Binding proteins for p53 protein and uses and kits thereof

Abstract

This invention provides a binding protein for P53, its applications, and a reagent kit, relating to the field of biotechnology. The binding protein provided by this invention can specifically recognize P53 protein and has a high affinity for it. It can be used to develop reagents or kits for the detection of P53 protein, and further for the diagnosis or prognostic assessment of diseases using P53 as a biomarker. This invention provides more and better antibody options for the detection of P53 and the diagnosis of diseases using P53 as a biomarker.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a binding protein of P53 protein, its applications, and a kit. Background Technology

[0002] p53 is a tumor suppressor gene that encodes a 393-amino acid nucleoprotein. Its protein band appears at 53 kDa, as indicated by the marker band, hence the name p53. The encoded protein exists in two subtypes: wild-type and mutant. The wild-type p53 induces apoptosis in cancer cells, thus preventing carcinogenesis, and also helps repair defects in cellular genes. Mutant p53, on the other hand, increases the risk of cancer.

[0003] The p53 protein is mainly distributed in the nucleus and cytoplasm, and can specifically bind to dopamine (DA). Its activity is regulated by post-translational modifications such as phosphorylation, acetylation, methylation, and ubiquitination. The normal biological function of p53 is to check for DNA damage sites during the G1 phase and monitor the integrity of the genome. If damage is found, the p53 protein inhibits DNA replication to provide sufficient time for repair. If repair fails, the p53 protein induces apoptosis. If the p53 gene mutates, its spatial conformation changes, and it loses its regulatory role in cell growth, apoptosis, and DNA repair. The p53 gene transforms from a tumor suppressor gene into an oncogene, leading to cell carcinogenesis.

[0004] The p53 gene is the gene most strongly associated with human tumors discovered to date. Studies have shown that more than half of solid tumor cells have gene mutations or loss of function in the p53 protein. If p53 gene mutations occur early in tumor development, it aids in early tumor diagnosis. Furthermore, individuals with p53 gene mutations often respond poorly to radiotherapy and chemotherapy and are more prone to metastasis, making it an indicator of treatment efficacy and prognosis. Therefore, immunohistochemical detection of mutant p53 plays a crucial role in the study of various malignant tumors.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a binding protein that specifically binds to the P53 protein, so as to solve the above-mentioned technical problems.

[0007] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a binding protein for P53 protein, the binding protein comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes: complementarity-determining region CDR1-VH as shown in SEQ ID NO.1, complementarity-determining region CDR2-VH as shown in SEQ ID NO.2, and complementarity-determining region CDR3-VH as shown in SEQ ID NO.3; The light chain variable region includes: complementarity-determining regions CDR1-VL as shown in SEQ ID NO.4, complementarity-determining regions CDR2-VL as shown in SEQ ID NO.5, and complementarity-determining regions CDR3-VL as shown in SEQ ID NO.6.

[0008] As a further technical solution, the heavy chain variable region includes: backbone region FR1-VH with amino acid sequence as shown in SEQ ID NO.7, backbone region FR2-VH with amino acid sequence as shown in SEQ ID NO.8, backbone region FR3-VH with amino acid sequence as shown in SEQ ID NO.9, and backbone region FR4-VH with amino acid sequence as shown in SEQ ID NO.10; The light chain variable region includes: backbone region FR1-VL with amino acid sequence as shown in SEQ ID NO.11, backbone region FR2-VL with amino acid sequence as shown in SEQ ID NO.12, backbone region FR3-VL with amino acid sequence as shown in SEQ ID NO.13, and backbone region FR4-VL with amino acid sequence as shown in SEQ ID NO.14.

[0009] As a further technical solution, the binding protein is an antibody or antigen-binding fragment.

[0010] As a further technical solution, the binding protein of the P53 protein is any one of F(ab')2, Fab', Fab, Fv and scFv.

[0011] As a further technical solution, a constant region is also included; Preferably, the constant region includes a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; Preferably, the light chain constant region is selected from the constant region of either the kappa (κ) chain or the lambda (λ) chain; Preferably, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Preferably, the constant region is derived from mice.

