A monoclonal antibody that recognizes trimethylation of the K159 site of the SOX7 protein

Abstract

The present application relates to the technical field of immunology, and specifically discloses the preparation and application of a monoclonal antibody that recognizes trimethylation of the K159 site of the SOX7 protein, wherein the monoclonal antibody sequence that recognizes trimethylation of the K159 site of the SOX7 protein includes a heavy chain variable region and a light chain variable region. The present invention also relates to an immunohistochemistry kit for marking trimethylation of the SOX7 protein after the monoclonal antibody is prepared, which can be used to diagnose MsPGN disease. The monoclonal antibody of the present invention can specifically bind to the SOX7 protein trimethylated at the K159 site, and has no cross-reaction with the SOX7 protein that is unmethylated (me0), monomethylated (me1) and dimethylated (me2) at the K159 site, thereby significantly improving the specificity, accuracy and reliability of the immunoassay.

Description

Technical Field

[0001] The present application relates to immunology and belongs to the technical field of MsPGN disease diagnosis. More specifically, it relates to a monoclonal antibody that recognizes trimethylation of the K159 site of the SOX7 protein and its application. Background Art

[0002] SRY-related HMG-box gene 7 (SOX7) is a high-mobility group-box (HMG-box) transcription factor. Its protein contains a conserved DNA-binding domain (containing the HMG box) and a transcriptional activation domain, which play corresponding biological functions. Literature reports indicate that SOX7 can bind to target gene promoters through its DNA-binding domain and enhance target gene transcription through its transcriptional activation domain. As a tumor suppressor gene, SOX7 can inhibit glioma cell proliferation through the Wnt / β-catenin signaling pathway. Aspirin induces SOX7 overexpression through the p38-MAPK pathway, mediating the growth of human colorectal cancer cells. Furthermore, SOX7 can promote angiogenesis through the Notch signaling pathway.

[0003] Rat Thy-1 nephritis (Thy-1N) is a well-established animal model for studying human mesangial proliferative glomerulonephritis (MsPGN). Injection of rats with antibodies against Thy-1 antigens allows the antibodies to bind to Thy-1 antigens expressed on the surface of rat glomerular mesangial cells (GMCs), subsequently activating complement and inducing an inflammatory response and GMC proliferation. Complement target cells are categorized as either fully lytic or sublytic. While sublytic C5b-9 does not cause cell lysis, it can trigger a variety of biological responses. Previous studies by our group have demonstrated that Thy-1N lesions are dependent on complement C5b-9, particularly sublytic C5b-9.

[0004] Our previous experiments showed that SOX7 expression increased significantly in the kidneys of Thy-1N rats and in rat GMCs stimulated with sublytic C5b-9 in vitro, reaching a peak at 3 hours. Furthermore, SOX7 expression was also high in renal sections from Thy-1N rats and patients with MsPGN. Further investigation of SOX7 PTMs revealed that hypermethylation of the renal SOX7 protein occurs at the earliest stages of the disease in both Thy-1N rats and patients with MsPGN. Specifically, trimethylation of lysine 159 (K159) in the SOX7 protein was significantly elevated, a trimethylation site unique to MsPGN. Therefore, rapid detection of SOX7 protein with K159 trimethylation would allow for early diagnosis of MsPGN, which would be crucial for the prevention and treatment of MsPGN.

[0005] Currently, there is no test kit for detecting K159 trimethylation of SOX7 protein. There is an urgent need for a monoclonal antibody that can specifically recognize K159 trimethylation of SOX7 protein to develop a highly sensitive and specific immunohistochemistry kit for the diagnosis of MsPGN disease. Summary of the Invention

[0006] In order to perform high-specificity and high-sensitivity detection on whether lysine 159 of SOX7 protein is trimethylated, the present application provides a monoclonal antibody that recognizes trimethylation of K159 of SOX7 protein and its application.

