Antibody for resisting P53 protein, tumor diagnosis product and application thereof

Abstract

The invention discloses an anti-P53 protein antibody, a tumor diagnosis product and application thereof, and relates to the technical field of antibodies. The heavy chain complementarity determining region comprises a heavy chain complementarity determining region in a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain complementarity determining region in a light chain variable region as shown in SEQ ID NO: 15. The antibody can be used for diagnosing diseases with P53 as a marker, more protein choices are provided for detection of P53 and diagnosis of diseases with P53 as a marker, and the types of monoclonal antibodies aiming at the P53 protein are enriched. In addition, the antibody provided by the invention can be used for preparing a tumor curative effect evaluation product, a prognosis evaluation product or a recurrence and metastasis monitoring product.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-P53 protein antibody, a tumor diagnosis product and applications thereof. Background Art

[0002] P53 is a tumor suppressor gene that encodes a nuclear protein composed of 393 amino acids. Its protein band appears at 53KDa as shown by the marker band, so it is named P53. The protein it encodes is divided into two subtypes: wild type and mutant type. The wild type causes cancer cells to apoptosis, thereby preventing canceration, and also has the function of helping cell genes repair defects; the mutant type of p53 will increase canceration. P53 is a very important anti-cancer gene.

[0003] The P53 protein is mainly distributed in the nucleoplasm of cells and can specifically bind to DNA. Its activity is regulated by post-translational modifications such as phosphorylation, acetylation, methylation, and ubiquitination. The normal biological function of P53 is to check DNA damage points in the G1 phase and monitor the integrity of the genome. If there is damage, the P53 protein prevents DNA replication to provide enough time for the damaged DNA to be repaired; if the repair fails, the p53 protein triggers cell apoptosis; if the P53 gene mutates, its spatial conformation changes, and it loses its regulatory effect on cell growth, apoptosis, and DNA repair. The p53 gene is transformed from a tumor suppressor gene to an oncogene, leading to cell cancer.

[0004] The P53 gene is the gene with the highest correlation with human tumors discovered so far. Studies have shown that more than half of solid tumor cells have gene mutations or loss of function of the P53 protein. If the p53 gene often mutates in the early stages of tumor development, it will help with early diagnosis of tumors. In addition, people with p53 gene mutations often have poor responses to radiotherapy and chemotherapy and are prone to metastasis. It can be used as an indicator of treatment efficacy and prognosis. Therefore, immunohistochemical detection of mutant P53 plays an important 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 the present invention is to provide an anti-P53 protein antibody, a tumor diagnosis product and its application to solve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides an anti-P53 protein antibody or an antigen-binding fragment thereof, which comprises a heavy chain complementary determining region in a heavy chain variable region as shown in SEQ ID NO: 14, and a light chain complementary determining region in a light chain variable region as shown in SEQ ID NO: 15.

[0009] In a second aspect, the present invention also provides the use of an antibody or an antigen-binding fragment thereof in the preparation of any of the following products:

[0010] (1) Tumor diagnostic products;

[0011] (2) Tumor auxiliary diagnosis products;

[0012] (3) Tumor efficacy evaluation products;

[0013] (4) Tumor prognosis assessment products;

[0014] (5) Products for monitoring tumor recurrence and metastasis;

[0015] (6) P53 protein detection products;

[0016] (7) P53 protein separation product or enrichment product.

[0017] In a third aspect, the present invention also provides a product for tumor diagnosis, auxiliary diagnosis, efficacy evaluation, prognosis evaluation or recurrence and metastasis monitoring, the product comprising: the above-mentioned antibody or antigen-binding fragment thereof.

[0018] In a fourth aspect, the present invention further provides a P53 protein detection product, a P53 protein separation product or an enrichment product, the product comprising: the above-mentioned antibody or antigen-binding fragment thereof.

[0019] In a fifth aspect, the present invention further provides a cell, which is a non-plant cell and expresses the above-mentioned antibody or antigen-binding fragment thereof.

