIPTION

[0021] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the present invention to those skilled in the art. The present invention will be limited only by the claims.

[0022] The present invention provides an ANP32a K236 lactoylated modified polypeptide, the amino acid sequence of the modified polypeptide is shown in SEQ ID NO.1, and the 7th position in the amino acid sequence is modified with a lactic acid group to be CGSQKR-(lactyl)K-REPDD.

[0023] The present invention also provides a rabbit polyclonal antibody (Ab3) that specifically targets the lactoylation modification at the K236 site of the ANP32a protein. The technical solution for this antibody includes the following core contents:

[0024] (1) Antigen design: A peptide containing K236 lactylation modification was chemically synthesized and compared with a non-modified control peptide and a negative peptide to ensure antigen specificity.

[0025] (2) Antibody preparation: New Zealand white rabbits were immunized with the modified polypeptides, and high-titer antibodies were obtained through multiple immunizations and serum titer detection. They were further purified by affinity chromatography to obtain high-purity polyclonal antibodies.

[0026] (3) Antibody specificity verification:

[0027] ELISA assay: In a 96-well plate coated with modified peptides, Ab3 still effectively binds to the target peptide at a dilution of 1:50,000, and the binding ability is more than 10 times stronger than that of the control peptides (unmodified and modified controls).

[0028] Dot blot analysis: Ab3 showed a strong signal at a dose of 4 ng of modified peptide, but had no binding to the unmodified peptide.

[0029] Western Blot analysis: Ab3 could specifically recognize the 29kDa ANP32a K236 lactylated protein band in NIH-3T3 cells (with or without sodium lactate treatment) and mouse brain tissue lysates.

[0030] Example 1: Antibody Preparation

[0031] 1. Antigen synthesis: Synthesize K236 lactyl modified peptide (CGSQKR-(lactyl)K-REPDD) and purify by HPLC. The specific preparation method is as follows:

[0032] 1.1 Peptide design

[0033] A polypeptide sequence containing K236 lactoylation modification was designed: CEGSQKR(lactyl)K-REPDDE (a cysteine ​​(C) was introduced at the N-terminus of the polypeptide for subsequent coupling, K represents lysine, and (lactyl)K represents lactoylation modification at the K236 site.), and was named Anp32a K236la polypeptide A; a polypeptide sequence containing K236 lactoylation modification: CGSQKR(lactyl)K-REPDD, was named Anp32a K236la polypeptide B; and a non-modified polypeptide sequence CPKKTESHHKAKGK, was named Anp32a non-modified polypeptide.

[0034] 1.2 Solid-Phase Peptide Synthesis (SPPS)

[0035] The Fmoc solid-phase synthesis strategy was used to gradually connect each amino acid on the Rink Amide resin. The lysine side chain protecting group (such as Mtt) was removed when the K236 site was synthesized. Subsequently, lactic acid and HATU activating agents were used in DMF in the presence of DIPEA for 2-4 hours for lactoylation modification. After the modification was completed, the standard synthesis process was continued until the peptide synthesis was completed.

[0036] 1.3 Peptide cleavage and preliminary purification

[0037] Use a mixture of TFA:TIS:water (95:2.5:2.5) to cleave the polypeptide from the resin and remove the side chain protecting groups. Add the cleavage solution dropwise to pre-cooled anhydrous ether to precipitate the polypeptide, recover it by centrifugation and wash it 2-3 times with cold ether to obtain the crude polypeptide.

[0038] 1.4 HPLC purification

[0039] Purification was performed by C18 reverse-phase high performance liquid chromatography (RP-HPLC) using 0.1% TFA aqueous solution and 0.1% TFA acetonitrile solution as the mobile phase, eluting under 5%-60% acetonitrile gradient conditions for 30 minutes and monitoring at a wavelength of 214 nm. The main peak was collected and lyophilized to obtain the purified polypeptide.

[0040] 1.5 Peptide identification

[0041] The molecular weight of the polypeptide is confirmed by mass spectrometry (such as MALDI-TOF or ESI-MS), and its purity is tested by HPLC, which is required to be ≥90%.

[0042] 2. Animal immunization: Modified polypeptide B was coupled with KLH and mixed with Freund's adjuvant and injected subcutaneously into New Zealand white rabbits. Boost immunization was performed every two weeks for a total of 4 times. The immunization method was to inject antigen (0.5 mg) into two points of the shoulder subcutaneous and two points of the hind leg muscle. A total of 4 immunizations were performed. Blood was collected 4 times after the fourth immunization. The immunization and blood collection time were as follows: Figure 1 shown.

[0043] 3. Serum purification: Collect high-titer serum and purify it through Protein A / G affinity chromatography to obtain IgG components.

[0044] 3.1 Serum collection

[0045] After the titer of the serum of the immunized animal reaches a high level, whole blood samples are collected by cardiac bleeding method, and after standing at room temperature for 1 hour, the serum is separated by centrifugation at 3000 rpm for 10 minutes at 4°C.

