A bclaf1 protein ser564 site phosphorylation antigen peptide, specific antibody and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a BCLAF1 protein Ser564 site phosphorylation antigen peptide, a specific antibody and a preparation method and application thereof. The application identifies BCLAF1 protein Ser564 as a key phosphorylation site through SIK2 kinase screening, and designs and synthesizes a phosphorylation antigen peptide based on the site. After immunizing animals, a polyclonal antibody specifically recognizing BCLAF1 Ser564 site phosphorylation modification is successfully prepared. The antibody has high sensitivity and strong specificity, is suitable for various detection platforms such as immunohistochemistry and immunoblotting, and can be used for evaluating tumor immunotherapy efficacy. The application provides a key tool for analyzing BCLAF1 phosphorylation regulation mechanism, and lays a theoretical foundation for developing an immune combined therapy strategy targeting a SIK2-BCLAF1-CCL5 axis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a phosphorylated antigenic peptide at the Ser564 site of the BCLAF1 protein, a specific antibody, its preparation method, and its application. Background Technology

[0002] BCL-2 associated transcription factor (BCLAF1) is a multifunctional protein with an arginine-serine-rich RS domain and a DNA-binding domain, participating in the regulation of gene transcription and post-transcriptional processing. Studies have shown that BCLAF1 is involved in various cell biological processes, such as apoptosis, proliferation, and DNA damage repair, and BCLAF1 gene mutations or abnormal protein expression are also closely related to the occurrence of human cancers.

[0003] The human BCLAF1 gene is located in the 6q22-23 region of chromosome 6. This gene contains 13 exons, with a total length of 2763 bp. Alternative splicing occurs at the 5' end of exon 5 and exon 11, while the remaining exons undergo constitutive splicing. Due to different alternative splicing methods, the BCLAF1 protein has four distinct protein isoforms, including the full-length protein (BCLAF1-L) and three truncated isoforms. The BCLAF1-L isoform is widely distributed in mammalian tissues and is also expressed in various tumor cell lines. In terms of protein sequence characteristics, BCLAF1 contains a basic zipper-like structure (bZIP) and a Myb-like DNA-binding domain, thus functioning as a transcription factor. Subsequent studies suggest that the most significant functional domain of BCLAF1 is the arginine-serine-rich RS domain located at the N-terminus of its open reading frame. Proteins containing RS domains are often involved in the splicing of mRNA precursors. For example, SR proteins are a family of proteins that contain RS domains and play an important role in mRNA precursor splicing. Their RS domains usually mediate protein-protein interactions.

[0004] Initially, B-cell lymphoma-2-associated transcription factor 1 (BCLAF1) was identified as a regulator of apoptosis and transcription. Subsequent studies have revealed its association with a variety of biological processes, including DNA damage responses, pre-mRNA splicing and processing, T cell activation, lung development, muscle cell proliferation and differentiation, autophagy, ischemia-reperfusion injury, and viral infection. In recent years, increasing evidence suggests that BCLAF1 can act as both a tumor promoter and a tumor suppressor in tumorigenesis, depending on the cellular environment and cancer type. Even within the same tumor type, the effects of BCLAF1 can be contradictory.

[0005] CD8 + T cells are a crucial type of immune cell in the tumor immune microenvironment. They are cytotoxic T cells that release pro-inflammatory factors and recognize and kill tumor cells. In patients with serous ovarian cancer, elevated CD8+ levels are observed. + The overall survival of the T cell group was significantly better than that of the low CD8 group. + The T-cell group was not related to age, pathological grade, or clinical stage. Furthermore, studies have found CD8... + The number of T cells showed a positive correlation with survival time, suggesting that their anti-tumor effect is dose-dependent. CD8+ cells were present in cancerous lesions. + For every certain threshold increase in T cell count, patient survival may be correspondingly prolonged. In the tumor immune microenvironment, the chemokine CCL5 acts as a CD8+ chemokine. + The "navigation signals" of T-cell anti-tumor immunity can directionally guide CD8 + T cells participate in anti-tumor immune responses. CD8 + T cells are the "main force" in the fight against tumors, playing a core role by directly killing tumor cells and reshaping the immune microenvironment. Therefore, in-depth research is needed on the regulatory network of CCL5 expression to provide new ideas and methods for immunotherapy of ovarian cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a phosphorylated antigenic peptide at the Ser564 site of the BCLAF1 protein, a specific antibody, its preparation method, and its applications. This not only enables in-depth research into the correlation of BCLAF1 phosphorylation modification in the evaluation of tumor immunotherapy efficacy, but also allows for screening upstream kinases that regulate phosphorylation at this site, thus elucidating the multiple biological functions of BCLAF1.

[0007] The technical solution provided by this invention is as follows:

[0008] This invention provides a phosphorylated antigenic peptide at the Ser564 site of the BCLAF1 protein, wherein the active amino acid sequence of the antigenic peptide contains CDDSNRPAS(pi)LTKDR, as shown in SEQ ID NO: 1, wherein S is phosphorylated.

[0009] The present invention also provides a specific antibody against phosphorylation at the Ser564 site of the BCLAF1 protein. The specific antibody is prepared by immunizing animals with the phosphorylated antigenic peptide at the Ser564 site of the BCLAF1 protein as described above, and the specific antibody is a polyclonal antibody.

