Lactylated antigen and antibody of CREB1 protein K122 site as well as preparation method and application of lactylated antigen and antibody

Abstract

The invention discloses a lactylated antigen and antibody of a CREB1 protein K122 site as well as a preparation method and application of the lactylated antigen and antibody, the lactylated antigen is a lactylated polypeptide, the amino acid sequence of the lactylated polypeptide is Ser Tyr Arg Lys Ile Leu Asn and is shown as SEQ ID NO: 1, and the lactylated site of the lactylated polypeptide is lysine. The lactylated antibody is prepared from the lactylated antigen through an immune animal body. The preparation method of the lactylated antibody comprises the following steps: S1, immunizing an animal model to a preset antiserum titer by adopting the lactylated antigen to obtain an antiserum sample; and S2, purifying the antiserum sample to obtain the lactylated antibody. The lactylated antigen and the antibody provided by the invention fill the blank that the lactylated antigen and the antibody of the CREB1 protein K122 site do not exist temporarily in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a lactic acid antigen and antibody at the K122 site of a CREB1 protein, and a preparation method and application thereof. Background Art

[0002] Cyclic AMP-responsive element-binding protein 1 (CREB1 / CREB) is a phosphorylation-dependent transcription factor that stimulates transcription after binding to the DNA cAMP response element (CRE). CREB1 is involved in a variety of cellular processes, such as synchronization of circadian rhythms, differentiation of adipocytes, and regulates the expression of apoptotic and inflammatory response factors in cardiomyocytes in response to ERFE-mediated activation of AKT signaling.

[0003] Post-translational modifications such as phosphorylation, lactylation, and ubiquitination play an important regulatory role in physiological and pathological processes mediated by cell signaling pathways. CREB1 plays an important role in epigenetic modification and signal transduction, so further research on the post-translational modification of CREB1 protein is of great significance. Based on previous studies, it was found that the 122nd lysine (K122) in the amino acid sequence of CREB1 is the site of protein lactylation modification, which may play an important role in regulating the function of CREB1 protein and upstream and downstream signal transduction.

[0004] Antibodies are important tools for studying protein functions and have been widely used in the study of the pathogenesis of autoimmune diseases, infectious diseases, tumors, disease diagnosis, and clinical treatment. However, there is currently no specific lactylated CREB1 K122 antibody available, which has seriously hindered the functional analysis of CREB1 protein and related molecular mechanism research. The specificity of antibodies mainly depends on the antigen, so a specific lactylated antigen of the CREB1 protein K122 site needs to be provided before antibody preparation. Summary of the invention

[0005] The purpose of the present invention is to provide a lactic acid antigen and antibody at the K122 site of CREB1 protein, and a preparation method and application thereof, so as to fill the gap of the prior art in that there is no specific lactic acid antigen and antibody at the K122 site of CREB1 protein.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a lactic acid antigen of the K122 site of the CREB1 protein, wherein the lactic acid antigen is a lactic acid polypeptide, and the amino acid sequence of the lactic acid polypeptide is Ser Tyr Arg Lys Ile Leu Asn, as shown in SEQ ID NO: 1, wherein the lactic acid site of the lactic acid polypeptide is lysine.

[0008] In the above technical solution, the lactylation site of the lactated polypeptide is site K122 of the CREB1 protein.

[0009] In a second aspect, the present invention provides a lactic acid antibody against the K122 site of the CREB1 protein, wherein the lactic acid antibody is prepared by immunizing an animal with the lactic acid antigen according to claim 1.

[0010] In a third aspect, the present invention provides a method for preparing a lactic acid antibody against the K122 site of the CREB1 protein, comprising the following steps:

[0011] S1: Immunizing an animal model with the lactated antigen according to claim 1 to a preset antiserum titer to obtain an antiserum sample;

[0012] S2: Purifying the antiserum sample to obtain the lactated antibody.

[0013] In the above technical solution, in S2, the preset antiserum titer is ≥1:64000; and / or the number of immunizations is ≥4 times.

[0014] In the above technical solution, in S2, the specific steps of purifying the antiserum sample to obtain the lactated antibody are:

[0015] The antiserum sample is affinity purified once using the lactated antigen shown in SEQ ID NO.1 to obtain a crude lactated antibody;

[0016] The crude lactic acid antibody is subjected to secondary affinity purification using the lactic acid antigen shown in SEQ ID NO.1 to obtain the lactic acid antibody.

