Anti-EZH2 protein antibody and application thereof

Abstract

The invention discloses an anti-EZH2 protein antibody and application thereof. A heavy chain variable region of the anti-EZH2 protein antibody contains the following complementarity determining regions: an HCDR1 with a nucleotide sequence as shown in a sequence SEQ ID NO: 1, an HCDR2 with a nucleotide sequence as shown in a sequence SEQ ID NO: 2 and an HCDR3 with a nucleotide sequence as shown in a sequence SEQ ID NO: 3; a light chain variable region sequence of the gene contains the following complementarity determining regions: an LCDR1 with a nucleotide sequence as shown in a sequence SEQ ID NO: 4, an LCDR2 with a nucleotide sequence as shown in a sequence SEQ ID NO: 6 and an LCDR3 with a nucleotide sequence as shown in a sequence SEQ ID NO: 5. The anti-EZH2 protein antibody provided by the invention can be specifically bound with EZH2 protein, has better binding activity and is used for detecting intraocular malignant tumors. According to the cascade amplification EZH2 protein detection product / method based on nano enzyme and TSA mediation provided by the invention, the expression of EZH2 protein in a pathological sample can be conveniently subjected to immunohistochemical research, and meanwhile, the tissue morphology and gene expression information of the sample can be obtained, so that the accuracy and comprehensiveness of pathological diagnosis are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a culture medium, a kit and an application thereof for culturing salivary gland tumor organoids. Background Art

[0002] Intraocular malignancies are highly malignant and seriously threaten patients' life and visual prognosis. Intraocular malignancies include retinoblastoma (RB) and uveal melanoma (UM), which mainly occur in children and adults, respectively. These tumors are difficult to detect in the early stages because they occur in the eye, often resulting in patients being in the late stages when symptoms appear, limiting the possibility of early diagnosis and treatment.

[0003] The treatment of RB and UM faces many challenges, including the lack of effective early screening methods and drug resistance. Current treatments for these intraocular malignancies mainly rely on surgery, radiotherapy, and chemotherapy, but these methods have certain limitations, especially for patients with advanced or recurrent disease.

[0004] In recent years, subunits of the polycomb repressive complex 2 (PRC2), especially enhancer of Zeste homolog 2 (EZH2), have attracted widespread attention as potential therapeutic targets. EZH2 is involved in gene silencing regulation under normal circumstances. However, in a variety of cancers, including breast cancer, bladder cancer, and prostate cancer, EZH2 is often overexpressed and is closely related to the occurrence, progression, and drug resistance of tumors. Our research team found that the expression of EZH2 was significantly increased in the retinal tissues of RB and UM patients, and its promotion of tumor cell proliferation suggests its potential role in these intraocular malignancies.

[0005] Although EZH2 has great potential as a therapeutic target, there is still a lack of specific antibodies and effective detection methods for it on the market. This not only limits the application of EZH2 in the diagnosis of intraocular malignancies, but also hinders its development and application as a therapeutic target. Therefore, the development of efficient antibodies against EZH2 and corresponding immunohistochemical detection methods has become an important step in solving the current challenges in the diagnosis and treatment of intraocular malignancies. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-EZH2 protein antibody and its application, wherein the anti-EZH2 protein antibody can specifically bind to EZH2 protein, has good binding activity, is used for the detection of intraocular malignant tumors, and has good clinical application scenarios and practical value; in addition, the EZH2 protein detection product / method based on nanozyme and TSA-mediated cascade amplification provided by the present invention, the detection results of which can be interpreted under an ordinary optical microscope, can conveniently perform immunohistochemical research on the expression of EZH2 protein in pathological samples, and simultaneously obtain the tissue morphology and gene expression information of the samples, thereby improving the accuracy and comprehensiveness of pathological diagnosis. .

[0007] In a first aspect, the present invention provides an anti-EZH2 protein antibody, wherein the heavy chain variable region contains the following complementarity determining regions: HCDR1 with a nucleotide sequence as shown in SEQ ID NO: 1, HCDR2 with a nucleotide sequence as shown in SEQ ID NO: 2, and HCDR3 with a nucleotide sequence as shown in SEQ ID NO: 3; and the light chain variable region sequence contains the following complementarity determining regions: LCDR1 with a nucleotide sequence as shown in SEQ ID NO: 4, LCDR2 with a nucleotide sequence as shown in SEQ ID NO: 6, and LCDR3 with a nucleotide sequence as shown in SEQ ID NO: 5.

[0008] The second aspect of the present invention provides a nucleic acid molecule comprising: a nucleotide sequence encoding the above-mentioned antibody.

[0009] The third aspect of the present invention provides a recombinant vector comprising the isolated nucleic acid molecule.

[0010] The fourth aspect of the present invention provides a recombinant host cell comprising the above nucleic acid molecule or the above recombinant vector.

