Neddylation modified DNA-pkcs protein polyclonal antibody and preparation method therefor

Abstract

A neddylation modified DNA-PKcs protein polyclonal antibody and a preparation method therefor, belonging to the technical field of bioengineering. On the basis of a DNA-PKcs protein sequence, a neddylation antigen polypeptide is designed and synthesized, and is then used for animal immunization to prepare a polyclonal antibody.

Description

Polyclonal antibody against ubiquitination-modified DNA-PKcs protein and preparation method thereof Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a polyclonal antibody against ubiquitination-modified neddylation DNA-PKcs protein and a preparation method thereof. Background Art

[0002] DNA-PKcs, along with ATM and ATR, belongs to the PI3KK (phosphoinositide 3-kinase (PI3K)-related kinase) family. They are serine / threonine protein kinases that share similar domain structures and many common structural features. Their kinase domains are located at the C-terminus of the protein, with the FAT (FRAP-ATM-TRRAP) domain and the FATC (FAT C-terminal) domain located upstream and downstream of the kinase domain, respectively. Due to their structural similarities, the three share many common substrates, some functional overlap, and play a crucial role in the DNA damage response.

[0003] DNA-PKcs, encoded by the prkdc gene, is evolutionarily conserved and expressed in virtually all mammalian cells. The DNA-PKcs protein consists of 4128 amino acid residues, primarily comprising a catalytic domain, a DNA-binding domain, and a Ku-binding domain. DNA-PKcs and ATM primarily respond to DSBs and are recruited to DNA break ends by the Ku70 / Ku80 and MRN complexes, respectively. ATR, on the other hand, primarily responds to DNA replication stress and is recruited to DNA break ends through interaction with ATRIP. Early biochemical studies revealed that DNA-PKs prefer serine or threonine (S / TQ) followed by glutamine when phosphorylating their substrate proteins, and this was later found to be the case for ATM and ATR.

[0004] DNA double-strand breaks (DSBs) are one of the most severe forms of DNA damage, requiring the most complex repair process and posing the greatest threat to genomic stability. If DSBs are not promptly repaired, they can affect the accurate transmission of genetic information and induce a variety of diseases, including cancer. However, during chemotherapy and radiotherapy, ionizing radiation or chemotherapy drugs can induce DSBs in cancer cells, leading to cell death. Following the occurrence of DNA double-strand breaks, a group of highly conserved PI3 (Phosphoinositide 3)-like kinases, including ATM, ATR, and DNA-PKcs, become activated within the cell. These kinases then phosphorylate hundreds of downstream substrates to regulate cell cycle checkpoints and DNA damage repair.

[0005] DNA-PKcs is a key upstream protein in the non-homologous end-joining (NHEJ) repair pathway. Upon DSB formation, it is rapidly recruited to the DNA break ends by the cyclic heterodimer Ku70 / Ku80, forming a DNA-PK complex and activating the kinase activity of DNA-PKcs. Activated DNA-PKcs can phosphorylate a variety of proteins, including histone H2AX, Chk2, P53, 53BP1, XRCC4, XLF, Ku70 / 80, and Artemis. It shares many substrates with ATM, such as H2AX and 53BP1, suggesting that ATM and DNA-PKcs share overlapping functions in DNA repair and embryonic development.

[0006] When double-strand breaks occur, the hallmark post-translational modification of DNA-PKcs is phosphorylation, with over 40 phosphorylation sites clustered. The most studied site is phosphorylation at Thr2609. Thr2609 is primarily phosphorylated by ATM and ATR. Phosphorylation of the Thr2609 cluster is crucial for NHEJ, as ablation of these sites by alanine substitution leads to severe radiosensitivity and reduced DNA end-joining ability in vitro. Furthermore, phosphorylation of the Thr2609 cluster and / or conformational changes in DNA-PKcs facilitate its binding to other DNA repair molecules.

[0007] Another well-characterized DNA-PKcs phosphorylation cluster is the Serine 2056 (Ser2056) cluster. Serine 2056 is a bona fide autophosphorylation site for DSBs in vivo, and phosphorylation of the Ser2056 cluster is important for NHEJ, as loss of phosphorylation of this cluster leads to increased radiosensitivity and reduced DSB repair efficiency.