[0046] 3.2 Protein A / G affinity chromatography

[0047] The collected serum was diluted 1:1 into equilibration buffer (PBS or Binding Buffer), filtered to remove impurities, and then loaded onto a Protein A / G affinity column pre-equilibrated with PBS for binding at a flow rate of 1 mL / min.

[0048] 3.3 Elution and recovery

[0049] After binding is completed, unbound proteins are removed by washing with PBS, and the IgG component is eluted with a low pH elution buffer (such as 0.1 M glycine-HCl, pH 2.7), and the eluate is immediately neutralized with a neutralizing solution (such as 1 M Tris-HCl, pH 9.0).

[0050] 3.4 Concentration and buffer exchange

[0051] The eluted IgG solution was concentrated using an ultrafiltration centrifuge tube and buffer exchanged into PBS to obtain a highly pure IgG fraction.

[0052] 3.5 Purity Identification

[0053] Part of the purified product was tested for purity by SDS-PAGE, and the IgG concentration was determined by BCA method to confirm that it met the requirements of subsequent experiments.

[0054] Example 2: ELISA detection

[0055] 1. Coating peptide

[0056] The modified peptides (Anp32a K236la peptide A, Anp32a K236la peptide B) and the unmodified control peptide (Anp32a unmodified peptide) were diluted to a consistent appropriate concentration (1 μg / mL), and 100 μL was added to a 96-well plate and incubated at 4°C overnight to complete coating.

[0057] 2. Closed treatment

[0058] The next day, the coating solution was discarded, and the cells were gently washed three times with PBS. 200 μL of 1% BSA-PBS blocking solution was added to each well and incubated at 37°C for 1 hour to block nonspecific binding.

[0059] 3. Antibody incubation

[0060] After discarding the blocking solution, different gradient dilutions of Ab3 antibody (dilution range 1:2000 to 1:4374000, diluent is 1% BSA-PBS) were added, 100 μL per well, and incubated at 37° C. for 1 hour.

[0061] 4. Secondary antibody incubation and color development

[0062] After incubation, wash the plate 3-5 times with PBST (PBS + 0.05% Tween-20), add HRP-labeled anti-mouse secondary antibody (e.g., 1:10,000 dilution), incubate at 37°C for 45 minutes, add TMB substrate solution after washing, react for 5-10 minutes, and then add stop solution (e.g., 2M sulfuric acid) to terminate the reaction.

[0063] 5. Result reading and analysis

[0064] The OD value of each well was measured at a wavelength of 450 nm using a microplate reader, and the curve was fitted using software such as GraphPad Prism to calculate the half-maximal effective concentration (EC50) of Ab3 for the modified polypeptide to evaluate the binding affinity ( Figure 2 ).

[0065] Figure 2 The results of the Ab3 antibody's ability to recognize modified peptides show its binding activity against coated Anp32a K236la peptide A (CEGSQKR-(lactyl)K-REPDDE), Anp32a K236la peptide B (CGSQKR-(lactyl)K-REPDD), and Anp32a unmodified peptide (CPKKTESHHKAKGK) at different dilutions (1:2K to 1:4374K). The results demonstrate that Ab3 can still effectively recognize at least one modified peptide at a dilution of 1:54K, and its recognition ability for modified peptides is over 10 times higher than that for unmodified peptides, demonstrating high specificity and sensitivity.

[0066] Example 3: Dot blot detection

[0067] 1. Protein sample preparation

[0068] The modified polypeptides (Anp32a K236la polypeptide A, Anp32a K236la polypeptide B) and the control unmodified polypeptide were prepared into 1 μg / μL solutions, diluted and prepared at different doses (1 ng, 4 ng, 16 ng, 64 ng), and stored at 4°C until use.

[0069] 2. Spot sample onto membrane

[0070] Take a pre-wetted PVDF membrane (or nitrocellulose membrane), use a micropipette to add 2-5 μL of each dose of peptide solution on the membrane, and air-dry at room temperature for 15-30 minutes to fix the protein.

[0071] 3. Closed treatment

[0072] The membrane was placed in 5% skim milk powder in PBS-T buffer and gently shaken at room temperature for 1 hour to block nonspecific binding sites.

[0073] 4. Primary Antibody Incubation

[0074] After blocking, the membrane was washed three times with PBS-T and incubated overnight at 4°C in the presence of Ab3 antibody (1:1,000 dilution in PBS-T) to promote specific binding.

[0075] 5. Secondary Antibody Incubation and Signal Detection

[0076] The next day, the membrane was washed five times with PBS-T, and then HRP-labeled anti-rabbit secondary antibody (1:5,000 dilution) was added and incubated at room temperature for 1 hour. The membrane was then washed five times and then treated with ECL luminescent solution. The luminescent signal was detected in an imaging system.

