An anti-SUMO2 antibody and its preparation method and application

Abstract

The present invention discloses an anti-SUMO2 antibody and a preparation method and application thereof. Specifically, a SUMO2 monoclonal antibody with high activity and higher affinity is efficiently prepared by using an in vitro culture method of B cells. The SUMO2 monoclonal antibody can specifically bind to SUMO2 of mammals, especially humans. The antibody can be used to detect SUMO2 protein and study diseases related to SUMO2 protein.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-SUMO2 antibody and a preparation method and application thereof. The antibody can specifically bind to the SUMO2 protein of mammals (especially humans). Background Art

[0002] SUMO2 refers to Small Ubiquitin-like Modifier 2, which is a small ubiquitinated protein belonging to the SUMO (Small Ubiquitin-like Modifier) ​​family. SUMO proteins are involved in many cellular processes, such as transcriptional regulation, protein stabilization, and stress response. SUMO2 can covalently bind to target proteins through the SUMOylation process, changing the function, localization, and interaction of target proteins. SUMO2 has certain similarities with other SUMO family members (such as SUMO1 and SUMO3), but also has its own specific functions and target proteins. Studying SUMO2 and its related SUMOylation process is of great significance for understanding the basic mechanisms of cell biology and the occurrence and development of certain diseases (such as cancer and neurodegenerative diseases).

[0003] SUMO2 antibodies are widely used to detect and study the expression and modification status of SUMO2 protein. At present, the literature has reported that the preparation steps of polyclonal antibodies for specific binding to SUMO2 are mainly to use SUMO2 fusion protein as antigen to immunize rabbits, prepare antiserum, and purify to obtain SUMO2 polyclonal antibodies; SUMO2 / SUMO3 rabbit polyclonal antibody is a polyclonal antibody used for a variety of biological detections, suitable for Western blot, immunofluorescence and other experiments, and has high specificity and affinity; In addition, there are ELISA kits specifically for SUMO2 protein, which can be used for quantitative analysis of SUMO2 protein in cell or tissue samples.

[0004] However, there is still a lack of specific SUMO family antibodies, especially high-affinity recombinant rabbit monoclonal antibodies. Therefore, it is necessary to further develop SUMO2 antibodies with higher activity and affinity for the research and application of related diseases. Summary of the invention

[0005] In order to overcome at least one problem existing in the prior art, the present invention utilizes an in vitro culture method of B cells to efficiently prepare highly active and higher affinity SUMO2 monoclonal antibodies, specifically involving three monoclonal antibodies that can specifically bind to human SUMO2 protein, which provide a powerful tool for detecting SUMO2 protein and studying diseases related to SUMO2 protein.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide an anti-SUMO2 antibody, which uses B cells obtained after immunization with antigens to culture in vitro to prepare SUMO2 monoclonal antibodies.

[0008] Furthermore, the anti-SUMO2 antibody includes at least one of antibody 016-1, antibody 016-3 and antibody 016-4.

[0009] Furthermore, the antibody 016-1, antibody 016-3 and antibody 016-antibody have high binding activity with SUMO2 protein; wherein, the EC50 of the three antibodies are 3.971, 7.617 and 10.67 nM, respectively.

[0010] Furthermore, the amino acid sequence of the heavy chain hypervariable region of antibody 016-1 is CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3; the amino acid sequence of the light chain hypervariable region of antibody 016-1 is CDR1 shown in SEQ ID NO.4, CDR2 shown in SEQ ID NO.5, and CDR3 shown in SEQ ID NO.6.

[0011] Furthermore, the amino acid sequence of the heavy chain hypervariable region of antibody 016-3 is CDR1 shown in SEQ ID NO.7, CDR2 shown in SEQ ID NO.8, and CDR3 shown in SEQ ID NO.9; the amino acid sequence of the light chain hypervariable region of antibody 016-3 is CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.11, and CDR3 shown in SEQ ID NO.12.