[0008] In addition to protein phosphorylation, protein ubiquitination is another important post-translational modification that occurs after DNA damage. Following DNA double-strand breaks, a group of E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases, including UBC13, RNF8, and RNF168, are recruited to the DNA damage site and ubiquitinate chromosomal proteins, such as histones H2A and H2B. Following DNA double-strand breaks, H2A is ubiquitinated by RNF168 at the K13 and K15 lysine sites. Increased expression of RBX1 protein promotes the neddylation and activity of cullin1, a key component of the Skp1-Cullin1-Fbox ubiquitin E3 ligase. This mediates the ubiquitination and degradation of EXO1 during the G1 phase. Increased DNA-PKcs activity leads to increased RBX1 protein expression, limiting the formation of ssDNA at DSB ends and inhibiting HR repair in G1 cells.

[0009] DNA-PKcs-related changes in tumors primarily include prkdc gene mutations and altered DNA-PKcs expression and activity. These changes in activity vary across different tumor tissues. Studies have found increased activity in colorectal cancer and gliomas, decreased activity in breast and cervical cancers, and both increased and decreased activity in esophageal cancer and lymphomas. Increased DNA-PKcs activity has beneficial effects on tumors. First, increased activity enhances the ability of precancerous cells to repair DNA damage through NHEJ, allowing them to escape death. However, this repair process is prone to errors, causing structural changes in DNA and promoting tumor formation. Second, the primary mechanism of action of DNA-damaging chemotherapies and radiotherapy commonly used in cancer treatment is to create lethal double-strand breaks in DNA molecules, thereby inducing tumor cell death. Increased DNA-PKcs activity can, to a certain extent, inhibit cell death, conferring resistance to chemoradiotherapy. Furthermore, cells that survive chemoradiotherapy in tumor tissues are often those with high DNA-PKcs activity, making them less sensitive to treatment and contributing to poor efficacy and prognosis. Therefore, DNA-PKcs is a potential target for tumor treatment. Detection of DNA-PKcs and use of its inhibitors to enhance the effects of radiotherapy and chemotherapy is a new strategy.

[0010] Ubiquitination (neddylation) is a process in which the ATP-dependent ubiquitin-like small protein Nedd8 (neural precursor cell expressed, developmentally downregulated 8) interacts with a lysine residue on a substrate protein through a series of catalytic actions, including the activating enzyme E1, the conjugating enzyme E2, and the ligating enzyme E3, resulting in covalent binding of its C-terminal glycine to the substrate protein. DNA-PKcs regulates the repair pathways of DSBs at multiple biochemical points in the cell cycle, but simply detecting phosphorylation at specific sites is insufficient to reflect the activation state and biological function of DNA-PKcs. Due to the lack of comprehensive detection methods and related research on other DNA-PKcs sites, there are also significant gaps in the understanding of neddylation at the DNA-PKcs K4007 site. Therefore, the development of a polyclonal antibody against neddylation at the K4007 site of the DNA-PKcs protein is crucial for evaluating the DNA-PKcs function in DNA break repair and its role in cancer.

[0011] Summary of the Invention

[0012] The present invention designs and synthesizes ubiquitination-like antigen polypeptides based on the DNA-PKcs protein sequence, which are then used for animal immunization to prepare polyclonal antibodies.

[0013] In a first aspect, the present invention provides a polyclonal antibody against a tumor DNA-PKcs protein, wherein the antigen amino acid sequence of the polyclonal antibody is shown in SEQ ID NO. 1.

[0014] SEQ ID NO. 1: CMDVFVLRGGKEPSFDWK.

[0015] In a second aspect, the present invention provides a method for preparing a polyclonal antibody against a tumor DNA-PKcs protein, the method comprising the following steps:

[0016] S1. Antigen peptide design;

[0017] S2. Immunize the animal with the antigen designed in step S1;

[0018] S3. After the animal is immunized in step S2, the animal serum is obtained and purified to obtain the polyclonal antibody against the tumor DNA-PKcs protein of the present invention.

[0019] Furthermore, the antigen polypeptide is designed to perform ubiquitination-like modification on the DNA-PKcs protein, wherein the ubiquitination-like modification is achieved by connecting the C-terminal carboxyl group of the ubiquitination-like protein to the ε-amino group of the lysine residue of the DNA-PKcs protein.