[0011] The fifth aspect of the present invention provides an antibody conjugate, which comprises the above-mentioned anti-EZH2 protein antibody; the antibody is labeled with a marker.

[0012] The sixth aspect of the present invention provides use of the above anti-EZH2 protein antibody or the above antibody conjugate in the preparation of a product for detecting EZH2 protein.

[0013] The seventh aspect of the present invention provides an EZH2 protein detection product based on nanozyme and TSA-mediated cascade amplification, comprising the above-mentioned antibody conjugate and a dual signal amplification system, wherein the dual signal amplification system comprises a nanozyme conjugate and a SA-TSA conjugate, wherein the SA-TSA conjugate is a streptavidin-tyramide conjugate.

[0014] In one embodiment, the nanozyme conjugate comprises a metal-organic framework nanozyme, and the metal-organic framework nanozyme is labeled with a marker.

[0015] The eighth aspect of the present invention provides an EZH2 protein detection method, using the above-mentioned EZH2 protein detection product, comprising the following steps:

[0016] The sample to be tested is mixed with the antibody conjugate and then incubated, and then washed;

[0017] Add nanozyme conjugates, incubate, and wash;

[0018] SA-TSA conjugate was added to catalyze TSA to covalently bind to the target protein, and a color developing solution was added to locate, qualitatively, and quantitatively detect the EZH2 protein in the sample.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The anti-EZH2 protein antibody provided by the present invention can specifically bind to the EZH2 protein and has good binding activity.

[0021] 2. The anti-EZH2 protein antibody provided by the present invention is used for the detection of intraocular malignant tumors, and has good clinical application scenarios and practical value. The antibody can not only be used for early diagnosis, but also assist in pathological analysis and formulation of treatment strategies. It has particularly important clinical application value for rare or difficult-to-diagnose ocular tumors, can significantly improve the accuracy and reliability of diagnosis, and provide patients with more effective treatment options.

[0022] 3. The EZH2 protein detection product / method based on nanozyme and TSA-mediated cascade amplification provided by the present invention allows researchers to conveniently conduct immunohistochemical studies on the expression of EZH2 protein in pathological samples, while obtaining the tissue morphology and gene expression information of the samples, thereby improving the accuracy and comprehensiveness of pathological diagnosis. In addition, the test results can be interpreted under an ordinary optical microscope, which facilitates the promotion of this technology to hospitals and regions with underdeveloped medical care. This method / product can quickly and visually detect a variety of intraocular malignant tumors, has the characteristics of simple operation, high sensitivity, and strong specificity, and has good clinical application scenarios and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the EZH2 protein detection method based on nanozyme and TSA-mediated cascade amplification provided in an embodiment of the present invention;

[0024] Figure 2 This is a graph showing the test results of mouse serum EZH2 antibody titer in Example 1;

[0025] Figure 3 This is a diagram showing the results of EZH2 detection in hybridoma supernatant in Example 1;

[0026] Figure 4This is a diagram showing the results of FACs detection of EZH2-positive cells in hybridoma supernatant in Example 1;

[0027] Figure 5 This is a purification characterization diagram of the EZH2 protein antibody protein in Example 2;

[0028] Figure 6 This is a transmission electron microscopy characterization image of the metal framework MOF nanozyme in Example 2;

[0029] Figure 7 An example diagram of using positive tissues / cells in Example 2 to verify the feasibility of the system;

[0030] Figure 8 This is a diagram showing the in situ detection of intraocular malignant tumor pathological tissue in Example 2. DETAILED DESCRIPTION

[0031] In this article, the range expressed by "a value to another value" is a summary expression method to avoid listing all the values ​​in the range one by one in the specification. Therefore, the description of a specific numerical range covers any value in the numerical range and a smaller numerical range defined by any value in the numerical range, just as if the arbitrary value and the smaller numerical range were explicitly written in the specification.

[0032] In this application, the use of "or" means "and / or" unless otherwise stated. In the case of multiple dependent claims, use of "or" refers to more than one of the preceding independent or dependent claims only in the alternative.

[0033] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0034] In order to solve the technical problem that there is still a lack of specific antibodies and effective detection methods for EZH2 protein on the market, the present invention provides an anti-EZH2 protein antibody, whose heavy chain variable region contains the following complementary determining regions: HCDR1 with a nucleotide sequence as shown in the sequence of SEQ ID NO: 1, HCDR2 with a nucleotide sequence as shown in the sequence of SEQ ID NO: 2, and HCDR3 with a nucleotide sequence as shown in the sequence of SEQ ID NO: 3; and its light chain variable region sequence contains the following complementary determining regions: LCDR1 with a nucleotide sequence as shown in the sequence of SEQ ID NO: 4, LCDR2 with a nucleotide sequence as shown in the sequence of SEQ ID NO: 6, and LCDR3 with a nucleotide sequence as shown in the sequence of SEQ ID NO: 5.