[0010] This invention also provides a method for preparing the above-described specific antibody against phosphorylation at the Ser564 site of the BCLAF1 protein, comprising the following steps:

[0011] Identify the Ser564 phosphorylation site of the anti-BCLAF1 protein;

[0012] The phosphorylated antigenic peptide at the Ser564 site of the BCLAF1 protein was designed based on the BCLAF1 target sequence and the determined Ser564 phosphorylation site of the anti-BCLAF1 protein.

[0013] The obtained phosphorylated antigenic peptide at site Ser564 of the BCLAF1 protein was coupled with a carrier protein to obtain the antigen.

[0014] Animals were immunized with the obtained antigens, and antiserum was collected. The antiserum was purified and identified to obtain a specific antibody against phosphorylation of BCLAF1 protein at the Ser564 site.

[0015] Furthermore, the Ser564 phosphorylation site of the anti-BCLAF1 protein was identified as including:

[0016] BCLAF1, a substrate protein downstream of SIK2 kinase that is phosphorylated by SIK2 kinase and participates in the regulation of CCL5 expression, was screened out by proteomic analysis and bioinformatics prediction.

[0017] The Ser564 site was verified to be a specific phosphorylation site of SIK2 kinase through in vitro kinase experiments and point mutation experiments.

[0018] Furthermore, the immunization method includes: after the animal's initial immunization, multiple booster immunizations are performed, with each immunization involving the injection of a mixture of the phosphorylated antigen peptide at the Ser564 site of the BCLAF1 protein and an adjuvant, and finally, the antiserum is collected.

[0019] Furthermore, the purification and identification of the antiserum includes: purifying the collected antiserum using affinity chromatography; and identifying the titer and specificity of the purified antibody using enzyme-linked immunosorbent assay (ELISA).

[0020] The present invention also provides the use of the above-described phosphorylated antigenic peptide of BCLAF1 protein at Ser564 site or the above-described specific antibody against phosphorylation of BCLAF1 protein at Ser564 site in the preparation of a formulation for detecting phosphorylation of BCLAF1 protein at Ser564 site.

[0021] The present invention also provides the application of the phosphorylated antigenic peptide at the Ser564 site of the BCLAF1 protein or the specific antibody against phosphorylated Ser564 site of the BCLAF1 protein described above in the preparation of antitumor immune products for regulating ovarian cancer.

[0022] Furthermore, the regulation involves SIK2 kinase phosphorylating the Ser564 site of the BCLAF1 protein, thereby inhibiting CCL5 expression and subsequently negatively regulating CD8.+ T cell infiltration and function affect the immune response to ovarian cancer.

[0023] The present invention also provides the use of the phosphorylated antigenic peptide at the Ser564 site of the BCLAF1 protein described above or the specific antibody against phosphorylated Ser564 site of the BCLAF1 protein described above in the preparation of products for the diagnosis and / or prevention of ovarian cancer.

[0024] The present invention also provides a diagnostic reagent or kit for detecting the phosphorylation level of BCLAF1 protein at the Ser564 site in ovarian cancer, comprising the specific antibody as described above.

[0025] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of ovarian cancer, comprising an active ingredient capable of inhibiting phosphorylation of the BCLAF1 protein at the Ser564 site, or comprising a specific antibody as described above.

[0026] Beneficial effects

[0027] This invention reveals for the first time the crucial regulatory role of phosphorylation at site S564 of the BCLAF1 protein in immune escape from ovarian cancer. The study found that phosphorylation at this site inhibits the expression of CCL5 chemokine, thereby weakening CD8+ expression in the tumor microenvironment. + T cell infiltration and dysfunction lead to impaired anti-tumor immune responses. Targeted intervention of SIK2 kinase can induce dephosphorylation and inactivation of BCLAF1 at S564, thereby restoring CCL5 secretion and enhancing CD8. + T cell recruitment and activation. This discovery reveals the potential value of the SIK2-BCLAF1-CCL5 regulatory axis in tumor immunotherapy and provides an important theoretical basis for developing novel immunotherapy strategies.

[0028] This invention successfully prepared a polyclonal antibody that specifically recognizes phosphorylation modification at the S564 site of human BCLAF1 protein. This antibody can detect the phosphorylation status of BCLAF1 protein at this site with high sensitivity and specificity, and can be widely used in various experimental platforms such as immunohistochemistry and Western blotting. Using this antibody, not only can the correlation of BCLAF1 phosphorylation modification in the evaluation of tumor immunotherapy efficacy be investigated in depth, but it can also be used to screen upstream kinases that regulate phosphorylation at this site, elucidating the multiple biological functions of BCLAF1. Furthermore, by qualitatively detecting the phosphorylation level at the S564 site, this antibody enables the auxiliary assessment of tumor immunotherapy sensitivity, providing a new technical means for patient stratification and the selection of personalized treatment plans, and has good scientific research value and clinical translation prospects. Attached Figure Description

[0029] Figure 1To target and inhibit SIK2 to increase CD8 in the ovarian cancer tumor microenvironment + T cell infiltration; where A represents the detection of CD8+ in two groups of subcutaneous tumors using flow cytometry. + The proportion of T cells; B represents the two groups of CD8. + T cells account for 45% of CD45 + A statistical chart showing the proportion of T cells.