[0020] In a sixth aspect, the present invention also provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, which comprises: culturing the above-mentioned cells, and isolating and purifying the antibody or antigen-binding fragment thereof from the culture product.

[0021] The present invention has the following beneficial effects:

[0022] The anti-P53 protein antibody provided by the present invention has high activity and high affinity with the P53 protein, and can be used to develop corresponding detection products, such as reagents, kits, chips, etc., for detecting the expression level of the P53 protein in related tumor tissues, and can improve the sensitivity and specificity of the detection. The antibody can be used to diagnose diseases with P53 as a marker. The present invention provides more protein options for the detection of P53 and the diagnosis of diseases with P53 as a marker, and enriches the types of monoclonal antibodies against the P53 protein.

[0023] In addition, the antibodies provided by the present invention can be used to prepare tumor efficacy evaluation products, prognosis evaluation products or recurrence and metastasis monitoring products, based on the high specific binding force between P53 protein and antibody and the level of P53 protein in the subject's sample, thereby achieving tumor efficacy evaluation, prognosis evaluation or recurrence and metastasis monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 is the antibody affinity curve;

[0026] Figure 2 This is the result of immunohistochemical staining of breast cancer and ovarian cancer sections;

[0027] Figure 3 This is a diagram showing the experimental results of immunohistochemical staining of breast cancer sections using the antibody provided by the present invention and a commercially available P53 antibody. DETAILED DESCRIPTION

[0028] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0029] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0030] The term "antigen binding fragment" refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antigen binding fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0031] The term "antibody" herein is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to P53 protein.

[0032] In the present invention, the term "complementarity determining region or complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment with its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0033] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0034] Typically, the variable region VH of the heavy chain of an antibody can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0035] The variable region VL of the antibody light chain can be obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0036] LCDR1 is CDR-L1, LCDR2 is CDR-L2, and LCDR3 is CDR-L3.

[0037] In the first aspect, the present invention provides an anti-P53 protein antibody or an antigen-binding fragment thereof, which includes a heavy chain complementarity determining region in a heavy chain variable region (VH) as shown in SEQ ID NO: 14, and a light chain complementarity determining region in a light chain variable region (VL) as shown in SEQ ID NO: 15.

[0038] VH, SEQ ID NO:14:

[0039] VQLQESGGGLVQPGRSMKLSCVASGFTFGNYWMNWVRQSPEKGLEWL AQIRLKSDNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYFCTAL DY WGQGTTVTVSS.

[0040] VL, SEQ ID NO:15:

[0041] DIELTQSPSLASQVRASPVSISCKSSQSLLDSDGKTHFNWLLQRPGQSPKR LIYLVSKLDSGVPDRFTGSGSGTDFTLKISKVEAEDLGVYYCWQGTHFPGT FGGGTKLEIK.

[0042] By inputting the above SEQ ID NO: 14 and SEQ ID NO: 15 sequences into the CDR labeling system, the corresponding CDR sequences can be obtained.

[0043] The amino acid sequences of the heavy chain complementary determining region and the light chain complementary determining region are first discovered and disclosed by the present invention, and are new sequences that can give the binding protein the ability to specifically bind to the P53 protein. Based on the good binding activity and affinity of the antibody, it can be used to develop detection products for detecting the P53 protein, such as detection reagents and kits. The use of the binding protein of the present invention to detect the P53 protein can improve the sensitivity and specificity of the detection. In addition, the antibody can be used to diagnose diseases with the P53 protein as a marker. It is conducive to early detection and early intervention treatment of the disease. The present invention provides more protein options for the detection of the P53 protein and the diagnosis of diseases with the P53 protein as a marker.

[0044] In a preferred embodiment of the present invention, the binding protein binds to the P53 protein with a K of ≤3.27×10 9 L / mol affinity binding.

[0045] The affinity constant is calculated by the formula K = (N-1) / (N*AB'-AB). AB', AB are the antibody concentrations (mol / L) that produce half the absorbance value at the corresponding antigen concentration AG. N = AG / AG' (AG>AG').

