Monoclonal antibody for recognizing trimethylation modification of SOX7 protein K159 site

Abstract

The invention relates to the technical field of immunology, and particularly discloses preparation and application of a monoclonal antibody for recognizing trimethylation modification of a K159 site of SOX7 protein, and the sequence of the monoclonal antibody for recognizing trimethylation of the K159 site of the SOX7 protein comprises a heavy chain variable region and a light chain variable region. The invention further relates to an immunohistochemical kit for marking SOX7 protein trimethylation after the monoclonal antibody is prepared, and the immunohistochemical kit can be applied to diagnosis of MsPGN diseases. The monoclonal antibody disclosed by the invention can be specifically combined with the SOX7 protein with the K159 site being trimethylated, and has no cross reaction with the SOX7 protein with the K159 site being not methylated (me0), monomethylated (me1) and dimethylated (me2), so that the specificity, accuracy and reliability of immunodetection are remarkably improved.

Description

Technical Field

[0001] The present application relates to immunology and belongs to the technical field of MsPGN disease diagnosis, and more specifically, 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 transcription factor containing a high mobility group-box (HMG-box). Its protein contains a conserved DNA binding domain (containing the HMG box) and a transcription activation domain, which exerts corresponding biological functions. Literature reports show that SOX7 can bind to the promoter of the target gene through its DNA binding domain and enhance the transcription of the target gene through its transcription activation domain. As a tumor suppressor gene, SOX7 can inhibit the proliferation of glioma cells through the Wnt / β-catenin signaling pathway. Aspirin induces SOX7 overexpression through the p38-MAPK pathway, mediating the growth of human colorectal cancer cells. In addition, SOX7 can also promote angiogenesis through the Notch signaling pathway.

[0003] Rat Thy-1 nephritis (Thy-1N) is a recognized animal model for studying human mesangial proliferative glomerulonephritis (MsPGN). After the rats are injected with antibodies against Thy-1 antigen, the antibodies can bind to the Thy-1 antigen on the surface of rat glomerular mesangial cells (GMC), and then activate complement, causing inflammatory reactions and GMC proliferative lesions. It is known that complement target cells can be divided into fully lytic and sublytic types. Although sublytic C5b-9 cannot cause cell lysis, it can trigger a variety of biological reactions. Previous studies by our research group have confirmed that Thy-1N lesions are complement C5b-9 dependent, especially sublytic C5b-9 dependent.

[0004] Our previous experiments showed that the expression of SOX7 in the renal tissue of Thy-1N rats and in vitro rat GMC stimulated by sublytic C5b-9 increased significantly, reaching a peak at 3h. In addition, SOX7 was also highly expressed in the renal sections of Thy-1N rats and MsPGN patients. In-depth exploration of SOX7 PTMs revealed that hypermethylation of SOX7 protein in the kidney occurred at the earliest stage in Thy-1N rats and MsPGN patients. Among them, the level of trimethylation of lysine 159 (K159) of SOX7 protein increased significantly, which is also a trimethylation site unique to MsPGN. Therefore, if the SOX7 protein with K159 trimethylation can be quickly detected, MsPGN can be diagnosed at an early stage, which is of great significance for the prevention and treatment of MsPGN.

[0005] At present, there is no 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 highly specific and sensitive detection on whether lysine 159 of SOX7 protein is trimethylated, the present application provides a monoclonal antibody for recognizing 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 only specifically recognizes and binds to SOX7 protein with trimethylation at 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 solution of the present invention is 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 the present application, the complementarity determining region (CDR) is located in the variable region of the antibody, and the heavy chain CDR and the light chain CDR cooperate with each other to form a surface complementary to the three-dimensional structure of the antigenic determinant, and then complementarily bind to the antigenic determinant. The amino acid sequence of the antibody CDR determines the specificity and affinity of the antibody for the antigen. The antibody with the above-mentioned CDR sequence of the present 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 trimethylation monoclonal antibody 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 SOX7 protein K159 site or the aforementioned immunohistochemical detection reagent that recognizes trimethylation of SOX7 protein K159 site in the preparation of a product for immunohistochemical detection of trimethylation of 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 scheme: 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 above-mentioned SOX7 protein K159 trimethylation detection kit can efficiently detect whether the 159th lysine of the SOX7 protein in the clinical sample is trimethylated, with accurate results, high sensitivity and easy 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 comprising: 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 monoclonal antibody cell supernatant 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 SOX7 protein in the present application only specifically recognizes and binds to SOX7 protein with trimethylation of K159, and does not bind to unmethylated, monomethylated and dimethylated SOX7 proteins, thereby ensuring the accuracy of the detection results, and can efficiently and accurately detect whether trimethylation occurs at lysine 159 of SOX7 protein.