[0017] In a fourth aspect, the present invention provides use of the above-mentioned lactic acid antigen or the above-mentioned lactic acid antibody in the preparation of a preparation for detecting lactic acid at the K122 site of human CREB1 protein.

[0018] In a fifth aspect, the present invention provides the use of the above-mentioned lactated antigen or the above-mentioned lactated antibody in the preparation of a pharmaceutical preparation for the diagnosis, treatment and prognosis of tumors, autoimmune diseases and cardiovascular diseases.

[0019] In a sixth aspect, the present invention provides a kit for the diagnosis, treatment and prognosis of tumors, autoimmune diseases and cardiovascular diseases, wherein the kit comprises the above-mentioned lactic acid antigen or the above-mentioned lactic acid antibody.

[0020] The beneficial effects of the present invention are as follows: the present invention provides a lactic acid antigen of the K122 site of the CREB1 protein, and the lactic acid analysis of the amino acid sequence of the CREB1 protein, combined with the specific analysis of the CREB1 protein, can clearly show that the No. 122 site of the CREB1 protein is a key site for lactic acid modification, and the site may play an important role in regulating the function of the CREB1 protein and upstream and downstream signal transduction, so the site is specifically designed to obtain the specific lactic acid antigen of the CREB1 protein, and the specific amino acid sequence of the lactic acid antigen is refined, and a lactic acid antibody with good specificity can be prepared by the lactic acid antigen, which is helpful to study the mechanism of action of lactic acid modification of a specific site of the CREB1 protein in the occurrence and development of a specific biological event or disease, and can also provide a potential target for the diagnosis and treatment of clinical diseases, and can also improve the accuracy of detecting the lactic acid level of the CREB1 protein by experiments such as western blot; at the same time, the lactic acid antigen can also be used to explore the relationship between CREB1 and autoimmune diseases, infectious diseases, tumors and other diseases, and has broad application prospects in disease diagnosis, treatment and prognosis determination. Therefore, the lactated antigen and antibody provided by the present invention fill the gap in the prior art in that there is no specific lactated antigen and antibody for the K122 site of the CREB1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic flow chart of a method for preparing a lactic acid antibody against the K122 site of CREB1 protein provided by the present invention;

[0022] Figure 2 A detailed schematic diagram of a method for preparing a lactic acid antibody against the K122 site of CREB1 protein provided by the present invention;

[0023] Figure 3 A schematic diagram of the actual process of a method for preparing a lactic acid antibody against the K122 site of the CREB1 protein provided by the present invention;

[0024] Figure 4 This is a graph showing the immunogenicity assessment results of the lactated polypeptide provided in Example 1 of the present invention;

[0025] Figure 5 This is a diagram showing the hydrophilicity evaluation results of the amino acid sequence of the lactated polypeptide provided in Example 1 of the present invention;

[0026] Figure 6This is a diagram showing the results of mass spectrometry and high performance liquid chromatography of the lactated polypeptide provided in Example 1 of the present invention, wherein: Figure 6 a is the mass spectrometry detection result of lactic acid peptide, Figure 6 b is a graph showing the results of HPLC detection of lactic acid polypeptide;

[0027] Figure 7 This is a graph showing the results of mass spectrometry and high performance liquid chromatography of the non-lactated polypeptide provided in Example 1 of the present invention, wherein: Figure 7 a is the mass spectrometry detection result of non-lactated peptides. Figure 7 b is a graph showing the results of high performance liquid chromatography detection of non-lactated polypeptides;

[0028] Figure 8 This is a graph showing the specificity of the CREB1 K122 lactic acid antibody verified by Western blotting technology provided in Example 4 of the present invention;

[0029] Fig. 9 This is a graph showing the specificity of the CREB1 K122 lactic acid antibody verified by immunofluorescence staining technology provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0030] 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 being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0031] The present invention provides a lactic acid antigen at the K122 site of a CREB1 protein. The lactic acid antigen is a lactic acid polypeptide. The amino acid sequence of the lactic acid polypeptide is Ser Tyr Arg Lys Ile Leu Asn, as shown in SEQ ID NO: 1, wherein the lactic acid site of the lactic acid polypeptide is the 122nd lysine site of the CREB1 protein.