[0020] Furthermore, the ubiquitination-modified protein is selected from one or more of LRGG, LALRGG, SUMO1, SUM02, SUM03, SUMO4, NEDD8, FAT10, FAT1, FUB1, UBL5, ISG15, UFM1, Urml, ATG12, ATG8, Apg12, MAP1LC3 and GABARAPL.

[0021] Furthermore, the ubiquitination-like protein is preferably NEDD8.

[0022] In a third aspect, the present invention provides a pharmaceutical composition of a polyclonal antibody against the tumor DNA-PKcs protein as described in the first aspect.

[0023] Furthermore, the pharmaceutical composition may further comprise an adjuvant and / or a pharmaceutically acceptable auxiliary material.

[0024] Furthermore, the composition can be prepared as a vaccine, a detection reagent or other biological diagnostic reagents.

[0025] In a fourth aspect, the present invention provides a use of the polyclonal antibody against the tumor DNA-PKcs protein as described in the first aspect in the preparation of a tumor-inhibiting drug.

[0026] Furthermore, the tumor includes but is not limited to small cell lung cancer, non-small cell lung cancer, ovarian cancer, endometrial cancer, breast cancer, head and neck cancer, thymoma, colorectal cancer, pancreatic cancer, prostate cancer, bladder cancer or melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the antibody development route and timeline;

[0028] Figure 2 Mass spectrometry detection results of Peptide 1;

[0029] Figure 3 Mass spectrometry detection results of Peptide 2;

[0030] Figure 4 Mass spectrometry detection results of Peptide 3;

[0031] Figure 5 Mass spectrometry detection results of Peptide 4;

[0032] Figure 6 Schematic diagram of antibody purification;

[0033] Figure 7 Dotblot test results;

[0034] Figure 8 Immunofluorescence staining results (red dots represent DNA-PKcs K4007 modification signals, blue dots represent DAPI staining);

[0035] Figure 9. Immunofluorescence staining results. DETAILED DESCRIPTION

[0036] The animals used in the present invention are three healthy New Zealand rabbits. The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0037] Example 1 Antigen Design and Synthesis

[0038] The custom antibody development route and timeline are shown in Figure 1.

[0039] 1. Antigen design

[0040] The DNA-PKcs protein is ubiquitinated by the ubiquitination protein NEDD8, that is, the C-terminal carboxyl group of the NEDD8 molecule is connected to the ε-amino group of the lysine site 4007 in the DNA-PKcs protein sequence, forming the ubiquitination sequence LRGG at position 4007.

[0041] Based on the sequence information of the ubiquitination-modified DNA-PKcs protein, peptides with no more than 10 amino acids were selected around site 4007, and two modified peptides and two non-modified peptides were designed and synthesized. The peptide information is shown in Table 1 below.

[0042] Table 1 Peptide information

[0043] 2. Mass spectrometry detection

[0044] The sequences of the four synthesized polypeptides are subjected to antigen mass spectrometry detection. The synthesized peptide mixture is ionized in a mass spectrometer to form charged ions. The electric field and magnetic field of the mass spectrometer separate the peptide ions with a specific mass to charge ratio (i.e., mass-to-charge ratio, M / Z). The separated ions are collected by the detector to determine the M / Z value of each ion. The M / Z of each peptide can be analyzed by the mass analyzer to obtain the M / Z spectrum of all peptides of the protein, i.e., the primary mass spectrum peak diagram of the protein. The ion selection device automatically selects peptide ions with larger intensity for secondary mass spectrometry analysis, outputs the secondary mass spectrum peak diagram of the selected peptide, and compares the primary mass spectrum peak diagram and the secondary mass spectrum peak diagram theoretically produced after the protein is digested with trypsin to identify the antigen required by the present invention.

[0045] The synthesized peptides were tested by mass spectrometry, and the results were satisfactory. The sequences of the four peptides were accurate, with the difference between the measured and theoretical masses of the four peptide segments within 10 ppm, indicating that the amino acid sequences were correct (see Figures 2-5, where the vertical axis represents the intensity of the ion peak and the horizontal axis represents the mass to charge ratio).

[0046] Example 2 Immunization of experimental animals

[0047] The two modified peptides were coupled to KLH and used for rabbit immunization.

