A lactation antigen and antibody at the K122 site of the CREB1 protein and its preparation method

Abstract

This invention provides a lactation antigen at the K122 site of the CREB1 protein, an antibody, and a method for preparing the same. The lactation antigen is a lactated polypeptide, the amino acid sequence of which is shown in SEQ ID No. 2 or 3, and the lactation site of the lactated polypeptide is lysine. This invention provides a site-specific antibody targeting the lactation modification of lysine at position 122 of CREB1, enabling precise identification of specific lactation sites in the CREB1 protein. Compared with mass spectrometry detection, the antibody of this invention can be directly applied to conventional experimental platforms, offering advantages such as ease of operation, low detection cost, and short experimental cycle. It is more suitable for large-scale sample and tissue sample processing and routine experimental verification, facilitating its widespread application in basic research and disease-related studies.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a lactated antigen of the K122 site of the CREB1 protein, an antibody thereon, and a method for preparing the same. Background Technology

[0002] CREB1 (cAMP response element-binding protein 1) is a classic transcription factor that binds to cAMP response elements (CREs) in the promoter regions of target genes, thereby regulating the transcriptional expression of these genes. Previous studies have shown that CREB1 plays a crucial role in cell proliferation, apoptosis inhibition, energy metabolism regulation, and tumor progression, often acting as a pro-cancer regulator in various tumors. Furthermore, CREB1 is involved in hepatic glucose metabolism, lipid metabolism, and energy homeostasis maintenance, playing a significant role in metabolic diseases and tumor metabolic reprogramming. Therefore, elucidating the post-translational modification state and dynamic changes of CREB1 is of great value in clarifying its functional regulatory mechanisms.

[0003] Lactation is an important post-translational modification of lysine residues discovered in recent years, and it has been recognized as a crucial bridge connecting glycolysis / lactic acid accumulation with gene expression, signal transduction, metabolic reprogramming, and tumor progression. With further research, non-histone lactation is considered to be significant in both quantity and function, targeting metabolic enzymes, transcription factors, and DNA repair proteins.

[0004] Currently, research on protein lactation modifications primarily relies on mass spectrometry identification and pan-lactation antibody detection. Mass spectrometry-based lactation site identification typically involves sample lysis, proteolysis, lactated peptide enrichment, liquid chromatography-tandem mass spectrometry analysis, and database comparison. This technology provides site-level information on protein lactation and is suitable for discovering new sites and studying modification profiles. However, mass spectrometry detection often suffers from high experimental costs, high technical barriers, long detection cycles, and strong equipment dependence, making it difficult to meet the rapid validation needs of large sample volumes, routine experiments, and subsequent clinical translation studies. On the other hand, pan-lactation antibodies can be used in experiments such as Western blot, immunoprecipitation, immunofluorescence, and immunohistochemistry to detect the overall lactation level of a sample. While these methods are relatively simple to operate and suitable for routine experimental platforms, they only reflect the overall lactation level and cannot distinguish specific proteins or accurately identify the modification status of specific lysine sites. Therefore, they cannot meet the needs of protein-specific site mechanism research and disease-related sample detection. Furthermore, using short peptides containing post-translational modification sites as immunogens to prepare site-specific antibodies is a common technique in the study of post-translational modifications such as phosphorylation, acetylation, and methylation. This type of method can identify the modification status of specific sites in specific proteins; however, dedicated detection tools for lactation modifications, especially for lactation modifications at specific sites in the CREB1 protein, are still relatively lacking.

[0005] Previous studies have identified lysine residue 122 of CREB1 as a lactation modification site for the first time in human hepatocellular carcinoma cell lines, indicating that this site has clear scientific research and potential translational value. However, without site-specific antibodies, further research cannot be conducted on endogenous detection, dynamic monitoring, mechanism studies, tissue sample comparison, or reagent kit development.

[0006] Therefore, there is an urgent need to establish a site-specific antibody and its preparation technology that can specifically recognize lactation modification of CREB1 K122 site while avoiding non-specific recognition of unmodified sites in the same sequence. This would meet the needs of conventional experimental platforms for repeated verification and functional studies of this modified site, and provide technical support for related basic research and industrial applications. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a lactated antigen and antibody at the K122 site of the CREB1 protein, and a method for their preparation.

[0008] This invention provides a lactated antigen at the K122 site of the CREB1 protein, wherein the lactated antigen is a lactated polypeptide, the amino acid sequence of which is shown in SEQ ID No. 2 or 3, and the lactation site of which is lysine.

[0009] In some embodiments, the lactation site of the lactated polypeptide is site 122 of the CREB1 protein.

