Tau protein-k274 lactylation modified polyclonal antibody and application thereof

Abstract

The application discloses a tau protein-K274 lactylation modified polyclonal antibody and application thereof, and relates to the field of biomedical technology. An immunogen of the antibody comprises a modified peptide segment of SEQ ID NO:1; the peptide segment comprises an amino acid sequence of SEQ ID NO:1 (VQIINKKlaLGS) or an epitope of tau protein in the amino acid sequence, and is combined with the tau protein; and the epitope of the tau protein comprises tau protein K268-280aa lactylation modification. The tau antibody specifically combined in the application has high sensitivity in diagnosis of Alzheimer's disease (AD), can sensitively detect K274la-tau levels in the brain and cells, and has a minimum detection limit of less than or equal to 10 pg. Moreover, the tau antibody specifically combined in the application has good specificity and does not cross-recognize non-lactylation tau or other modified forms.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polyclonal antibody targeting the lactation modification of lysine 274 (K274la) of tau protein and its application in the preparation of diagnostic devices for tau-related neurodegenerative diseases, particularly in the diagnosis of Alzheimer's disease (AD). Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized primarily by memory impairment and cognitive decline. Early diagnosis is crucial for intervention and treatment. Current methods mainly rely on cerebrospinal fluid Aβ42 and phosphorylated tau (pTau181, pTau217) detection or PET imaging. For example, publication number CN 120040585 A discloses the preparation of an anti-p-Tau217 antibody and its application in an Alzheimer's disease detection kit. However, these technologies suffer from drawbacks such as high invasiveness and high cost. Summary of the Invention

[0003] To address the aforementioned issues, this invention proposes a polyclonal antibody modified with lactation of tau protein-K274 and its application. This antibody can specifically recognize tau, exhibiting high specificity, without cross-recognizing non-lactated tau or other modified forms, and demonstrating high sensitivity.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solution: A polyclonal antibody modified with tau protein-K274 lactation, wherein the immunogen of the antibody comprises the modified peptide of SEQ ID NO:1: The peptide comprises the amino acid sequence of SEQ ID NO:1 (VQIINKKlaLGS) or the tau protein epitope within the amino acid sequence binds to the tau protein; The epitope of the tau protein includes tau protein K274 lactation modification.

[0005] Furthermore, the K274 lactation modification is a 268-280aa (H, M100%) lactation modification, and its amino acid sequence is HQPGGGK(lac)VQIINK.

[0006] The specific amino acid sequence of the modified peptide segment of SEQ ID NO:1 contained in the antibody immunogen is as follows: SEQ ID NO:1 >NP_005901.2 microtubule-associated protein tau isoform 2 [Homosapiens] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTR EPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHK PGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL.

[0007] This invention also provides a method for preparing a polyclonal antibody modified by tau protein-K274 lactation, comprising the following steps: Step (1): Based on the human Tau protein sequence SEQ ID NO:1, synthesize a peptide containing the K274 lactation modification site as an immunogen.

[0008] Step (2), peptide purification and identification: purification was performed using C18 reversed-phase high-performance liquid chromatography (RP-HPLC); Step (3): Inject the purified peptide from step (2) into the animal for immunization, collect the animal antiserum, and obtain the target tau protein-K274 lactation-modified polyclonal antibody (K274la polyclonal antibody) after affinity purification.

[0009] Furthermore, the method for collectively identifying the peptides at the K274 lactation modification site in step (1) is as follows: i. Rink amide resin is swelled in DMF. DMF is a commonly used swelling solvent. The swelling time is 1 hour (to ensure the resin is fully swollen). The swollen resin is then mixed with a buffer containing the antigen peptide (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) and incubated at room temperature for 2 hours. Unbound peptides are washed away with the same buffer. Unreacted sites are blocked for 1 hour with blocking buffer (0.2 M glycine, pH 8.0). The resin is then washed three times alternately with high / low pH buffer (0.1 M sodium acetate, pH 4.0 + 0.1 M Tris-HCl, pH 8.0) to remove non-covalently bound substances.

