H4K31 crotonylation modified antigen epitope peptide, polyclonal antibody as well as preparation method and application of H4K31 crotonylation modified antigen epitope peptide and polyclonal antibody

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an H4K31 crotonylation modified antigen epitope peptide, a polyclonal antibody and a preparation method and application of the H4K31 crotonylation modified antigen epitope peptide. The invention provides an H4K31 crotonylation modified antigen epitope peptide, the amino acid sequence of the H4K31 crotonylation modified antigen epitope peptide is shown as SEQ ID NO.1 in a sequence table, and the ninth amino acid is subjected to crotonylation modification. According to verification of the invention, the antigen epitope peptide can be used for preparing an effective polyclonal antibody at domestic rabbits, and the obtained antibody can specifically recognize H4K31 crotonylation modification in animal cells and tissues, and can be used for detecting the H4K31 crotonylation level of animal and human samples.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an H4K31 crotonylated modified antigenic epitope peptide, a polyclonal antibody, its preparation method, and its uses. Background Technology

[0002] Current research on crotonylation modification mainly focuses on crotonylation proteomics and immunoprecipitation, using pan-antibodies against crotonylation. However, omics assays are time-consuming and expensive, unsuitable for rapid detection of large batches of samples and clinical applications. In recent years, antibodies targeting certain important sites have been developed, and crotonylation antibodies targeting sites such as H2BK34, H3K4, and H3K18 have begun to appear on the market. This is mainly due to the importance of specific sites to bodily functions and related scientific progress. Recent studies have found that H4K31 crotonylation levels are closely related to the body's immune function, and H4K31 is expected to become a promising target. However, there are currently no antibodies on the market specifically targeting H4K31 crotonylation. Therefore, providing an antibody that can be applied to scientific research and clinical sample detection with high efficiency and specificity to recognize H4K31 crotonylation modification has become an urgent problem to be solved in this field. Products related to H4K31 crotonylation will have significant market potential. Summary of the Invention

[0003] The purpose of this invention is to provide an H4K31 crotonylated modified antigenic epitope peptide, a polyclonal antibody, and its preparation method and uses, thereby solving the bottleneck problem in the development of H4K31 crotonylated antibodies.

[0004] To achieve the above objectives, the present invention first provides the following technical solution: an H4K31 crotonylated modified antigenic epitope peptide, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing, and the 9th amino acid is modified by crotonylation.

[0005] Furthermore, the present invention provides a recombinant antigen containing the aforementioned antigenic epitope peptide.

[0006] Furthermore, the present invention provides a biological material containing a nucleic acid sequence encoding the said antigenic epitope peptide or the said recombinant antigen.

[0007] Furthermore, the present invention provides the use of the said antigenic epitope peptide, the said recombinant antigen, or the said biological material in the preparation of antibodies for detecting H4K31 crotonylation modification.

[0008] Furthermore, the present invention also provides a method for preparing a polyclonal antibody to detect H4K31 crotonylation modification, wherein the antigen epitope peptide or recombinant antigen is used to immunize animals.

[0009] Preferably, the antigenic epitope peptide or recombinant antigen is covalently coupled to hemocyanin KLH, and then the animal is immunized.

[0010] Preferably, the antigenic epitope peptide or recombinant antigen is covalently coupled to hemocyanin KLH at a ratio of 1:1 to 1:10.

[0011] Furthermore, the present invention also provides a polyclonal antibody prepared by the aforementioned preparation method.

[0012] Furthermore, the present invention also provides the use of the aforementioned polyclonal antibody in the preparation of products for detecting H4K31 crotonylation levels.

[0013] Preferably, the product includes a reagent kit, a biochip, a test strip, or a biological reagent.

[0014] This invention provides an H4K31 crotonylation-modified antigenic epitope peptide. As verified by this invention, this antigenic epitope peptide can be used to prepare effective polyclonal antibodies in rabbits. The obtained antibodies can specifically recognize H4K31 crotonylation modification in animal cells and tissues, and can be used to detect the H4K31 crotonylation level in animal and human samples. Attached Figure Description