[0030] Figure 2 To target and inhibit SIK2 to promote CD8 + The function of T cells; among them, A is the detection of GZMB in two groups of subcutaneous tumors using flow cytometry. + CD8 + The proportion of T cells; B represents the two groups of GZMB. + CD8 + T cells account for a significant portion of CD8. + A statistical chart showing the proportion of T cells.

[0031] Figure 3 SIK2 inhibits CD8 + T cell infiltration and function are involved in the immune regulation of ovarian cancer; among them, A shows the tumor burden at three different time points using small animal in vivo imaging technology; B is a statistical graph of the peritoneal tumor burden of three groups of mice on day 28.

[0032] Figure 4 Inhibiting SIK2 can promote the secretion of CCL5.

[0033] Figure 5 CCL5-mediated SIK2-CD8 + T cell chemotaxis; where A is the detection of CD8 in two groups of subcutaneous tumors using flow cytometry. + The proportion of T cells; B represents the two groups of CD8. + T cells account for 45% of CD45 + A statistical chart showing the proportion of T cells.

[0034] Figure 6 To confirm through co-immunoprecipitation that SIK2 can bind to the transcriptional repressor BCLAF1; where A is the set of SIK2-interacting proteins, the set of proteins whose phosphorylation level is affected by SIK2, and the set of candidate transcription factors for CCL5, the intersection of the three was found that BCLAF1 meets the three common conditions; B is to verify the binding of SIK2 and BCLAF1 through Co-IP experiment.

[0035] Figure 7 In vitro kinase experiments revealed that SIK2 can phosphorylate the transcriptional repressor BCLAF1 at site S564.

[0036] Figure 8Chromatin immunoprecipitation experiments confirmed that BCLAF1 can bind to the CCL5 promoter region.

[0037] Figure 9 To investigate how targeted inhibition of BCLAF1 can promote the transcription of the CCL5 gene, A was used to detect the gene silencing effect of BCLAF1 siRNA transfected into SKOV3 cells by Western blot; B was used to detect the expression level of CCL5 mRNA after knocking down BCLAF1 by PCR experiment; and C was used to detect the level of CCL5 transcriptional activity after knocking down BCLAF1 by luciferase reporter assay.

[0038] Figure 10 Knockout of SIK2 affects phosphorylation at the S564 site of BCLAF1. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0040] The experimental materials used in the embodiments of this invention are:

[0041] Human ovarian cancer cell line SKOV3 and mouse ovarian cancer cell line ID8 were purchased from ATCC. SIK2 knockout ID8, parental SKOV3 cells, and LUC-tagged cells were constructed by Shanghai Aibosi Biotechnology Co., Ltd. SKOV3-SIK2 - / - -LUC, ID8-SIK2 - / - -LUC cells were cultured in DMEM medium containing 10% FBS, 100 U / mL penicillin and streptomycin (double antibiotics), and 5 μg / mL Luromycin. SKOV3 and ID8 cells were cultured in 1640 medium containing 10% FBS, 100 U / mL penicillin and streptomycin (double antibiotics).

[0042] The experiment used 3-5 week old female nude mice and C57BL / 6 mice, all purchased from Nanjing Jicui Pharmaceutical Co., Ltd. All mice were housed in the animal facility of Soochow University.

[0043] Table 1 Main Reagents

[0044]

[0045] Example 1: Determination of phosphorylation sites of BCLAF1 protein

[0046] 1. SIK2 deficiency affects CD8+ in the tumor microenvironment. + The effect of T cells

[0047] To investigate the effect of SIK2 on CD8 + The influence of T cell infiltration and function in the tumor microenvironment was investigated in this embodiment. A mouse model of ovarian cancer cells with SIK2 gene knockout was constructed, and tumor-infiltrating lymphocytes were analyzed by flow cytometry.

[0048] 1.1 Experimental Methods

[0049] (1) Animal model construction: Healthy female C57BL / 6 mice were randomly divided into control and experimental groups. They were subcutaneously inoculated with ID8-Control (control group) and ID8-SIK2 mice in the logarithmic growth phase, respectively. - / - (Experimental group) Ovarian cancer cells were used to establish a subcutaneous xenograft model.

[0050] (2) Preparation of spleen single-cell suspension: Control mice were sacrificed, spleens were aseptically removed, ground and filtered through a sieve, centrifuged, and red blood cells were lysed to obtain spleen single-cell suspension, which was used as a staining control sample.

[0051] (3) Preparation of single-cell suspension of tumor tissue: Tumor-bearing mice were sacrificed, the tumor was dissected, cut into small pieces, ground, and filtered through a sieve. The obtained cells were digested with type I collagenase, digestion was stopped, and the suspension was filtered to obtain a single-cell suspension of tumor tissue.

[0052] (4) T cell flow cytometry analysis: Tumor single cell suspensions from the control group and experimental group were taken and activated with cell stimulants. Subsequently, staining with live / dead dyes, staining with cell surface antibodies (CD45-APC, CD4-FITC, CD8-APC), and staining with cell membrane permeability and intracellular factors (GZMB-FITC) were performed. After washing and resuspending, the cells were analyzed by flow cytometry, and the levels of CD8+ in the two groups were compared. + The proportion of T cells and the expression level of GZMB.