[0010] ii. Take the standard Fmoc-protected L-amino acid (Fmoc-AA-OH), remove the amino protecting group using 20% ​​piperidine / DMF solution, and set aside; iii. Amino acid coupling: Each Fmoc-AA-OH with the amino protecting group removed is activated with an activator and gradually coupled together. The coupling time is 1.5–5 h. iv. Introducing lactate modification: During coupling synthesis to the K274 site, Fmoc-AA-OH with the amino protecting group removed was replaced with Fmoc-Lys(L-lactyl)-OH to obtain the lactated modified peptide. v. Terminal Deprotection and Lysis: The lactated modified peptide obtained in step iv is lysed using a TFA lysis buffer for 2–3 hours, simultaneously removing side-chain protecting groups and releasing the peptide to obtain the peptide with the K274 lactated modification site. TFA lysis buffer formulation: TFA:water (H2O):triisopropylsilane (TIPS):ethylene dithiol (EDT) = 94%:2.5%:2.5%:1%. Mix the above reagents in the specified ratio (prepare fresh), pre-cool in an ice bath to reduce side effects, transfer the lactated modified peptide obtained in step iv to a lysis tube, lyse with the buffer for 2–3 hours, filter to remove resin, and wash the resin with a small amount of TFA (1–2 mL). Combine the filtrates, add 10 times the volume of ice-cold diethyl ether to precipitate the peptide. Centrifuge (4000 rpm, 5 min), discard the supernatant, and collect the white precipitate. Wash the precipitate 2–3 times with cold diethyl ether to remove residual TFA and impurities. Vacuum dry to obtain the crude peptide.

[0011] Furthermore, the activator mentioned in step iii is DIC / HOBt or HBTU / DIPEA.

[0012] Furthermore, the ratio of the TFA pyrolysis mixture in step v is TFA:water:TIPS:EDT = 94%:2.5%:2.5%:1%.

[0013] This invention also discloses the use of a tau protein-K274 lactation-modified polyclonal antibody in the preparation of diagnostic kits for neurodegenerative diseases.

[0014] This invention also discloses a diagnostic kit for detecting neurodegenerative diseases, the kit comprising a polyclonal antibody at a concentration of 1 mg / ml and a dilution ratio of 1:1000-5000, for Western blot analysis and ELISA detection; the enzyme-labeled secondary antibody is a horseradish peroxidase (HPR)-labeled anti-rabbit or anti-mouse secondary antibody at a concentration of 1 mg / ml and a dilution ratio of 1:5000-10000.

[0015] Furthermore, the diagnostic kit is used to detect K274la-tau levels in a sample.

[0016] Furthermore, the sample is a human or animal brain protein or cell lysate.

[0017] This invention discloses a polyclonal antibody modified with K274 lactation of tau protein and its application. Its advantages include: the tau antibody specifically binds to this invention, exhibiting high sensitivity in diagnosing Alzheimer's disease (AD), sensitively detecting K274la-tau levels in the brain and cells, with a detection limit ≤10 pg. Furthermore, it demonstrates good specificity, without cross-recognition of non-lactated tau or other modified forms. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the ELISA result of the purified antibody titer detection of this invention; Figure 2 This is the level of K274la-tau in the Alzheimer's disease (AD) model as shown by Western blot analysis. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to embodiments. 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.