[0048] The immunogen was diluted with saline and then mixed with the corresponding Freund's adjuvant at a 1:1 ratio. The antigen and adjuvant were thoroughly mixed to form a stable emulsion. The antigen mixture was then drawn up with a syringe and injected into the rabbits' shoulders and hind legs at two subcutaneous sites, with approximately 1 / 4 of the immunogen volume injected into each site. This ensures the immunogen persists and enhances the immune response. Each rabbit was immunized four times: on days 1, 21, 28, and 35.

[0049] The first blood draw: on the 45th day, 30 mL of whole blood was collected and centrifuged. The supernatant was collected after centrifugation and sent to the laboratory for serum screening test, including ELISA; the second / third / fourth blood draw: on the 50th day, 65th day, and 70th day, 3 times of whole blood were collected, each time 20 mL, and the supernatant was collected after centrifugation and sent to the laboratory for serum screening test.

[0050] Example 3 ELISA method to determine the calibration of antibodies

[0051] Two SPF experimental grade New Zealand white rabbits were immunized with the antigen peptides peptide 1 and peptide 2 respectively. The rabbits immunized with peptide 1 were labeled R1 and the rabbits immunized with peptide 2 were labeled R2. After multiple immunizations, ELISA screening was performed to preliminarily evaluate the titer and specificity of the antiserum.

[0052] 3.1 Serum screening ELISA test

[0053] Antiserum is added at varying dilutions to a 96-well microtiter plate coated with an antigen-modified peptide or an unmodified control peptide. Binding of the peptide and antiserum to the plate is detected using an enzyme-labeled secondary antibody and TMB chromogenic substrate. The titer of the antiserum is determined by the antiserum dilution ratio at which the absorbance at 450 nm (OD450) is approximately 1. Serum titers are shaded gray.

[0054] The ELISA test results are shown in Table 2 below. The titer of R1 rabbit serum recognizing modified peptide 1 is about 1:54K, the titer recognizing modified peptide 2 is about 1:6K, the titer recognizing unmodified peptide 3 is about 1:6K, and basically does not recognize unmodified peptide 4. The titer of R2 rabbit serum recognizing modified peptide 1 and modified peptide 2 is between 1:54K and 1:486K, and the titer recognizing unmodified peptide 3 is between 1:486K, and basically does not recognize unmodified peptide 4. In general, the antisera of R1 and R2 rabbits both showed good titers and were purified.

[0055] Table 2 Serum ELISA test results

[0056] Example 4: Antibody purification using affinity chromatography column

[0057] 4.1 Prepare protein A affinity column:

[0058] Typically, 5 mL or 10 mL of Protein A filler is prepared. Equal volumes of filler and PBS buffer are mixed and stirred, and air is removed from the filler. Slowly add Protein A filler to the glass column to prepare the chromatography column. Avoid drying out the column during this process. After priming, equilibrate the column with 10 volumes of pre-chilled PBS buffer.

[0059] 4.2 Protein A affinity chromatography:

[0060] 4.2.1 After filtering the serum, load it onto the equilibrated protein A column. To detect the binding efficiency of the antiserum to the filler, retain the load effluent.

[0061] 4.2.2 Wash the column with PBS buffer and elute with 150 mM glycine buffer. Collect the eluate and adjust the pH to 7 by adding neutralizing buffer.

[0062] 4.2.3 Load the crude IgG obtained after protein A purification onto the equilibrated antigen peptide affinity chromatography column to specifically enrich the target antibody.

[0063] 4.2.4 Removal of non-specific antibodies:

[0064] The target antibody obtained in the previous step is loaded onto the well-equilibrated picolinyl-modified and isonicotinoic acid-acylated affinity chromatography columns, and the effluent is directly collected to remove non-specific antibody components.

[0065] 4.2.5 Antibody Storage:

[0066] Determine the protein content. Add 10% glycerol to preserve the antibody, and store the purified antibody in aliquots at 2°C-8°C.

[0067] The schematic diagram of antibody purification is shown in Figure 6. After the serum is filtered through a filter, it is loaded onto a balanced protein A chromatography column to obtain ET1. ET1 is loaded onto an Antigen Peptides chromatography column to obtain ET2. ET2 is loaded onto a non-modified control peptides chromatography column to obtain FT3.

[0068] Example 5: ELISA for antibody quality control detection

[0069] Sufficient rabbit serum was collected for multi-step affinity purification. The purified antibodies were then tested by ELISA and Dot Blot.