[0012] Secondly, the present invention provides a biomaterial selected from any one of the following: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a binding protein of said P53 protein; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing a binding protein of the p53 protein.

[0013] Thirdly, the present invention provides a method for preparing a binding protein of P53 protein, which is obtained by the aforementioned cell fermentation.

[0014] Fourthly, the present invention provides the application of the above-mentioned P53 protein binding protein in the preparation of tumor diagnostic or P53 protein detection products.

[0015] As a further technical solution, the product is used to detect p53 protein in tissue cells.

[0016] Fifthly, the present invention provides a kit for tumor diagnosis or P53 protein detection, comprising a binding protein of the P53 protein.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The binding protein provided by this invention can specifically recognize the P53 protein and has a high affinity for it. It can be used to develop reagents or kits for the detection of the P53 protein, and further for the diagnosis or prognostic assessment of diseases using P53 as a biomarker. This invention provides more protein options for the detection of P53 and the diagnosis of diseases using P53 as a biomarker, and enriches the types of monoclonal antibodies against the P53 protein. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 Image showing the results of immunohistochemical staining of breast and ovarian cancer sections; Figure 2 This image shows the results of immunohistochemical staining of breast cancer sections using the antibody of this invention compared to those of other companies. Detailed Implementation

[0020] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0021] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0022] The term "binding protein" broadly refers to all proteins / protein fragments containing complementarity-determining regions, particularly antibody or antigen-binding fragments. "Antigen-binding fragments" include Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0023] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable region can be called "VH," and the light chain variable region can be called "VL." These domains are usually the most variable parts of the antibody and contain antigen-binding sites. The variable regions of both the heavy and light chains consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held tightly together by the FRs to form the variable region. Typically, the VL / VH variable regions of the heavy and light chains are obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0024] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0025] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0026] In a first aspect, the present invention provides a binding protein for P53 protein, the binding protein comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes: complementarity-determining region CDR1-VH as shown in SEQ ID NO.1, complementarity-determining region CDR2-VH as shown in SEQ ID NO.2, and complementarity-determining region CDR3-VH as shown in SEQ ID NO.3; The light chain variable region includes: complementarity-determining regions CDR1-VL as shown in SEQ ID NO.4, complementarity-determining regions CDR2-VL as shown in SEQ ID NO.5, and complementarity-determining regions CDR3-VL as shown in SEQ ID NO.6.

[0027] The amino acid sequences of the above 6 complementarity-determining regions are shown in Table 1.

[0028] Table 1

[0029] The binding protein provided by this invention possesses high activity and a high affinity for the p53 protein through six complementarity-determining regions composed of the aforementioned specific amino acid sequence. It can be used to develop corresponding detection reagents or kits for detecting the p53 protein. This binding protein can be used to diagnose diseases using p53 as a biomarker. This invention provides more protein options for the detection of p53 and the diagnosis of diseases using p53 as a biomarker, enriching the types of monoclonal antibodies against the p53 protein.

[0030] In a preferred embodiment of the present invention, the binding protein provided by the present invention and the P53 protein have a K ≤ 3.27 × 10⁻⁶ ratio. 9 Affinity binding at L / mol.

[0031] In some optional embodiments, the heavy chain variable region includes: backbone region FR1-VH with amino acid sequence as shown in SEQ ID NO.7, backbone region FR2-VH with amino acid sequence as shown in SEQ ID NO.8, backbone region FR3-VH with amino acid sequence as shown in SEQ ID NO.9, and backbone region FR4-VH with amino acid sequence as shown in SEQ ID NO.10; The light chain variable region includes: backbone region FR1-VL with amino acid sequence as shown in SEQ ID NO.11, backbone region FR2-VL with amino acid sequence as shown in SEQ ID NO.12, backbone region FR3-VL with amino acid sequence as shown in SEQ ID NO.13, and backbone region FR4-VL with amino acid sequence as shown in SEQ ID NO.14.

[0032] The amino acid sequences of the above backbone regions are shown in Table 2.