[0007] The monoclonal antibody provided in the present application that recognizes trimethylation of SOX7 protein K159 specifically recognizes and binds only to SOX7 protein with trimethylation of lysine 159, and does not bind to unmethylated, monomethylated, and dimethylated SOX7 proteins. It has good specificity, high sensitivity, and accurate results, and can be used in actual clinical testing.

[0008] The technical solutions of the present invention are as follows:

[0009] The first object of the present invention is to provide a monoclonal antibody that recognizes trimethylation at the K159 site of the SOX7 protein, wherein the monoclonal antibody that recognizes trimethylation at the K159 site of the SOX7 protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 as shown in the amino acid sequence of SEQ ID NO.1, a CDR2 as shown in the amino acid sequence of SEQ ID NO.2, and a CDR3 as shown in the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises a CDR1 as shown in the amino acid sequence of SEQ ID NO.4, a CDR2 as shown in the amino acid sequence of SEQ ID NO.5, and a CDR3 as shown in the amino acid sequence of SEQ ID NO.6.

[0010] In this application, the complementarity determining regions (CDRs) are located in the variable region of the antibody, and through the mutual cooperation of the heavy chain CDR and the light chain CDR, a surface complementary to the three-dimensional structure of the antigenic determinant is formed, and then complementary binding to the antigenic determinant is formed. The amino acid sequence of the antibody CDR determines the specificity and affinity of the antibody for the antigen. Antibodies with the above-mentioned CDR sequences of this application can specifically recognize and bind to the SOX7 protein with trimethylation of lysine at position 159, without binding to unmethylated, monomethylated and dimethylated SOX7 proteins, thereby achieving efficient detection of trimethylation of SOX7 protein K159, with high sensitivity and accurate results.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody that recognizes trimethylation at the K159 site of the SOX7 protein is shown in SEQ ID NO.7.

[0012] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody that recognizes trimethylation at the K159 site of the SOX7 protein is shown in SEQ ID NO.8.

[0013] The second object of the present invention is to provide an immunohistochemical detection reagent for identifying trimethylation of the K159 site of the SOX7 protein, comprising the aforementioned monoclonal antibody against trimethylation of the K159 site of the SOX7 protein.

[0014] The third object of the present invention is to provide the use of the aforementioned monoclonal antibody that recognizes trimethylation of the SOX7 protein K159 site or the aforementioned immunohistochemical detection reagent that recognizes trimethylation of the SOX7 protein K159 site in the preparation of a product for immunohistochemical detection of trimethylation of the SOX7 protein K159 site in tissue cells.

[0015] Furthermore, the tissue cells are renal tissues of MsPGN patients and MsPGN animal models (ie, Thy-1 nephritis rats).

[0016] The present application also provides a SOX7 protein K159 trimethylation detection kit, which adopts the following technical solution: a SOX7 protein K159 trimethylation detection kit, the SOX7 protein K159 trimethylation detection kit comprising the monoclonal antibody for recognizing SOX7 protein K159 trimethylation as described in the first aspect. In the present application, the SOX7 protein K159 trimethylation detection kit can efficiently detect whether lysine 159 of the SOX7 protein in clinical samples is trimethylated, with accurate results, high sensitivity, and ease of use.

[0017] The present application also provides a method for preparing a monoclonal antibody that recognizes trimethylation of SOX7 protein K159. The method for preparing a monoclonal antibody that recognizes trimethylation of SOX7 protein K159 comprises: artificially synthesizing an antigen polypeptide, immunizing mice with the polypeptide, fusing mouse spleen cells with SP2 / 0 cells, obtaining a monoclonal cell line by limiting dilution method, and obtaining a cell supernatant of a monoclonal antibody that recognizes trimethylation of SOX7 protein K159.

[0018] In summary, this application has the following beneficial effects:

[0019] 1. The monoclonal antibody for recognizing trimethylation of K159 of the SOX7 protein in the present application specifically recognizes and binds only to SOX7 proteins with trimethylation of K159, and does not bind to unmethylated, monomethylated, and dimethylated SOX7 proteins, thereby ensuring the accuracy of the test results and enabling efficient and accurate detection of whether trimethylation of lysine 159 of the SOX7 protein occurs.