[0015] Figure 1 The OD value of the serum collected on day 47 in Example 1 was detected by ELISA; in the figure, K is 1000; Ab1 is the serum obtained by injecting modified peptide A into rabbits; Ab3 is the serum obtained by injecting modified peptide B into rabbits; Figure 2 The results of Dot Blot analysis of the purified antibody in Example 1 are shown below; channel 1 is H4K31Cr modified polypeptide A immobilized on the solid membrane; channel 2 is H4K31Cr modified polypeptide B immobilized on the solid membrane; and channel 3 is H4K31 unmodified polypeptide immobilized on the solid membrane. Figure 3 The results of Western blot analysis of NIH-3T3 cell proteins using purified antibodies in Example 1 are shown; the primary antibody dilution ratio is 1:2K, the secondary antibody dilution ratio is 1 / 10000, and the exposure time is 60 seconds. Figure 4 The results of Western blot analysis of Raw264.7 cell proteins using purified antibody Ab3 in Example 1 are shown; the primary antibody was diluted 1:500, the secondary antibody was diluted 1 / 10000, and the exposure time was 60 seconds. Detailed Implementation

[0016] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0018] Example 1: Preparation of Antibody (1) Antigen design and preparation: The inventors designed and synthesized two modified polypeptide sequences, named H4K31Cr modified polypeptide A and H4K31Cr modified polypeptide B, and ensured their immunogenicity and specificity. Simultaneously, an unmodified H4K31 polypeptide was synthesized for antibody detection. Specifically: H4K31Cr modified polypeptide A (SEQ ID NO.2): CRDNIQGIT-(Crotonyl)K-PAIRRL; corresponding to positions 24-38 of the H4 protein, where position 31 (K) is crotonylated; H4K31Cr modified peptide B (SEQ ID NO.1): CDNIQGIT-(Crotonyl)K-PAIRR; corresponding to positions 25-37 of the H4 protein, and also modified at position 31 (K); H4K31 unmodified polypeptide (SEQ ID NO.3): CRDNIQGITKPAIRRL; corresponds to positions 24-38 of the H4 protein, with position 31 (K) unmodified.

[0019] (2) Coupling of modified peptides with carriers: H4K31Cr modified peptide A and H4K31Cr modified peptide B were covalently coupled with hemocyanin KLH at a concentration ratio of 1:5.

[0020] (3) Immunization of rabbits: Two New Zealand rabbits were immunized with the conjugated polypeptide along with the adjuvant, for a total of four rabbits. On day 1, the complete Freund's adjuvant and the recombinant antigen were mixed at a volume ratio of 1:1 and injected subcutaneously into both shoulders and intramuscularly into both hind legs. The antigen injection dose was 300-800 μg to ensure the immunogen could persist and thus enhance the immune response. On day 14, the incomplete Freund's adjuvant and the antigen were mixed at a volume ratio of 1:1 and injected subcutaneously into both shoulders and intramuscularly into both hind legs. The antigen injection dose was 150-400 μg. From day 15 to day 21, the incomplete Freund's adjuvant and the antigen were mixed at a volume ratio of 1:1 and injected subcutaneously into both shoulders and intramuscularly into both hind legs. 150-400 μg; On days 22-28, mix the incomplete Freund's adjuvant and antigen at a 1:1 volume ratio and inject subcutaneously into both shoulders and intramuscularly into both hind legs, with an antigen injection dose of 150-400 μg; On days 29-35, mix the incomplete Freund's adjuvant and antigen at a 1:1 volume ratio and inject subcutaneously into both shoulders and intramuscularly into both hind legs, with an antigen injection dose of 150-400 μg; On day 40, mix the incomplete Freund's adjuvant and antigen at a 1:1 volume ratio and inject subcutaneously into both shoulders and intramuscularly into both hind legs, with an antigen injection dose of 150-400 μg.

[0021] (4) Antibody purification: After four immunizations, blood samples were collected on days 28, 35, 47, and 52. Serum antibody titers were detected using ELISA, and Western blotting was performed on the serum. First, the antigen was diluted with coating buffer and added to the ELISA plate at a rate of 50 μg / well. Coating was completed overnight at 4°C. The plate was then washed three times with 1×TBST. Next, 1% BSA blocking buffer was added, and the plate was incubated at 37°C for 1 h, followed by three washes with 1×TBST. The antibody was then purified according to… Figure 2 Adjust the dilution volume. Add the solutions sequentially to the microplate, incubate at 37°C for 1.5 h, and wash three times. Dilute the secondary antibody to 1:10K with 1% BSA blocking buffer, incubate at 37°C for 45 min, and wash three times. Add TMB chromogenic buffer 5, then stop the display reaction with 1M sulfuric acid. Read the data using a microplate reader. Results are as follows: Figure 1 As shown, the serum serum reached the target concentration on day 47, and the antibodies at a dilution ratio of 1:54K could still recognize the modified antigen, resulting in an OD450 > 1.0. However, the Ab1 antibody prepared from H4K31Cr modified peptide A showed a significantly reduced ability to recognize unmodified peptides at dilution ratios below 1:54K, and the Ab3 antibody prepared from H4K31Cr modified peptide B showed a significantly reduced ability to recognize unmodified peptides at dilution ratios below 1:6K. On day 52, whole blood was collected from rabbits, and antibody affinity purification was performed using an affinity purification column conjugated with modified peptides.