[0033] Table 2

[0034] In some alternative implementations, the binding protein is an antibody or an antigen-binding fragment.

[0035] In some alternative embodiments, the binding protein of the P53 protein is any one of F(ab')2, Fab', Fab, Fv, and scFv.

[0036] Those skilled in the art will readily understand from the description of the present invention that the aforementioned antigen-binding fragments can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0037] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0038] In some alternative implementations, a constant region is also included; In some alternative implementations, the constant region includes a heavy chain constant region and a light chain constant region; In some alternative implementations, the heavy chain constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; In some alternative implementations, the light chain constant region is selected from the constant region of either the kappa (κ) chain or the lambda (λ) chain; In some alternative implementations, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; In some alternative implementations, the constant region is derived from mice.

[0039] Secondly, the present invention provides a biomaterial selected from any one of the following: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a binding protein of said P53 protein; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing a binding protein of the p53 protein.

[0040] In some alternative implementations, the cells include hybridoma cells.

[0041] Thirdly, the present invention provides a method for preparing a binding protein of P53 protein, which is obtained by the aforementioned cell fermentation and purification.

[0042] This preparation method is simple and efficient, and can obtain a large amount of P53 protein binding protein through fermentation.

[0043] In some alternative embodiments, the preparation method includes: transfecting host cells with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cells under suitable conditions to express the binding protein. The host cells may also be transfected with one or more expression vectors, which may contain DNA encoding at least a portion of the binding protein, either alone or in combination. The binding protein can be isolated from culture medium or cell lysates using conventional protein and peptide purification techniques, including ammonium sulfate precipitation, chromatography (such as ion exchange, gel filtration, affinity chromatography, etc.), and / or electrophoresis.

[0044] Constructing suitable vectors containing the target coding and regulatory sequences can be performed using standard ligation and restriction techniques known in the art. Isolated plasmids, DNA sequences, or synthetic oligonucleotides are cut, tailed, and re-ligated as needed. Mutations can be introduced into the coding sequence using any method to produce variants of the invention; these mutations can include deletions, insertions, substitutions, etc.

[0045] Based on the disclosure of the amino acid sequence of the antibody or its functional fragment in this invention, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, hybridoma cells) to prepare the antibody or its functional fragment, for example, by isolating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention.

[0046] Fourthly, the present invention provides the application of the above-mentioned P53 protein binding protein in the preparation of tumor diagnostic or P53 protein detection products.

[0047] The P53 protein binding protein provided by this invention can specifically recognize the P53 protein, and therefore can be used for the detection of the P53 protein and tumors using the protein as a marker.

[0048] In some alternative embodiments, the product is used to detect p53 protein in tissue cells. The tissue cells are at least one of the following: colon, liver, pancreas, ovary, endometrium, cervix, lung, thyroid gland, mammary epithelium, bronchus, bladder, and renal pelvis.

[0049] In some alternative implementations, the tumor includes, but is not limited to, epithelial tumors; In some alternative embodiments, the epithelial tumor includes, but is not limited to, papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, or squamous cell carcinoma.

[0050] In some alternative embodiments, the gastrointestinal cancer is selected from esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, small bowel cancer, colorectal cancer, or anal cancer, wherein the colorectal cancer is selected from colon cancer or rectal cancer.

[0051] In some alternative implementations, adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid carcinoma, salivary gland carcinoma, or pancreatic carcinoma; In some alternative implementations, the adenoma includes, but is not limited to, cystic adenoma, fibroadenoma, pleomorphic adenoma, or polypoid adenoma.

[0052] In some alternative implementations, breast cancer includes, but is not limited to, ductal carcinoma, epithelial carcinoma, or lobular carcinoma. Breast cancer, preferably stage II to IV and / or poorly differentiated aggressive ductal carcinoma, comedo carcinoma, and medullary carcinoma (preferably grade 2).

[0053] Fifthly, the present invention provides a kit for tumor diagnosis or P53 protein detection, comprising a binding protein of the P53 protein.