[0046] In a preferred embodiment of the present invention, the heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of CDR-H1 and CDR-H2 are shown in SEQ ID NOs: 1-2, respectively, and the amino acid sequence of CDR-H3 is LDY; the light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, and their amino acid sequences are shown in SEQ ID NOs: 3-5, respectively.

[0047] name sequence serial number CDR-H1 NYWMN SEQ ID NO:1 CDR-H2 QIRLKSDNYATHYAESVKG SEQ ID NO:2 CDR-H3 LDY CDR-L1 KSSQSLLDSDGKTHFN SEQ ID NO:3 CDR-L2 LVSKLDS SEQ ID NO:4 CDR-L3 WQGTHFPGT SEQ ID NO:5

[0048] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a heavy chain framework region, and / or a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 10-13, for example, the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4, which are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs: 10-13, for example.

[0049] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs:6-9, for example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs:6-9.

[0050]

[0051]

[0052] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region, the constant region comprises a heavy chain constant region, and / or a light chain constant region, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0053] In a preferred embodiment of the present invention, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0054] In a preferred embodiment of the present invention, the species of origin of the constant region is mouse.

[0055] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of an antibody.

[0056] The antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds.

[0057] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0058] In a second aspect, the present invention also provides the use of an antibody or an antigen-binding fragment thereof in the preparation of any of the following products:

[0059] (1) Tumor diagnostic products;

[0060] (2) Tumor auxiliary diagnosis products;

[0061] (3) Tumor efficacy evaluation products;

[0062] (4) Tumor prognosis assessment products;

[0063] (5) Products for monitoring tumor recurrence and metastasis;

[0064] (6) P53 protein detection products;

[0065] (7) P53 protein separation product or enrichment product.

[0066] In a preferred embodiment of the present invention, the tumor is a tumor with overexpression of P53 protein or mutation of p53 gene.

[0067] In a preferred embodiment of the present invention, the tumor with p53 gene mutation is a tumor with p53 gene deletion or p53 missense mutation;

[0068] In a preferred embodiment of the present invention, the solid tumor is selected from epithelial tumors;

[0069] In a preferred embodiment of the present invention, the epithelial tumor is selected from gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma or squamous cell carcinoma;

[0070] In a preferred embodiment of the present invention, gastrointestinal cancer is selected from esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, small intestine cancer, colon cancer, rectal cancer, colorectal cancer and anal cancer; squamous cell carcinoma is one or more of oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer, esophageal squamous cell carcinoma, lip squamous cell carcinoma, uterine squamous cell carcinoma, vaginal squamous cell carcinoma and skin squamous cell carcinoma.

[0071] In an alternative embodiment, the adenocarcinoma includes but is not limited to lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0072] In an alternative embodiment, the adenoma includes but is not limited to a cystadenoma, a fibroadenoma, a pleomorphic adenoma or a polypoid adenoma.

[0073] In an alternative embodiment, breast cancer includes but is not limited to breast epithelial cancer;

[0074] Lung cancer, preferably includes small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma) and large cell lung cancer.

[0075] The squamous cell carcinoma is one or more of oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer, esophageal squamous cell carcinoma (eg esophageal squamous cell carcinoma), lip squamous cell carcinoma, uterine squamous cell carcinoma, vaginal squamous cell carcinoma and skin squamous cell carcinoma.

[0076] Oral squamous cell carcinoma (OSCC) is also called oral squamous cell carcinoma. Examples of oral squamous cell carcinoma include but are not limited to tongue squamous cell carcinoma.

[0077] In other embodiments, the squamous cell carcinoma may also be squamous cell carcinoma formed by squamous metaplasia in the bronchus, bladder, renal pelvis and other parts.

[0078] In a preferred embodiment of the present invention, the tumor is one or more of breast cancer, prostate cancer, liver cancer, ovarian cancer, colorectal cancer, gastric cancer and colon cancer.

[0079] Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which the corresponding target object can be qualitatively identified.

[0080] In an optional embodiment, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent agents, and nanoparticle markers.

