Antibody for resisting CK14 protein and application thereof

Abstract

The invention discloses an anti-CK14 protein antibody and application thereof, and relates to the technical field of antibodies. The heavy chain complementary determining region comprises a light chain complementary determining region in a light chain variable region as shown in SEQ ID NO: 15 and a heavy chain complementary determining region in a heavy chain variable region as shown in SEQ ID NO: 16. The antibody provided by the invention can be used for diagnosing diseases taking the CK14 as a marker, and provides more protein choices for detection of the CK14 and diagnosis of the diseases taking the CK14 as the marker.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an antibody against CK14 protein and an application thereof. Background Art

[0002] At present, CK14 (cytoceratin, cytokeratin 14) is a differentiation-specific protein belonging to the acidic cytokeratin family. The expression of CK14 is limited to the basal cells of myoepithelial and keratinized stratified epithelial cells of various origins, and it is one of the proteins that make up the cytoskeleton. Studies have shown that CK14 is highly expressed in malignant tumors such as lung squamous cell carcinoma and breast invasive ductal carcinoma. In addition, studies have shown that the high expression of CK14 is related to the carcinogenesis, development and prognosis of esophageal squamous cell carcinoma and cervical cancer.

[0003] In clinical practice, immunohistochemistry (IHC) pathology experiments are often used to detect protein expression in tumor cells. The accuracy and sensitivity of IHC experiments are determined by the quality of monoclonal antibodies that specifically bind to proteins. Therefore, developing a monoclonal antibody against CK14 with high binding specificity is of great significance for IHC experiments to detect CK14 expression levels.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-CK14 protein antibody and its application to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides an anti-CK14 protein antibody or an antigen-binding fragment thereof, which comprises a light chain complementary determining region in a light chain variable region as shown in SEQ ID NO: 15, and a heavy chain complementary determining region in a heavy chain variable region as shown in SEQ ID NO: 16.

[0008] In a second aspect, the present invention further provides a use of an antibody or an antigen-binding fragment thereof in the preparation of any of the following products:

[0009] (1) Tumor diagnostic products;

[0010] (2) Tumor efficacy evaluation products;

[0011] (3) Tumor prognosis assessment products;

[0012] (4) Products for monitoring tumor recurrence and metastasis;

[0013] (5) CK14 protein detection products;

[0014] (6) CK14 protein separation product or enrichment product.

[0015] In a third aspect, the present invention also provides a tumor diagnosis, efficacy evaluation product, prognosis evaluation product or recurrence and metastasis monitoring product, the product comprising: the above-mentioned antibody or its antigen-binding fragment.

[0016] In a fourth aspect, the present invention further provides a CK14 protein detection product, a CK14 protein separation product or an enrichment product, the product comprising: the above-mentioned antibody or antigen-binding fragment thereof.

[0017] 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.

[0018] 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.

[0019] The present invention has the following beneficial effects:

[0020] The present invention finds that an antibody with specific six complementary determining regions has good activity and can maintain high binding specificity and affinity with the CK14 antigen. This means that the binding protein can be used to detect CK14, which helps to improve the sensitivity and specificity of the detection. The antibody can be used to diagnose diseases with CK14 as a marker. The present invention provides more protein options for the detection of CK14 and the diagnosis of diseases with CK14 as a marker.

[0021] The antibody specifically binding to CK14 polypeptide provided by the present invention can be used to prepare tumor diagnostic reagents, kits or chips. The tumor diagnostic marker is CK14 (cytoceratin, cytokeratin 14). Based on the activity of the antibody specifically binding to CK14, the expression level of CK14 in related tumor tissues is detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 The results of immunohistochemistry experiments on esophageal squamous cell carcinoma samples, (A) is the CK14 monoclonal antibody 10E1, and B is the CK14 antibody of Maixin Company;

[0024] Figure 2 The second batch of immunohistochemistry experiments and neutralization experiments on esophageal squamous cell carcinoma samples are shown in Figure 1. (A) is the CK14-10E1 antibody produced in this example, (B) is the CK14 antibody produced by Maixin Company, and (C) is the neutralization experiment result of the CK14-10E1 antibody. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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".