[0012] Furthermore, the amino acid sequence of the heavy chain hypervariable region of antibody 016-4 is CDR1 shown in SEQ ID NO.13, CDR2 shown in SEQ ID NO.14, and CDR3 shown in SEQ ID NO.15; the amino acid sequence of the light chain hypervariable region of antibody 016-4 is CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, and CDR3 shown in SEQ ID NO.18.

[0013] Furthermore, the amino acid sequence of the heavy chain framework region of antibody 016-1 is FR1 shown in SEQ ID NO.19, FR2 shown in SEQ ID NO.20, FR3 shown in SEQ ID NO.21, and FR4 shown in SEQ ID NO.22; the amino acid sequence of the light chain framework region of antibody 016-1 is FR1 shown in SEQ ID NO.23, FR2 shown in SEQ ID NO.24, FR3 shown in SEQ ID NO.25, and FR4 shown in SEQ ID NO.26.

[0014] Furthermore, the amino acid sequence of the heavy chain framework region of antibody 016-3 is FR1 shown in SEQ ID NO.27, FR2 shown in SEQ ID NO.28, FR3 shown in SEQ ID NO.29, and FR4 shown in SEQ ID NO.30; the amino acid sequence of the light chain framework region of antibody 016-3 is FR1 shown in SEQ ID NO.31, FR2 shown in SEQ ID NO.32, FR3 shown in SEQ ID NO.33, and FR4 shown in SEQ ID NO.34.

[0015] Furthermore, the amino acid sequence of the heavy chain framework region of antibody 016-4 is FR1 shown in SEQ ID NO.35, FR2 shown in SEQ ID NO.36, FR3 shown in SEQ ID NO.37, and FR4 shown in SEQ ID NO.38; the amino acid sequence of the light chain framework region of antibody 016-4 is FR1 shown in SEQ ID NO.39, FR2 shown in SEQ ID NO.40, FR3 shown in SEQ ID NO.41, and FR4 shown in SEQ ID NO.42.

[0016] Furthermore, antibody 016-1 comprises at least one of the following amino acid sequences: a heavy chain variable region as shown in SEQ ID NO.43, and a light chain variable region as shown in SEQ ID NO.44.

[0017] Furthermore, antibody 016-3 comprises at least one of the following amino acid sequences: a heavy chain variable region as shown in SEQ ID NO.45, and a light chain variable region as shown in SEQ ID NO.46.

[0018] Furthermore, antibody 016-4 comprises at least one of the following amino acid sequences: a heavy chain variable region as shown in SEQ ID NO.47, and a light chain variable region as shown in SEQ ID NO.48.

[0019] Furthermore, the amino acid sequence of the heavy chain constant region HC of antibody 016-1, antibody 016-3, and antibody 016-4 includes SEQ ID NO.49, and the amino acid sequence of the light chain constant region LC of antibody 016-1, antibody 016-3, and antibody 016-4 includes SEQ ID NO.50.

[0020] It is to be understood that the above amino acid sequence encompasses an amino acid sequence that has at least 80% identity (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity) with itself and has the same function, which may contain mutations such as deletions, insertions and / or substitutions, wherein the sequence of the heavy chain or light chain variable region differs from the reference sequence only in conservative amino acid substitutions.

[0021] The second aspect of the present invention is to provide a biomaterial related to any anti-SUMO2 antibody described in the first aspect, which is selected from one of the following A) to B):

[0022] A) a nucleic acid molecule encoding the anti-SUMO2 antibody according to any one of the first aspects;

[0023] B) An expression cassette, a recombinant vector, or a recombinant cell line containing the nucleic acid molecule described in A).

[0024] Furthermore, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of antibody 016-1 is the nucleotide sequence shown in SEQ ID NO.51 or a nucleotide sequence different from SEQ ID NO.51 due to the degeneracy of the genetic code.

[0025] Furthermore, the nucleotide sequence encoding the amino acid sequence of the light chain variable region of antibody 016-1 is the nucleotide sequence shown in SEQ ID NO.52 or a nucleotide sequence different from SEQ ID NO.52 due to the degeneracy of the genetic code.