[0035] In the present invention, the "antibody" refers to a structure formed by the association of two heavy chains (H chains) and two light chains (L chains) stabilized by a pair of disulfide bonds. The heavy chain is composed of a heavy chain variable region VH, a heavy chain constant region CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain is composed of a light chain variable region VL and a light chain constant region CL. Among them, the variable region fragment (Fv) composed of VH and VL directly participates in antigen binding and is a region that gives antibodies diversity. In addition, the antigen binding region composed of VL, CL, VH, and CH1 is called the Fab region, and the region composed of the hinge region, CH2, and CH3 is called the Fc region. In the variable region, the region that directly contacts the antigen changes particularly greatly and is called the complementarity-determining region (CDR). The part with fewer mutations outside the CDR is called the framework region (FR).

[0036] Functional variants of the antibodies provided by the present invention are also included in the protection scope of the present invention, and the functional variants refer to proteins having obvious or significant sequence identity or similarity compared to the parent antibody (the antibody provided in the first aspect of the present invention), and the functional variants retain the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the antibodies (parent antibodies) described herein, which retain the ability to recognize the target antigen (EZH2 protein) to a similar extent, to the same extent, or to a higher extent than the parent antibody. With reference to the parent antibody, the functional variant may, for example, have at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95% or more homology with the parent antibody in terms of nucleotide sequence.

[0037] The second aspect of the present invention provides a nucleic acid molecule comprising: a nucleotide sequence encoding the above-mentioned antibody.

[0038] In some embodiments, the nucleic acid molecule includes any of the following nucleic acid molecules: a nucleic acid molecule encoding the antibody described in the second aspect of the invention, a nucleic acid molecule encoding the heavy chain variable region of the antibody described in the first aspect of the invention, and a nucleic acid molecule encoding the light chain variable region of the antibody described in the first aspect of the invention.

[0039] Those skilled in the art can easily mutate the nucleotide sequence corresponding to the antibody provided in the first aspect of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 80% or more homology with the nucleotide sequence corresponding to the antibody or bispecific antibody of the present invention, as long as they encode the antibody described in the first aspect of the present invention, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention, and are also included in the protection scope of the present invention.

[0040] The third aspect of the present invention provides a recombinant vector comprising the isolated nucleic acid molecule.

[0041] In some embodiments, any vector capable of delivering nucleic acid can be suitable for use in the present invention. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a plasmid, a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0042] The fourth aspect of the present invention provides a recombinant host cell comprising the above nucleic acid molecule or the above recombinant vector.

[0043] In some embodiments, the host cell includes yeast cells, mammalian cells, or any other cells suitable for preparing antibodies. In some embodiments, suitable host cells include prokaryotic microorganisms, such as Escherichia coli. The host cell can also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells, etc. Vertebrate cells can also be used as host cells.

[0044] In the present invention, host cells that can be used to express antibodies are well known to those skilled in the art, and many host cells are available from companies. These host cells include, but are not limited to, various yeasts and many other types of cell lines.

[0045] In some embodiments, the recombinant vector described in the third purpose can be introduced into the host cell using technology well known in the art. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses. For bacterial cells, suitable techniques can include calcium chloride conversion, electroporation and transfection using bacteriophage. After introduction, nucleic acid expression can be caused or allowed, for example, by culturing host cells under the condition of gene expression.

[0046] The fifth aspect of the present invention provides an antibody conjugate, which comprises the above-mentioned anti-EZH2 protein antibody; the antibody is labeled with a marker.

[0047] In some embodiments, the label refers to any substance that can be used to assist the antibody in detecting the EZH2 protein. For example, detectable labels include, but are not limited to, streptavidin, biotin, luciferase, luciferin, and the like.

[0048] The sixth aspect of the present invention provides use of the above anti-EZH2 protein antibody or the above antibody conjugate in the preparation of a product for detecting EZH2 protein.

[0049] The present invention also provides a metal organic framework (MOF) nanozyme, which is a type of nanomaterial with biocatalytic function, which can catalyze the substrate of natural enzymes based on specific nanostructures and serve as a substitute for enzymes. It has both the physical and chemical properties of the nanomaterial itself and the catalytic function similar to that of enzymes, and has the advantages of both natural enzymes and artificial enzymes. Among them, the nanostructure not only gives the nanozyme efficient catalytic function, but also makes the nanozyme more stable than natural enzymes and easy to mass produce. In addition, the unique multi-enzyme activity of the nanozyme will provide functional molecules for designing cheap, stable, and various new catalytic cascade reactions. Further, the nanozyme described in the present invention has superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity.

[0050] In some embodiments, any substance having peroxidase activity can be suitable for the present invention. In some embodiments, the catalytic substance used is a nanozyme. In some embodiments, the catalytic substance used for the carrier can be a variety of nanozymes, HRP, or any combination thereof.