[0010] The present invention also provides an antibody that recognizes lactation modification at the K122 site of the CREB1 protein, obtained by immunizing an animal with the lactation antigen.

[0011] In some embodiments, the antibody is capable of recognizing lactation modification at the K122 site of the endogenous CREB1 protein in a cell or tissue sample.

[0012] In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a recombinant antibody, or an antigen-binding fragment.

[0013] The present invention also provides a method for preparing the antibody, the method comprising the following steps: (1) The lactated polypeptide of claim 1 or 2 is coupled with a carrier protein to prepare an immunogen; (2) Immunize the experimental animals with the immunogen described in step (1) to obtain antiserum; (3) The antiserum is purified to obtain the antibody.

[0014] In some embodiments, the carrier protein in step (1) includes one or more of keyhole hemocyanin, bovine serum albumin, ovalbumin, hemocyanin, and thyroglobulin.

[0015] In some implementations, the experimental animals in step (2) include one or more of rabbits, mice, rats, sheep, horses, chickens, or donkeys.

[0016] The present invention also provides a kit for detecting the level of lactation modification at the K122 site of the CREB1 protein, comprising the antibody.

[0017] The present invention also provides the use of the lactated antigen in the preparation of lactated antibodies at the K122 site of the CREB1 protein.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) This invention provides a site-specific antibody targeting the lactation modification of lysine at position 122 of CREB1 (CREB1 K122la), which can achieve precise identification of specific lactation sites in the CREB1 protein. Compared with mass spectrometry detection, the antibody of this invention can be directly applied to conventional experimental platforms, and has the advantages of simple operation, low detection cost, and short experimental cycle. It is more suitable for large-scale sample, tissue sample and routine experimental verification, and is convenient for promotion and application in basic research and disease-related research.

[0019] (2) Compared with existing pan-lactation antibodies that can only reflect the overall lactation level of the sample and cannot distinguish specific proteins and specific modification sites, the antibody of the present invention has both protein specificity and site specificity. It can specifically recognize the lactation modification status of lysine at position 122 of the CREB1 protein, thereby directly answering the key scientific question of whether CREB1 has undergone lactation at position K122. It provides an effective technical tool for the study of the functional mechanism of CREB1 lactation, the study of disease-related molecular markers, and the monitoring of dynamic modifications.

[0020] (3) Compared with low-specificity antibodies obtained by conventional peptide immunization, the present invention improves the modification dependence on the K122la site through a purification process that removes non-specific antibodies from unmodified peptides. The ELISA titer of antibody Ab3 is greater than 54,000, the detection limit of Dot blot is 4 ng, and the signal for modified peptides is significantly stronger than that for unmodified peptides. An endogenous band of approximately 37 kDa can be detected in Western blot, indicating that the present invention has a clear, reproducible, and superior technical effect compared to existing methods. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a mass spectrometry result of the CREB1 peptide (lactyl)K-ILNDLSSDAPGVPR in Example 1 of the present invention.

[0023] Figure 2 This is the mass spectrometry detection result of CREB1 K122la polypeptide A in Example 1 of the present invention.

[0024] Figure 3 This is the mass spectrometry detection result of CREB1 K122la polypeptide B in Example 1 of the present invention.

[0025] Figure 4 This is the mass spectrometry detection result of the unmodified peptide CREB1 K122la in Example 1 of the present invention.

[0026] Figure 5 This is a preliminary screening result of immunoblotting of serum proteins in Example 1 of the present invention (HeLa cells).

[0027] Figure 6 This is a preliminary screening result of immunoblotting of serum proteins in Example 1 of the present invention (HepG2 cells).

[0028] Figure 7 This is a diagram showing the results of the purified antibody spot blot detection in Example 2 of the present invention.

[0029] Figure 8 This is a graph showing the antibody-protein immunoblotting detection results of Example 2 of the present invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0032] Example 1: Design, synthesis and validation of antigenic peptides Based on the CREB1 protein sequence (as shown in SEQ ID No. 1), two modified peptides and one unmodified control peptide were designed with lysine at position 122 as the core: CREB1 K122la polypeptide A: RRPSYR-(lactyl)K-ILNDLSC (SEQ ID No. 2).

[0033] CREB1 K122la polypeptide B: CRRPSYR-(lactyl)K-ILND (SEQ ID No. 3).

[0034] CREB1 K122la unmodified peptide: RRPSYRKILNDLSC (SEQ ID No. 4).