[0020] 2. The monoclonal antibody in this application that recognizes trimethylation of SOX7 protein K159 has high sensitivity and strong affinity with the antigen. The OD450 value of the ELISA detection of SOX7 polypeptide with trimethylation of K159 can reach 3.148, which can be used to detect actual samples with good accuracy.

[0021] 3. The preparation method of the monoclonal antibody against trimethylation of SOX7 protein K159 in this application is simple and efficient, with low production cost and high antibody purity. It has the potential for factory production and can be further combined with other reagents to make corresponding kits. It is easy to use, has low detection cost, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The titer of SOX7 protein K159 trimethylation (SOX7-K159me3) antibody in the serum of immunized mice; Figure 1 A is an ELISA test using SOX7 protein K159 unmethylated (SOX7-K159me0) polypeptide as substrate antigen. Figure 1 B is an ELISA test using SOX7 protein K159 unmethylated (SOX7-K159me1) polypeptide as substrate antigen. Figure 1 C is an ELISA test using SOX7 protein K159 unmethylated (SOX7-K159me2) polypeptide as substrate antigen. Figure 1 D is an ELISA test using SOX7-K159me3 not conjugated with KLH polypeptide as substrate antigen.

[0023] Figure 2Verification of the binding ability and specificity of SOX7-K159me3 monoclonal antibodies; Figure 2 A is the binding ability of SOX7-K159me3 monoclonal antibody, Figure 2 B is the verification of the specificity of the SOX7-K159me3 monoclonal antibody.

[0024] Figure 3 Sequencing map of the light and heavy chain variable regions of the SOX7-K159me3 monoclonal antibody; Figure 3 A is the map of the heavy chain variable region of the SOX7-K159me3 monoclonal antibody. Figure 3 B is the map of the light chain variable region of the SOX7-K159me3 monoclonal antibody.

[0025] Figure 4 IHC staining effect of SOX7-K159me3 monoclonal antibody in rat kidney tissue sections.

[0026] Figure 5 IHC staining effect of SOX7-K159me3 monoclonal antibody in renal tissue sections of MsPGN patients.

[0027] Figure 6 SOX7-K159me3 monoclonal antibody was used to examine SOX7-K159me3 methylation after GMC stimulation by sublytic C5b-9. DETAILED DESCRIPTION

[0028] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0029] Example 1 Immunogen Preparation

[0030] This embodiment provides an immunogen.

[0031] The immunogen is an immune complex consisting of a trimethylated peptide at position 159 of the SOX7 protein and KLH (hemocyanin), and its immune effect was verified using a commercial antibody. The amino acid sequence of the SOX7 protein is NSIGRGPLGEKEDRGEYAPGA (SEQ ID NO. 9).

[0032] The steps for preparing the above-mentioned immunogen are as follows: synthesize the above-mentioned polypeptide, trimethylate the lysine at the K159 position, and add a cysteine ​​at the C-terminus during synthesis for coupling with KLH to obtain KLH-polypeptide, which is the immunogen. The specific operations were commissioned to Nanjing GenScript Biotechnology Co., Ltd. (see Table 1, No. 5).

[0033] Table 1

[0034]

[0035] Example 2 Hybridoma Cell Preparation

[0036] This embodiment provides a method for preparing hybridoma cells.

[0037] The above preparation method uses the KLH-polypeptide (immunogen) prepared in Example 1 to immunize animals, prepare hybridoma cells and screen them, and the steps are as follows:

[0038] (1) Animal immunization.

