Anti-gata3 recombinant rabbit monoclonal antibody and application thereof

Abstract

The application belongs to the technical field of immunochemistry, and particularly relates to an anti-GATA3 recombinant rabbit monoclonal antibody and application thereof. The anti-GATA3 recombinant rabbit monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:8. The amino acid sequence of the light chain variable region is shown as SEQ ID NO:9. The application also relates to nucleotide sequences SEQ ID NO:6 and SEQ ID NO:7 encoding the anti-GATA3 recombinant rabbit monoclonal antibody, a nucleic acid molecule, an expression vector or a recombinant plasmid, a host cell, a preparation method of the antibody, and application of the antibody in a GATA3 protein detection method or device. The GATA3 recombinant rabbit monoclonal antibody of the application has the characteristics of good specificity and strong positive signal, and is easier to score in IHC staining, and is more accurate in distinguishing cancer.

Description

Technical Field

[0001] This invention belongs to the field of immunochemical technology, and particularly relates to an anti-GATA3 recombinant rabbit monoclonal antibody and its applications, especially in immunohistochemical detection. Background Technology

[0002] GATA3 (GATA-binding protein 3) is a key member of the GATA transcription factor family. It is a highly conserved zinc finger transcription factor with a molecular weight of approximately 48 kDa, primarily located in the cell nucleus. The core function of GATA3 protein is to regulate gene transcription by binding to the regulatory regions of target genes, thereby participating in cell development, differentiation fate determination, and tissue phenotype maintenance. Its function exhibits significant tissue specificity.

[0003] GATA3 expression exhibits extremely strict tissue specificity, with high expression primarily in two major systems: epithelial tissues such as the breast and kidneys, and hematopoietic immune cells such as T lymphocytes and NK cells. In non-target tissues, GATA3 is expressed at low levels or absently. This strict spatiotemporal expression specificity endows GATA3 with extremely high pathological diagnostic value, making it a core immunohistochemical marker for the clinical differential diagnosis of epithelial tumors (especially breast cancer), and also serving as an auxiliary indicator for prognostic assessment of some tumors. Abnormal GATA3 protein expression (high expression / deletion) is closely related to tumor occurrence and development; its nuclear expression characteristics and tissue specificity make it a core marker for the differential diagnosis of epithelial tumors in clinical pathology, and also an auxiliary indicator for prognostic assessment of some tumors.

[0004] Currently, the detection of GATA3 protein mainly relies on immunohistochemistry. However, existing anti-GATA3 antibodies have limited ability to capture low-abundance targets, often resulting in false negatives due to insufficient sensitivity and missed detection of weakly positive samples. This deficiency particularly affects the accurate identification of early-stage tumors, poorly differentiated tumors, and some metastatic lesions, limiting the clinical efficacy of GATA3 as a diagnostic marker. Therefore, developing an anti-GATA3 antibody reagent that can efficiently identify GATA3 protein in tissue samples, has higher sensitivity and stronger specificity, and can adapt to routine clinical sample processing conditions is of significant practical importance and clinical application value for improving the diagnostic accuracy of epithelial tumors and promoting in-depth basic and clinical research related to GATA3. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a widely applicable and accurately identifiable anti-GATA3 recombinant rabbit monoclonal antibody and its application. The present invention also relates to the nucleotide sequence encoding the anti-GATA3 recombinant rabbit monoclonal antibody, the recombinant plasmid or expression vector, the preparation method, and the application of the anti-GATA3 recombinant rabbit monoclonal antibody in GATA3 protein detection methods or devices.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a recombinant rabbit monoclonal antibody against GATA3, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9.

[0010] This recombinant rabbit monoclonal antibody against GATA3 (GATA3 rabbit-derived antibody) can be used for immunohistochemical detection, and can identify and detect the expression of GATA3 protein on tumor cells or immune cells with high specificity and high sensitivity.

[0011] The anti-GATA3 monoclonal antibody was obtained through recombinant expression in mammalian cells. Specifically, the anti-GATA3 recombinant rabbit monoclonal antibody provided by this invention was produced through rabbit hybridoma fusion screening and eukaryotic expression in 293 cells. In preparing the anti-GATA3 monoclonal antibody, the antigen used to immunize the rabbits (New Zealand White rabbits) was a synthetic polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1, and it was obtained through artificial chemical synthesis. After immunizing the rabbits, cell fusion and clonal screening were performed to obtain a positive hybridoma cell line that could efficiently secrete monoclonal antibodies. Molecular cloning technology was used to obtain the nucleotide sequences encoding the heavy chain and light chain amino acid sequences of the antibody. The nucleotide sequences were constructed on a eukaryotic expression vector, transfected into the 293 cell line using a transfection reagent, and the cell supernatant was collected and purified by protein A column affinity chromatography to obtain the rabbit monoclonal antibody. Immunohistochemical detection showed that the antibody specifically recognizes the GATA3 protein.