[0054] The kit provided by this invention includes a binding protein for P53 protein. Based on the specific recognition ability of the binding protein for P53 protein, it can detect P53 protein and tumors using this protein as a marker.

[0055] In some alternative embodiments, the binding protein of the P53 protein is labeled with a detectable marker.

[0056] Detectable markers refer to substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc. These properties enable qualitative or quantitative detection of the corresponding target.

[0057] In some alternative implementations, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0058] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0059] In some optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar dyes, Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar dyes) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar dyes).

[0060] In some alternative embodiments, the enzymes that catalyze the color development of the substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0061] In some alternative implementations, the radioactive isotope includes, but is not limited to, [other types of radioactive isotopes]. 212Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0062] In some alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0063] In some alternative implementations, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids. Nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0064] In some alternative embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0065] In some alternative implementations, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0066] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0067] Example 1 This embodiment describes the preparation of a monoclonal antibody specifically targeting the P53 protein.

[0068] 1. Preparation of antigen.

[0069] First, primer sequences for the P53 protein were designed. Then, PCR was performed to obtain the target gene (wild type). The target gene was then embedded in a plasmid. The plasmid was then introduced into DH5α competent cells using electroporation. The competent cells were then inoculated onto antibiotic-containing plates and placed in a 37°C oven for incubation. This process screened for E. coli that had been introduced with the target gene. The E. coli were then sonicated and purified by filtration using a His-tagged nickel column to obtain the P53 protein, i.e., the antigen (amino acid sequence as shown in SEQ ID NO. 15).

[0070] MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSSDSDGLAPPQHLIRVEGNL RVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACCPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSDHHHHHHH (SEQ ID NO.15).

[0071] 2. Immunize the mice.

[0072] Mice were immunized with an immunogen using a standard method. The immunogen was the aforementioned His-tagged recombinant human P53 protein, which also served as a detection antigen for serum titer and hybridoma screening. High-purity antigen increases the chance of obtaining the desired monoclonal antibody while reducing the screening workload. Five mice were immunized, each receiving 50 μg of MRP1 antigen. An antigen-protein solution was prepared using PBS. Appropriate amounts of antigen protein, PBS, and Freund's adjuvant were placed in a syringe, the syringe outlet was plugged, and the solution was emulsified thoroughly on an emulsifier to form a stable water-in-oil solution. Tail blood serum titers were measured 7-10 days after the primary and secondary immunizations. Good titers were obtained after 2-4 booster immunizations. Mice with high serum titers were selected for immunization, followed by intraperitoneal final immunization and cell fusion.

[0073] 3. Perform hybridoma cell fusion and screening.

[0074] The following preparations need to be made before cell fusion: (1) Culture mouse myeloma cells SP2 / 0 to the logarithmic growth phase; (2) One negative mouse was sacrificed the day before fusion. The peritoneal trophoblast cells of the mouse were injected into the peritoneal cavity of the mouse in a sterile environment with HAT medium and plated on a 96-well plate with 100 μL per well. These cells have a promoting effect on the growth of hybridoma cells.

[0075] Immunized mice were sacrificed, and spleens were harvested under sterile conditions. Spleen B cells and SP2 / 0 myeloma cells were chemically fused using PEG. Appropriate HAT medium was added according to the number of cells to be plated. Finally, the fused cells were plated on feeder cell culture plates at 100 μL per well.

[0076] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, the supernatant from each well is collected, and hybridoma cells are screened using ELISA with the p53 protein antigen. The method is as follows: Coat an ELISA plate with 100 μL of PBS solution containing 2 μg / ml P53 protein antigen and incubate at 37°C for two hours. Wash the plate three times with PBST, then add 150 μL / well of PBS solution containing 3% skim milk powder and incubate overnight at 4°C. Wash the plate three more times, add 80 μL / well of hybridoma supernatant, incubate at 37°C for 1 hour, and then wash three more times. Add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted 1:7000, incubate at 37°C for 45 minutes, wash three times, and blot dry. Add 100 μL / well of TMB chromogenic buffer, incubate at room temperature for 5-10 minutes, stop the reaction with 2M sulfuric acid solution, and measure the absorbance at 450 nm for each well. Select positive hybridoma cells.