[0077] 6. Data recording and analysis

[0078] The signal intensities of peptides at different doses were recorded. The results showed that Ab3 antibody could generate binding signals with modified peptide A and peptide B, with a stronger binding ability to peptide B in a dose-dependent manner. However, there was basically no significant binding to the control non-modified peptide, further verifying that Ab3 has good specificity and high affinity for modified peptides ( Figure 3 ).

[0079] Figure 3The results of the Ab3 antibody's recognition of modified peptides in a Dot blot assay show the binding of Ab3 to modified peptides (Anp32a K236la peptide A and Anp32a K236la peptide B) and unmodified peptides at varying doses (1 ng to 64 ng) on ​​a solid phase membrane. The results demonstrate that Ab3 generates binding signals with both modified peptides A and B, with particularly strong and dose-dependent binding to peptide B. However, there is essentially no significant binding to the unmodified control peptide, further confirming Ab3's excellent specificity and high affinity for modified peptides.

[0080] Example 4: Western Blot Verification

[0081] 1. Sample preparation

[0082] NIH-3T3 cell lysate, NIH-3T3 cell lysate treated with 25 mM sodium lactate (Lac Na) for 24 hours, mouse brain tissue lysate, and mouse testis tissue lysate were prepared. The lysates were extracted with RIPA lysis buffer with protease inhibitors. After lysis for 30 minutes, the cells were centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected for protein determination and adjusted to a consistent level.

[0083] 2. Protein electrophoresis and membrane transfer

[0084] 20–30 μg of protein from each sample was added to 4× loading buffer, denatured at 95°C for 5 minutes, and loaded onto a 12% SDS-PAGE gel for electrophoresis separation. The protein was then transferred to a PVDF membrane (300 mA constant current transfer for 1.5 hours).

[0085] 3. Closed treatment

[0086] After transfer, the PVDF membrane was placed in 5% skim milk powder PBS-T buffer and gently shaken at room temperature for 1 hour to block nonspecific binding sites.

[0087] 4. Primary Antibody Incubation

[0088] After blocking, the membrane was washed three times with PBS-T and placed in a solution containing Ab3 antibody (1:500 diluted in PBS-T) and incubated at 37°C for 2 hours to promote specific binding.

[0089] 5. Secondary Antibody Incubation

[0090] After the primary antibody incubation, the sections were washed five times with PBS-T for 5 minutes each time, and then HRP-labeled anti-rabbit secondary antibody (1:10,000 dilution in PBS-T) was added and incubated at room temperature for 45 minutes.

[0091] 6. Signal detection

[0092] After incubation, the membrane was washed again with PBS-T 5 times for 5 minutes each time, ECL luminescent solution was added to treat the membrane surface, and the protein band signal was detected using an imaging system with exposure for 60 seconds.

[0093] 7. Data recording and analysis

[0094] The results showed that Ab3 antibody detected a band of approximately 29 kDa in NIH-3T3, sodium lactate-treated NIH-3T3, and mouse brain tissue lysates, corresponding to the target protein Anp32a K236la; however, no obvious band was observed in mouse testis lysate, suggesting that Anp32a K236la may not be expressed in this tissue or its expression level is low ( Figure 4 ).

[0095] Figure 4 Western blot analysis of Ab3 in NIH-3T3 cells, lactate-treated NIH-3T3 cells, mouse brain tissue, and mouse testis tissue lysates shows that at a 1:500 antibody dilution, Ab3 specifically recognized a protein band of approximately 29 kDa in NIH-3T3, lactate-treated NIH-3T3, and mouse brain tissue lysates. This band corresponds to the target protein, Amp32a K236la. No distinct band was detected in mouse testis tissue, suggesting that Amp32a K236la may not be expressed in this tissue or its expression level is extremely low. This result confirms the specific recognition ability of Ab3 for the Amp32a K236la protein.

[0096] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An ANP32a K236 lactoylated modified polypeptide, characterized in that: The amino acid sequence of the modified polypeptide is shown in SEQ ID NO. 1, and position 7 of the amino acid sequence is modified with a lactic acid group, namely CGSQKR-(lactyl)K-REPDD.

2. A rabbit polyclonal antibody that specifically recognizes ANP32a K236 lactylated modification, characterized in that: The rabbit polyclonal antibody is prepared by immunizing an animal using the modified polypeptide according to claim 1 as an immunogen.

3. The rabbit polyclonal antibody according to claim 2, characterized in that: The animals are New Zealand white rabbits.

4. The rabbit polyclonal antibody according to claim 2, characterized in that: The specific process of immunizing animals is as follows: the modified polypeptide is mixed with Freund's adjuvant, and subcutaneously injected into New Zealand white rabbits. The immunization is boosted once every two weeks for a total of 4 times. The serum is collected and purified to prepare the rabbit polyclonal antibody.

5. The rabbit polyclonal antibody according to claim 4, characterized in that: The purification was performed by Protein A / G affinity chromatography.

6. Use of the rabbit polyclonal antibody according to any one of claims 2 to 5 in the preparation of a kit or reagent for detecting lactoylation modification at K236 of ANP32a protein.

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