[0081] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0082] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.60, Cy3.61, Cy3.62, Cy3.63, Cy3.77, Cy3.78, Cy3.79, Cy3. 5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0083] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0084] In an alternative embodiment, the radioactive isotopes include but are not limited to 212 Bi, 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 Such as 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.

[0085] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0086] In an optional embodiment, the nanoparticle marker includes but is 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.

[0087] In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0088] In alternative embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0089] In a preferred embodiment of the present invention, in application (7), the detection of P53 protein is to label tissues and / or cells with binding protein and then detect the labeled tissues and / or cells.

[0090] The antibody chip refers to a chip formed by fixing the above-mentioned antibodies or antigen-binding fragments thereof on a carrier.

[0091] The antibody provided by the present invention can be used as a coating antibody or a detection antibody.

[0092] In a third aspect, the present invention also provides a product for tumor diagnosis and auxiliary diagnosis, the product comprising: the above-mentioned antibody or antigen-binding fragment thereof.

[0093] In a preferred embodiment of the present invention, the product is a reagent or a kit.

[0094] In a fourth aspect, the present invention further provides a P53 protein detection product, a P53 protein separation product or an enrichment product, the product comprising: the above-mentioned antibody or antigen-binding fragment thereof;

[0095] In a preferred embodiment of the present invention, the P53 protein detection product is a reagent or a kit;

[0096] In a preferred embodiment of the present invention, the P53 protein separation product or enrichment product is a magnetic bead, a kit or a separation column.

[0097] For example, the above-mentioned antibody is coated on magnetic beads for separation and enrichment of P53 protein. In one embodiment, the antibody is coated on a filler and filled in a separation column; it is used for affinity separation and enrichment of P53 protein. Therefore, the above-mentioned antibody or its antigen-binding fragment has a good application prospect in the preparation of P53 protein enrichment products.

[0098] In a fifth aspect, the present invention further provides a cell, which is a non-plant cell and expresses the above-mentioned antibody or antigen-binding fragment thereof.

[0099] The host cell is selected from mammalian cells; the mammalian cell is selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, Hela cells, MDCK cells, Hep-2 cells and Per6 cells. Among them, 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

[0100] In a sixth aspect, the present invention also provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, which comprises: culturing the above-mentioned cells, and isolating and purifying the antibody or antigen-binding fragment thereof from the culture product.

[0101] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0102] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0103] Example 1

[0104] This example is about the preparation of monoclonal antibody specific to P53 protein.

[0105] 1. Preparation of Antigen

[0106] First, the primer sequence of P53 protein is designed, and then PCR is performed to obtain the target gene, and then the target gene is embedded in the plasmid, and then the plasmid is allowed to enter DH5α competent cells by electroporation, and then the competent cells are inoculated on a plate containing antibiotics and placed in a 37-degree oven to shake the bacteria, thereby screening out Escherichia coli with the introduced target gene, and then the Escherichia coli is broken by ultrasound, and then filtered and purified with a nickel column with a his tag, and finally the P53 protein, i.e., the antigen, is obtained.

[0107] 2. Immunize mice.

[0108] Immunize mice with immunogens using a common method. The immunogen is a recombinant human P53 protein with a His tag, and is also used as a detection antigen to measure and screen serum titers and hybridomas. High-purity antigens can increase the chance of obtaining the desired monoclonal antibody and reduce the amount of screening. Immunize 5 mice, each with 50ug of MRP1 antigen. Prepare the antigen protein solution with PBS. Put an appropriate amount of antigen protein, PBS, and Freund's adjuvant into a syringe, plug the outlet of the syringe with a stopper, and stir on an emulsifier to fully emulsify, so that the antigen adjuvant forms a stable oil-in-water solution. The first tail blood serum titer test is performed 7-10 days after the primary and secondary immunizations, and a good titer is obtained after 2 to 4 booster immunizations. Select mice with high serum titers for intraperitoneal final immunization for cell fusion.

[0109] 3. Perform hybridoma cell fusion and screening.