[0077] Multiple strains were selected from the ELISA-positive fusion wells and then subjected to immunohistochemical staining (IHC). Positive wells were then selected for subsequent experiments.

[0078] ELISA and IHC assays were used to select fusion cells that showed positive binding, and these cells were cloned using the limiting dilution method. Each positive cell line was seeded into 48 / 96-well plates and cultured further. A second round of screening was performed using ELISA and IHC to identify hybridomas that specifically recognize p53 protein and can block p53 binding. These hybridomas were then subcloned using the limiting dilution method to obtain a single-clone cell line, 4G5. This single-clone cell line was then expanded, and approximately 1 × 10⁻⁶ cells were collected. 6 One cell was injected into selected mice (the mice needed to be injected with paraffin oil into their peritoneum one week in advance), and after a waiting period of 7-10 days, the mice produced ascites. The ascites was collected for antibody purification. After purification, a mouse monoclonal antibody specifically against P53 protein was obtained.

[0079] Example 2 The binding ability of purified antibody p53-4G5 to p53 protein was determined by immunohistochemistry (IHC).

[0080] Ovarian serous carcinoma, gastric antral adenocarcinoma, or breast cancer slides were baked in a 60°C oven for 60 minutes. The slides were then soaked in xylene I for 15 minutes, followed by xylene II for another 15 minutes. They were then soaked in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes, respectively. Finally, they were soaked in ddH2O for 5 minutes and washed three times. Antigen retrieval was performed using a pressure cooker (boiling method). 10 mmol / L citrate buffer (pH 6.0) was added to the pressure cooker to submerge the slides. The mixture was heated to boiling. The slides were placed on a heat-resistant slide rack and placed in the pressure cooker. The lid was closed, the pressure valve was engaged, and the mixture was heated. The pressure was maintained for 4 minutes. After the time was up, the vent valve was opened to release the pressure. Once the pressure returned to zero, the lid was opened, the inner pot was removed, and the mixture was allowed to cool to room temperature.

[0081] After the solution cools to room temperature, remove the sections (about 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the sections in 20 ml of 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes, wash 3 times; add one drop of goat serum blocking solution (about 25 μl) to each tissue group, incubate in a humidified chamber at room temperature for 45 minutes; soak in PBST for 5 minutes, wash 3 times.

[0082] For comparison, Roche's p53 antibody was added to the processed tissue sections, while the remaining sections were added to P53-4G5 purified antibody. Incubation was performed overnight in a humidified chamber at 4°C; after removal from the 4°C freezer, incubation was carried out at room temperature for 60 minutes; after gentle rinsing with PBST, the sections were soaked for 5 minutes, and washed three times; 25 μL of HRP-labeled Long Island Biotechnology secondary antibody (CAT#:) was added to each tissue group, and incubation was carried out at room temperature for 45 minutes; washing was performed; DAB chromogenic solution was prepared, and after reacting in the dark for 10-15 minutes, it was added to the sections, and chromogenic reaction was carried out for 1-5 minutes; the chromogenic reaction was terminated with distilled water; 50 μL of hematoxylin staining solution was added to each tissue group, and staining was carried out for 5-10 minutes, followed by rinsing with distilled water; the sections were then placed in 1% hydrochloric acid-ethanol for decolorization for 2-3 seconds, quickly removed, and placed in distilled water to terminate the reaction, followed by inversion in PBST (pH 8.0) for 5-10 minutes; and then soaked in 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and anhydrous ethanol for 5 minutes respectively. Soak in xylene for 10 minutes, then replace with xylene and soak for another 10 minutes; add neutral resin and mount the slide, then cover with a coverslip; photograph under a microscope. Figure 1 , Figure 2 As shown.

[0083] Immunohistochemical neutralization assay: A certain amount of P53 antigen protein was mixed with purified antibody and neutralized at 37°C for 1 hour. Following the immunohistochemical assay procedure, the primary antibody was added to the lung adenocarcinoma section and incubated overnight at 4°C. Immunohistochemical staining and imaging were then performed using the same procedure.