[0053] 1.2 Experimental Results

[0054] Studies have found that targeted inhibition of SIK2 reduces CD8 levels in the ovarian cancer tumor microenvironment. + T cell infiltration was significantly increased. Figure 1 Furthermore, after targeting and inhibiting SIK2, CD8 in the tumor microenvironment... + The functional marker GZMB of T cells was also significantly increased, indicating that CD8 + Enhanced T cell function Figure 2 ).

[0055] 2. Verification of anti-tumor effects depends on CD8 + T cells

[0056] To confirm whether SIK2 negatively regulates CD8 + T-cell-mediated anti-tumor immunity was investigated by constructing an ovarian cancer peritoneal xenograft model in immunocompetent mice (C57BL / 6). The tumor burden was assessed using in vivo imaging technology, and the CCL5 secretion level was detected by ELISA to explore its molecular mechanism.

[0057] 2.1 Experimental Methods

[0058] (1) Animal grouping and treatment: 3-5 week old female C57BL / 6 mice were intraperitoneally inoculated with luciferase-labeled ID8-Luc-Control or ID8-Luc-SIK2- / - ovarian cancer cells (2×10⁻⁶). 6 Cells / mouse). One week after inoculation, tumor formation was confirmed by small animal in vivo imaging. Tumor-bearing mice were randomly divided into three groups (n=5): Control group (inoculated with ID8-Luc-Control cells), SIK2 group, and control group (inoculated with ID8-Luc-Control cells). - / - Group (inoculated with ID8-Luc-SIK2) - / - (cells) and SIK2 - / - +anti-CD8α group (inoculated with ID8-Luc-SIK2) - / - Cells, and combined with intraperitoneal injection of CD8 + T-cell clearance antibody anti-CD8α (BioXcell, BE0004-1-25MG, dosage 10mg / kg, twice weekly).

[0059] (2) Tumor growth monitoring: Starting from the date of grouping, the tumor luciferase activity of mice in each group was detected and recorded weekly using a small animal in vivo imaging system to assess tumor growth. Mice were sacrificed on day 28 of the experiment, and tumor tissue was collected for subsequent analysis.

[0060] (3) ELISA detection of CCL5 secretion: Follow the instructions of the ELISA kit to detect and analyze ID8-Luc-Control and ID8-SIK2. - / - Changes in CCL5 in cell culture supernatant.

[0061] 2.2 Experimental Results

[0062] (1) In vivo luciferin activity display: Compared with the control group, SIK2 - / - Tumor growth was significantly inhibited in the group, demonstrating the anti-tumor effect of SIK2 knockout in immune-intelligent mice; importantly, SIK2 - / - After combined anti-CD8α treatment, the anti-tumor effect was significantly weakened or even disappeared, proving that SIK2 regulates CD8. +T cells participate in the immune regulation of ovarian cancer. Figure 3 ).

[0063] (2) ELISA results showed that knocking out SIK2 significantly increased CCL5 secretion. Figure 4 This indicates that in ovarian cancer, knocking out SIK2 expression can promote CCL5 secretion.

[0064] 3. Identification and Functional Verification of CCL5

[0065] It is known that the chemokine CCL5 can mediate CD8 + The directed migration of T cells to tumor sites activates the regulatory axis of specific anti-tumor immune responses. This study aims to verify the role of CCL5 in regulating CD8 downstream of SIK2. + In this embodiment, a stable cell line overexpressing CCL5 and ID8 was constructed to investigate the role of T cell chemotaxis, and its effect on CD8 was analyzed using a subcutaneous xenograft model. + The effect of T cell infiltration.

[0066] 3.1 Experimental Methods

[0067] (1) Construction of stable cell lines: CCL5 overexpressing lentivirus (OE-CCL5) and its control lentivirus (OE-NC) were purchased from Shanghai Jikai Gene Co., Ltd. ID8 cells were seeded in 24-well plates and cultured at 37°C for 24 h. Viral supernatant and polybrene were added at a multiplicity of infection (MOI) of 20. The medium was changed 24 hours after infection, and the transfection efficiency was observed under a fluorescence microscope after 72 hours. Subsequently, the cells were screened with 3 µg / mL puromycin for 2 weeks to obtain a stable ID8 cell line overexpressing CCL5 (ID8-CCL5) and its control cell line (ID8-NC), which were then expanded and cultured for subsequent experiments.

[0068] (2) Animal Model and Flow Cytometry Analysis: Healthy female C57BL / 6 mice were randomly divided into two groups: a control group and an experimental group, which were subcutaneously inoculated with ID8-blank cells in the logarithmic growth phase. A subcutaneous xenograft tumor model was established. Tumor tissue single-cell suspensions were prepared according to the method described in Example 1, and CD8+ cells in the tumor tissues of the two groups of mice were analyzed by flow cytometry. + The infiltration rate of T cells and their effector functional activity characteristics.

[0069] 3.2 Experimental Results

[0070] Flow cytometry analysis showed that, compared with the control group inoculated with ID8-blank cells, the tumor tissue of mice in the experimental group inoculated with ID8-CCL5 cells contained CD8... +The significantly increased proportion of T cells suggests that CCL5 can mediate SIK2 inhibition of CD8 in ovarian cancer. + T cell chemotaxis ( Figure 5 ).