[0032] By clarifying the specific lactylation site of the lactated polypeptide, the key lactylation modification site in the CREB1 protein can be identified. Since site 122 of the CREB1 protein may play an important role in regulating the function of the CREB1 protein and upstream and downstream signal transduction, specific design is carried out for this site, and specific antigens for the CREB1 protein can be clearly obtained, which is a basis for the subsequent acquisition of specific antibodies for the CREB1 protein.

[0033] Based on a general inventive concept, the present invention provides a lactic acid antibody against the K122 site of CREB1 protein. The lactic acid antibody is prepared by immunizing an animal body with a lactic acid antigen.

[0034] like Figure 1 As shown, the present invention provides a method for preparing a lactic acid antibody of the K122 site of the CREB1 protein, comprising the following steps:

[0035] S1. Immunize the animal model with lactated antigen to a preset antiserum titer to obtain an antiserum sample;

[0036] S2. Purify the antiserum sample to obtain lactic acid antibodies.

[0037] It should be noted that the preset antiserum titer can be detected by an indirect ELISA method.

[0038] It should be noted that the purification method can be carried out by antigen-antibody affinity purification.

[0039] In some optional embodiments, the preset antiserum titer is ≥1:64000, and the number of immunizations is ≥4 times.

[0040] By controlling the specific number of immunizations and the specific preset antiserum titer, the antiserum titer in the animal model can reach 1:64000 or more through multiple immunizations, thereby making it clear that the lactic acid antibody has good specificity.

[0041] In some optional embodiments, such as Figure 2 As shown, in S2, the specific steps of purifying the antiserum sample to obtain the lactic acid antibody are:

[0042] S201. Affinity purification is performed on the antiserum sample using the lactated antigen shown in SEQ ID NO.1 to obtain a crude lactated antibody;

[0043] S202. Using the lactated antigen shown in SEQ ID NO.1, the crude lactated antibody is subjected to secondary affinity purification to obtain the lactated antibody.

[0044] By refining the specific steps of purification, the antiserum samples are affinity purified multiple times using non-lactated antigens and lactated antigens in sequence, so that the antiserum samples can be initially purified using non-phosphoantigens, and then the antiserum samples are further purified using lactated antigens, so that specific antibodies can be obtained.

[0045] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0046] Embodiment 1: Figure 3 A schematic diagram showing a method for preparing a lactic acid antibody against the K122 site of the CREB1 protein

[0047] like Figure 3 As shown, the CREB1 protein K122 site lactic acid antibody is prepared, and the specific steps include:

[0048] 1. The immunogenicity and hydrophilicity of the antigen peptide sequence (SEQ ID NO: 1) were evaluated using the Immune Epitope Database (IEDB). Figure 4 and Figure 5 As shown, the results were all good, which indicated that SEQ ID NO: 1 had the potential to develop antigen peptides.

[0049] 2. According to the design of the hapten synthetic peptide, a lactic acid group was added to the K122 site to obtain a lactic acid antigen (lactated polypeptide), and then the quality of the synthetic lactic acid polypeptide was detected by HPLC and MASS. Figure 6 a is the mass spectrometry detection result of lactic acid peptide, Figure 6 b is the result of HPLC detection of lactic acid polypeptide. The results show that the sequence of the synthesized lactic acid polypeptide is correct, and the purity is greater than 90%, and the total amount meets the requirements. Subsequently, the lactic acid polypeptide is coupled with hemocyanin (KLH) to obtain the whole antigen, and then the whole antigen is used for immunization of rabbits (lK122-KLH).

[0050] Correspondingly, a non-lactated peptide corresponding to K122 was synthesized, and the quality of the synthesized peptide was then tested using HPLC and MASS. Figure 7 a is the mass spectrometry detection result of non-lactated peptides. Figure 7 b is the result of HPLC detection of non-lactated peptides. The results show that the sequence of the synthesized non-lactated peptide is correct, the purity is greater than 90%, and the total amount meets the requirements; each peptide is packaged and stored in a -80℃ refrigerator for subsequent antibody purification steps.

[0051] Example 2: Preparation of antiserum using the whole antigen (lK122-KLH) according to the multi-point immunization rabbit method

[0052] 1. Preparation of negative serum:

[0053] The experiment selected healthy female New Zealand white rabbits, 4 months old, with an average weight of 2.1 kg, and injected at multiple points on the back. 3 mL of blood was collected from the ear vein of the New Zealand white rabbit used for injection into a blood collection tube, and hemostasis was stopped by pressing with a cotton ball; the blood was then placed at room temperature for about 1 hour, and the blood was allowed to coagulate to form a blood clot, and then placed at 4°C for 2 hours to allow the serum to precipitate, centrifuged at 3000 rpm for 15 minutes, and the supernatant was aspirated, marked as negative control serum, and packaged and stored at -80°C for testing.