[0029] 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 CK14 protein.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The variable region VL of the light chain of an antibody 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.

[0034] LCDR1 is CDR-L1, LCDR2 is CDR-L2, and LCDR3 is CDR-L3.

[0035] In a first aspect, the present invention provides an anti-CK14 protein antibody or an antigen-binding fragment thereof, which comprises a light chain complementary determining region in a light chain variable region as shown in SEQ ID NO: 15, and a heavy chain complementary determining region in a heavy chain variable region as shown in SEQ ID NO: 16.

[0036] Light chain variable region: SEQ ID NO: 15:

[0037] DIVMSQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSN LESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK.

[0038] Heavy chain variable region: SEQ ID NO: 16:

[0039] EAQLQQSGAELVKPGASVKLSCRASGYIFRSYDIHWVRQRPEQGLEWIGWIFPGDGS TKYNEEFKGKATLTTDKSSSTAYMHLNRLTSEDSAVYFCARDGSRYIDYWGQGTTVTVSS.

[0040] By inputting the above SEQ ID NO: 15 and SEQ ID NO: 16 sequences into the CDR labeling system, the corresponding CDR sequences can be obtained.

[0041] 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. They are new sequences that can give the binding protein the ability to specifically bind to the CK14 protein. Based on the good binding activity and affinity of the antibody, it can be used to develop detection products for detecting the CK14 protein, such as detection reagents and kits. The detection reagents include but are not limited to immunohistochemical staining (IHC) related reagents and reagents with detectable markers; the kits include but are not limited to: ELISA kits, Elisopt kits, microfluidic chip kits, etc. Using the binding protein of the present invention to detect CK14 protein can improve the sensitivity and specificity of the detection. In addition, the antibody can be used to diagnose diseases with CK14 protein as a marker. It is conducive to early detection and early intervention treatment of diseases. The present invention provides more protein options for the detection of CK14 protein and the diagnosis of diseases with CK14 protein as a marker.

[0042] In a preferred embodiment of the present invention, the binding protein and CK14 polypeptide are K D ≤3.2525×10 9 L / mol affinity binding.

[0043] For example, K D ≤8×10 10 L / mol affinity binding, such as K D ≤9×10 10 L / mol, K D ≤7×10 10 L / mol, K D ≤6×10 10 L / mol, K D ≤5×10 10 L / mol, K D ≤4×10 10 L / mol, K D ≤3×10 10 L / mol, K D ≤2×10 10 L / mol or K D ≤1×10 10 L / mol affinity binding.

[0044] In a preferred embodiment of the present invention, the heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs: 9-11, respectively; 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: 12-14, respectively.

[0045] CDR-VH1: SYDIH; SEQ ID NO: 9;

[0046] CDR-VH2: WIFPGDGSTKYNEEFKG; SEQ ID NO: 10;

[0047] CDR-VH3: DGSRYIDY; SEQ ID NO: 11;

[0048] CDR-VL1:RASKSVSTSGYSYMH;SEQ ID NO:12;

[0049] CDR-VL2: LVSNLES; SEQ ID NO: 13;

[0050] CDR-VL3: QHIRELTRS; SEQ ID NO: 14.

[0051] 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:5-8, 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:5-8.

[0052] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 1-4, for example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, 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: 1-4.

[0053] The sequences of SEQ ID NO: 1-8 are shown in the following table:

[0054]

[0055] 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;

[0056] 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;

[0057] In a preferred embodiment of the present invention, the species of origin of the constant region is mouse;

[0058] 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.

[0059] 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.

[0060] 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.

[0061] In a second aspect, the present invention further provides a use of an antibody or an antigen-binding fragment thereof in the preparation of any of the following products:

[0062] (1) Tumor diagnostic products;

[0063] (2) Tumor efficacy evaluation products;

[0064] (3) Tumor prognosis assessment products;

[0065] (4) Products for monitoring tumor recurrence and metastasis;

[0066] (5) CK14 protein detection products;

[0067] (6) CK14 protein separation products or enrichment products;

[0068] In a preferred embodiment of the present invention, the tumor is a tumor that overexpresses CK14 protein.