[0020] 2. The monoclonal antibody for recognizing trimethylation of K159 of SOX7 protein in the present application has high sensitivity and strong affinity with the antigen. The OD450 value of 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 trimethylated monoclonal antibody of SOX7 protein K159 in the present application is simple and efficient, with low production cost and high antibody purity, and has the potential for factory production. It can also be further combined with other reagents to form a corresponding kit, which 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 the SOX7 protein K159 unmethylated (SOX7-K159me0) polypeptide as the substrate antigen. Figure 1 B is an ELISA test using the SOX7 protein K159 unmethylated (SOX7-K159me1) polypeptide as the substrate antigen. Figure 1 C is an ELISA test using the SOX7 protein K159 unmethylated (SOX7-K159me2) polypeptide as the substrate antigen. Figure 1 D is the ELISA detection of SOX7-K159me3 without KLH-coupled peptide as substrate antigen.

[0023] Figure 2Verification of the binding ability and specificity of the SOX7-K159me3 monoclonal antibody; 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 heavy chain variable region map of 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 sublytic C5b-9 stimulation of GMC. DETAILED DESCRIPTION

[0028] The present invention is further explained below with reference to the 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 comprising a polypeptide trimethylated with lysine 159 of the SOX7 protein and coupled with KLH (hemocyanin), and its immune effect is verified using a commercial antibody. The polypeptide amino acid sequence of the SOX7 protein is NSIGRGPLGEKEDRGEYAPGA (SEQ ID NO.9).

[0032] The steps for preparing the above immunogen are as follows: synthesize the above polypeptide, and trimethylate the lysine at the K159 site. During the synthesis, add a cysteine ​​at the C-terminus for coupling with KLH to obtain KLH-polypeptide, which is the immunogen. The specific operation was entrusted 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] KLH-peptide was used to immunize male Balb / c mice aged 8 to 12 weeks (Experimental Animal Center of Nanjing Medical University). For the first immunization, 400 μg of KLH-peptide was injected into each mouse, diluted to a volume of 200 μL with saline, mixed with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple points on the back after sufficient emulsification. The second immunization was performed 2 weeks apart, with a dose of 400 μg of KLH-peptide injected into each mouse, diluted to a volume of 200 μL with saline, mixed with an equal volume of Freund's incomplete adjuvant, and injected after sufficient emulsification. The third immunization was performed 4 weeks apart, and the method was the same as the second immunization. 7 days after the third immunization, 50 μL of blood was collected from the tail vein, and serum was obtained after centrifugation. The serum antibody titer was detected by indirect ELISA. The steps were as follows: 100 μL of 0.5 μg / mL SOX7 protein K159 site unmethylated (me0), monomethylated (me1), dimethylated (me2) and trimethylated (me3) modified peptides (specific operations were entrusted to Nanjing GenScript Biotechnology Co., Ltd., see Table 1 No. 1-4) were coated on the ELISA test plate, incubated overnight at 4°C, and the coating solution was removed the next day and washed 4 times with PBST. 3% BSA was added and allowed to stand at 37°C for 1h. The gradient diluted serum was added to the ELISA test wells and incubated at 37°C for 1h. After washing 4 times with PBST, 100 μL of HRP-goat anti-mouse IgG secondary antibody (Biosharp, BL001A) diluted 1:10000 was added to each well and incubated at 37°C for 1h. After washing with PBST for 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 me3 polypeptide at the K159 site of the SOX7 protein could only specifically bind to the polypeptide antigen at the K159 site of the SOX7 protein me3, and did not bind to the polypeptide antigens at the K159 site of the SOX7 protein me0, me1 and me2.

[0041] (2) Cell fusion.