[0110] The following preparations need to be done before cell fusion:

[0111] (1) Cultivate mouse myeloma cells SP2 / 0 to the logarithmic growth phase;

[0112] (2) One day before fusion, a negative mouse was killed and HAT culture medium was injected into the mouse peritoneal cavity in a sterile environment to obtain mouse peritoneal trophoblast cells. The cells were plated on a 96-well plate, with 100 ul per well. These cells have a promoting effect on the growth of hybridoma cells.

[0113] The immunized mice were killed, and the spleens were taken out under sterile conditions. The spleen B cells and SP2 / 0 myeloma cells were fused with PEG chemical. According to the number of cells to be plated, appropriate HAT medium was added, and finally the fused cells were plated on the trophoblast cell culture plate, with 100ul per well.

[0114] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, the supernatant of each well is collected and hybridoma cells are screened using ELISA method with P53 protein antigen. The method is as follows:

[0115] Coat the ELISA plate with 100ul 2ug / ml PBS solution of P53 protein antigen at 37℃ for two hours. Wash the plate three times with PBST, add 150ul / well of 3% skim milk powder in PBS solution and block overnight at 4℃. Wash the plate three times again, add 80ul / well of hybridoma supernatant, incubate at 37℃ for 1 hour, and then wash the plate three times. Add 1:7000 diluted horseradish peroxidase-labeled goat anti-mouse secondary antibody at 100ul / well, incubate at 37℃ for 45 minutes, wash the plate three times, and pat dry. Add 100ul / well of TMB colorimetric solution, color at room temperature for 5-10 minutes, stop with 2M sulfuric acid solution, and measure the absorbance of each well at 450nm. Select the positive hybridoma cells.

[0116] From the ELISA-positive fusion wells, select multiple strains and then perform immunohistochemistry (IHC) staining, and select the positive wells for subsequent experiments.

[0117] The fusion cells with positive binding were selected by ELISA and IHC experiments, cloned by limiting dilution method, and each well of the positive strain was plated on a 48 / 96-well plate for further culture. A second round of screening was performed by ELISA and IHC methods to screen out hybridomas that specifically recognized p53 protein and could block p53 binding. The monoclonal cell line 4G5 was obtained by limiting dilution method subcloning. The monoclonal cell line was expanded and about 1×10 6 The cells were injected into selected mice (mice were injected with paraffin oil in the peritoneal cavity one week in advance), and then after 7-10 days of waiting, the mice produced ascites, which was collected for antibody purification. After purification, mouse monoclonal antibodies specific to the P53 protein were obtained.

[0118] Example 2

[0119] The binding ability of purified antibody p53-4G5 to p53 protein was determined based on immunohistochemistry (IHC).

[0120] Place the ovarian serous carcinoma or breast cancer slices in a 60℃ constant temperature oven and bake for 60 minutes. Soak the slices in xylene I for 15 minutes, replace with xylene II and soak for 15 minutes, soak 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; soak in ddH2O for 5 minutes and wash 3 times; use a pressure cooker to perform antigen repair (boiling method). Add 10mmol / L citrate buffer (pH6.0) enough to submerge the slices in the pressure cooker, heat to boiling, place the slices on a heat-resistant slice rack, put them in the pot, cover the pot, buckle the pressure valve, continue heating, set the pressure to maintain for 4 minutes, open the air valve to release air after the time is up, open the pot cover after the pressure returns to zero, take out the inner pot and let it cool at room temperature.

[0121] After the solution cooled to room temperature, take out the slices (about 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the slices in 20ml 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes and wash 3 times; add one drop of goat serum blocking solution (about 25ul) to each tissue group, incubate in a wet box at room temperature for 45 minutes; soak in PBST for 5 minutes and wash 3 times.