[0051] The present invention also provides a nanozyme conjugate, which includes a metal organic framework (MOF) nanozyme and a marker directly or indirectly coupled to the nanozyme conjugate, wherein the detectable marker includes but is not limited to: streptavidin, biotin, luciferase, luciferin, etc.

[0052] The seventh aspect of the present invention provides an EZH2 protein detection product based on nanozyme and TSA-mediated cascade amplification, comprising the above-mentioned antibody conjugate and a dual signal amplification system, wherein the dual signal amplification system comprises a nanozyme conjugate and a SA-TSA conjugate, wherein the SA-TSA conjugate is a streptavidin-tyramide conjugate.

[0053] In one embodiment, the nanozyme conjugate comprises a metal-organic framework nanozyme, and the metal-organic framework nanozyme is labeled with a marker.

[0054] The eighth aspect of the present invention provides an EZH2 protein detection method, using the above-mentioned EZH2 protein detection product, comprising the following steps:

[0055] Process the samples to be tested according to the routine operation of immunohistochemistry;

[0056] The sample to be tested is mixed with the antibody conjugate and then incubated, and then washed;

[0057] Add nanozyme conjugates, incubate, and wash;

[0058] SA-TSA conjugate was added to catalyze TSA to covalently bind to the target protein, and a color developing solution was added to locate, qualitatively, and quantitatively detect the EZH2 protein in the sample.

[0059] Through the dual amplification strategy mediated by nanozymes and TSA, the EZH2 protein in tissue and cell specimens is located, qualitatively and quantitatively detected, and the detection results can be observed under an ordinary optical microscope.

[0060] The product for detecting EZH2 protein in the present invention can be a detection kit, a detection test strip, or a detection chip. In other embodiments, the detection kit includes, but is not limited to, an ELISA detection kit, an immunofluorescence detection kit, a chemiluminescence detection kit, a radioimmunoassay kit, a flow sorting detection kit, an IHC detection kit, and a colloidal gold immunoassay kit.

[0061] The samples to be tested in the present invention include but are not limited to liquids, such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or solid or semi-solid samples, such as tissues, feces and the like, or solid tissues commonly used in histological diagnosis. The present invention has no particular limitation on the samples to be tested, as long as there are samples to be tested for the detection of EZH2 protein.

[0062] The present invention is further described in detail below through specific examples.

[0063] The specific steps of the tissue section immunohistochemistry experiment in the following examples are as follows:

[0064] 1. Western blot

[0065] 1.1 Total cell protein extraction:

[0066] ① Collect cells: adherent cells were cultured in a 10 cm dish. When the cells grew to 90%, the culture medium was aspirated and the cells were washed twice with PBS. Suspended cells were directly collected into a 15 ml centrifuge tube, centrifuged at 800 rpm for 5 min, the supernatant was discarded, the cell pellet was resuspended with PBS, transferred to a 1.5 ml Eppendorf centrifuge tube, centrifuged at 800 rpm for 5 min, and the supernatant was discarded.

[0067] ② Protein lysis: Add protein lysis buffer (RIPA) containing 1 mM PMSF to the adherent cells in a 10 cm dish to cover the bottom of the dish, lyse on ice for 30 min, collect the cells with a scraper into a 1.5 ml Eppendorf centrifuge tube, centrifuge at 4°C, 12000 rpm for 30 min, and take the supernatant; for suspended cells, resuspend the cells directly with protein lysis buffer containing 1 mM PMSF, lyse on ice for 30 min, centrifuge at 4°C, 12000 rpm for 30 min, and take the supernatant.

[0068] ③ Protein quantification and denaturation: Use the BCA assay kit to quantify the protein. First, prepare a BSA solution of standard concentration, draw a protein concentration standard curve based on the OD590 nm measurement value, and then calculate the protein sample concentration based on the standard curve and the absorbance value of the protein sample. According to the volume of the protein sample, add 4×SDS gel loading buffer, mix well, heat in boiling water for 10 minutes to denature the protein, and freeze at -80℃.

[0069] 1.2 Polyacrylamide gel electrophoresis:

[0070] ① Preparation of SDS-PAGE separation gel: The preparation method of 10% separation gel is as follows: 4.0 ml of double distilled water, 3.3 ml of 30% acrylamide, 2.5 ml of 1.5 M Tris (pH 8.8), 100 μl of 10% SDS, 100 μl of 10% ammonium persulfate, and 10 μl of N,N,N',N'-tetramethylethylenediamine (TEMED) are added to 10 ml system in sequence, and the mixture is quickly inverted to mix; the preparation method of 7% separation gel is as follows: 5.1 ml of double distilled water, 2.3 ml of 30% acrylamide, 2.5 ml of 1.5 M Tris (pH 8.8), 100 μl of 10% SDS, 100 μl of 10% ammonium persulfate, and 10 μl of TEMED are added to 10 ml system in sequence, and the mixture is quickly inverted to mix.