[0042] Place the freshly removed spleen of the immune mouse on a cell sieve, crush and filter, mix with SP2 / 0 cells (myeloma cells) at a quantitative ratio of 3:1, centrifuge at 1000rpm for 5 minutes at room temperature, and discard the supernatant. Place the centrifuge tube containing the centrifuged cells in a 37°C warm water bath, and slowly add 1mL of polyethylene glycol solution (Sigma, P7181) while stirring the cells. Let it stand in the water bath for 1 minute, slowly add 10mL of serum-free DMEM culture medium (Gibco, 12100046), centrifuge at 1000rpm for 5 minutes, and discard the supernatant. Add serum-containing DMEM culture medium, and inoculate the cells into a 96-well cell culture plate, inoculating 150μL per well to a cell density of 1×10 5 100 μL / well, and culture in an incubator at 37°C and 5% CO2. After 24 hours, add 50 μL of culture medium containing HAT (Basalmedia, S912JV). Thereafter, change the medium every 3 to 5 days.

[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 the monoclonal antibody that specifically recognizes and binds to the SOX7 protein K159 site me3 is screened by indirect ELISA method, and the steps are the same as described in (1). When the OD450 value is greater than 0.5, it is judged as a positive clone. The obtained positive clone is screened for subclones by limiting dilution method.

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

[0046] After three subcloning and indirect ELISA screening, hybridoma cell lines that specifically recognize the SOX7 protein K159 site me3 antibody were selected, and the clone numbers were named P-1, P-2, P-3, P-4, P-5, P-6 and P-7. The affinity of the antibodies secreted by the above 7 hybridoma cell lines to the SOX7 protein K159 site me3 peptide not coupled to KLH was detected by indirect ELISA method, and the steps were the same as described in (1). The results are shown in Table 2.

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

[0048] Table 2

[0049]

[0050] As can be seen from Table 2, the antibody secreted by the hybridoma cell line P-2 has the strongest affinity for the trimethylated polypeptide of SOX7 protein K159. Therefore, the antibody secreted by P-2 was selected as the monoclonal antibody for recognizing 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 is the monoclonal antibody trimethylation of SOX7 protein K159 obtained in Example 2, and its binding ability, sensitivity and specificity are tested.

[0054] 1. Dot Blot Detection

[0055] The specific operation is as follows: dilute the peptides of me0, me1, me2 and me3 at the K159 site of the customized SOX7 protein that are not coupled with KLH; cut the cellulose acetate membrane (NC membrane) of appropriate size, use a pipette to absorb 3μL of peptide solutions of different concentrations, drop them on the NC membrane, and wait for the droplets to completely air-dry; put the NC membrane in 5% skim milk or 5% BSA for blocking at room temperature for 1h; primary antibody incubation: put the NC membrane in a TBST incubation box with antibody, and incubate it on a horizontal shaker at 4℃ overnight; then take the NC membrane out of the incubation box, wash it in TBST, and wash it on a horizontal shaker 3 times, each time for 10min; secondary antibody incubation and washing: according to the species of the primary antibody to be incubated, prepare the corresponding secondary antibody solution with blocking solution, and the configuration ratio is carried out according to the secondary antibody instructions. Incubate at room temperature for 45min or at 4℃ for 1-2h. Then, the NC membrane was taken out from the incubation box, placed in TBST, and washed three times on a horizontal shaker, each time for 10 minutes; Exposure: the luminescent solution was prepared according to the instructions, the residual TBST buffer on the NC membrane was absorbed with filter paper, the luminescent solution was evenly added, and after incubation for 30 seconds, it was placed in an 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 the trimethylation of SOX7 protein K159 in the present application has a good binding ability to K159 trimethylated polypeptides. The monoclonal antibody that recognizes the trimethylation of SOX7 protein K159 in the present application hardly binds to other methylated polypeptides. The above results show that the monoclonal antibody that recognizes the trimethylation of SOX7 protein K159 in the present 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 methods

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

[0058] (1) Transfection: 48 h after the rat SMYD4 overexpression plasmid with Flag tag (constructed by Nanjing Kress Biotechnology Co., Ltd.) and rat SMYD4 overexpression plasmid with 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 min, scrape the cells and transfer them to an EP tube, centrifuge at 12,000 rpm for 10 min, discard the precipitate, transfer the supernatant to a clean EP tube, and detect the protein concentration using a onedrop OD1000 micro-spectrophotometer; take 300 μg of total protein from each group, add 50 μl 2×SDS-PAGE loading buffer, boil for 8–10 min, 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℃ for 2h; centrifuge at 4℃ and 1000g for 10min, discard the precipitate, collect the supernatant into another clean centrifuge tube; add 1μg of primary antibody (HA antibody) and incubate on a shaker at 4℃ overnight; add 40μl of Protein G Agarose, continue to incubate on a shaker at 4℃ for 2h; centrifuge at 4℃ and 1000g for 10min, 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 4℃ and 1000g for 10min, 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 8min, and store at -20℃. The resulting sample is the IP group.