[0122] Add Roche's p53 antibody to the processed tissue sections for comparison, and add P53-4G5 purified antibody to the remaining sections. Incubate in a 4℃ wet box overnight; take out from the 4℃ refrigerator and incubate at room temperature for 60 minutes; rinse gently with PBST and soak for 5 minutes, wash 3 times; add 25ul of HRP-labeled Long Island Company secondary antibody (CAT#:) to each tissue group, incubate at room temperature for 45 minutes; wash; prepare DAB color development solution, react in the dark for 10-15 minutes, then add it to the sections and color for 1-5 minutes; stop the color development reaction with distilled water; add 50ul of hematoxylin stain to each tissue group, stain for 5-10 minutes, and rinse with distilled water; put the sections in 1% hydrochloric acid-ethanol for decolorization for 2-3 seconds, then quickly take them out and put them in distilled water to stop, and then put them in PBST (pH8.0) for anti-blueing for 5-10 minutes; soak in 75% ethanol for 5 minutes: 85% ethanol for 5 minutes; soak in 95% ethanol for 5 minutes: soak in anhydrous ethanol for 5 minutes. Soak in xylene for 10 minutes, then change to xylene and soak for another 10 minutes; add neutral gum to seal the slide, then cover the slide with a glass slide; photograph the slide under a microscope. Figure 2 , Figure 3 shown.

[0123] Immunohistochemical neutralization experiment: Take a certain amount of P53 antigen protein and mix it with purified antibody, and neutralize it at 37℃ for 1 hour. According to the immunohistochemical experimental steps, add it as the primary antibody to the lung adenocarcinoma section, incubate it at 4℃ overnight, and then follow the same steps for immunohistochemical staining and filming.

[0124] Figure 2 , Figure 3 This is an immunohistochemical experiment of P53, and the test specimens are ovarian cancer and breast cancer tissues from different patients. Figure 2 A is ovarian cancer tissue, B is breast cancer tissue, and the antibody used is the p53-4G5 antibody produced by the present invention. Figure 3 is breast cancer tissue, A is the p53-4G5 antibody of the present invention, Figure 3 In the middle, B is a P53 antibody from a domestic biological company. Figure 3 The staining results show that the antibody provided by the present invention has stronger staining than the antibody of a biological company. It can be seen that the anti-P53 protein monoclonal antibody P53-4G5 provided in Example 1 of the present invention can recognize and bind to the P53 protein in human cells, and in the subsequent IHC staining experiment, the cell nuclei in ovarian cancer tissues appear brown, which proves that the p53-4G5 antibody provided by the present invention has application prospects in preparing kits.

[0125] Example 3

[0126] In this example, the affinity and sensitivity of the monoclonal antibody prepared in the above example 1 were tested.

[0127] 1. The recombinant human p53 protein in Example 1 was plated at 3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L.

[0128] 2. Adjust the concentration of the monoclonal antibody prepared in Example 1 to 10 -7 mol / L level (1*10 -7 Up to 5*10 -7 Then dilute it in multiples of 1:2-1:256 and add it to the wells with different antigen coating amounts.

[0129] 3. Add secondary antibody and develop color with TMB. Measure the absorbance at 450nm. The absorbance data is shown in Table 1.

[0130] 4. According to the antigen-antibody binding S curve, calculate the antibody concentration at half absorbance under different antigen concentrations. In this way, there are four antibody concentrations (mol / L). 9.924*10 -10 mol / L、6.611*10 -10 mol / L、4.32*10 -10 mol / L, 2.766*10-10 mol / L.

[0131] 5. Substitute the formula K = (N-1) / (N*AB'-AB) to calculate the affinity constant. AB', AB are the antibody concentrations that produce half the absorbance value at the corresponding antigen concentration AG (3mg / L, 1.5ml / L, 0.75mg / L, 0.375mg / L). N = AG / AG' (AG>AG').

[0132] 6. When N = 2, three K values ​​can be obtained (x10 9 L / mol) 3.032, 4.929, 8.251. When N = 4, we can get two K values ​​of 4.078 and 6.737. When N = 8, we can get a K value of 5.736. The average value of the 6 K values ​​is 5.46*10 9 .

[0133] P53 antibody affinity curve Figure 1 shown.

[0134] According to ELISA at OD 450 The test results were used to calculate the half absorbance value anti-N protein antibody concentration at each antigen coating concentration. The results are as follows:

[0135] Antigen coating concentration (AG) (ug / m) <![CDATA[Half absorbance antibody concentration (AB) (10 -9 mol / L)]]> 3 0.9924 1.5 0.6611 0.75 0.432 0.3725 0.2766

[0136]

[0137] P53 antibody affinity curve Figure 1 shown.