[0021] Example 1

[0022] A method for preparing a polyclonal antibody modified by tau protein-K274 lactation includes the following steps: Step (1), peptide design and synthesis: Based on the human Tau protein sequence (SEQ ID NO:1), a short peptide containing the K274 site was designed and synthesized; Synthesis platform: C-terminal amide peptides were constructed on Rink Amide resin using Fmoc solid-phase peptide synthesis (SPPS) technology. Raw material preparation: Standard Fmoc-protected L-amino acids (Fmoc-AA-OH); specially modified amino acids Fmoc-Lys(L-lactyl)-OH (L-lactic acid introduced at the ε site); Synthesis steps: i. Resin pre-activation: Rink amide resin swells in DMF; ii. Take the standard Fmoc-protected L-amino acid (Fmoc-AA-OH), remove the Fmoc protection, and use a 20% piperidine / DMF solution to remove the amino protecting group; iii. Amino acid coupling: Each Fmoc-AA-OH with the amino protecting group removed is activated and gradually coupled with an activator DIC / HOBt or HBTU / DIPEA of equal amount to the Fmoc-AA-OH. The coupling time is 4 hours. iv. Introduce lactic acid modification: When coupling to the K274 site, replace the Fmoc-AA-OH with the amino protecting group removed with the specially modified amino acid Fmoc-Lys(L-lactyl)-OH; v. Terminal deprotection and lysis: Use TFA lysis mixture (TFA:water:TIPS:EDT = 94:2.5:2.5:1) for 2–3 hours to lyse, while removing side chain protecting groups and releasing peptides; Step (2), Peptide Purification and Identification: Purification was performed using C18 reversed-phase high-performance liquid chromatography (RP-HPLC). The molecular weight of the peptide and the correctness of the lactate modification site were confirmed using MALDI-TOF MS or LC-MS. Keyhole Limpet Hemocyanin (KLH) was selected as the C-terminus of the peptide for subsequent experiments.

[0023] Step (3) Animal immunization: Select healthy guinea pigs and administer the first subcutaneous injection (containing Freund's complete adjuvant), followed by booster immunizations every 2 weeks (containing Freund's incomplete adjuvant), for a total of 4–6 immunizations.

[0024] Step (4): Collect antiserum for antibody purification. The affinity chromatography column was thoroughly washed sequentially with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h. Guinea pig antiserum was collected as the sample to be purified. 10 mL of the sample was placed in a 50 mL centrifuge tube and filtered through a 0.45 μm pore size, 25 mm diameter microporous membrane. The filtered sample was loaded onto the column at a flow rate of 40 mL / h, and this process was repeated once. The column was then washed with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, the column was connected to a protein analyzer, and the instrument's transmittance (T setting) was adjusted to 100 during the washing process. The absorbance of the protein analyzer (1A range) was 0. At this point, the HD-A computer acquisition unit on the desktop was turned on, and the full-screen range was set to 5. The antibody was eluted with glycine solution (pH 2.7, 0.2M) at a rate of 40 mL / h. The green elution recording button was pressed to start elution. Antibody collection began when the instrument reading started to rise, yielding a polyclonal antibody modified with tau protein-K274 lactation (K274la polyclonal antibody). During polyclonal antibody collection, the pH of the antibody was adjusted to approximately 7 using 1M sodium bicarbonate, and the highest peak value of the elution was recorded.

[0025] Example 2

[0026] A diagnostic kit for the detection of neurodegenerative diseases, comprising: a polyclonal antibody at a concentration of 1 mg / ml and a dilution ratio of 1:1000-5000; and an enzyme-labeled secondary antibody (HRP-labeled) at a concentration of 1 mg / ml and a dilution ratio of 1:5000-10000 for Western blot analysis and ELISA testing.

[0027] The detection method of this kit is ELISA (enzyme-linked immunosorbent assay). The specific steps are as follows: Plate coating: Dilute the tau protein-K274 lactated peptide antigen to 1 μg / ml with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μl to each well of the polystyrene plate, incubate overnight at 4°C, discard the solution in the well the next day, and wash once with 1xPBST washing buffer at 180 μl per well.

[0028] Blocking: Add 150 μl of 1% BSA (prepared with PBST) to each well for blocking, and incubate at 37°C for 1 hour. Then discard the blocking solution.