[0069] In a preferred embodiment of the present invention, the tumor is selected from lung squamous cell carcinoma, lung adenocarcinoma, breast cancer or esophageal squamous cell carcinoma.

[0070] In a preferred embodiment of the present invention, the breast cancer is invasive ductal carcinoma of the breast.

[0071] In a preferred embodiment of the present invention, the products in applications (1)-(5) are reagents, test kits, test strips, antibody chips, antibody probes or detectors; the separation products or enrichment products in application (6) are magnetic beads, test kits or separation columns.

[0072] The antibody chip refers to a chip formed by fixing the above-mentioned antibodies or antigen-binding fragments thereof on a carrier.

[0073] In a preferred embodiment of the present invention, in applications (1)-(5), the antibody or its antigen-binding fragment is labeled with a detectable marker.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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, Cy3.21, 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.).

[0078] 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.

[0079] 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, 105Rh, 177 Lu, 172 Lu and 18 F.

[0080] 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.

[0081] 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.

[0082] In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0083] In alternative embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0084] In a preferred embodiment of the present invention, in the application (1)-(5), the tissue and / or cell is labeled with an antibody or an antigen-binding fragment thereof, and the labeled tissue and / or cell is detected;

[0085] In a preferred embodiment of the present invention, after the antibody or its antigen-binding fragment labels the tissue and / or cell, the secondary antibody is incubated with the labeled tissue and / or cell, and the incubation product is detected;

[0086] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is labeled with at least one of the following markers: a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent agent, and a nanoparticle marker.

[0087] In a third aspect, the present invention further provides a tumor diagnosis, efficacy evaluation product, prognosis evaluation product or recurrence and metastasis monitoring product, the product comprising: the above-mentioned antibody or antigen-binding fragment thereof;

[0088] In a preferred embodiment of the present invention, the product is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector.

[0089] Reagents include but are not limited to immunohistochemical staining (IHC) related reagents and reagents with detectable markers; kits include but are not limited to ELISA kits, Elisopt kits, microfluidic chip kits, etc.

[0090] In a fourth aspect, the present invention further provides a CK14 protein detection product, a CK14 protein separation product or an enrichment product, the product comprising: the above-mentioned antibody or antigen-binding fragment thereof.

[0091] In one embodiment, the CK14 protein detection product, CK14 protein separation product or enrichment product comprises a solid phase carrier, on which the above-mentioned antibody or antigen-binding fragment thereof is coated. The solid phase carrier includes but is not limited to glass, microplate, membrane, and a substrate with an activated group.

[0092] In a preferred embodiment of the present invention, the CK14 protein detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector;

[0093] In a preferred embodiment of the present invention, the CK14 protein separation product or enrichment product is a magnetic bead, a kit or a separation column.

[0094] For example, the above-mentioned antibody is coated on magnetic beads for separation and enrichment of CK14 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 CK14 protein. Therefore, the above-mentioned antibody or its antigen-binding fragment has a good application prospect in the preparation of CK14 protein enrichment products.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0099] Example 1

[0100] This example is about the preparation of specific anti-CK14 monoclonal antibody.

[0101] 1. Animal (mice) immunization.

[0102] Immunize mice with immunogens using a common method. The immunogen is the CK14 polypeptide synthesized by Gill Biotech, 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 CK14 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 place it on an emulsifier to stir and 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 immunized mice with high serum titers for final intraperitoneal immunization and perform cell fusion.

[0103] 2. Hybridoma cell fusion and screening.

[0104] Preparations need to be made before cell fusion: 1. Cultivate mouse myeloma cells SP2 / 0 to the logarithmic growth phase; 2. Sacrifice a negative mouse one day before fusion, inject HAT culture medium into the mouse peritoneal cavity in a sterile environment, take mouse peritoneal trophoblast cells and plate them on a 96-well plate, 100ul per well. These cells promote the growth of hybridoma cells. Sacrifice the immune mouse, take the spleen in a sterile environment, use PEG to chemically fuse spleen B cells and SP2 / 0 myeloma cells, add appropriate HAT culture medium according to the number of cells to be plated, and finally plate the fused cells on the trophoblast cell culture plate, 100ul per well.