[0054] 2. Animal immunization (total time is about 49 days):

[0055] (1) Dissolve the lK122-KLH antigen polypeptide powder in sterile 1×PBS, draw the antigen solution with one sterile syringe, draw an equal amount of Freund's complete adjuvant (CFA) with another syringe, connect the two with a plastic tube, and pump back and forth repeatedly to mix the lK122-KLH peptide with CFA until a completely emulsified emulsion is formed. When dropped into water, it will not spread (the first main injection uses Freund's complete adjuvant, and the subsequent booster injections use Freund's incomplete adjuvant, both of which are fully mixed with an equal volume of antigen before injection).

[0056] (2) The first immunization was performed by subcutaneous injection on both sides of the thin and loose skin of the neck and back, intramuscular injection on both sides of the gluteal muscles and thighs, intradermal injection on both sides of the waist, injection on the rabbit paw pads, etc. The antigen emulsion was injected at multiple sites and multiple points. The first main injection dose was 1.6 mg antigen polypeptide / experimental rabbit.

[0057] (3) A second booster immunization was performed 14 days after the first immunization. Freund's incomplete adjuvant (IFA) was used instead of CFA as the immune adjuvant to prepare the antigen emulsion and injected in the same manner as the first immunization. 0.8 mg of antigen polypeptide was injected per experimental rabbit for booster injection.

[0058] (4) A third booster immunization was performed 28 days after the first immunization. Freund's incomplete adjuvant (IFA) was used instead of CFA as the immune adjuvant to prepare the antigen emulsion and injected in the same manner as the first immunization. The total amount of antigen in this booster immunization was about 0.8 mg.

[0059] (5) Blood collection after the third booster immunization: On the 29th day, 1 mL of blood was collected from the ear vein. The blood was placed at room temperature for about 1 hour to allow the blood to coagulate and form a blood clot. The blood was placed at 4°C for 2 hours to allow the serum to precipitate. The blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant was aspirated. The antiserum titer was detected by ELISA.

[0060] (6) The fourth booster immunization was carried out 42 days after the first immunization. The immunization antigen was Freund's incomplete adjuvant + lK122-KLH antigen polypeptide. Elisa test was performed after the fourth immunization. If the titer reached 256K or above, the rabbit was finally released. If the titer did not reach 256K, 6 to 7 more injections were given.

[0061] (7) Final bloodletting: On the 49th day, the titer of the antiserum tested by ELISA reached the requirement, and whole blood was collected from the carotid artery. The beaker used to collect the blood was sealed and left to stand at room temperature overnight to allow the blood clot to shrink. The next day, the precipitated serum was dispensed into 50 mL centrifuge tubes under aseptic operation, centrifuged at 3000 rpm for 15 min, and the supernatant was taken and dispensed into 1 mL / tube, labeled as post-immune antiserum (about 51 mL in total), and stored at -80°C.

[0062] Example 3: Antiserum purification, preparation of affinity purified antibodies and ELISA titer detection

[0063] 1. Preparation of affinity purified antibodies:

[0064] (1) The lactated antigen peptide 1K122 and the non-lactated antigen peptide K122 prepared in Example 1 were respectively coupled to bovine serum albumin (BSA) by the glutaraldehyde method and used as fillers 1K122-BSA and K122-BSA of affinity purification chromatography columns;

[0065] (2) Preparation of lactated synthetic peptide chromatography column and non-lactated synthetic peptide chromatography column: Dissolve 1K122-BSA and K122-BSA in coupling buffer, and the concentration is 0.7 mg / mL. Take 7 mg of 1K122-BSA and K122-BSA solution and add them to 5 mL of resin, mix them well, and then add them to the chromatography column and mix them at room temperature for 15 minutes. Place the chromatography column upright at room temperature for 30 minutes, remove the caps at the upper and lower ends of the chromatography column, collect the outflowing liquid, and rinse the column with 3 times the volume of resin coupling buffer. Cover the bottom cover of the chromatography column, add 50mM L-Cysteine ​​HCl to the coupling buffer, mix well, and then add an equal volume of buffer to the chromatography column. Mix well at room temperature for 15min, let it stand for 30min, then remove the bottom cover to release the coupling buffer, wash the chromatography column with 6 volumes of 1M NaCl, and then rinse the chromatography column with 2 volumes of storage buffer, cover the cover, add 1 volume of storage buffer, and store at 4℃ for later use;