[0071] ② Glue filling: Before preparing the separation gel, wash, dry and clamp the glass plates in advance. After mixing the separation gel, quickly pour it into the gap between the two glass plates for filling until it reaches the upper edge of the green baffle. Cover the surface of the solution with 1ml of isopropanol for pressing to make the gel surface flat. Let it stand at room temperature for 40 minutes. After the separation gel is completely solidified, pour out the upper layer of isopropanol, wash the top of the gel several times with double distilled water, and wipe off the liquid.

[0072] ③ Prepare stacking gel and pour it: The method for preparing 5ml system stacking gel is as follows: add 2.85ml of double distilled water, 0.85ml of 30% acrylamide, 1.25ml of 1.5M Tris (pH 6.8), 50μl of 10% SDS, 50μl of 10% ammonium persulfate, and 5μl of TEMED in this order, and quickly invert to mix; pour the stacking gel directly on the solidified separation gel. After pouring the stacking gel, immediately insert a pre-cleaned comb and let it stand at room temperature for 30 minutes. After the stacking gel is completely solidified, use it for SDS-PAGE electrophoresis.

[0073] ④ Loading: Fix the prepared gel plate on the electrophoresis device, check whether the glass plate and the plastic clamp are clamped tightly, add 1000ml Tris-glycine electrophoresis buffer (25mM Tris, 250mM glycine, 0.1% SDS), remove the comb, and add the protein sample and marker to the wells in order, adding 20μg of protein sample to each well.

[0074] ⑤ Electrophoresis: Perform electrophoresis at 80V first. After the bromophenol blue dye begins to enter the separation gel (about 30 minutes), increase the voltage to 120V and continue electrophoresis until the bromophenol blue reaches the bottom of the separation gel, then turn off the power.

[0075] 1.3 Protein transfer:

[0076] ① Prepare electrophoresis transfer buffer in advance and place it in a 4°C refrigerator to cool; after SDS-PAGE electrophoresis, remove the stacking gel and sample-free area, and soak the gel, wet transfer sponge, and filter paper in electrophoresis transfer buffer.

[0077] ②Cut a piece of polyvinylidene fluoride membrane (PVDF) of the same size as the gel and immerse it in 100% methanol for activation.

[0078] ③ Place the electrophoresis transfer clamp in the electrophoresis transfer buffer, and place the sponge, filter paper, PVDF membrane, gel, filter paper, and sponge in order from the anode to the cathode; be careful to remove the bubbles, clamp the clamp and place it in the transfer tank, add 1000ml of electrophoresis transfer buffer, put it in the ice box, and transfer at a constant voltage of 80V for 50-100min depending on the size of the target protein.

[0079] 1.4 Antibody labeling:

[0080] ① Blocking: Place the PVDF membrane after protein transfer in BSA blocking solution prepared with 5% TBS, and shake on a horizontal shaker at room temperature for 1 hour.

[0081] ② Incubate with antibodies: Take out the PVDF membrane and place it in the antibody solution prepared with blocking solution. Note that the liquid in the hybridization bag must cover the PVDF membrane on both sides. Incubate at 4°C overnight.

[0082] 1.5Odyssey infrared imaging system scanning detection:

[0083] The washed PVDF filter was placed in an Odyssey infrared laser imaging scanner for scanning. All grayscale value analyses of western blots were performed using Bandscan 5.0 software.

[0084] 2. Tissue Section Immunohistochemistry Experiment

[0085] After the tissue is sliced, place it in a wet box and drip PBS on the slice. After absorbing PBS from one end, drip the diluted target antibody onto the slice, cover the sample, cover the wet box, incubate at room temperature for 1 hour, wash 3 times with PBS, take the cover glass, drip 1 drop of Gelvatol in the center, press the reverse transfer slide on the cover glass, and place it at room temperature away from light for 30 minutes to solidify. Observe and record the results under a microscope.

[0086] Example 1 Method for constructing anti-EZH2 protein antibodies

[0087] 1.1 Animal immunization

[0088] 10 Balb / c mice were immunized using commercial recombinant human EZH2 protein (AntibodySystem, YHH22901) according to the conventional method described in the literature. Each mouse was immunized subcutaneously (SC) 4 times using recombinant human EZH2 protein. For the first immunization, 100ul (75ug) of recombinant human EZH2 solution was mixed with 100ul of complete Freund's adjuvant, and for other immunizations, 100ul (75ug) of recombinant human EZH2 solution was mixed with 100ul of incomplete Freund's adjuvant.