[0105] 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 the hybridoma cells are screened by ELISA method with CK14 polypeptide antigen. The method is as follows: Coat the enzyme-labeled plate with 100ul 2ug / ml CK14 polypeptide antigen in PBS solution at 37℃ for two hours. After washing the plate three times with PBST, add 150ul / well of 3% skim milk powder in PBS solution and block it at 4℃ overnight. 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 1:8000 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 positive hybridoma cells.

[0106] Select the ELISA-positive fusion wells and then perform immunohistochemistry (IHC), and select the positive wells for subsequent experiments. The experimental steps are as follows:

[0107] (1) Slice processing: Place the esophageal squamous cell carcinoma slices in a 60°C thermostat and bake for 60 minutes. Soak the slices in xylene I for 15 minutes, then replace with xylene II and soak for 15 minutes. Soak the slices 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. Soak in ddH2O for 5 minutes and wash three times. Perform antigen repair in a pressure cooker (boiling method). Add 10 mmol / L citrate buffer (pH 6.0) to the pressure cooker to cover the slices. Heat to boiling. Place the slices on a heat-resistant slice rack and put them in the pot. Cover the pot with a lid, close the pressure valve, continue heating, set the pressure to maintain for 4 minutes, open the vent valve to release air after the time is up, and open the lid to remove the inner pot after the pressure returns to zero and let it cool at room temperature. 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.

[0108] (2) Incubation of tissue sections with antibodies:

[0109] Add the CK14 antibody of Maixin Company to the processed tissue sections for comparison, and add the antibodies secreted by the above positive hybridoma cells to the other 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 Changdao Company secondary antibody 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-blue 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 with a coverslip; photograph the slide under a microscope.

[0110] 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 method to screen out hybridomas that specifically recognized CK14 polypeptide and could block CK14 binding. The monoclonal cell line 10E1 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 CK14 polypeptide were obtained.

[0111] Figure 1 For the immunohistochemistry experiment of CK14 monoclonal antibody 10E1, Figure 2 For the immunohistochemical neutralization experiment of CK14, Figure 1 and Figure 2 The specimens tested were all esophageal squamous cell carcinoma tissues, the difference being that they came from different parts of the esophageal squamous cell carcinoma tissues. Figure 1 A is the CK14-10E1 antibody produced in this example, and B is the CK14 antibody produced by Maixin Company. Figure 2 A is the CK14-10E1 antibody produced in this example, and B is the CK14 antibody produced by Maixin Company.

[0112] Depend on Figure 1 and Figure 2 The staining results show that there is no significant difference in the staining site and staining intensity between the CK14-10E1 antibody and the Maixin antibody. This proves that the CK14 provided by the present invention has an application prospect in preparing a kit.

[0113] Example 2

[0114] DNA cloning and sequencing were performed, and variable region gene sequencing of anti-CK14 monoclonal antibody was performed.

[0115] 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 Trizol reagent 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-freeddH2O. Store the resulting RNA solution at -70℃ or use it for subsequent experiments.

[0116] 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.

[0117] 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 + cDNA 2ul + 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 + ddH2O21ul. 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):

[0118] Step 1 - pre-denaturation at 94°C for 4 min;

[0119] Step 2 - denaturation at 94°C for 30 seconds;

[0120] Step 3-annealing at 55°C for 45 seconds;

[0121] Step 4 - extension 72°C, 60 sec;

[0122] Step 5-72°C, 10 min;

[0123] Step 6 - Store at 4°C.

[0124] The PCR products were analyzed by 1% agarose gel electrophoresis, and the DNA segment bands of corresponding sizes (VH is about 375bp, VL is about 325bp) 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 it 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.

[0125] The light chain variable region is shown in SEQ ID NO:15, and the heavy chain variable region is shown in SEQ ID NO:16.