[0066] (3) Incubate 10 mL of antiserum with the affinity purification column overnight. Pre-wash with HCl at pH 5.0 to remove impurities; then elute with 0.15 Mglycine (pH 2.5) buffer and quickly neutralize with 10x PBS buffer to prepare affinity purified antibodies;

[0067] (4) passing the affinity purified antibody through a non-lactic acid affinity adsorption column to remove antibodies against the non-lactated polypeptide and obtain a lactated site-specific antibody;

[0068] The concentration of purified antibody was detected by Bradford method. The results are shown in Table 1.

[0069] Table 1 Concentration of each purified antibody

[0070] project Concentration (mg / ml) Volume(ml) Mass (mg) M40316-A 0.501 0.4 0.2004 M40316-B 0.3 0.4 0.12

[0071] As shown in Table 1, the yield of affinity purified antibodies is normal.

[0072] 2. Affinity purification antibody ELISA titer detection:

[0073] (1) Antigen coating: Lactated antigen peptide lK122 and non-lactated antigen peptide K122 were diluted to 6 μg / mL with 0.05 mol / L carbonate (pH = 9.6), 100 μL / well, and incubated at 4°C overnight;

[0074] (2) Washing the plate: Wash the plate three times with 0.05% Tween-20 (PBST), 3 min each time;

[0075] (3) Blocking: Add 150 μl of 5% skim milk powder (PBST) to each well and block at 37°C for 60 min;

[0076] (4) Washing the plate: Wash three times with 0.05% Tween-20 (PBST), 3 min each time;

[0077] (5) Adding primary antibody: dilute affinity purified antibody at 1:1000, then dilute in equal proportions, and incubate at 37°C for 1 h;

[0078] (6) Washing the plate: Wash three times with 0.05% Tween-20 (PBST), 3 min each time;

[0079] (7) Add secondary antibody: horseradish enzyme-labeled goat anti-rabbit IgG (H+L), catalog number: 116154, antibody company: Jackson. Dilute at a volume ratio of 1:8000 and incubate at 37°C for 45 min;

[0080] (8) Washing the plate: Wash the plate five times with 0.05% Tween-20 (PBST), 3 min each time;

[0081] (9) Color development: Add 100 μL / well of substrate solution (TMB), react for 5 min to 10 min, and finally add 100 μL of 2 mol / L sulfuric acid to terminate the reaction;

[0082] (10) OD value measurement: The OD value was measured using an enzyme marker at a wavelength of 450 nm. The results are shown in Table 2.

[0083] Table 2 Affinity purification antibody ELISA titer test results

[0084]

[0085] Example 4: Application of CREB1 protein K122 site lactylation antibody in Western blot experiments

[0086] The highly specific lactic acid antibody of the CREB1 protein K122 site prepared by the present invention can be used to detect the difference in the lactic acid level of the CREB protein in cells by Western blot experiment, and the specific steps include:

[0087] (1) One day in advance, a 96-well plate was coated with anti-CD3 antibody and anti-CD28 antibody and placed in a 4°C refrigerator overnight. Naive CD4+T cells were extracted from the spleen and lymph nodes of wt mice with a NOD background, resuspended in T cell complete medium containing IL-2 and TGF-β, and plated into a 96-well plate coated with CD3 CD28 overnight. Cell protein was extracted 72 hours later, and cells were lysed using RIPA Lysis Buffer at 4°C. The cell lysate was centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was transferred to a new EP tube, 5×SDS loading buffer was added, and boiled at 100°C for 10 minutes. To prevent protein degradation, the operation should be performed on ice as much as possible.