[0026] Furthermore, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of antibody 016-3 is the nucleotide sequence shown in SEQ ID NO.53 or a nucleotide sequence different from SEQ ID NO.53 due to the degeneracy of the genetic code.

[0027] Furthermore, the nucleotide sequence encoding the amino acid sequence of the light chain variable region of antibody 016-3 is the nucleotide sequence shown in SEQ ID NO.54 or a nucleotide sequence different from SEQ ID NO.54 due to the degeneracy of the genetic code.

[0028] Furthermore, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of antibody 016-4 is the nucleotide sequence shown in SEQ ID NO.55 or a nucleotide sequence different from SEQ ID NO.55 due to the degeneracy of the genetic code.

[0029] Furthermore, the nucleotide sequence encoding the amino acid sequence of the light chain variable region of antibody 016-4 is the nucleotide sequence shown in SEQ ID NO.56 or a nucleotide sequence different from SEQ ID NO.56 due to the degeneracy of the genetic code.

[0030] Furthermore, the nucleotide sequence encoding the amino acid sequence of the heavy chain constant region HC of antibody 016-1, antibody 016-3, and antibody 016-4 is the nucleotide sequence shown in SEQ ID NO.57 or a nucleotide sequence different from SEQ ID NO.57 due to the degeneracy of the genetic code.

[0031] Furthermore, the nucleotide sequence encoding the amino acid sequence of the light chain constant region LC of antibody 016-1, antibody 016-3, and antibody 016-4 is the nucleotide sequence shown in SEQ ID NO.58 or a nucleotide sequence different from SEQ ID NO.58 due to the degeneracy of the genetic code.

[0032] It is understandable that the difference between the nucleotide sequence and the degeneracy of the genetic code means that: based on the degeneracy of the codon, while ensuring that the coding sequence remains unchanged, in addition to the nucleic acid sequence defined above, several nucleic acid sequences that can encode the same heavy chain sequence or light chain sequence (for example, conservative nucleotide sequence variants are derived from the degeneracy of the genetic code and silent variants, and nucleotide substitutions, deletions and additions are also included), all of which are within the scope of protection of the present invention.

[0033] Furthermore, the expression cassette, recombinant vector, and recombinant cell line can be used to express the above-mentioned heavy chain sequence, light chain sequence, or monoclonal antibody.

[0034] In the above-mentioned biological materials, the expression cassette containing the nucleic acid molecule encoding the antibody refers to a DNA capable of expressing the antibody in a host cell, and the DNA may include not only a promoter for initiating transcription of the antibody gene, but also a terminator for terminating transcription of the antibody gene.

[0035] In the above-mentioned biological materials, a vector is a nucleic acid carrier into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect host cells to express the genetic material elements it carries in the host cells. The vector can be a plasmid, cosmid, phage or virus vector, specifically a pTT5 expression vector, a PEI co-transfection light chain expression plasmid and a heavy chain expression plasmid, etc.

[0036] In the above biological materials, host cells refer to cells into which vectors are introduced, including prokaryotic cells, fungal cells, insect cells, animal cells, etc., such as Escherichia coli, yeast cells, S2 fruit fly cells, BHK cells, CHO cells, HEK 293 cells. The above expression cassettes, recombinant vectors, and recombinant cell lines can all be prepared by conventional methods in the art.

[0037] The amino acid sequences of the heavy chains and light chains of the above-mentioned antibody 016-1, antibody 016-3 and antibody 016-4 and the nucleotide sequences of their encoding nucleic acid molecules are shown in the following table:

[0038] Table 1 - Antibody sequence information

[0039]

[0040]

[0041]

[0042]

[0043] The third aspect of the present invention is to provide a product containing any anti-SUMO2 antibody described in the first aspect, or containing any biological material described in the second aspect.

[0044] Furthermore, the product includes a kit containing an anti-SUMO2 antibody or biological materials related thereto, and a drug conjugate containing an anti-SUMO2 antibody. The above product may also include other detection reagents or reaction reagents.