[0071] 4. Screening and discovery of phosphorylated substrate candidate proteins

[0072] To identify substrate proteins downstream of SIK2 that may be phosphorylated and involved in regulating CCL5 expression, this embodiment combines proteomic data with bioinformatics analysis for screening.

[0073] 4.1 Data Sources

[0074] (1) SIK2 interaction protein profile data: The tag-vector control vector and the tag-SIK2 overexpression vector were transfected into SKOV3 cells, respectively. Immunoprecipitation was performed using tag antibody (Flag antibody) to enrich the protein complex that binds to SIK2. The precipitate was separated by SDS-PAGE, stained with Coomassie brilliant blue, and the target band was excised and sent to Hangzhou Jingjie Biotechnology Co., Ltd. for mass spectrometry identification to obtain the SIK2 interaction protein profile.

[0075] (2) Phosphorylation modification proteomic data: Proteomic and phosphorylation modification proteomic analyses were performed on SIK2 knockout and control cells (completed by Hangzhou Jingjie Biotechnology Co., Ltd.) to obtain proteomic data showing significant differences in phosphorylation levels after SIK2 knockout.

[0076] 4.2 Bioinformatics prediction and cross-validation: The set of candidate transcription factors that may regulate CCL5 gene expression was predicted using JASPAR software, and cross-referenced with the protein profile data obtained above to further screen potential phosphorylated substrate candidate proteins.

[0077] 4.3 Screening results: The transcriptional repressor BCLAF1 was screened as a key candidate molecule. The amino acid sequence of the BCLAF1 gene can be found in Swiss-Prot (Gene ID: 9774).

[0078] 5. Verify the interaction between BCLAF1 and SIK2.

[0079] To confirm the direct protein-protein interaction between SIK2 and BCLAF1, this example uses a co-immunoprecipitation (Co-IP) experiment for verification.

[0080] 5.1 Experimental Methods

[0081] After obtaining the target protein using SKOV3 cell protein lysis buffer, it was co-precipitated with BCLAF1 specific antibody or IgG isotype control at the recommended titer ratio and incubated with continuous shaking at 4°C for 16 hours. The next day, protein A / G-beads were added and incubated at room temperature for 2 hours to enrich the immune complex. After incubation, unbound protein was removed by washing, and 1× loading buffer was added to the precipitate, which was then boiled at 100°C for 8 minutes for denaturation. Finally, SDS-PAGE gel electrophoresis and Western blotting were used to observe whether SIK2 protein was specifically co-precipitated by BCLAF1 antibody.

[0082] 5.2 Experimental Results

[0083] Immunoprecipitation results showed that BCLAF1-specific antibody could effectively coprecipitate SIK2 protein, while control IgG did not exhibit this phenomenon. Figure 6 This indicates that BCLAF1 and SIK2 proteins have a specific interaction within the cell.

[0084] 6. Phosphorylation modification of BCLAF1 by SIK2 and site identification

[0085] To determine whether SIK2 directly phosphorylates BCLAF1 and to identify its specific phosphorylation site, an in vitro protein kinase experiment was performed in this embodiment.

[0086] 6.1 Experimental Methods

[0087] Purified recombinant SIK2 kinase protein (Abcam, catalog number: ab89856) was co-incubated with BCLAF1 peptide in an ATP-containing reaction system. Kinase activity was assessed by detecting the amount of ADP produced in the reaction. Two substrates were used: wild-type BCLAF1 peptide (Kesheng Jingtai Biotechnology Co., Ltd., corresponding amino acids 553-577, sequence: PVPLDDSNRPASLTKDRLLASTLVH (SEQ ID NO: 6)) and its S564 mutant peptide (Kesheng Jingtai Biotechnology Co., Ltd., S564A, sequence: PVPLDDSNRPATLTKDRLLASTLVH (SEQ ID NO: 7)). SIK2 kinase and the above peptides were mixed at molar ratios of 1:1, 1:10, 1:100, 1:1000, and 1:10000, and reaction buffer and ATP were added. The mixture was incubated at room temperature for 30 minutes. The ADP signal generated in the reaction was detected using an ADP assay kit, and the chemiluminescence values ​​were read using a microplate reader. The data were then analyzed.

[0088] 6.2 Experimental Results

[0089] In vitro protein kinase assays showed that SIK2 phosphorylates wild-type BCLAF1 peptide (aa553-577) in a dose-dependent manner; while the S564A mutant peptide showed almost no phosphorylation signal under the same conditions. Figure 7 This indicates that serine residue 564 (S564) of the BCLAF1 protein is the in vitro direct phosphorylation site of SIK2 kinase.

[0090] 7. Verify the direct binding of BCLAF1 to the CCL5 gene promoter.

[0091] To investigate whether BCLAF1 acts as a transcription factor to directly regulate the expression of the CCL5 gene, we used chromatin immunoprecipitation (ChIP) to detect whether the BCLAF1 protein binds to the promoter region of the CCL5 gene in vivo.