[0088] (2) SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis): Clamp the glass plate with polyacrylamide gel as needed, quickly add the prepared 12% concentration SDS polyacrylamide separation gel into the gap of the glass plate to 2 / 3 of the glass plate, and seal the gel with isopropanol. After the separation gel solidifies, pour out the isopropanol and absorb the residual liquid with filter paper. Add the prepared 5% mass concentration stacking gel to the separation gel and immediately insert the comb. After the gel is completely solidified, pull out the comb and rinse the sample wells with water. Fix the electrophoresis device, add Tris-glycine electrophoresis buffer, and add 20μL of sample and pre-stained marker in sequence. Connect the electrophoresis device to the power supply, adjust the voltage to 80V, and when bromophenol blue runs to the separation gel, adjust the voltage to 110V until bromophenol blue runs to the bottom of the separation gel, and turn off the power;

[0089] (3) Transfer: Remove the gel, remove the accumulated gel and excess parts, and completely soak it in the newly prepared transfer buffer; cut 4 pieces of filter paper and 1 PVDF membrane of equal size to the gel, and make a mark on one corner of the membrane with a pencil; soak the PVDF membrane in 100% methanol for about 1 min to activate the PVDF membrane, and then soak it in transfer buffer; lay the cathode plate of the transfer device flat, place a layer of sponge soaked in transfer buffer, and neatly place two layers of filter paper soaked in transfer buffer on the sponge, and use a glass rod to drive out bubbles; place the transfer clip with the black side facing down in the transfer buffer, and place the black sponge, two layers of filter paper, gel, membrane (activated with methanol for 1 min in advance, mark the front and back in the upper right corner, and the reverse side is attached to the membrane), two layers of filter paper and black sponge in order from bottom to top, ensuring that there are no bubbles between the membrane and the gel and that they are completely attached; clamp the gel with a transfer clip. Place the fixed transfer clip into the transfer tank, fill the transfer tank with transfer solution, and finally place the transfer tank in a foam box filled with ice. Transfer the membrane for 2.5 hours at a constant current of 180mA (black to black, red to red, pay attention to the direction of the electrodes);

[0090] (4) Blocking: Take out the electroporated PVDF membrane, wash it once with TBST, and block it with 5% milk on a shaker at room temperature for 1 h;

[0091] (5) Primary antibody incubation: dilute the CREB1 protein K122 site lactic acid antibody 1:1000 with primary antibody diluent; remove the blocked PVDF membrane, rinse with TBST for 2 minutes, and then incubate in the primary antibody at 4°C overnight (16 hours)

[0092] (6) Washing: Wash with TBST 5 times, 5 min each time;

[0093] (7) Secondary antibody incubation: Place the PVDF membrane in a horseradish peroxidase-labeled secondary antibody (1:5000) diluted in 5% milk and incubate on a shaker at room temperature for 1 h.

[0094] (8) Washing: Wash with TBST 5 times, 5 min each time;

[0095] (9) Development: Mix ECL AB solution in a volume ratio of 1:1 (0.5 mL each), pour evenly onto the PVDF membrane, and wet and dry the color development box with clean toilet paper. Place the membrane in the box and remove bubbles. After 5 minutes of color development, take it to a dark room, cut a film of corresponding size and stick it on the membrane. Determine the exposure time according to the fluorescence intensity.

[0096] (10) After developing the film, place it in the developer for 1-3 minutes, rinse it with clean water, place it in the fixer for about 9 minutes, and finally dry it in an oven. Determine the target band based on the marker and analyze the results.

[0097] Test results such as Figure 8As shown, this indicates that in the immunoprecipitation product, the K122 site lactic acid antibody (CREB1K122) can recognize the CREB1 protein in the lactic acid modified state at the K122 site, but cannot recognize the CREB1 protein in the non-lactated state at the K122 site, proving that the lactic acid antibody has good specificity. Example 5: Application of the CREB1 protein K122 site lactic acid antibody in immunofluorescence staining experiments

[0098] The highly specific lactic acid antibody against the K122 site of the CREB1 protein prepared in the present application can be used for Western blot experiments to detect the difference in the lactic acid level of the CREB protein in cells. The specific steps include:

[0099] (1) One day in advance, raw cells were plated in a 24-well plate with a cell slide and cultured in complete medium containing 50 mM sodium L-lactate;

[0100] (2) After 24 hours, the culture medium was removed and PBS was added to wash the cells;

[0101] (3) Add 1 ml of 4% paraformaldehyde to the wells to fix the cells at room temperature for 20 min;

[0102] (4) Remove the paraformaldehyde and wash with PBS three times, 5 min each time;

[0103] (5) Add 0.5% Triton X-100 (prepared in PBS) to the wells and permeabilize for 20 min at room temperature. Wash with PBS for 5 min after completion.