[0045] Furthermore, the above product can be applied to conventional detection methods to detect SUMO2 protein, including ELISA, Western blot, immunofluorescence, immunoprecipitation, etc.

[0046] The fourth aspect of the present invention is to provide a method for preparing the anti-SUMO2 antibody described in any one of the first aspects, comprising the steps of: transfecting a host cell with a recombinant vector containing a nucleic acid molecule of the anti-SUMO2 antibody described in any one of the first aspects; culturing the transfected host cells and collecting the supernatant, and purifying to obtain the anti-SUMO2 antibody.

[0047] Furthermore, the preparation method specifically comprises the steps of:

[0048] S1, New Zealand white rabbits were immunized with SUMO2 antigen, spleens were taken out three days after immunization, and rabbit lymphocytes were isolated;

[0049] S2, using biotin-labeled SUMO2 protein to co-incubate with successfully immunized rabbit lymphocytes, and sorting out the rabbit memory B cells;

[0050] S3. Collect B cell positive clones, extract total RNA and reverse transcribe it into cDNA, amplify the light chain variable region and heavy chain variable region sequences by PCR, connect and transform the antibody fragments to obtain a recombinant plasmid with the correct sequence (constructed on the pTT5 expression vector containing the corresponding heavy chain constant region and light chain constant region for sequencing); transfect cells (293F cells) with the recombinant plasmid to express the monoclonal antibody, and further purify to obtain the anti-SUMO2 antibody.

[0051] Furthermore, the preparation method of the SUMO2 antigen is as follows: 293T cells are collected, total RNA is extracted and reversely transcribed into cDNA, the SUMO2 gene sequence is amplified by PCR method, and constructed into a prokaryotic cell expression vector with a GST tag to obtain a recombinant plasmid with a correct sequence, and the recombinant plasmid is transfected into Escherichia coli to express and obtain the GST-SUMO2 antigen.

[0052] The fifth aspect of the present invention is to provide an application of an anti-SUMO2 antibody as described in any one of the first aspect, or a biomaterial as described in any one of the second aspect, which is selected from at least one of the following applications: application in detecting SUMO2 protein for non-diagnostic purposes, application in preparing products for diagnosing and treating diseases related to SUMO2 protein, and application in studying cell cycle and cell biology.

[0053] Furthermore, the diseases associated with the SUMO2 protein include autoimmune liver disease, primary biliary cholangitis, pancreatic ductal adenocarcinoma, etc.

[0054] The sixth aspect of the present invention provides a method for detecting SUMO2 protein in a sample for non-diagnostic purposes, comprising the following steps: contacting the sample to be tested with any anti-SUMO2 antibody described in the first aspect to confirm the presence or level of SUMO2 protein in the sample to be tested.

[0055] The above-mentioned test samples cover a variety of sample types obtained from subjects and can be used in diagnosis or detection, including but not limited to blood and other liquid samples of biological origin, solid tissue samples, covering clinical samples, cells in culture medium, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples, etc.

[0056] Furthermore, the anti-SUMO2 antibody is used as a coating antibody and an enzyme-labeled secondary antibody (detection antibody).

[0057] It is understandable that in practical applications, antibody 016-1, antibody 016-3 or antibody 016-4 can be used alone, or a combination of two or three antibodies can be used in combination to detect SUMO2 protein.

[0058] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0059] The present invention utilizes an in vitro culture method of B cells to develop SUMO2 monoclonal antibodies, thereby avoiding the loss of antibody diversity during myeloma fusion and the problem that the antibody light and heavy chain pairing caused by phage display technology is no longer a natural combination, and antibodies with good diversity and high specificity can be prepared.