[0092] 7.1 Experimental Methods

[0093] ChIP experiments were performed in cells overexpressing Myc-BCLAF1. The specific steps included: cross-linking cells with formaldehyde to fix the protein-DNA complex; lysing cells and extracting chromatin; digesting the chromatin with micrococcal nuclease to cut it into fragments of appropriate length. Subsequently, Myc tag-specific antibody (experimental group) was added for immunoprecipitation to enrich chromatin fragments bound to BCLAF1 protein; rabbit-derived normal IgG was used as a negative control, and the original lysate without immunoprecipitation was used as a positive control. After elution and decross-linking, the enriched products were purified and the DNA fragments were recovered. Real-time quantitative PCR (qPCR) was used with specific primers designed for the CCL5 gene promoter region (F: 5'-CTAGCCCGGACCAAATTGTTG-3' (SEQ ID NO: 2), R: 5'-GCCAGCCACTATTCCACTGT-3' (SEQ ID NO: 3)) to detect the content of CCL5 promoter DNA in the enriched products.

[0094] 7.2 Experimental Results

[0095] ChIP and qPCR results showed that, compared with the negative control IgG, the Myc antibody was significantly enriched in the promoter DNA fragment of the CCL5 gene. Figure 8 This indicates that the Myc-tagged BCLAF1 protein can specifically bind to the promoter region of the CCL5 gene within the cell.

[0096] 8. Verify the inhibitory function of BCLAF1 on CCL5 gene transcription.

[0097] To elucidate the functional effects of BCLAF1 binding to the CCL5 promoter, we used a luciferase reporter gene assay to detect the effect of altered BCLAF1 protein expression levels on CCL5 promoter transcriptional activity. siBCLAF1#1:5'-GCGAUACAGUUCUAGGUCUTT-3' (SEQ ID NO: 4); siBCLAF1#2:5'-CAUCAGCCACAUCCGGUGATT-3' (SEQ ID NO: 5).

[0098] 8.1 Experimental Methods

[0099] A luciferase reporter plasmid carrying the CCL5 gene promoter sequence was co-transfected into cells with a BCLAF1 knockdown vector (siBCLAF1#1, siBCLAF1#) or a control vector (NC). After culturing for 48 hours, cells were collected and lysed with passive lysis buffer (PLB). After vortexing and sonication on ice, the cells were centrifuged at 4°C, and the supernatant was collected. 20 μL of the sample was added to each well of a 96-well plate, with three replicates per group. 15 μL of luciferase substrate was added to each well under dark conditions, and luciferase activity was immediately detected using a microplate reader. The transcriptional activity of the CCL5 promoter was assessed by relative luminescence intensity.

[0100] 8.2 Experimental Results

[0101] Results of luciferase reporter gene assays showed that knocking down BCLAF1 promoted CCL5 transcription, further demonstrating that BCLAF1 can inhibit CCL5 transcription by binding to the promoter sequence of the CCL5 gene. Figure 9 ).

[0102] 9. Design and synthesize the phosphorylated antigenic peptide at site S564 of the BCLAF1 protein.

[0103] To clarify that SIK2 can phosphorylate the evolutionarily conserved site BCLAF1 S564, the BCLAF1S564 peptide CDDSNRPAS(pi)LTKDR (SEQ ID NO: 1) was designed and synthesized (Hefei Kesheng Jingtai Biotechnology Co., Ltd.).

[0104] Example 2: Preparation of a specific BCLAF1 protein S564 phosphorylation antibody

[0105] 1. Peptide conjugated with KLH (antigen)

[0106] 1.1 Dissolve 20 mg KLH in 2 mL of 5 mM EDTA aqueous solution.

[0107] 1.2 Weigh 8 mg of Sulfo-SMCC and dissolve it completely in 50 μL of DMSO, then add 150 μL of 1×PBS and mix well.

[0108] 1.3 Add the Sulfo-SMCC solution dropwise to KLH while gently shaking (vigorous shaking will produce a precipitate), and let stand at room temperature for 1 hour.

[0109] 1.4 Place the activated KLH solution into a dialysis bag, clamp it with a dialysis clamp, and dialyze for 1 h in 2 L of 1×PBS at 4 °C with magnetic stirring.

[0110] 1.5 Replace with fresh 1×PBS and dialyze for 2 hours, repeating once. Place the activated and dialyzed KLH into a 15mL imported centrifuge tube, label the tube with the reagent name, time, and concentration, and store at 4°C.

[0111] 1.6 Weigh 4 mg of peptide, dissolve it in 50 μL of DMSO, add 200 μL of 1×PBS, mix quickly, and then immediately add KLH at a ratio of peptide:KLH = 1 mg: 680 μg. Incubate overnight at 4°C or react at room temperature for 2 h.

[0112] 1.7 Place the cross-linked KLH-peptide complex into a dialysis bag, clamp it with a dialysis clamp, and dialyze overnight in 4L of 1×PBS at 4°C with magnetic stirring.

[0113] 1.8 Transfer the dialyzed KLH-peptide into a clean 1.5mL centrifuge tube, aliquot according to the immunization dose, and store at -20℃.

[0114] 2. Experimental procedure for antibody synthesis

[0115] 2.1 Animal Immunization Program

[0116] 2.1.1 Animal Selection: Select healthy animals with glossy fur and free movement. After selecting the animals, pre-raise them for about two weeks. The purpose is to eliminate any unsuitable animals to ensure the smooth progress of later experiments.