[0089] 1.2 Antigen-specific ELISA to detect mouse serum titers

[0090] Antigen-specific ELISA was used to determine the anti-EZH2 titer in the sera of immune mice. An EZH2 solution with a concentration of 1ug / ml was coated in a 96-well plate, 100ul per well, and incubated overnight at 4°C. The coating solution was then washed off, and a blocking solution (protein-free blocking buffer) was added to each well, and incubated at room temperature for 2 hours. The mouse serum was pre-diluted 100 times in PBSA (PBS solution containing 1% BSA) and diluted continuously at 1:2 for 10 dilution gradients. After washing the coating solution, the diluted serum was added to the plate wells and incubated at room temperature for 1 hour. After washing the 96-well plate with PBST, 100ul / well of a 1 / 30000 diluted secondary antibody was added, and the plate was incubated at room temperature for 45 minutes. The plate was washed three times with PBST, and 20ul / well of TMB equilibrated at room temperature was added. The plate was incubated at room temperature for 10 minutes, and the absorbance was measured at 650nm. The results are shown in the figure. Figure 2 shown by Figure 2 It can be seen that the concentration of EZH2 antibodies in serum is high.

[0091] 1.3 Anti-human EZH2 hybridoma production

[0092] Mouse spleen was taken, and the isolated spleen cells were fused with myeloma cells P3X63Ag8.653 using the electrofusion method. The fused hybridoma cells were inoculated in a 384-well plate, cultured for 14 days, coated with human EZH2 recombinant protein (1ug / ml, pH9.6, 0.1M NaHCO3), incubated at 4°C overnight; blocked with 4% skim milk powder-PBS, incubated at 37°C for 2 hours; washed three times with PBST (0.05% Tween20-PBS), added with hybridoma clone culture supernatant, and incubated at 37°C for 1 hour. Wash three times with PBST (0.05% Tween20-PBS), add HRP-goat anti-mouse IgG, dilute 1:20000, and incubate at 37°C for 1 hour; then wash five times with PBST (0.05% Tween20-PBS), add TMB colorimetric solution, color for 10 minutes in dark, and read A650 absorbance with a microplate reader. The results are as follows: Figure 3 shown by Figure 3 It was found that the EZH2 antibody was present in the supernatant.

[0093] The flow cytometry binding capacity of EZH2-overexpressing cells was detected using a 96-well plate. 2E5 cells were used in each test well, and 20ul of hybridoma supernatant was added. After incubation at 4°C for 1 hour, the cells were washed with PBS. A secondary antibody of FITC-labeled anti-human antibody diluted 1000 times was added, and the cells were resuspended at 100mL / well. After incubation at 4°C for 1 hour, the cells were washed with PBS and analyzed on a flow cytometer. A total of multiple secretion-positive EZH2 antibodies were obtained. The results are as follows Figure 4 shown by Figure 4 It was found that a plurality of secretion-positive EZH2 antibodies were obtained.

[0094] The hybridoma cell clones that specifically recognized human EZH2 obtained by the combined experimental screening were single-celled by the limiting dilution method. After two rounds of subcloning, each hybridoma cell clone obtained secreted only one antibody. After the hybridoma cells were expanded and cultured, the total RNA of the cells was extracted according to the instructions of the RNAfast200 kit; the total RNA of the hybridoma cells was reverse transcribed into cDNA using 5× PrimeScript RTMaster Mix; the antibody light chain variable region VL and heavy chain variable region VH sequences were amplified using degenerate primers and Extaq PCR reagents; the PCR amplification products were purified using the PCR clean-up Gel extraction kit; the amplified PCR products were connected to the T vector and transformed into Escherichia coli competent cells according to the instructions of the pClone007 Simple Vector Kit kit. After strain amplification and plasmid extraction, the antibody light chain variable region VL and heavy chain variable region VH sequences were amplified using degenerate primers (Anke Krebber.1997) and Extaq PCR reagents to obtain the monoclonal antibody variable region sequences (Table 1).

[0095] Table 1 summarizes the nucleotide sequence information of the variable region sequences of the anti-EZH2 protein antibodies of the present invention.

[0096]

[0097] 1.4 Antibody Screening

[0098] Take 10ug of the above commercially available human EZH2 recombinant protein and incubate it with SA magnetic beads at 4 degrees overnight. Wash it with PBS-Tween (0.5% v / v) and PBS for 5 rounds, add it to 1ml of phage, rotate and mix it for about 10 minutes. Wash it with PBS-Tween (0.5% v / v) and PBS for 15 rounds to remove unbound phage. The washed magnetic beads are used to infect logarithmic phase Escherichia coli TG1 bacteria and rescue them for the next round of enrichment screening. The second round of screening will reduce the input antigen to 5ug, and the third round to 2ug. After three rounds of enrichment screening, take a small amount of TG1 Escherichia coli from each round to coat the LB plate, culture it overnight at 37 degrees, pick a single clone to 96-well U-shaped plate for IPTG induction expression, take the supernatant for ELISA detection screening (ELISA detection kit), and the ELISA positive clones are sequenced to verify the antibody sequence (same as Table 1).