[0126] Experimental Example 1

[0127] This experimental example tests the affinity and sensitivity of the antibody prepared in Example 1 above.

[0128] 1. The antigen in Example 1 was coated on plates at 3 mg / L, 1.5 mg / L, 0.75 mg / L and 0.375 mg / L respectively.

[0129] 2. Adjust the antibody concentration 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.

[0130] 3. Add secondary antibody and develop color with TMB. Measure the absorbance at 450nm. The measured data are shown in Table 1.

[0131] 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).

[0132] 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').

[0133] 6. When N = 2, we can get three K values ​​of 1.385, 2.702, and 5.882. When N = 4, we can get two K values ​​of 2.051 and 4.225. When N = 8, we can get a K value of 3.267. The average value of the six K values ​​is 3.2525×10 9 L / mol.

[0134] Table 1 Statistics of absorbance values ​​under different treatments.

[0135]

[0136] Experimental Example 2

[0137] In this experimental example, an immunohistochemical neutralization experiment was performed: a certain amount of CK14 antigen was mixed with the purified CK14-10E1 antibody obtained by screening in Example 1, and the neutralization reaction was carried out at 37°C for 1 hour. According to the immunohistochemical experimental steps, the primary antibody was added dropwise to the esophageal squamous cell carcinoma sections, and the sections were kept at 4°C overnight. After that, immunohistochemical staining was performed according to the same steps as in Example 1, and the sections were photographed.

[0138] Figure 2 Figure C is the result of the neutralization experiment. In the neutralization test, the antigen reaction combined with the CK14-10E1 antibody, and the antibody could not bind to the antigen in the cancer tissue, so that the tissue was almost not stained. It can be seen that the anti-CK14 polypeptide monoclonal antibody CK14-10E1 provided in the embodiment of the present invention can recognize and bind to the CK14 polypeptide in human cells, and in the subsequent IHC staining experiment, the cell cytoplasm in the lung adenocarcinoma tissue is brown, and there is no significant difference with the CK14 antibody of Maixin Company.

[0139] 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-CK14 protein antibody or an antigen-binding fragment thereof, characterized in that: It includes a light chain complementarity determining region in the light chain variable region as shown in SEQ ID NO:15, and a heavy chain complementarity determining region in the heavy chain variable region as shown in SEQ ID NO:

16.

2. The anti-CK14 protein 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, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs: 9-11, respectively; 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: 12-14, respectively.

3. The anti-CK14 protein 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: 5-8, in sequence; 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: 1-4, in sequence; 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 efficacy evaluation products; (3) Tumor prognosis assessment products; (4) Products for monitoring tumor recurrence and metastasis; (5) CK14 protein detection products; (6) CK14 protein separation products or enrichment products; Preferably, the tumor is a tumor that overexpresses CK14 protein; Preferably, the tumor is selected from lung squamous cell carcinoma, lung adenocarcinoma, breast cancer or esophageal squamous cell carcinoma; Preferably, the breast cancer is invasive ductal carcinoma of the breast.

5. The use according to claim 4, characterized in that: The products in the applications (1)-(5) are reagents, test kits, test strips, antibody chips, antibody probes or detectors; the separation products or enrichment products in the application (6) are magnetic beads, test kits or separation columns; Preferably, in the applications (1)-(5), the antibody or antigen-binding fragment thereof is labeled with a detectable marker.

6. The use according to claim 5, characterized in that: In the applications (1)-(5), the tissue and / or cell is labeled with the antibody or antigen-binding fragment thereof, and the labeled tissue and / or cell is detected; Preferably, after the antibody or antigen-binding fragment thereof labels tissues and / or cells, the labeled tissues and / or cells are incubated with a secondary antibody to detect the incubation product; Preferably, the antibody or antigen-binding fragment thereof is labeled with at least one of the following markers: a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent agent, and a nanoparticle marker.

7. A tumor 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 CK14 protein detection product, CK14 protein separation product or 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 CK14 protein detection product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; Preferably, the CK14 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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