[0039] Male Balb / c mice aged 8 to 12 weeks (Nanjing Medical University Laboratory Animal Center) were immunized with KLH peptide. For the first immunization, each mouse was injected with 400 μg of KLH peptide, diluted to 200 μL with saline, mixed with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple points on the back after thorough emulsification. A second immunization was performed two weeks later with 400 μg of KLH peptide per mouse, diluted to 200 μL with saline, mixed with an equal volume of Freund's incomplete adjuvant, and injected subcutaneously after thorough emulsification. A third immunization was performed four weeks later using the same method as the second. Seven days after the third immunization, 50 μL of blood was collected from the tail vein and centrifuged to obtain serum. Serum antibody titers were assessed by indirect ELISA. The following steps were performed: 100 μL of 0.5 μg / mL SOX7 protein K159 modified peptides (specific procedures were commissioned by Nanjing GenScript Biotechnology Co., Ltd., see Table 1, Nos. 1-4) were coated on ELISA plates. The plates were incubated overnight at 4°C. The next day, the coating solution was removed and the plates were washed four times with PBST. 3% BSA was added and the plates were incubated at 37°C for 1 hour. Serially diluted serum was added to the ELISA wells and incubated at 37°C for 1 hour. After washing four times with PBST, 100 μL of a 1:10,000 dilution of HRP-goat anti-mouse IgG secondary antibody (Biosharp, BL001A) was added to each well and incubated at 37°C for 1 hour. After washing with PBST 4 times, TMB (Elabscience, E-IR-R201) was added for color development, and the OD value at 450 nm was measured and the results were analyzed. Figure 1 shown.

[0040] Figure 1 The titer of SOX7-K159me3 antibodies in the serum of mice immunized with the KLH-peptide prepared in Example 1 was detected. The results showed that the serum antibodies of mice immunized with the SOX7 protein K159 site me3 polypeptide could only specifically bind to the SOX7 protein K159 site me3 polypeptide antigen, and did not bind to the SOX7 protein K159 site me0, me1 and me2 polypeptide antigens.

[0041] (2) Cell fusion.

[0042] The freshly removed spleen of the immune mouse was placed on a cell sieve, crushed and filtered, mixed with SP2 / 0 cells (myeloma cells) at a quantitative ratio of 3:1, centrifuged at 1000 rpm for 5 minutes at room temperature, and the supernatant was discarded. The centrifuge tube containing the centrifuged cells was placed in a 37°C warm water bath, and 1 mL of polyethylene glycol solution (Sigma, P7181) was slowly added while stirring the cells. Let it stand in the water bath for 1 minute, slowly add 10 mL of serum-free DMEM medium (Gibco, 12100046), centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add serum-containing DMEM medium and inoculate the cells into a 96-well cell culture plate, inoculating 150 μL per well to a cell density of 1×10 5 Each well was cultured in a 37°C, 5% CO2 incubator. After 24 hours, 50 μL of HAT (Basalmedia, S912JV) culture medium was added. The medium was changed every 3-5 days thereafter.

[0043] (3) Cell screening.

[0044] When the cell density reaches about 50%, the cell supernatant is collected and the hybridoma cell line that can secrete a monoclonal antibody that specifically recognizes and binds to the SOX7 protein K159 site me3 is screened by indirect ELISA, using the same steps as described in (1). When the OD450 value is greater than 0.5, it is determined to be a positive clone. The obtained positive clones are screened for subclones by limiting dilution method.

[0045] (4) Establishment of hybridoma cell lines.

[0046] After three rounds of subcloning and indirect ELISA screening, hybridoma cell lines that specifically recognized antibodies targeting the SOX7 protein K159 site me3 were selected and designated P-1, P-2, P-3, P-4, P-5, P-6, and P-7, respectively. The affinity of the antibodies secreted by the seven hybridoma cell lines for the SOX7 protein K159 site me3 peptide not conjugated to KLH was tested by indirect ELISA, using the same steps as described in (1). The results are shown in Table 2.

[0047] Table 2 shows the affinity test results of the antibodies secreted by 7 hybridoma cell lines and the peptide trimethylated at the K159 site of the SOX7 protein that is not coupled to KLH. The results show that the OD450 value of the ELISA test of the SOX7-K159me3 antibody secreted by the hybridoma cell lines can reach up to 3.148.