[0104] (6) Blocking solution (10% BSA 100ul + donkey serum 50ul + 850ul PBS) at room temperature for 1h;

[0105] (7) Primary antibody incubation: add Creb lactic acid antibody (diluted at 1:100 using primary antibody diluent) to immerse the stained cell slides and incubate overnight (14-16 hours);

[0106] (8) Place at room temperature for 30 min, then wash with 1x PBS for 5 min x 3 times;

[0107] (9) Add rabbit 594 fluorescent secondary antibody (diluted 1:200 using secondary antibody diluent) and incubate at room temperature for 1 h;

[0108] (10) Wash with 1x PBS, 5 min x 3 times;

[0109] (11) Add DAPI dye to immerse the cell slides for 2-5 min, and wash with 1x PBS for 5 min x 3 times;

[0110] (12) Sealing the slides and adding an anti-fluorescence quenching agent to seal the slides;

[0111] (13) Observe and take photos under a fluorescence microscope.

[0112] In summary, the present invention provides a lactic acid antigen at the K122 site of the CREB1 protein, through which a lactic acid antibody with good specificity can be prepared. Therefore, the lactic acid antigen provided by the present invention fills the gap in the prior art that there is no specific lactic acid antigen at the K122 site of the CREB1 protein.

[0113] At the same time, the present invention provides a lactic acid antibody for the K122 site of the CREB1 protein. In practical applications, the lactic acid antibody is convenient for studying the correlation between lactic acid modification of a specific site of the CREB1 protein and specific biological events such as immune cell activation, cell metabolic reprogramming, cell cycle, etc.

[0114] At the same time, the lactic acid antibody of the K122 site of the CREB1 protein provided by the present invention can also be used to study the lactic acid polyclonal antibody of the K122 site of the CREB1 protein, which is helpful to explore the various biological functions of the CREB1 protein.

[0115] In addition, the lactic acid antibody against the K122 site of the CREB1 protein provided by the present invention is helpful to explore the mechanism of action of the lactic acid modification of the CREB1 protein in the occurrence and development of autoimmune diseases, infectious diseases, tumors and other diseases, and can also be used to evaluate changes in tumor metabolic levels under different treatment conditions, providing potential targets for the diagnosis and treatment of clinical tumor diseases.

[0116] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A lactic acid antigen at the K122 site of CREB1 protein, characterized in that: The lactated antigen is a lactated polypeptide, and the amino acid sequence of the lactated polypeptide is Ser Tyr Arg Lys Ile Leu Asn, as shown in SEQ ID NO: 1, wherein the lactated site of the lactated polypeptide is lysine.

2. The lactated antigen according to claim 1, characterized in that: The lactic acid site of the lactated polypeptide is site K122 of the CREB1 protein.

3. A lactic acid antibody against the K122 site of CREB1 protein, characterized in that: The lactated antibody is prepared by immunizing an animal with the lactated antigen according to claim 1.

4. A method for preparing a lactic acid antibody against the K122 site of CREB1 protein, characterized in that: The following steps are involved: S1: Immunizing an animal model with the lactated antigen according to claim 1 to a preset antiserum titer to obtain an antiserum sample; S2: Purifying the antiserum sample to obtain the lactated antibody.

5. The preparation method according to claim 4, characterized in that: In S2, the preset antiserum titer is ≥1:64000; and / or the number of immunizations is ≥4 times.

6. The preparation method according to claim 4, characterized in that: In S2, the specific steps of purifying the antiserum sample to obtain the lactated antibody are: The antiserum sample is affinity purified once using the lactated antigen shown in SEQ ID NO.1 to obtain a crude lactated antibody; The crude lactic acid antibody is subjected to secondary affinity purification using the lactic acid antigen shown in SEQ ID NO.1 to obtain the lactic acid antibody.

7. Use of the lactated antigen according to claim 1 or the lactated antibody according to claim 3 in the preparation of a preparation for detecting lactation at the K122 site of human CREB1 protein.

8. Use of the lactated antigen according to claim 1 or the lactated antibody according to claim 3 in the preparation of a pharmaceutical preparation for the diagnosis, treatment and prognosis of tumors, autoimmune diseases and cardiovascular diseases.

9. A kit for diagnosis, treatment and prognosis of tumors, autoimmune diseases and cardiovascular diseases, characterized in that: The kit comprises the lactated antigen according to claim 1 or the lactated antibody according to claim 3.

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