[0099] Example 2 Preparation of anti-human EZH2 antibody protein and its application in in situ detection of intraocular malignant tumors

[0100] 2.1 Preparation of anti-human EZH2 antibody protein

[0101] The heavy chain variable region sequence (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) of the anti-human EZH2 monoclonal antibody and the heavy chain constant region sequence of the publicly published human monoclonal antibody IgG1 subclass were spliced ​​together and constructed into a mammalian cell expression vector; the light chain variable region sequence (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6) of the anti-human EZH2 monoclonal antibody and the light chain constant region sequence of the publicly published human monoclonal antibody κ subclass were spliced ​​together and constructed into a mammalian cell expression vector. The constructed heavy chain vector and light chain vector of the anti-human α chimeric antibody were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified using a Protein A purification kit (Abcam, ab102784) to obtain the anti-human EZH2 chimeric antibody protein.

[0102] 2.2 Preparation of Antibody Conjugates

[0103] The anti-human EZH2 antibody protein was mixed with N-hydroxysuccinimide-streptavidin (NHS-SA) to prepare an antibody conjugate.

[0104] The anti-EZH2 antibody protein was mixed with NHS-SA at a molar ratio of 1:3 and incubated at 37°C for 2 h; then, an ultrafiltration tube was used to separate the cross-linked EZH2 antibody protein from NHS-SA.

[0105] The concentration of the antibody conjugate was determined using Bradford and adjusted to 2 μM by centrifugation / dilution.

[0106] 2.3 Preparation of nanozyme conjugates

[0107] In this embodiment, the MOF nanozyme is synthesized by the solution heat method. First, the metal ion copper ion and the organic ligand pyridine are dissolved in the organic solvent chloroform at a molar ratio of 1:2 to form a mixed solution. Then, the mixed solution is packaged into a sealed container and heated at 60°C for 90 minutes. During the heating process, the metal ions and the organic ligands undergo a coordination reaction to form MOF crystals.

[0108] The nanozyme was mixed with thiol-biotin SH-PEG-Biotin to prepare a nanozyme conjugate.

[0109] Nanozyme and SH-PEG-Biotin were mixed at a molar ratio of 1:3 and incubated at 37°C for 2 h; then, an ultrafiltration tube was used to separate the cross-linked nanozyme and SH-PEG-Biotin.

[0110] 2.4 Preparation of SA-TSA conjugate

[0111] Streptavidin SA was mixed with N-hydroxysuccinimide-tyramide (NHS-TSA) to prepare SA-TSA conjugate.

[0112] SA and NHS-TSA were mixed at a molar ratio of 1:3 and incubated at 37°C for 2 h; then, the cross-linked SA and NHS-TSA were separated using an ultrafiltration tube.

[0113] 2.5 Binding of Antibody Conjugates to Target Antigens

[0114] The paraffin sections were placed in xylene for 20 min, then placed in 95% anhydrous ethanol for 10 min, and rinsed with deionized water to remove the wax tissue.

[0115] Take the antibody conjugate prepared in 2.2 and drop it onto the dewaxed glass slide (1 cm 2 40 μL was added dropwise). The slides were incubated at 37°C for 30 min, and then washed with 1× PBS.

[0116] 2.6 Binding of Nanozyme Conjugates to Antibody Conjugates

[0117] Take the nanozyme conjugate prepared in 2.3 and drop it onto the glass slide (1 cm 2 40 μL was added dropwise). The slides were incubated at 37°C for 30 min, and then washed with 1× PBS.

[0118] 2.7SA-TSA covalently modifies neighboring proteins

[0119] Take the SA-TSA prepared in 2.4 and drop it onto the glass slide cleaned in 2.6 (1 cm 2 40 μl was added dropwise). The slides were incubated at 37°C for 30 min, and then washed with 1× PBS.

[0120] 2.8DAB color development

[0121] Prepare DAB colorimetric solution at a ratio of DAB A solution: DAB B solution = 1:20. Then add it dropwise onto the glass slide (1 cm 2 Add 40 μL). Let the color develop for 2 to 5 minutes. Put the sections into tap water to terminate the reaction.

[0122] 2.9 Counterstaining, differentiation, dehydration, transparency, and sealing

[0123] The following steps are routine immunohistochemistry operations:

[0124] After staining, the sections were rinsed in tap water for 2 minutes, stained in hematoxylin for 10 seconds to 60 seconds, and rinsed in running water for 2 to 5 minutes. Differentiation solution (75% ethanol: concentrated hydrochloric acid = 99:1) was used for differentiation for 2 seconds, and then rinsed in running water for 2 minutes.

[0125] Soak the slices in 85% ethanol for 3 minutes, then in 95% ethanol for 3 minutes, then in anhydrous ethanol for 3 minutes, then in anhydrous ethanol for 3 minutes, then take out the slices and air dry them. Put the slices in xylene to make them transparent, and seal them with neutral resin and coverslips. Observe under a microscope.