[0029] Sample loading: Add 50 μl of a certain diluted polyclonal antibody (dilute the sample to be tested according to a certain ratio) to the above-mentioned sealed reaction wells.

[0030] Meanwhile, set up negative control wells (1% BSA), incubate at 37°C for 30 min, and wash 3 times with 1xPBST washing buffer at 150 μl per well.

[0031] Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP (diluted with 1% BSA) at 50 μl / well to the wells of the ELISA plate, incubate at 37°C for 45 min, and wash 3 times with 1xPBST buffer at 150 μl / well.

[0032] Add substrate solution for color development: Add 50 μl of the temporarily prepared TMB substrate solution to each reaction well and react at 37 °C for 5 min.

[0033] To terminate the reaction, add 50 μl of 1M sulfuric acid to each well.

[0034] Plate reading: Place the ELISA plate in a preheated ELISA reader (450nm) for reading. The result is the OD value. Each sample is repeated twice, and the data is saved for analysis. Results are shown in Table 1. Table 1. ELISA results for detecting purified antibody titer.

[0035] 1. Antibody titer assessment Signal comparison between modified and unmodified peptides: Within the dilution range of 1:250 to 1:4000, the OD values ​​of modified peptides were significantly higher than those of unmodified peptides (e.g., 1.610 vs. 0.199 at 1:250), indicating that the antibody specifically binds to the lactation-modified epitope (K274la). As the dilution ratio increases (e.g., above 1:16000), the signal of modified peptides gradually approaches that of unmodified peptides and the background (1% BSA), indicating that a specific signal can still be detected at antibody titers between 1:4000 and 1:16000.

[0036] 2. Valence determination: The effective signal threshold is usually defined as OD value ≥ twice the background value (1% BSA).

[0037] Modified peptide group: OD≈0.06 (background≈0.01) at 1:16000, still meeting the titer requirement; Unmodified peptide group: the signal at all dilution ratios is close to the background, indicating that the antibody has almost no cross-reactivity with the unmodified peptide.

[0038] 3. Antibody specificity analysis High specificity: The OD value of the antibody against the modified peptide (1.610) at low dilution (e.g., 1:250) is much higher than that against the unmodified peptide (0.199), indicating that it can significantly distinguish between the lactated and unmodified forms of K274.

[0039] Example 3

[0040] Methods and efficacy of polyclonal antibodies modified with tau protein-K274 lactation in the detection of Alzheimer's disease (AD). The detection was performed using Western blotting, and the specific steps included: ① Protein extraction: Tissue protein extraction and tissue processing were performed. Fresh brain tissue (approximately 0.01 g) was taken, washed twice with pre-cooled PBS, and the water was aspirated. 50-100 μL of ice-cold lysis buffer (containing protease inhibitors) was added, and homogenized until no tissue lumps remained. After homogenization, the mixture was placed on ice for 10-30 minutes, gently tapping to mix occasionally. The mixture was then centrifuged at 4°C (12000 rpm, 15 min). The supernatant was collected, aliquoted, and stored at -20°C.

[0041] Tissue protein extraction was performed, and neuronal cells were treated with different methods, transfected with tau and tau K274R plasmids, and then treated with lactate (lac). After 24 h, the cell or tissue samples were lysed with RIPA lysis buffer (containing protease inhibitors), rotated and incubated for 30 min, centrifuged and the supernatant was collected to obtain protein samples, and the protein concentration was determined (BCA method).

[0042] ② SDS-PAGE protein separation: Take 30–50 µg of the protein sample obtained in step ①, add 5× loading buffer, and boil for 5 minutes to denature. Load the sample onto a 12% SDS-PAGE gel and separate the proteins by electrophoresis according to the Tau size (50–70 kDa).

[0043] ③ Protein transfer: Transfer the protein obtained in step ② from the gel to a PVDF membrane using a wet transfer system. The transfer conditions are typically 100 V for 1.5 h.