[0117] 2.1.2 Preparation before the experiment: Mark the animals as RB12483 / RB12484 / RB12485.

[0118] 2.1.3 Antigen preparation:

[0119] 2.1.3.1 Remove the antigen from the -20℃ freezer and thaw it at room temperature, avoiding repeated freeze-thaw cycles. Label the syringe with the project number and animal number.

[0120] 2.1.3.2 Extract the antigen (the antigen should be completely mixed). The initial immunization dose is 0.1~1.0 mg (depending on the species and weight of the immunized animal), 0.5 mL / animal. The antigen dose for the second to fourth immunizations is halved.

[0121] 2.1.3.3 Adjuvant should be drawn at a 1:1 volume ratio to antigen. Complete adjuvant is used for the first immunization, while incomplete adjuvant is used for the second to fourth immunizations. The adjuvant must be thoroughly mixed before being drawn into the syringe.

[0122] 2.1.3.4 After connecting the two syringes with the syringe connecting tube, complete emulsification is performed. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37°C water.

[0123] 2.1.4 Immunization: Animals were given multiple subcutaneous injections, with 0.2 mL at each point.

[0124] Immunization schedule: The second immunization is administered 14 days after the first immunization, with a 7-day interval between the second and third immunizations. A small serum sample is collected from the middle ear artery 7 days after the third immunization. If the sample passes the test, a booster immunization is administered 7 days later, and whole blood can be collected 7 days after the booster immunization.

[0125] 2.1.4.1 Procedure for collecting small serum samples

[0126] The rabbit was secured in a frame, and its ear was gently tapped to dilate the central auricular artery. The area was disinfected with 75% alcohol. The rabbit's ear was held still with the left hand, and the syringe was held in the right hand. The needle was inserted into the central auricular artery at the distal 1 / 3 of its length, parallel to the artery and directed towards the heart. 8 mL of blood was collected at a time, and pressure was applied with a cotton ball to stop the bleeding.

[0127] 2.1.4.2 Whole blood collection procedure

[0128] (1) Capture the animals to be immunized, check the ear tag number and weigh them, and anesthetize them by intravenous injection of 1 ml of 3% sodium pentobarbital per kilogram.

[0129] (2) After anesthesia, the immunized animal is placed with its abdomen facing upward, and its limbs are fixed on a stainless steel mesh frame for cardiac blood collection.

[0130] (3) After blood collection, place the centrifuge tube containing the blood in a 37°C water bath for 15-30 minutes, then remove it, cool it, and place it in a 4°C refrigerator. Wait for the blood to separate automatically, and then transfer the supernatant into a clean 50mL centrifuge tube.

[0131] (4) Centrifuge at 12000 rpm for 2 min, transfer the supernatant to a clean centrifuge tube, add 100 μL of 10% sodium thimerosal solution (final concentration 0.02%) to 50 mL of supernatant, mix well, and store at -20℃.

[0132] 2.2 Rabbit serum titer detection (ELISA detection)

[0133] 2.2.1 Plate coating: Dilute the known antigen to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μL to each reaction well of the polystyrene plate, incubate overnight at 4°C, and the next day, discard the solution in the wells and wash once with 1xPBST washing buffer at 180 μL per well.

[0134] 2.2.2 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.

[0135] 2.2.3 Sample addition: Add 50 μL of the diluted test sample (dilute the test sample according to a certain ratio) to the above-mentioned sealed reaction wells. Also, set up negative control wells (1% BSA). Incubate at 37°C for 30 min, then wash three times with 150 μL of 1xPBST washing buffer per well.

[0136] 2.2.4 Add enzyme-labeled antibody: Add 50 μL of freshly diluted secondary antibody-HRP (diluted with 1% BSA) to each well of the ELISA plate, incubate at 37°C for 45 min, and wash three times with 150 μL of 1×PBST buffer per well.

[0137] 2.2.5 Adding substrate solution for color development: Add 50 μL of the temporarily prepared TMB substrate solution to each reaction well and incubate at 37 °C for 5 min.

[0138] 2.2.6 Termination of reaction: Add 50 μL of 1M sulfuric acid to each reaction well.

[0139] 2.2.7 Plate reading: Place the ELISA plate in a preheated ELISA reader (450nm) to read the data, save the data, and perform analysis.

[0140] The ELISA test results are shown in Table 2:

[0141] Table 2 Results of rabbit serum titer detection

[0142]

[0143]

[0144] 2.3 Antibody purification

[0145] 2.3.1 The affinity chromatography column was thoroughly washed with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0146] 2.3.2 Take 10 mL of the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0147] 2.3.3 Load the filtered serum sample at a flow rate of 40 mL / h, and repeat once.

[0148] 2.3.4 Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance (T setting) to 100.

[0149] 2.3.5 When the absorbance reading of the protein detector (1A range) is 0, turn on the HD-A computer acquisition device on the computer desktop and adjust the full-screen range to 5. Elute the antibody with glycine solution (pH 2.7, 0.2M) at a rate of 40mL / h. Press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0150] 2.3.6 During antibody collection, the pH of the antibody was adjusted to around 7 with 1M Tris-HCl in a timely manner, and the highest peak value of the elution peak was recorded.