[0138] Table 1 Statistics of absorbance values ​​at 450nm.

[0139]

[0140] Example 4

[0141] In this example, DNA cloning and sequencing were performed on the mouse monoclonal antibodies screened out in the above example 1, and variable region genes of the anti-human P53 monoclonal antibodies were sequenced.

[0142] Use Trizol reagent to extract total RNA from mouse monoclonal cell lines. Collect cells cultured in 9cm dishes into 1.5ml centrifuge tubes and aspirate the supernatant. Add 1ml of total RNA extraction reagent (Trizol) and blow the cells evenly to lyse the cells. Place the lysed sample or homogenate at room temperature for 5-10min to completely separate the nucleoprotein from the nucleic acid. Add 0.2ml of chloroform, shake vigorously for 15sec, and place at room temperature for 3min. Centrifuge at 12000rpm and 4℃ for 10min. Transfer the upper aqueous phase to a clean centrifuge tube, add an equal volume of isopropanol, mix well, and place at room temperature for 20min. Centrifuge at 12000rpm and 4℃ for 10min, and discard the supernatant. Add 1ml of 75% ethanol to wash the precipitate. Centrifuge at 12000rpm and 4℃ for 3min, and discard the supernatant. Dry at room temperature for 5-10min. Add 30-50ulRNase-free ddH2O. Store the resulting RNA solution at -70℃ or use it for subsequent experiments.

[0143] AMV first-strand cDNA synthesis kit was used to reverse transcribe total RNA into cDNA. The experimental system was configured as follows: 6ul total RNA + 1ul Oligo dT + 4ul RNase-free water (a total of 11ul). Gently mix and centrifuge for 3-5s. The reaction mixture was pre-denatured at 65°C for 5min, ice-bathed for 30s, then centrifuged for 3-5s, and then ice-bathed for 2min. In an ice bath, 4ul of 5x buffer + 1ul of dNTP mixture + 1ul of RNase inhibitor + 1ul of reverse transcriptase (a total of 20ul system) was added, gently mix and centrifuged for 3-5s, and cDNA synthesis was completed on a PCR instrument at 42°C for 50 minutes and 85°C for 5min. Random primers are suitable for the synthesis of short-chain cDNAs below 500bp. The transcribed RNA template does not require a poly(A) tail, and the 5' end region can be transcribed.

[0144] PCR amplification of light and heavy chains. For amplifying the variable region sequence of the antibody light chain, configure the PCR reaction system: 2x Taq enzyme buffer 25ul + FP-VL 1ul + RP-VL 1ul + cDNA2ul + ddH2O 21ul. For amplifying the variable region sequence of the antibody heavy chain, configure the PCR reaction system: 2x Taq enzyme buffer 25ul + FP-VH 1ul + RP-VH 1ul + cDNA2ul + ddH2O 21ul. The temperature cycle for PCR amplification of the heavy and light chain variable regions is as follows (steps 2 to 4 are repeated 35 cycles):

[0145] Step 1 - pre-denaturation at 94°C for 4 min;

[0146] Step 2 - denaturation at 94°C for 30 seconds;

[0147] Step 3-annealing at 55°C for 45 seconds;

[0148] Step 4 - extension 72°C, 60 sec;

[0149] Step 5-72°C, 10 min;

[0150] Step 6 - Store at 4°C.