[0048] Table 2

[0049]

[0050] As shown in Table 2, the antibody secreted by the hybridoma cell line P-2 has the strongest affinity for the trimethylated peptide of SOX7 protein K159. Therefore, the antibody secreted by P-2 was selected as the monoclonal antibody recognizing the trimethylated SOX7 protein K159, thereby ensuring that the antibody only specifically recognizes and binds to the SOX7 protein with trimethylated K159, thereby ensuring the accuracy of subsequent test results.

[0051] Example 3

[0052] This example detects the binding ability, sensitivity and specificity of the monoclonal antibody against trimethylation of SOX7 protein K159 provided in the present application.

[0053] Specifically, the monoclonal antibody used to recognize trimethylation of SOX7 protein K159 was the monoclonal antibody trimethylation of SOX7 protein K159 obtained in Example 2, and its binding ability, sensitivity and specificity were tested.

[0054] 1. Dot Blot Detection

[0055] The specific steps are as follows: Dilute custom peptides targeting the K159 sites of the SOX7 protein, me0, me1, me2, and me3, that are not conjugated to KLH. Cut a cellulose acetate membrane (NC membrane) of appropriate size, pipette 3 μL of peptide solutions of varying concentrations onto the NC membrane, and allow the droplet to air-dry completely. Block the NC membrane in 5% skim milk or 5% BSA for 1 hour at room temperature. Incubate the NC membrane with primary antibodies in a TBST incubation box containing the antibody overnight at 4°C on a horizontal shaker. Remove the NC membrane from the incubation box and wash it in TBST, then wash it three times for 10 minutes each on a horizontal shaker. Incubate and wash with secondary antibodies: Prepare the appropriate secondary antibody solution using blocking buffer based on the species of the primary antibody being incubated, using the appropriate ratios according to the secondary antibody's instructions. Incubate at room temperature for 45 minutes or at 4°C for 1-2 hours. Then, the NC membrane was removed from the incubation box, placed in TBST, and washed three times on a horizontal shaker, each time for 10 minutes; Exposure: Prepare the luminescent solution according to the instructions, use filter paper to absorb the residual TBST buffer on the NC membrane, evenly add the luminescent solution, incubate for 30 seconds, and then place it in the exposure machine for exposure.

[0056] The binding ability test results of the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in this application are as follows Figure 2 As shown in A. Figure 2From the binding ability test results of the monoclonal antibody in A, it can be seen that the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in this application has a good binding ability to K159 trimethylated polypeptides. The monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in this application hardly binds to other methylated polypeptides. The above results show that the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in this application has a stronger affinity for K159 trimethylated polypeptides and has better specificity. It hardly binds to other methylated polypeptides, has higher detection accuracy, and has higher value in practical application. 2. Detection by Immunoprecipitation (IP) and Western Blot

[0057] 1.IP specific operations are as follows:

[0058] (1) Transfection: 48 h after the rat SMYD4 overexpression plasmid with a Flag tag (constructed by Nanjing Kress Biotechnology Co., Ltd.) and the rat SMYD4 overexpression plasmid with an HA tag (constructed by Anhui General Biotechnology Co., Ltd.) were transfected into H293T cells by Lipofectamine 2000;

[0059] (2) Sample collection: Place the cells on ice, add 200 μl IP lysis buffer to each dish to lyse the protein, place on ice for 20 minutes, scrape the cells and transfer them to EP tubes, centrifuge at 12000 rpm for 10 minutes, discard the precipitate, and transfer the supernatant to a clean EP tube. Detect the protein concentration using a onedrop OD1000 microspectrophotometer; take 300 μg of total protein from each group, add 50 μl 2×SDS-PAGE loading buffer, boil for 8-10 minutes, and the resulting sample is the input group, which is stored in a -20°C refrigerator; take 1 mg of total protein from each group, add IP lysis buffer containing PMSF to make up to 600 μl, then add 1 μl IgG of the same species as the primary antibody, and then add 30 μl Protein G Agarose, incubate on a shaker at 4°C for 2 hours; centrifuge at 1000g at 4°C for 10 minutes, discard the precipitate, and collect the supernatant into another clean centrifuge tube; add 1μg of primary antibody (HA antibody) and incubate on a shaker at 4°C overnight; add 40μl of Protein G Agarose and continue incubating on a shaker at 4°C for 2 hours; centrifuge at 1000g at 4°C for 10 minutes, carefully remove the supernatant without touching the precipitate, add 1ml of PBS containing PMSF to wash the beads, and repeat this step 3 times; centrifuge at 1000g at 4°C for 10 minutes, carefully remove the supernatant, and keep the remaining liquid in each EP tube roughly equal; add 100μl of 2×SDS-PAGE loading buffer, mix well, boil for 8 minutes, and store at -20°C. The resulting sample is the IP group.