[0084] Figure 1 , Figure 2 The immunohistochemical assay for P53 was performed on ovarian and breast cancer tissues from different patients. Figure 1 In the diagram, A represents ovarian cancer tissue, and B represents breast cancer tissue. The antibody used is the p53-4G5 antibody produced in this invention. Figure 2 The tissue in question is breast cancer tissue, and A represents the p53-4G5 antibody used in this invention. Figure 2 In the text, B represents a P53 antibody from a domestic biotechnology company. Figure 2 The staining results show that the antibody provided by this invention stains more strongly than the antibody from a certain biotechnology company. This confirms that the p53-4G5 antibody provided by this invention has the potential for application in preparing reagent kits.

[0085] It can be seen that the anti-P53 protein monoclonal antibody P53-4G5 provided in Example 1 of this invention can recognize and bind to the P53 protein in human cells. In the subsequent IHC staining experiment, it makes the cell nuclei of ovarian cancer tissue brown, and the color is stronger than that of the p53 antibody of a certain domestic biological company.

[0086] Example 3 In this embodiment, the mouse monoclonal antibody screened in Example 1 above was cloned and sequenced to obtain the variable region gene sequence of the anti-human P53 monoclonal antibody.

[0087] Total RNA was extracted from mouse monoclonal cell lines using Trizol reagent. Cells cultured in 9cm dishes were transferred to 1.5ml centrifuge tubes, and the supernatant was aspirated. 1ml of total RNA extraction reagent (Trizol) was added, and the cells were lysed by pipetting. The lysed sample or homogenate was incubated at room temperature for 5-10 min to allow complete separation of nucleoproteins and nucleic acids. 0.2ml of chloroform was added, and the mixture was vigorously vortexed for 15 seconds and incubated at room temperature for 3 min. The mixture was centrifuged at 12000 rpm at 4°C for 10 min. The upper aqueous phase was transferred to a clean centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 20 min. The mixture was centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was discarded. The precipitate was washed with 1ml of 75% ethanol. The mixture was centrifuged at 12000 rpm at 4°C for 3 min, and the supernatant was discarded. The mixture was dried at room temperature for 5-10 min. 30-50ul of RNase-free ddH2O was added. The obtained RNA solution was stored at -70°C or used for subsequent experiments.

[0088] Total RNA was reverse transcribed into cDNA using the AMV first-strand cDNA synthesis kit. The experimental configuration was as follows: 6 μL total RNA + 1 μL Oligo dT + 4 μL RNase-free water (total 11 μL). After gentle mixing, centrifuge for 3-5 seconds. The reaction mixture was pre-denatured at 65°C for 5 minutes, then incubated on ice for 30 seconds, centrifuged for 3-5 seconds, and then incubated on ice for 2 minutes. While in the ice bath, 4 μL of 5X buffer + 1 μL of dNTP mixture + 1 μL of RNase inhibitor + 1 μL of reverse transcriptase (total 20 μL) was added. After gentle mixing, centrifuge for 3-5 seconds, and incubate at 42°C for 50 minutes and 85°C for 5 minutes on a PCR instrument to complete cDNA synthesis. Random primers are suitable for the synthesis of short-strand cDNAs under 500 bp. The transcribed RNA template does not require a poly(A) tail and can transcribe the 5' end region.

[0089] PCR amplification of the light and heavy chains. For amplifying the variable region sequence of the antibody light chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VL + 1 μL RP-VL + 2 μL cDNA + 21 μL ddH2O. For amplifying the variable region sequence of the antibody heavy chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VH + 1 μL RP-VH + 2 μL cDNA + 21 μL ddH2O. The temperature cycling for PCR amplification of the variable regions of the heavy and light chains was as follows (steps 2 to 4 were repeated 35 times): Step 1 - Pre-denaturation: 94℃, 4 min; Step 2 - Denaturation at 94°C for 30 seconds; Step 3 - Annealing at 55°C for 45 seconds; Step 4 - Extend at 72°C for 60 seconds; Step 5: 72℃, 10 min; Step 6 - Store at 4℃.