[0151] The PCR products were analyzed by 1% agarose gel electrophoresis, and the DNA segment bands of corresponding sizes (VH is about 355bp, VL is about 335bp) were cut out, and DNA was extracted using the SanPrep column DNA gel recovery kit. The process is briefly described as follows: cut the gel block containing the target fragment from the agarose gel and weigh it; add 3-6 times the weight of the gel block buffer B2, and sol it in a 50℃ water bath for 5-10 minutes; transfer the sol solution into the adsorption column, centrifuge at 8000g for 30 seconds; pour out the liquid in the collection tube; add 500ul wash solution to the column, centrifuge at 9000g for 30 seconds, and pour out the liquid in the collection tube; repeat the addition of wash solution once and pour out the liquid; centrifuge the adsorption column at 9000g for 1 minute; place the adsorption column in a clean 1.5ml centrifuge tube, add 15-40ul Elution Buffer to the center of the adsorption membrane, let it stand at room temperature for 1 minute, and centrifuge for 1 minute. The prepared DNA solution is obtained, and the PCR product is purified and sequenced to obtain the variable region sequence of the antibody.

[0152] The amino acid sequences of VH and VL of mouse anti-human P53 protein monoclonal antibody secreted by monoclonal cell line P53-4G5 are shown in the following table:

[0153]

[0154]

[0155] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-P53 protein antibody or an antigen-binding fragment thereof, characterized in that: It includes a heavy chain complementarity determining region in the heavy chain variable region as shown in SEQ ID NO:14, and a light chain complementarity determining region in the light chain variable region as shown in SEQ ID NO:

15.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of CDR-H1 and CDR-H2 are shown in SEQ ID NO: 1-2 respectively, and the amino acid sequence of CDR-H3 is LDY; the light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, and their amino acid sequences are shown in SEQ ID NO: 3-5 respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region, and / or a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 10-13, respectively; the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 6-9, respectively; Preferably, the antibody or antigen-binding fragment thereof further comprises a constant region, wherein the constant region comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the κ type or λ type light chain constant region; Preferably, the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Preferably, the species of origin of the constant region is mouse; Preferably, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of the antibody.

4. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of any of the following products: (1) Tumor diagnostic products; (2) Tumor auxiliary diagnosis products; (3) Tumor efficacy evaluation products; (4) Tumor prognosis assessment products; (5) Products for monitoring tumor recurrence and metastasis; (6) P53 protein detection products; (7) P53 protein separation products or enrichment products; Preferably, the tumor is a tumor with overexpression of P53 protein or mutation of p53 gene; Preferably, the tumor with p53 gene mutation is a tumor with p53 gene deletion or p53 missense mutation; Preferably, the tumor is a solid tumor or a hematological tumor; Preferably, the solid tumor is selected from epithelial tumors, and the blood tumor is selected from leukemia; Preferably, the epithelial tumor is selected from gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma or squamous cell carcinoma; Preferably, the gastrointestinal cancer is selected from esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, small intestine cancer, colon cancer, rectal cancer, colorectal cancer and anal cancer; the squamous cell carcinoma is one or more of oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer, esophageal squamous cell carcinoma, lip squamous cell carcinoma, uterine squamous cell carcinoma, vaginal squamous cell carcinoma and skin squamous cell carcinoma; Preferably, the tumor is one or more of breast cancer, prostate cancer, liver cancer, ovarian cancer, colorectal cancer, gastric cancer and colon cancer.

5. The use according to claim 4, characterized in that: The product in the applications (1)-(6) is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector; the separation product or enrichment product in the application (7) is a magnetic bead, a test kit or a separation column; Preferably, in the applications (1)-(6), the antibody or antigen-binding fragment thereof is labeled with a detectable marker.

6. The use according to claim 4, characterized in that: In the application (7), the P53 protein detection is to label tissues and / or cells with the binding protein and then detect the labeled tissues and / or cells.

7. A tumor diagnosis, auxiliary diagnosis, efficacy evaluation product, prognosis evaluation product or recurrence and metastasis monitoring product, characterized in that: The product comprises: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Preferably, the product is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector.

8. A P53 protein detection product, a P53 protein separation product or an enrichment product, characterized in that: The product comprises: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; Preferably, the P53 protein detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; Preferably, the P53 protein separation product or enrichment product is magnetic beads, a kit or a separation column.

9. A cell, characterized in that The cell is a non-plant cell, which expresses the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: It includes: Cultivate the cell according to claim 9, and separate and purify the antibody or antigen-binding fragment thereof from the culture product.

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