[0060] 2. Western Blot specific operations are as follows:

[0061] (1) Preparation of SDS-PAGE gel: The concentration of the separation gel to be prepared depends on the molecular weight of the protein to be detected. The larger the molecular weight of the protein to be detected, the lower the concentration of the separation gel to be prepared. When preparing the lower layer of separation gel, it must be fully stirred and poured into the glass plate, and the liquid surface must be flattened with isopropyl alcohol or distilled water. After the separation gel is completely solidified, pour out the isopropyl alcohol, clean it with distilled water and dry it. Then prepare the concentrated gel, stir it thoroughly and pour it into the glass plate, insert a gel comb that is suitable for the pore size and number of holes, and pull out the comb vertically when the concentrated gel is completely solidified;

[0062] (2) Preparation for electrophoresis: Prepare 1× electrophoresis buffer and set up the SDS-PAGE gel plate in the electrophoresis tank; Loading: Adjust the protein loading amount according to the protein content and antibody titer to be detected, and load the sample according to the experimental requirements; Electrophoresis: The starting voltage is 45V. After the protein sample completely enters the separation gel, increase the voltage to 120V until the loading completely runs out of the bottom of the SDS-PAGE gel;

[0063] (3) Transfer (wet transfer): Cut the PVDF membrane (5.5cm×5.5cm) and filter paper (9cm×6cm) in advance. First, overlap 3 pieces of filter paper and place them in the electrotransfer solution to soak them, then place them on the transfer sponge to remove all bubbles; soak the PVDF membrane in methanol for about 30 seconds to activate it, then place it on the filter paper to remove all bubbles. Open the SDS-PAGE gel plate, cut off the excess gel, turn it upside down on the PVDF membrane, and remove all bubbles; then overlap 3 pieces of filter paper and place them in the electrotransfer solution to soak them, place them on the gel, and remove all bubbles, then place another layer of sponge, fasten the electrotransfer clamp (i.e. "black gel and white membrane"), place it in the electrotransfer tank and fill it with electrotransfer solution. Transfer the membrane at a constant current of 200mA. The transfer time depends on the molecular weight of the target protein and the concentration of the SDS-PAGE gel.

[0064] (4) Blocking: Prepare the blocking solution (5% skim milk or 5% BSA) first, and prepare it immediately before use. After the transfer is completed, remove the PVDF membrane, pick up the PVDF membrane with tweezers, and use scissors to cut off the upper right corner as a mark, then place it in an incubation box with blocking solution, and block it on a horizontal shaker at room temperature for 1 hour or at 4°C overnight;

[0065] (5) Primary antibody incubation and washing: Cut the PVDF membrane according to the molecular weight of the desired target protein and place it in the prepared primary antibodies containing Flag, HA, β-actin (internal reference) and SOX7-K159me3. The antibody dilution ratio and dilution reagent are based on the antibody instructions (the SOX7-K159me3 antibody is the cell supernatant secreted by the P-2 cell line). Incubate at room temperature at 4°C overnight. Then remove the PVDF membrane from the incubation tube and wash it in TBST. Wash it three times on a horizontal shaker for 10 minutes each time.