[0044] ④ Blocking: Block with 5% BSA in TBST for 1 h at room temperature. The TBST is prepared as TBST: TBS + 0.1% Tween-20.

[0045] ⑤ Primary antibody incubation: The polyclonal antibody obtained in Example 1 was used as the primary antibody and diluted with 5% BSA at a ratio of 1:500. It was then incubated overnight at 4°C (gentle shaking is recommended).

[0046] ⑥ Wash the membrane: Wash the membrane 3 times with TBST, 5 minutes each time.

[0047] ⑦ Secondary antibody incubation: Use HRP-labeled anti-rabbit or anti-mouse secondary antibody (1:5000 diluted with TBST) and incubate at room temperature for 1 hour.

[0048] ⑧ Wash the membrane: Wash the membrane 3 times with TBST, 5 minutes each time.

[0049] ⑨ Chemiluminescence detection: Developed using ECL chemiluminescence solution, the image is recorded using an imaging system, and the results are as follows: Figure 1 and Figure 2 As shown.

[0050] from Figure 1 In a Western blot analysis, K274la polyclonal antibody was found to be significantly reduced in an Alzheimer's disease (AD) model, and this reduction was negatively correlated with total tau levels. Figure 2 As can be seen, the K274la polyclonal antibody has good recognition of lactate-treated wild-type tau, but almost no recognition of K274R mutant, proving that its specificity is highly dependent on K274 lactation.

[0051] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0052] Finally, it should be noted that the embodiments disclosed in this invention are merely preferred embodiments of this invention and are only used to illustrate the technical solutions of this invention, not to limit it. Although this invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this invention.

Claims

1. A polyclonal antibody that specifically binds to tau protein-K274 lactation-modified protein, characterized in that, The amino acid sequence of the immunogen of the antibody is HQPGGGK(lac)VQIINK.

2. A method for preparing a polyclonal antibody that specifically binds to tau protein-K274 lactation modification according to claim 1, comprising the following steps: Step (1): Based on the human Tau protein sequence SEQ ID NO:1, synthesize a peptide containing the K274 lactation modification site as an immunogen; the amino acid sequence of the peptide is HQPGGGK(lac)VQIINK; Step (2), peptide purification and identification: purification was performed using C18 reversed-phase high-performance liquid chromatography; Step (3): Inject the purified peptide from step (2) into the animal to immunize it, and collect the animal antiserum. After affinity purification, the target polyclonal antibody is obtained.

3. The method for preparing a polyclonal antibody that specifically binds to tau protein-K274 lactation modification according to claim 2, characterized in that: The method for synthesizing the peptide at the K274 lactation modification site in step (1) is as follows: i. Rink amide resin swells in DMF; ii. Take the L-amino acid Fmoc-AA-OH protected by standard Fmoc, remove the amino protecting group using 20% ​​piperidine / DMF solution, and set aside for later use; iii. Amino acid coupling: Each Fmoc-AA-OH with the amino protecting group removed is activated with an activator and gradually coupled together. The coupling time is 1.5–5 h. iv. Introducing lactate modification: During coupling synthesis to the K274 site, Fmoc-AA-OH with the amino protecting group removed was replaced with Fmoc-Lys(L-lactyl)-OH to obtain the lactated modified peptide. v. Terminal deprotection and cleavage: The lactated modified peptide obtained in step iv is cleaved using TFA cleavage mixture for 2–3 hours, while removing the side chain protecting group and releasing the peptide to obtain the peptide with the K274 lactated modification site.

4. The method for preparing a polyclonal antibody that specifically binds to tau protein-K274 lactation modification according to claim 3, characterized in that: The activator mentioned in step iii is DIC / HOBt or HBTU / DIPEA.

5. The method for preparing a polyclonal antibody that specifically binds to tau protein-K274 lactation modification according to claim 3, characterized in that: The weight ratio of the TFA pyrolysis mixture in step v is TFA:water:TIPS:EDT = 94%:2.5%:2.5%:1%.

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