[0070] 5.1ELISA test:

[0071] 5.1.1 Antigen coating: Dilute the antigen with coating solution and add 50 μg / well of the ELISA plate. Incubate in a refrigerator at 4°C overnight or in a 37°C oven for 2 hours.

[0072] 5.1.2 Washing: Take out the ELISA plate coated the day before and wash three times with 1×TBST.

[0073] 5.1.3 Blocking: Add 1% BSA blocking solution to the cleaned ELISA plate, incubate at 37°C for 1 hour, and then wash 1-3 times.

[0074] 5.1.4 Primary Antibody Incubation: Prepare a 3-fold serial dilution of the antibody starting at 1:1K. Adjust the dilution volume based on the actual situation. Add the cells to the ELISA plate in sequence, incubate at 37°C for 1.5 hours, and then wash 1-3 times.

[0075] 5.1.5 Secondary antibody incubation: Dilute the secondary antibody to 1:10K with 1% BSA blocking solution, incubate at room temperature or 37°C for 45 minutes, and then wash 1-3 times.

[0076] 5.1.6 Color development: Add TMB colorimetric solution and 5-10 minutes later, terminate the development reaction with 1M sulfuric acid and read the data using a microplate reader.

[0077] The antibodies purified from the sera of rabbits R1 and R2 were labeled as Ab1 and Ab2 (Ab2 was discarded because it strongly recognized unmodified peptide 3). The ELISA test results are shown in Table 3: Ab1 antibody recognized modified peptide 1 with a titer of approximately 1:1458K, and did not recognize unmodified peptides 3 and 4.

[0078] Table 3 Antibody ELISA test results

[0079] Example 6: Dot Blot for Antibody Quality Control

[0080] 6.2Dotblot detection:

[0081] 6.2.1 Spotting: Spot the uncross-linked antigen peptide onto the PVDF membrane in a gradient of 1 ng, 4 ng, 16 ng, and 64 ng.

[0082] 6.2.2 Blocking: After the membrane surface is dry, add blocking solution and block at room temperature for 60 minutes.

[0083] 6.2.3 Washing: Wash with 1× TBST for 10 min.

[0084] 6.2.4 Primary Antibody Incubation: Dilute the antibody with 2.5-5% skim milk powder, incubate at room temperature for 2 h, and then wash three times with 1× TBST, each time for 5-10 min.

[0085] 6.2.5 Secondary Antibody Incubation: Select the corresponding mouse or rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45 minutes to 1 hour, and then wash three times with 1× TBST, each time for 5-10 minutes.

[0086] 6.2.6 Add color developing substrate to the washed membrane and expose it to light.

[0087] The results are shown in Figure 7. As the sample amount gradually increased from 1 ng to 64 ng, the gray value of the Dot blot experimental results gradually increased, indicating that the purified antibody was successfully constructed and can be used for laboratory monitoring.

[0088] Example 7: Antibody quality control detection by immunofluorescence staining

[0089] 7.1 BEAS-2B cells were digested and counted and then cultured on glass slides for 24 hours.

[0090] 7.2 BEAS-2B cells were treated with 10 μM VP-16 for 4 hours. VP-16 caused damage to the intracellular DNA.

[0091] 7.3 Remove the culture medium from the cells, wash them twice with PBS, and fix them with 4% paraformaldehyde at room temperature for 15 minutes.

[0092] 7.4 Wash once with PBS and then perforate with 0.5% Triton-X100 for 10 minutes.

[0093] Block with 7.510% FBS at room temperature for 1 hour.

[0094] 7.6 Dilute the DNA-PKcs K4007 site antibody at 1:500 and incubate the cells at room temperature for 1 hour.

[0095] 7.7 Wash three times with PBS to remove non-specific binding.

[0096] 7.8 Incubate with rhodamine TRITC-labeled goat anti-rabbit secondary antibody at room temperature for 30 minutes.

[0097] 7.9 Wash three times with PBS to remove non-specific binding.

[0098] 7.10 Mount the slides with ProLong Gold antifade mounting medium (containing the nuclear dye DAPI).

[0099] 7.11 Observe with a confocal microscope and analyze the results statistically.

[0100] The results are shown in FIG8 . After BEAS-2B cells were treated with 10 μM VP-16 for 4 hours, the antibody against the neddylation modification of the DNAPKcs K4007 site was able to recognize foci formed by the neddylation modification of DNA-PKcs.