[0126] Figure 5 This is a purification characterization diagram of the EZH2 protein antibody protein. Western Blot shows that the antibody recognizes EZH2, which is highly expressed in RB and UM cells and lowly expressed in normal cells.

[0127] Figure 6 Transmission electron microscopy characterization of metal framework MOF nanozymes. Figure 6 The results showed that the synthesized MOF material was uniform in size, with an average particle size of about 250 nm and a typical polyhedral structure.

[0128] Figure 7The figures are examples of using positive tissues / cells to verify the feasibility of the system. Part (a) shows that EZH2 is highly expressed in human UM tissues; Part (b) shows that EZH2 is lowly expressed in human eye tissues.

[0129] Figure 8 This is an example of in situ detection of pathological tissue of intraocular malignant tumors. Immunohistochemistry shows that EZH2 is significantly overexpressed in RB tissue.

[0130] According to the above test results, it can be known that the anti-EZH2 protein antibody provided by the present invention can specifically bind to the EZH2 protein and has good binding activity.

[0131] The anti-EZH2 protein antibody provided by the present invention is used for the detection of intraocular malignant tumors, and has good clinical application scenarios and practical value. Specifically, for example, the antibody can not only be used for early diagnosis, but also assist in pathological analysis and formulation of treatment strategies. It has particularly important clinical application value for rare or difficult-to-diagnose ocular tumors, can significantly improve the accuracy and reliability of diagnosis, and provide patients with more effective treatment options.

[0132] The EZH2 protein detection product / method based on nanozyme and TSA-mediated cascade amplification provided by the present invention allows researchers to conveniently conduct immunohistochemical studies on the expression of EZH2 protein in pathological samples, and simultaneously obtain the tissue morphology and gene expression information of the samples, thereby improving the accuracy and comprehensiveness of pathological diagnosis. The method / product can quickly and visually detect a variety of intraocular malignant tumors, has the characteristics of simple operation, high sensitivity, and strong specificity, and has good clinical application scenarios and practical value.

[0133] The above disclosure is only the preferred embodiment of the present invention. The preferred embodiment does not describe all the details in detail, nor does it limit the invention to the specific implementation method described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present invention. The present invention is limited only by the claims and their full scope and equivalents.

[0134] Under the guidance of the present invention and the above-mentioned embodiments, it is easy for those skilled in the art to foresee that each raw material or its equivalent substitute, each processing method or its equivalent substitute listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of each raw material and processing method can realize the present invention, and the embodiments are not listed one by one here.

Claims

1. An anti-EZH2 protein antibody, characterized in that: Its heavy chain variable region contains the following complementarity determining regions: HCDR1 with a nucleotide sequence as shown in SEQ ID NO: 1, HCDR2 with a nucleotide sequence as shown in SEQ ID NO: 2, and HCDR3 with a nucleotide sequence as shown in SEQ ID NO: 3; its light chain variable region sequence contains the following complementarity determining regions: LCDR1 with a nucleotide sequence as shown in SEQ ID NO: 4, LCDR2 with a nucleotide sequence as shown in SEQ ID NO: 6, and LCDR3 with a nucleotide sequence as shown in SEQ ID NO:

5.

2. A nucleic acid molecule, characterized in that It comprises: a nucleotide sequence encoding the antibody according to claim 1.

3. A recombinant vector, characterized in that: It comprises the isolated nucleic acid molecule according to claim 2.

4. A recombinant host cell, characterized in that Comprising the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 3.

5. An antibody conjugate, characterized in that: The antibody conjugate comprises the anti-EZH2 protein antibody according to claim 1; the antibody is labeled with a marker.

6. Use of the anti-EZH2 protein antibody according to claim 1 or the antibody conjugate according to claim 5 in preparing a product for detecting EZH2 protein.

7. EZH2 protein detection product based on nanozyme and TSA-mediated cascade amplification, characterized in that: It comprises the antibody conjugate and the dual signal amplification system as described in claim 5, wherein the dual signal amplification system comprises a nanozyme conjugate and a SA-TSA conjugate.

8. The EZH2 protein detection product based on nanozyme and TSA-mediated cascade amplification according to claim 7, characterized in that: The nanozyme conjugate comprises a metal organic framework nanozyme, and the metal organic framework nanozyme is marked with a marker.

9. A method for detecting EZH2 protein, using the EZH2 protein detection product according to any one of claims 7-8, characterized in that: The following steps are involved: The sample to be tested is mixed with the antibody conjugate and then incubated, and then washed; Add nanozyme conjugates, incubate, and wash; SA-TSA conjugate is added to catalyze TSA to covalently bind to the target protein, and a color developing solution is added to locate, qualitatively, and quantitatively detect the EZH2 protein in the sample.

Images