[0060] 2. The specific operation of Western Blot is 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 is necessary to fully stir it and pour it into the glass plate, and use isopropanol or distilled water to flatten the liquid surface. After the separation gel is completely solidified, pour out the isopropanol, clean it with distilled water and absorb it dry. 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 solution 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 45 V. After the protein sample completely enters the separation gel, increase the voltage to 120 V 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 filter papers and place them in the electro-transfer solution to wet 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 filter papers and place them in the electro-transfer solution to wet them, place them on the gel, and remove all bubbles. Then place another layer of sponge, fasten the electro-transfer clamp (i.e. "black gel and white membrane"), place it in the electro-transfer tank and fill it with electro-transfer 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, use tweezers to pick up the PVDF membrane 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 target protein, and place it in the prepared primary antibodies dissolved with Flag, HA, β-actin (internal reference) and SOX7-K159me3. The antibody dilution ratio and dilution reagent are based on the antibody instructions (SOX7-K159me3 antibody is the cell supernatant secreted by the P-2 cell line), and incubate at room temperature at 4°C overnight. Then remove the PVDF membrane from the incubation tube, wash it in TBST, and wash it on a horizontal shaker 3 times, 10 minutes each time;

[0066] (6) Secondary antibody incubation and washing: According to the species of the primary antibody to be incubated (see the instructions for the primary antibody), prepare the corresponding secondary antibody solution with blocking solution, and the configuration ratio is based on 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, each time for 10 minutes;

[0067] (7) Exposure: Prepare the luminescent solution according to the instructions, use filter paper to absorb the residual TBST buffer on the PVDF membrane, evenly add the luminescent solution, incubate for 30 seconds, and then place it in an 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 the monoclonal antibody secreted by the hybridoma cell line P-2 that recognizes the 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 as follows: Figure 3 A; the 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 in the heavy chain variable region is GMGVG as shown in SEQ ID NO.1; 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 trimethylation of SOX7 protein K159. The above-mentioned immunohistochemical detection kit for trimethylation of SOX7 protein K159 includes the monoclonal antibody for recognizing trimethylation of SOX7 protein K159 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 in Example 5 was used to perform immunohistochemical detection of the trimethylation level of lysine 159 in kidney tissue sections of 5 Thy-1N rats, and kidney tissue sections of NRS rats were used as negative controls. The specific operation of the detection is as follows:

[0078] Dewaxing and hydration of paraffin sections: 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, 70% alcohol for 5 min; 3% H2O2 to block endogenous peroxidase at room temperature for 10 min; rinse with tap water for 2 min, rinse with distilled water twice; antigen retrieval (pH 6.0): boil in a water bath for 10 min, then cool naturally to room temperature; rinse with PBS buffer 3 times, 3 min each time; add primary antibody (i.e., the mouse anti-SOX7 protein K159 trimethylated antibody prepared in Example 1-4) in a 4°C refrigerator overnight; rinse with PBS buffer 3 times, 3 min each time; add corresponding secondary antibody, incubate at room temperature for 30 min; rinse with PBS buffer 3 times, 3 min each time; DAB color development, hematoxylin counterstaining of cell nuclei, step-by-step alcohol dehydration, xylene transparent, neutral resin sealing; microscopy.

[0079] The results are as follows Figure 4 As shown, the monoclonal antibody for recognizing 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, and the results were as follows: Figure 5 As shown, the monoclonal antibody for recognizing trimethylation of SOX7 protein K159 in the present application 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 to 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 stimulated GMC.

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

[0085] In summary, the monoclonal antibody for recognizing trimethylation of SOX7 protein K159 constructed in the present application only recognizes and binds to SOX7 protein with trimethylation of K159, and has similar specificity and accuracy to commercially available antibodies, but has stronger binding ability to antigens and has 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody that recognizes trimethylation of the K159 site of 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 for recognizing trimethylation of SOX7 protein K159 site according to claim 1, 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 for recognizing trimethylation of SOX7 protein K159 site according to claim 1, 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 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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