[0035] (lactyl)K represents lactated lysine; the C-terminal cysteine ​​is used for coupling with the carrier protein. Peptide A can be used for immunization, purification, and detection; peptide B can be used for parallel validation and supplemental screening; and the unmodified peptide is used for control detection and removal of nonspecific antibodies.

[0036] The specific sequences and theoretical molecular weights are shown in Table 1.

[0037] Table 1

[0038] The synthesized modified peptide A, modified peptide B, and unmodified control peptide were all confirmed by mass spectrometry. The difference between the measured mass and the theoretical mass was within 10 ppm, indicating that the peptide sequence and modification status were correct.

[0039] The above three polypeptides were detected by mass spectrometry, and the results are as follows: Figures 1-4 As shown, the sequences of the three peptides are accurate. The vertical axis represents the intensity of the ion peak, and the horizontal axis represents the mass-to-charge ratio. The difference between the measured and theoretical masses of the three peptides is within 10 ppm, indicating that the sequences are error-free.

[0040] (2) Peptide conjugation and immunogen preparation Modified peptide A and modified peptide B were conjugated to keyhole hemocyanin to form immunogens. The conjugated immunogens were diluted with physiological saline and emulsified with the corresponding adjuvant CFA at a 1:1 ratio to form a stable emulsion for later use.

[0041] (3) Animal immunization Four SPF-grade New Zealand white rabbits were selected as immunization animals. Rabbits immunized with CREB1 K122la peptide A were labeled R1 and R2; rabbits immunized with peptide B were labeled R3 and R4.

[0042] The immunization schedule is as follows: primary immunization is administered on day 1; booster immunizations are administered on days 21, 28, and 35. The injections are administered subcutaneously in both shoulders and intramuscularly in both hind legs at multiple points to enhance the immune response.

[0043] The blood collection procedure is as follows: On day 45, approximately 30 mL of whole blood was collected for the first time; subsequently, approximately 20 mL of blood was collected on days 50, 65, and 70. After centrifugation, the serum was collected for initial screening and subsequent purification.

[0044] (4) Initial serum screening Four SPF experimental-grade New Zealand white rabbits underwent multiple immunizations, and small amounts of serum were collected for ELISA testing to preliminarily assess the titer and specificity of the antiserum. Rabbits immunized with CREB1 K122la peptide A were designated R1 and R2, respectively, while those immunized with CREB1 K122la peptide B were designated R3 and R4. The ELISA results are shown in Tables 2 and 3.

[0045] Table 2

[0046] Table 3

[0047] The results showed that the serum of all four rabbits responded to the modified peptide, and the initial screening titer of the modified peptide reached about 1:4K (OD>1), indicating that the immunization was successful.

[0048] (5) Western blot initial screening Endogenous CREB1 K122la protein was detected using rabbit serum after immunization, and the results are as follows: Figures 5-6 As shown in the figure, bands with a theoretical molecular weight of approximately 37 kDa were detected at R1 and R3. Based on the combined results of ELISA and WB, serum from three rabbits (R1 and R3) was selected for purification.

[0049] (6) Antibody purification Antibody purification involves the following steps: The first step is Protein A affinity purification. The screened serum is loaded onto a Protein A column, and crude purified IgG is collected.

[0050] The second step is affinity enrichment of the modified peptide. Crude pure IgG is loaded onto an affinity column conjugated with CREB1 K122la modified peptides to enrich antibodies that recognize the modified peptides.

[0051] The third step is to remove non-specific antibodies. The antibody obtained in the previous step is loaded onto an unmodified homologous peptide affinity column, and the eluent is collected to remove antibody components that mainly recognize peptide backbones rather than lactation sites.

[0052] After purification, the antibody derived from R1 was labeled Ab1, and the antibody derived from R3 was labeled Ab3.

[0053] Example 2: Antibody Quality Control and Performance Verification Sufficient rabbit serum was collected and purified using Protein A and immunogenic peptide column affinity chromatography. The purified antibodies from R1 and R3 were labeled Ab1 and Ab3, respectively. The purified antibodies were then analyzed by ELISA, Dot Blot, and Western blotting.

[0054] (1) ELISA verification In 96-well ELISA plates coated with antigen-modified peptides and control peptides, antibodies were added and incubated at different dilution ratios (e.g., 1:54K, 1:162K). Then, enzyme-labeled secondary antibody and TMB substrate were applied, and the binding of the peptides and antibodies was detected by colorimetric analysis. The results are shown in Table 4.