[0151] 2.3.7 After antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tubing connected to the collector with purified water.

[0152] 2.3.8 Wash the affinity chromatography column sequentially with 20 mL of 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store at 4°C.

[0153] 2.4 Antibody titer detection (ELISA detection)

[0154] The purified antibody RB12484-IgG was tested by ELISA. The ELISA test procedure was the same as in 2.2. The results are shown in Table 3.

[0155] Table 3. RB12484-IgG antibody titer detection

[0156]

[0157] Example 3: Application of a specific BCLAF1 protein Ser564 site phosphorylation antibody for detection

[0158] To verify whether the antibody prepared in this invention can specifically recognize the phosphorylation state of the S564 site of the endogenous BCLAF1 protein in cells, and to confirm that this phosphorylation modification is regulated by SIK2 kinase, this embodiment uses a Western blot experiment for detection.

[0159] 1. Experimental Methods

[0160] (1) Cell protein extraction: Control group cells and SIK2 gene knockout cells were taken, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. An appropriate amount of RIPA lysis buffer containing protease and phosphatase inhibitors was added, and the cells were lysed on ice for 15 minutes. The lysate was collected with a cell scraper and transferred to a 1.5 mL centrifuge tube. The tube was centrifuged at 4 °C and 12500 rpm for 10 minutes, and the supernatant was transferred to a new centrifuge tube. 5× loading buffer was added, and the mixture was boiled at 100 °C for 8 minutes to denature the proteins. The tubes were then stored at -20 °C for later use.

[0161] (2) Protein concentration determination: Follow the instructions of the BCA protein concentration assay kit to prepare the reaction system, add the test sample and standard to the 96-well plate, incubate at 37°C for 30 minutes, measure the absorbance at 570 nm with an ELISA reader, calculate the sample protein concentration according to the standard curve, and determine the amount of sample to be loaded for subsequent electrophoresis.

[0162] (3) SDS-PAGE electrophoresis: Prepare SDS-PAGE gel, assemble the electrophoresis apparatus and add electrophoresis buffer. Take an equal amount of protein sample and add it to the sample well. First, electrophore at a constant voltage of 80V until bromophenol blue enters the separating gel. Then adjust the voltage to 120V and continue electrophoresis until the target protein is well separated.

[0163] (4) Transfer: Activate the PVDF membrane in methanol and assemble the transfer clamp (sponge-filter paper-gel-PVDF membrane-filter paper-sponge) in the transfer solution. Place the transfer clamp into the transfer tank, add pre-cooled transfer solution, and perform wet transfer in constant flow mode in an ice bath. The transfer time is determined according to the molecular weight of the target protein.

[0164] (5) Immunoassay: After transfer, the PVDF membrane was immersed in TBST blocking buffer containing 5% skim milk powder and blocked at room temperature for 1.5 hours. After TBST washing, the membrane was cut and the BCLAF1 pS564 phosphorylated specific antibody prepared in Example 2 of this invention was added and incubated overnight at 4°C. The primary antibody was recovered the next day, and the membrane was washed three times with TBST for 8 minutes each time. HRP-labeled secondary antibody (abcam, ab6721) was added and incubated at room temperature for 90 minutes, followed by three TBST washes. Finally, ECL chemiluminescent solution was added, and the reaction was carried out at room temperature in the dark for 1 minute. The signal was acquired using a gel imaging system.

[0165] 2. Experimental Results

[0166] Western blot analysis showed that in control cells, a distinct specific band was detected by the BCLAF1 pS564 phosphorylation-specific antibody; however, in SIK2 knockout cells, the signal intensity of this band was significantly reduced. Figure 10This indicates that the antibody prepared in this invention can specifically recognize the phosphorylation state of the S564 site of the BCLAF1 protein, and that the phosphorylation modification at this site is positively regulated by SIK2 kinase.

[0167] The phosphorylation antibody provided by this invention can be used in practical applications to detect the post-transcriptional phosphorylation modification of human BCLAF1 protein in immunological experiments such as WB (Western blot) and ELISA, thereby exploring the significance of human BCLAF1 protein phosphorylation modification in tumor-related diseases.

[0168] The transcriptional repression activity of BCLAF1 protein depends on phosphorylation modification. After phosphorylation, it can inhibit the transcription of downstream CCL5. Therefore, phosphorylated antibodies are convenient for studying the effect of phosphorylation modification of human BCLAF1 protein at Ser564 on specific biological events such as the transcriptional expression of chemokines, thereby exploring its role in the occurrence and development of diseases such as tumors.

[0169] This invention is a phosphorylated polyclonal antibody prepared targeting the Ser564 site of the human BCLAF1 protein. It helps to study the kinase activity that mediates its phosphorylation, explore the potential cell signaling pathways of the BCLAF1 protein, and thus discover potential targets for the diagnosis and treatment of clinical tumor diseases.

[0170] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A BCLAF1 protein Ser564 site phosphorylation antigen peptide, characterized in that, The amino acid sequence of the antigen peptide is CDDSNRPAS(pi)LTKDR, as shown in SEQ ID NO: 1, wherein S is modified by phosphorylation.

2. Use of the antigen peptide of claim 1 in the preparation of a preparation for detecting phosphorylation at the Ser564 site of human BCLAF1 protein.

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