[0060] The SUMO2 monoclonal antibody prepared by the present invention has the following advantages over the polyclonal antibody in the prior art: it has single specificity, generates less non-specific background signals during detection and analysis, and improves the sensitivity and specificity of the experiment; it can be repeatedly prepared and mass-produced in vitro; it has high affinity and can more effectively bind to the target antigen. The above-mentioned SUMO2 monoclonal antibody has high activity and affinity, and can be better applied to the detection of SUMO2 protein and the study of diseases related to it. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0062] Figure 1 This is a schematic diagram of the results of detecting the titer of immune serum by conventional Elisa method in one embodiment of the present invention;

[0063] Figure 2 This is a schematic diagram of the results of screening and identifying B cell positive clones in one embodiment of the present invention;

[0064] Figure 3 This is a schematic diagram of the results of the detection of the binding activity between the SUMO2 antibody and the SUMO2 protein in one embodiment of the present invention. DETAILED DESCRIPTION

[0065] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0066] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0067] Example 1 - Preparation of anti-SUMO2 antibody (monoclonal antibody)

[0068] In this example, a rabbit monoclonal antibody that specifically binds to SUMO2 protein was prepared by using an in vitro culture method of B cells, and the specific steps include:

[0069] (1) Preparation of SUMO2 antigen;

[0070] The human SUMO2 gene was amplified by PCR and inserted into the prokaryotic expression vector pGEX. A GST tag was added to the N-terminus, and the successfully sequenced plasmid was transformed into the Escherichia coli BL21 strain. Take 3mL of 2YT+Carb+2% glucose medium, pick a single clone, and culture overnight at 37℃, 220rpm. Transfer 1:100 to 200mL, and collect the bacteria until OD=0.5. Induce with 1mM IPTG, culture at 30℃ for 16 hours. Centrifuge at 4℃, 8000rpm for 10 minutes, collect the bacteria, freeze-thaw cycle of lysis buffer to lyse the cells, release the protein, centrifuge at 4℃, 12000rpm for 30 minutes to remove the debris, and collect the supernatant. Use GST affinity column for purification, and use elution buffer containing reduced glutathione to elute the GST-SUMO2 fusion protein bound to the column. This part of the experiment was completed by the research group of Professor Weng Jiemin from the School of Life Sciences, East China Normal University.

[0071] (2) Animal immunization;

[0072] In order to obtain rabbit monoclonal antibodies that recognize SUMO2 antigen, this embodiment uses the GST-SUMO2 fusion protein prepared in step (1) as an antigen to immunize New Zealand white rabbits. The first immunization is 200 μg. For the first immunization, Freund's complete adjuvant and an equal volume of antigen are mixed and emulsified, and the immunization is injected at multiple points on the back. After an interval of two weeks, a booster immunization is performed. The amount of antigen used for the booster immunization is 100 μg. Freund's incomplete adjuvant and an equal volume of antigen are mixed and emulsified, and the immunization is injected at multiple points on the back. The interval between immunizations is two weeks. After four immunizations, the titer of the immune serum is detected by conventional Elisa method. The results are shown in Table 1. Figure 1 . The specific steps include: the coating concentration of SUMO2 protein is 1μg / mL, blank is the rabbit serum before immunization, NO.1 is the serum of rabbit No. 1 after four immunizations, and NO.2 is the serum of rabbit No. 2 after four immunizations. The starting dilution ratio of the three samples is 1:500, followed by multiple dilutions, a total of 12 concentrations. After the antigen and antibody are incubated for 3 hours, a 1:20000 dilution of anti-rabbit-IgG-HRP antibody is added. After incubation for 1 hour, the OD450 absorbance value is detected by an enzyme marker. The serum of rabbit No. 1 is still positive after dilution of 1:60000 (defined as positive if it is three times greater than the background reading), and rabbit No. 1 is selected for subsequent experiments. Three days before screening antibodies, rabbit No. 1 is intraperitoneally injected with 50μg of antigen protein for a shock immunization. This time, the antigen does not need to be emulsified with an adjuvant, and the buffer is PBS. The spleen is taken three days later.

[0073] (3) Spleen cell isolation;

[0074] The spleen of the rabbit was surgically removed and placed in a sterile cell culture dish. The spleen was rinsed with DPBS containing 100U / mL penicillin and 100μg / mL streptomycin. The spleen was cut into pieces with surgical scissors and gently ground into single cells with a syringe core. Finally, the cell suspension was filtered with a 100μm cell sieve to collect the single cell filtrate. Centrifuged at 1200rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in RPMI-1640 containing 5% fetal bovine serum.