[0066] (6) Secondary antibody incubation and washing: Prepare the corresponding secondary antibody solution with blocking buffer according to the species of the primary antibody (see the instructions for the primary antibody). The proportion of the secondary antibody solution should be determined according to the instructions for the secondary antibody. Incubate at room temperature for 45 minutes or at 4°C for 1-2 hours. Then, remove the PVDF membrane from the incubation tube, place it in TBST, and wash it three times on a horizontal shaker for 10 minutes each time.

[0067] (7) Exposure: Prepare the luminescent solution according to the instructions, remove the residual TBST buffer on the PVDF membrane with filter paper, evenly add the luminescent solution, incubate for 30 seconds, and then place it in the exposure machine for exposure.

[0068] The specific results of the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in this application are as follows Figure 2 As shown in B, when SMYD4 and SOX7 were overexpressed simultaneously, the trimethylation level of SOX7 protein K159 site increased significantly, while the trimethylation level of SOX7 protein K159 site decreased significantly after transfection of SMYD4 and SOX7 protein K159 site mutant plasmids. Figure 2 It can be seen from the specific detection results of the monoclonal antibody in B that the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in the present application can specifically detect the level of trimethylation of SOX7 protein K159 site.

[0069] Example 4

[0070] This example provides the amino acid sequence of a monoclonal antibody secreted by the hybridoma cell line P-2 that recognizes trimethylation of K159 of the SOX7 protein.

[0071] The steps for determining the amino acid sequence of the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 are as follows: The hybridoma cell line P-2 screened in Example 2 was cut by RT-PCR and then sent to Anhui General Biological Co., Ltd. for antibody sequence determination and analysis. After sequence determination and analysis, the heavy chain variable region (including CDR1, CDR2 and CDR3) of the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in this application was determined to be as follows: Figure 3 As shown in A; Sequencing of the light chain variable region (including CDR1, CDR2 and CDR3) is as shown in Figure 3 As shown in B.

[0072] The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO.1: GMGVG; the amino acid sequence of CDR2 is shown in SEQ ID NO.2: QDFWKREYWPLIAAGSKN; the amino acid sequence of CDR3 is shown in SEQ ID NO.3: YYEHEGCGSETGY; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7: VGDIDNVLRMHTQLQEHRILLSLAVSAGEKVTMSCTSGMGVGWIRQFPGNKLEWMGQDF WKREYWPLIAAGSKNKATLTVDKSTAYMELRSLTSEDSAVYYCVRYYEHEGCGSETGYWG QGTSVTVSSGYWSAPQPPPS.

[0073] The amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO.4: QASKTVSTVGYSLVG; the amino acid sequence of CDR2 is shown in SEQ ID NO.5: IVVGLES; the amino acid sequence of CDR3 is shown in SEQ ID NO.6: KHIVELTTSRGVFT; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8: LGISTMETDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISYQASKTVSTVGYSLVG WNQQKPGQPPRLLIYIVVGLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCKHIVELTTS RGVFTFISGTKLELKKTFFGAK.

[0074] Example 5

[0075] This embodiment provides an immunohistochemical detection kit for SOX7 protein K159 trimethylation, which comprises the monoclonal antibody for recognizing SOX7 protein K159 trimethylation prepared in Examples 1-4, HRP goat anti-mouse IgG antibody, TMB, and a washing reagent.

[0076] Example 6

[0077] In this example, the immunohistochemical detection kit for SOX7 protein K159 trimethylation described in Example 5 was used to perform immunohistochemical detection of lysine 159 trimethylation in kidney tissue sections from five Thy-1N rats. Kidney tissue sections from NRS rats were used as negative controls. The specific detection procedures were as follows:

[0078] Paraffin sections were dewaxed and hydrated: xylene I and II for 10 min each; 100% alcohol I and II for 5 min each; 95% alcohol I and II for 5 min each; 90% alcohol for 5 min; 80% alcohol for 5 min, and 70% alcohol for 5 min; endogenous peroxidase was blocked with 3% H2O2 at room temperature for 10 min; tap water was rinsed for 2 min, and distilled water was rinsed twice; antigen retrieval (pH 6.0): boiled in a water bath for 10 min, then cooled to room temperature naturally; rinsed with PBS buffer three times, each for 3 min; primary antibody (i.e., the mouse anti-SOX7 protein K159 trimethylated antibody prepared in Example 1-4) was added and incubated in a 4°C refrigerator overnight; rinsed with PBS buffer three times, each for 3 min; the corresponding secondary antibody was added dropwise and incubated at room temperature for 30 min; rinsed with PBS buffer three times, each for 3 min; DAB was used for color development, hematoxylin was used for counterstaining of cell nuclei, dehydrated with alcohol step by step, transparentized with xylene, and mounted with neutral resin; and examined under a microscope.

[0079] The results are as follows Figure 4 As shown, the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in the present application can specifically detect the level of trimethylation of SOX7 protein K159 site in Thy-1N rat kidney tissue sections.

[0080] Example 7

[0081] In this example, the immunohistochemical detection kit for SOX7 protein K159 trimethylation in Example 5 was used to perform immunohistochemical detection of the trimethylation level of lysine 159 in renal tissue sections of 20 clinical MsPGN patients, and adjacent renal sections of clear renal cell carcinoma were used as negative controls. The specific operation of the detection was referred to Example 6. The results are shown in FIG. Figure 5 As shown, the monoclonal antibody in this application that recognizes trimethylation of SOX7 protein K159 can specifically detect the level of trimethylation of SOX7 protein K159 site in renal tissue sections of clinical MsPGN patients, indicating that it has good accuracy and specificity and can be applied in actual clinical detection.

[0082] Example 8

[0083] In this example, the prepared monoclonal antibody against trimethylation of SOX7 protein K159 was used to detect trimethylation of SOX7 protein K159 after sublyticC5b-9 stimulation of GMC.

[0084] Detection method: Western Blot, see Example 3 "II, 2" for details. Figure 6As shown, SOX7 protein K159 can be trimethylated after 3 hours of stimulation of GMC cells by sublytic C5b-9, indicating that the monoclonal antibody that recognizes trimethylation of SOX7 protein K159 in the present application can specifically detect trimethylation of SOX7 protein K159 in GMC cells stimulated by sublytic C5b-9.

[0085] In summary, the monoclonal antibody constructed in the present application that recognizes trimethylation of SOX7 protein K159 only recognizes and binds to SOX7 protein with trimethylation of K159, has similar specificity and accuracy to commercially available antibodies, but has stronger binding ability to antigens and no obvious post-translational modification. The detection results of actual samples are very accurate and can be used in related theoretical research and actual clinical testing.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that recognizes trimethylation of the K159 site of the SOX7 protein, characterized in that: The monoclonal antibody that recognizes trimethylation at the K159 site of the SOX7 protein includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3, and the light chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.4, a CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.

6.

2. The monoclonal antibody according to claim 1 that recognizes trimethylation of the K159 site of the SOX7 protein, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody that recognizes trimethylation at the K159 site of the SOX7 protein is shown in SEQ ID NO.

7.

3. The monoclonal antibody according to claim 1 that recognizes trimethylation of the K159 site of the SOX7 protein, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody that recognizes trimethylation at the K159 site of the SOX7 protein is shown in SEQ ID NO.

8.

4. An immunohistochemical detection reagent for identifying trimethylation of the K159 site of the SOX7 protein, characterized in that: The invention comprises the trimethylated monoclonal antibody against the K159 site of the SOX7 protein according to any one of claims 1 to 3.

5. Use of the monoclonal antibody recognizing trimethylation at K159 of SOX7 protein according to any one of claims 1 to 3 or the immunohistochemical detection reagent recognizing trimethylation at K159 of SOX7 protein according to claim 4 in the preparation of a product for immunohistochemical detection of trimethylation at K159 of SOX7 protein in tissue cells.

6. The use according to claim 5, characterized in that The tissue cells are kidney tissues of MsPGN patients and MsPGN animal models.

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