[0101] Example 8: Immunoprecipitation-Western blot for antibody quality control

[0102] 8.1 Culture BEAS-2B cells and treat them with 10 μM VP-16.

[0103] 8.2 Collect VP-16-treated BEAS-2B cells at 0h, 1h, 4h, and 8h respectively.

[0104] 8.3BEAS-2B cells were lysed using RIPA lysis buffer.

[0105] 8.4 Lyse protein and perform BCA quantification.

[0106] 8.5 Incubate the proteins at different time points with DNA-PKcs antibody at 4°C overnight.

[0107] 8.6 Add Protein A / G column material and continue incubation for 4 hours.

[0108] 8.7 Centrifuge and discard the supernatant. Wash three times with lysis buffer.

[0109] 8.8 Add 5X loading buffer and heat at 95 degrees for 5 minutes to denature the protein.

[0110] 8.9 Separate each protein group by SDS-PAGE electrophoresis.

[0111] 8.10 Transfer the proteins separated by PAGE gel to PVDF membrane.

[0112] 8.115% skim milk was used to block PVDF for 1 hour.

[0113] 8.12 Incubate the PVDF membrane with DNA-PKcs K4007 modified antibody and DNA-PKcs antibody respectively.

[0114] 8.13After washing three times with TBST, incubate with HRP-labeled secondary antibody.

[0115] 8.14 Add HRP substrate and expose to light for development.

[0116] The results are shown in Figure 9. After BEAS-2B cells were treated with 10 μM VP-16, the neddylation modification of the DNA-PKcs K4007 site increased over time as the DNA damage time prolonged.

Claims

1. A polyclonal antibody against tumor DNA-PKcs protein, the antigen amino acid sequence of the polyclonal antibody is shown in SEQ ID NO.1, SEQ ID NO. 1: CMDVFVLRGGKEPSFDWK.

2. A method for preparing a polyclonal antibody against a tumor DNA-PKcs protein, the method comprising the following steps: S1. Antigen peptide design; S2. Immunize the animal with the antigen designed in step S1; S3. After the animal is immunized in step S2, the animal serum is obtained and purified to obtain the polyclonal antibody of the tumor DNA-PKcs protein of the present invention.

3. The method for preparing a polyclonal antibody against tumor DNA-PKcs protein according to claim 2, characterized in that: The antigen polypeptide is designed to perform ubiquitination-like modification on the DNA-PKcs protein, wherein the ubiquitination-like modification refers to connecting the C-terminal carboxyl group of the ubiquitination-like protein to the ε-amino group of the lysine residue of the DNA-PKcs protein.

4. The method for preparing a polyclonal antibody against tumor DNA-PKcs protein according to claim 2, characterized in that: The ubiquitination-modified protein is selected from one or more of LRGG, LALRGG, SUMO1, SUM02, SUM03, SUMO4, NEDD8, FAT10, FAT1, FUB1, UBL5, ISG15, UFM1, Urml, ATG12, ATG8, Apg12, MAP1LC3 and GABARAPL.

5. The method for preparing a polyclonal antibody against tumor DNA-PKcs protein according to claim 4, characterized in that: The ubiquitination-like protein is selected from NEDD8.

6. A pharmaceutical composition of the polyclonal antibody against tumor DNA-PKcs protein as claimed in claim 1.

7. The pharmaceutical composition of polyclonal antibodies against tumor DNA-PKcs protein according to claim 6, characterized in that: The pharmaceutical composition may further comprise an adjuvant and / or a pharmaceutically acceptable auxiliary material.

8. The pharmaceutical composition of polyclonal antibodies against tumor DNA-PKcs protein according to claim 6, characterized in that: The composition can be prepared as a vaccine, a detection reagent or other biological diagnostic reagent.

9. Use of the polyclonal antibody against tumor DNA-PKcs protein as claimed in claim 1 in the preparation of tumor suppressor drugs.

10. Use of the polyclonal antibody against tumor DNA-PKcs protein according to claim 9 in the preparation of tumor suppressor drugs, characterized in that: The tumor is selected from small cell lung cancer, non-small cell lung cancer, ovarian cancer, endometrial cancer, breast cancer, head and neck cancer, thymoma, colorectal cancer, pancreatic cancer, prostate cancer, bladder cancer or melanoma or melanoma.

Images