[0075] (4) B cell culture and identification;

[0076] Take 50 μg of 1 mg / mL SUMO2 protein, add 3 μL of 20 mM NHS-activated Biotin, mix gently and react at room temperature for 30 minutes, then add 2 μL of 2M Tris-HCl (pH 8.0) buffer and mix gently to terminate the reaction.

[0077] 50 μg of biotinylated SUMO2 protein was successfully immunostained with 1×10 8Rabbit lymphocytes with a certain number of cells were co-incubated, and rabbit memory B cells were obtained by anti-Biotin magnetic bead sorting. In a 96-well cell culture plate, culture was carried out using B cell culture medium at 37°C and 5% CO2. After 14 days of culture, the binding activity of the clone supernatant was detected at the protein level by ELISA, and clones that were more than 3 times the background were judged to be positive. The specific steps include: the coating concentration of SUMO2 protein is 1μg / mL, 100μL / well is coated at 4°C overnight, washed twice with PBST, 3% BSA 100μL / well is added for blocking for 1 hour, and then 100μL of B cell supernatant is added. After incubation at room temperature for 3 hours, it is washed 3 times with PBST, and a 1:20000 diluted anti-rabbit-IgG-HRP antibody is added. After incubation for 1 hour, the OD450 absorbance value is detected by an enzyme reader. Results are shown in Figure 2 The OD values ​​of the three clones with the highest binding activity were 0.537, 0.353 and 0.238, respectively, and the background reading was 0.048. The three clones with the highest binding activity (named Antibody 016-1, Antibody 016-3 and Antibody 016-4) were selected for subsequent experiments.

[0078] (5) Cloning of genes encoding rabbit monoclonal antibodies;

[0079] B cell positive clones were collected, and total RNA was extracted from the three positive clones with the highest reads using RNAiso Plus (TAKARA, D9108A). TM II 1st Strand cDNA Synthesis Kit (TAKARA, 6210b) was used to reverse transcribe into cDNA, and the light chain variable region and heavy chain variable region sequences were amplified by PCR method, and constructed into the pTT5 expression vector containing the corresponding heavy chain constant region and light chain constant region for sequencing to obtain the correct sequence. The sequencing results were analyzed by VBASE2 (http: / / www.vbase2.org / vbscAb.php), and the light and heavy chain variable region sequences of antibody 016-1, antibody 016-3 and antibody 016-4 were obtained. The variable region and constant region sequences of the above-mentioned antibodies are shown in Table 1 above.

[0080] (6) Expression and purification of monoclonal antibodies;

[0081] Collect the 293F cells in the logarithmic growth phase with good growth status, inoculate them into a 250mL cell culture flask, add 50mL culture medium and culture until the cell density reaches 2×10 6 / mL, and PEI was co-transfected with 30 μg of each of the light chain expression plasmid and the heavy chain expression plasmid. The cell supernatant was collected on the 7th day after transfection, centrifuged and filtered using a 0.45 μM filter. The antibody was purified by Protein A medium and the SUMO2 antibody was replaced by dialysis into PBS pH 7.2 buffer. The antibody concentration and purity were determined by measuring the absorbance by Nanodrop, and the purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.

[0082] Example 2- Detection of the binding activity of anti-SUMO2 antibodies and SUMO2 protein

[0083] In this example, the binding activity of the SUMO2 antibody obtained in Example 1 to the SUMO2 protein was verified by an ELISA method. The specific steps included: the coating concentration of the SUMO2 protein was 1 μg / mL, the starting concentrations of the antibodies 016-1, 016-3, and 016-4 were 1000 nM, four-fold dilutions were used, a total of 12 concentrations, and after the antigen and antibody were incubated for 3 hours, a 1:20000 dilution of anti-rabbit-IgG-HRP antibody was added, and after incubation for 1 hour, the OD450 absorbance was detected by an ELISA instrument. The results are as follows: Figure 3 As shown, the EC50 values ​​of the three antibodies were analyzed to be 3.971, 7.617 and 10.67 nM, respectively.