[0055] Table 4

[0056] The results showed that both Ab1 and Ab3 could recognize at least one modified peptide at a dilution of at least 1:50K, and did not recognize unmodified negative peptides. It is recommended that antibody Ab3 be used at an ELISA positive dilution greater than 54,000, and that the recognition signal for modified peptides be 10 times stronger than that for unmodified peptides.

[0057] (2) Dot blot verification Modified and unmodified peptides at different doses (1 ng, 4 ng, 16 ng, 64 ng) were immobilized on a solid membrane, incubated with antibodies, and then enzyme-labeled secondary antibody and chemiluminescent substrate were added to detect the binding of peptides and antibodies.

[0058] Figure 7 The results showed that Ab1 showed no signal for unmodified peptides, while Ab3 showed almost no binding to unmodified peptides but significant binding to modified peptides. The recommended Dot blot detection limit for antibody Ab3 is 4 ng, and its signal for modified peptides is more than 10 times stronger than that for unmodified peptides.

[0059] (3) Western blot verification Ab1 and Ab3 were used to detect HeLa, NIH / 3T3 cell lysates and HeLa+Lac Na (100 mM, 24 h) and NIH / 3T3+Lac Na (25 mM, 24 h) lysates treated with sodium lactate, respectively.

[0060] Figure 8 The results showed that both Ab1 and Ab3 could be detected at approximately 37 kDa, and the signal changes were significant before and after sodium lactate treatment, indicating that the band represented the target protein CREB1 K122la. The recommended antibody Ab3 can stably recognize endogenous CREB1 K122la.

[0061] The antibody was stored in PBS buffer containing 50% glycerol and 0.01% sodium azide as a preservative. The antibody was aliquoted and stored at -20°C, avoiding repeated freeze-thaw cycles to ensure long-term stable preservation of its biological activity.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0063] sequence list SEQ ID No.1 MTMESGAENQQSGDAAVTEAENQQMTVQAQPQIATLAQVSMPAAHATSSAPTVTLVQLPN GQTVQVHGVIQAAQPSVIQSPQVQTVQISTIAESEDSQESVDSVTDSQKRREILSRRPSY RKILNDLSSDAPGVPRIEEEKSEEETSAPAITTVTVPTPIYQTSSGQYIAITQGGAIQLA NNGTDGVQGLQTLTMTNAAATQPGTTILQYAQTTDGQQILVPSNQVVVQAASGDVQTYQI RTAPTSTIAPGVVMASSPALPTQPAEEAARKREVRLMKNREAARECRRKKKEYVKCLENR VAVLENQNKTLIEELKALKDLYCHKSD SEQ ID No.2 RRPSYR-(lactyl)K-ILNDLSC SEQ ID No.3 CRRPSYR-(lactyl)K-ILND SEQ ID No.4 RRPSYRKILNDLSC。

Claims

1. A lactated antigen at the K122 site of the CREB1 protein, characterized in that, The lactation antigen is a lactation polypeptide, the amino acid sequence of which is shown in SEQ ID No. 2 or 3, and the lactation site of which is lysine.

2. The lactic acidified antigen according to claim 1, characterized in that, The lactation site of the lactated polypeptide is site 122 of the CREB1 protein.

3. An antibody that recognizes lactation modification at the K122 site of the CREB1 protein, characterized in that, The lactic acidified antigen described in claim 1 or 2 is obtained by immunizing an animal.

4. The antibody according to claim 3, characterized in that, The antibody can recognize lactation modification at the K122 site of the endogenous CREB1 protein in cell or tissue samples.

5. The antibody according to claim 3, characterized in that, The antibody is a polyclonal antibody, a monoclonal antibody, a recombinant antibody, or an antigen-binding fragment.

6. The method for preparing the antibody according to any one of claims 3 to 5, characterized in that, The preparation method includes the following steps: (1) The lactated polypeptide of claim 1 or 2 is coupled with a carrier protein to prepare an immunogen; (2) Immunize the experimental animals with the immunogen described in step (1) to obtain antiserum; (3) The antiserum is purified to obtain the antibody.

7. The preparation method according to claim 7, characterized in that, The carrier protein in step (1) includes one or more of keyhole hemocyanin, bovine serum albumin, ovalbumin, hemocyanin, and thyroglobulin.

8. The preparation method according to claim 7, characterized in that, The experimental animals mentioned in step (2) include one or more of the following: rabbit, mouse, rat, sheep, horse, chicken or donkey.

9. A kit for detecting the level of lactation modification at the K122 site of CREB1 protein, characterized in that, Includes the antibody as described in any one of claims 3 to 5.

10. The use of the lactated antigen according to claim 1 or 2 in the preparation of a lactated antibody at the K122 site of the CREB1 protein.

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