[0090] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA bands of corresponding sizes were excised (approximately 355 bp for VH and approximately 335 bp for VL). DNA extraction was performed using the SanPrep column DNA gel extraction kit. The procedure is briefly described as follows: A gel block containing the target fragment was excised from the agarose gel and weighed; 3-6 times the weight of the gel block was added to buffer B2, and the gel was dissolved in a 50°C water bath for 5-10 minutes; the dissolved gel was transferred to an adsorption column and centrifuged at 8000g for 30 seconds; the liquid in the collection tube was discarded; 500 μL of wash solution was added to the column, and the column was centrifuged at 9000g for 30 seconds, and the liquid in the collection tube was discarded; the wash solution was added again, and the liquid was discarded; the well adsorption column was centrifuged at 9000g for 1 minute; the adsorption column was placed in a clean 1.5 ml centrifuge tube, and 15-40 μL of Elution Buffer was added to the center of the adsorption membrane. After standing at room temperature for 1 minute, the column was centrifuged for 1 minute. The prepared DNA solution was obtained, and the PCR product was purified and sequenced to obtain the variable region sequence of the antibody.

[0091] The amino acid sequences of the mouse anti-human P53 protein monoclonal antibody secreted by the monoclonal cell line P53-4G5 are shown in Tables 1 and 2.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein that binds to p53 protein, characterized in that, The binding protein includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes: complementarity-determining region CDR1-VH as shown in SEQ ID NO.1, complementarity-determining region CDR2-VH as shown in SEQ ID NO.2, and complementarity-determining region CDR3-VH as shown in SEQ ID NO.3; The light chain variable region includes: complementarity-determining regions CDR1-VL as shown in SEQ ID NO.4, complementarity-determining regions CDR2-VL as shown in SEQ ID NO.5, and complementarity-determining regions CDR3-VL as shown in SEQ ID NO.

6.

2. The P53 protein-binding protein according to claim 1, characterized in that, The heavy chain variable region includes: backbone region FR1-VH with amino acid sequence as shown in SEQ ID NO.7, backbone region FR2-VH with amino acid sequence as shown in SEQ ID NO.8, backbone region FR3-VH with amino acid sequence as shown in SEQ ID NO.9, and backbone region FR4-VH with amino acid sequence as shown in SEQ ID NO.10; The light chain variable region includes: backbone region FR1-VL with amino acid sequence as shown in SEQ ID NO.11, backbone region FR2-VL with amino acid sequence as shown in SEQ ID NO.12, backbone region FR3-VL with amino acid sequence as shown in SEQ ID NO.13, and backbone region FR4-VL with amino acid sequence as shown in SEQ ID NO.

14.

3. The P53 protein binding protein according to claim 1, characterized in that, The binding protein is an antibody or antigen-binding fragment.

4. The P53 protein-binding protein according to claim 1, characterized in that, The binding protein of the P53 protein is any one of F(ab')2, Fab', Fab, Fv, and scFv.

5. The P53 protein-binding protein according to claim 1, characterized in that, It also includes the constant region; Preferably, the constant region includes a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; Preferably, the light chain constant region is selected from the constant region of either the kappa (κ) chain or the lambda (λ) chain; Preferably, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Preferably, the constant region is derived from mice.

6. A biomaterial, characterized in that, The biomaterial is selected from any one of ac: a. Nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a binding protein of the P53 protein according to any one of claims 1-5; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing a binding protein of the P53 protein of any one of claims 1-5.

7. A method for preparing a binding protein to p53 protein, characterized in that, It is obtained by cell fermentation as described in claim 6.

8. The use of the P53 protein binding protein according to any one of claims 1-5 in the preparation of tumor diagnostic or P53 protein detection products.

9. The application according to claim 8, characterized in that, The product is used to detect p53 protein in tissue cells.

10. A kit for tumor diagnosis or P53 protein detection, characterized in that, Includes the binding protein of the P53 protein as described in any one of claims 1-5.

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