[0084] It can be seen from the above examples that the three SUMO2 monoclonal antibodies screened and obtained in the present invention have strong binding ability with SUMO2 protein, and can be better applied to the detection of SUMO2 protein and the research of related diseases.

[0085] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-SUMO2 antibody, characterized in that The anti-SUMO2 antibody includes antibody 016-1; wherein the amino acid sequence of the heavy chain hypervariable region of antibody 016-1 is CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3; the amino acid sequence of the light chain hypervariable region of antibody 016-1 is CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, and CDR3 shown in SEQ ID NO.

6.

2. The anti-SUMO2 antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain framework region of the antibody 016-1 is FR1 shown in SEQ ID NO. 19, FR2 shown in SEQ ID NO. 20, FR3 shown in SEQ ID NO. 21, and FR4 shown in SEQ ID NO. 22; the amino acid sequence of the light chain framework region of the antibody 016-1 is FR1 shown in SEQ ID NO. 23, FR2 shown in SEQ ID NO. 24, FR3 shown in SEQ ID NO. 25, and FR4 shown in SEQ ID NO.

26.

3. The anti-SUMO2 antibody according to claim 1 or 2, characterized in that The antibody 016-1 comprises at least one of the following amino acid sequences: a heavy chain variable region as shown in SEQ ID NO. 43, and a light chain variable region as shown in SEQ ID NO.

44.

4. The anti-SUMO2 antibody according to claim 3, characterized in that The amino acid sequence of the heavy chain constant region HC of the antibody 016-1 includes SEQ ID NO. 49, and the amino acid sequence of the light chain constant region LC of the antibody 016-1 includes SEQ ID NO.

50.

5. A biomaterial related to the anti-SUMO2 antibody according to any one of claims 1 to 4, characterized in that: The biological material is selected from one of the following A) to B): A) a nucleic acid molecule encoding the anti-SUMO2 antibody according to any one of claims 1 to 4; B) An expression cassette, a recombinant vector, or a recombinant cell line containing the nucleic acid molecule described in A).

6. The biomaterial according to claim 5, characterized in that The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of antibody 016-1 is the nucleotide sequence shown in SEQ ID NO. 51 or a nucleotide sequence that is different from SEQ ID NO. 51 due to the degeneracy of the genetic code; The nucleotide sequence encoding the amino acid sequence of the light chain variable region of antibody 016-1 is the nucleotide sequence shown in SEQ ID NO. 52 or a nucleotide sequence that is different from SEQ ID NO. 52 due to the degeneracy of the genetic code.

7. The biomaterial according to claim 5, characterized in that The nucleotide sequence encoding the amino acid sequence of the heavy chain constant region HC of antibody 016-1 is the nucleotide sequence shown in SEQ ID NO. 57 or a nucleotide sequence different from SEQ ID NO. 57 due to the degeneracy of the genetic code; The nucleotide sequence encoding the amino acid sequence of the light chain constant region LC of antibody 016-1 is the nucleotide sequence shown in SEQ ID NO. 58 or a nucleotide sequence different from SEQ ID NO. 58 due to the degeneracy of the genetic code.

8. A product comprising the anti-SUMO2 antibody according to any one of claims 1 to 4, or the biomaterial according to any one of claims 5 to 7.

9. A use of the anti-SUMO2 antibody according to any one of claims 1 to 4, or the biomaterial according to any one of claims 5 to 7, characterized in that: The application includes application in preparing reagents for detecting SUMO2 protein for non-diagnostic purposes.

10. A method for detecting SUMO2 protein in a sample for non-diagnostic purposes, characterized in that: The method comprises the following steps: contacting a sample to be tested with the anti-SUMO2 antibody according to any one of claims 1 to 4 to confirm the presence or level of SUMO2 protein in the sample to be tested.

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