[0022] Example 2: Antibody Detection (1) Application Detection: The purified antibody was subjected to Dot Blot detection. Uncrosslinked antigen peptides were spotted onto a PVDF membrane in gradients of 1 ng, 4 ng, 16 ng, and 64 ng. After the membrane surface dried, 5% skim milk blocking buffer was added and the membrane was blocked at room temperature for 60 min. The antibody was added to 5% skim milk at a dilution ratio of 1:2K, and the PVDF membrane was incubated at room temperature for 2 h. After washing three times with TBST, commercial goat anti-rabbit HRP secondary antibody at a working concentration of 1:10K was added, and the membrane was incubated for 1 h, followed by washing three times with TBST. Then, the chromogenic substrate working solution was added, and the membrane was exposed to detect the binding of peptides and antibodies. The Dot Blot results are as follows: Figure 2 As shown, Ab1 and Ab3 antibodies bind strongly to modified peptides, but have virtually no binding with unmodified peptides.

[0023] NIH-3T3 cell proteins were detected using Western blotting with Ab1, Ab3 antibodies, and a commercially available H4 antibody. A 10% PAGE separating gel was prepared, and denatured proteins were separated by gel electrophoresis. The proteins were then transferred to a PVDF membrane using a wet transfer method. The membrane was blocked with 5% skim milk blocking buffer at room temperature for 60 min. Antibody was added to 5% skim milk at a 1:500 dilution, and the PVDF membrane was incubated at room temperature for 2 h. After three washes with TBST, a 1:10K concentration of commercially available goat anti-rabbit HRP secondary antibody was added, and the membrane was incubated for 1 h, followed by three washes with TBST. Finally, the chromogenic substrate was added, and the membrane was exposed to light.

[0024] The results are as follows Figure 3 As shown, the commercially available H4 antibody obtained a positive band at the H4 histone site. Of the two prepared antibodies, only the Ab3 antibody prepared from H4K31Cr modified peptide B obtained a positive band at the H4 histone site; the Ab1 antibody prepared from H4K31Cr modified peptide A failed to obtain a positive band at the H4 histone site. The Ab3 antibody was used to detect proteins in LPS- and butyrate- and succinate-treated Raw264.7 cells using Western blotting. The results are as follows... Figure 4 As shown, Ab3 antibody can detect positive bands. Therefore, Ab3 antibody can be used in ELISA, Dot Blot, Western Blot and other detection applications and related kit development for detecting the crotonylation modification level of the 32nd amino acid (H4K31) of H4 protein.

Claims

1. An H4K31 crotonylated modified antigenic epitope peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1, and the 9th amino acid is modified by crotonylation.

2. A recombinant antigen, characterized in that: It contains the antigenic epitope peptide as described in claim 1.

3. A biomaterial, characterized in that: It contains a nucleic acid sequence encoding the antigenic epitope peptide of claim 1 or the recombinant antigen of claim 2.

4. Use of the antigenic epitope peptide of claim 1, the recombinant antigen of claim 2, or the biomaterial of claim 3 in the preparation of antibodies for detecting H4K31 crotonylation modification.

5. A method for preparing a polyclonal antibody to detect H4K31 crotonylation modification, characterized in that, Immunize animals with the antigenic epitope peptide of claim 1 or the recombinant antigen of claim 2.

6. The preparation method according to claim 5, characterized in that, The antigenic epitope peptide or recombinant antigen is covalently coupled to hemocyanin KLH, and then the animal is immunized.

7. The preparation method according to claim 6, characterized in that, The antigenic epitope peptide or recombinant antigen is covalently coupled to hemocyanin KLH at a ratio of 1:1 to 1:

10.

8. A polyclonal antibody for detecting H4K31 crotonylation modification prepared by the preparation method according to any one of claims 5-7.

9. Use of the polyclonal antibody according to claim 8 for detecting H4K31 crotonylation modification in the preparation of a product for detecting H4K31 crotonylation levels.

10. The use according to claim 9, characterized in that: The products include reagent kits, biochips, test strips, or biological reagents.

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