[0012] The anti-GATA3 monoclonal antibody can recognize recombinant GATA3 antigen protein and GATA3 molecules on tumor cells and immune cells; the anti-GATA3 monoclonal antibody can also be used in immunohistochemical pathological diagnostic agents.

[0013] Secondly, the present invention provides a coding gene for encoding the above-mentioned anti-GATA3 recombinant rabbit monoclonal antibody.

[0014] Preferably, the encoding gene includes a DNA sequence as shown in SEQ ID NO:6 or a complementary sequence thereto, for encoding the heavy chain variable region of the anti-GATA3 recombinant rabbit monoclonal antibody; and a DNA sequence as shown in SEQ ID NO:7 or a complementary sequence thereto, for encoding the light chain variable region of the anti-GATA3 recombinant rabbit monoclonal antibody.

[0015] Thirdly, the present invention provides a nucleic acid molecule comprising a coding gene for encoding the anti-GATA3 recombinant rabbit monoclonal antibody.

[0016] Fourthly, the present invention provides an expression vector or recombinant plasmid comprising the above-described nucleic acid molecule.

[0017] Fifthly, the present invention provides a host cell that has been transformed or transfected with the above-mentioned expression vector or recombinant plasmid.

[0018] In a sixth aspect, the present invention provides a method for preparing a recombinant rabbit monoclonal antibody against GATA3, wherein the above-mentioned expression vector or recombinant plasmid is used to transform or transfect the above-mentioned host cells, the transformed or transfected cells are cultured, the cell supernatant is collected and purified, and the recombinant rabbit monoclonal antibody against GATA3 is obtained.

[0019] Seventhly, the application of the aforementioned anti-GATA3 recombinant rabbit monoclonal antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid in the preparation of a GATA3 protein molecule detection device. The detection device includes, but is not limited to, reagent kits, antibody chips, etc.

[0020] Eighthly, the present invention also provides a GATA3 detection kit, which includes the above-mentioned anti-GATA3 recombinant rabbit monoclonal antibody and immunohistochemical detection reagent.

[0021] Preferably, the GATA3 detection kit comprises: anti-GATA3 recombinant rabbit monoclonal antibody, horseradish peroxidase-labeled secondary antibody, EDTA-1 retrieval solution, catalase blocking solution, 3,3'-diaminophenylhydrazine concentrate, 3,3'-diaminophenylhydrazine buffer, hematoxylin, and blueing solution.

[0022] The immunohistochemical testing process includes dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, 3,3'-diaminophenylhydrazine (DAB) staining, counterstaining, dehydration, mounting, and microscopic examination.

[0023] (III) Beneficial Effects

[0024] The recombinant rabbit monoclonal antibody against GATA3 provided by this invention exhibits high specificity and sensitivity in binding to GATA3 protein molecules. It can specifically recognize and detect the expression of GATA3 protein on cells, showing high positive expression when detecting GATA3 protein, while significantly reducing background interference. This effectively solves the problems of "non-specific staining" and "narrow interpretation window" that are common with traditional antibodies. Immunohistochemical assays of various tissues have shown that this antibody can be applied to immunohistochemistry (IHC), indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody chip preparation, and other detection and screening fields, facilitating accurate assessment and detection results. The 255B6B8 clone of the recombinant rabbit monoclonal antibody against GATA3 of this invention, due to its high specificity and strong positive signal, is easier to score in IHC staining, making it more accurate for detecting and differentiating cancers. Attached Figure Description

[0025] Figure 1 These are immunohistochemical staining results of the 255B6B8 anti-GATA3 monoclonal antibody prepared in this invention and a commercially available antibody in tonsil, breast cancer, and kidney tissues. The concentration of the 255B6B8 anti-GATA3 monoclonal antibody used was 1 μg / mL. Image a shows the immunohistochemical staining results of the 255B6B8 clone GATA3 antibody in the tonsil; image b shows the immunohistochemical staining results of the commercially available antibody in the tonsil (images a and b are at the same magnification); image c shows the immunohistochemical staining results of the 255B6B8 clone GATA3 antibody in breast cancer; image d shows the immunohistochemical staining results of the commercially available antibody in breast cancer (images c and d are at the same magnification); image e shows the immunohistochemical staining results of the 255B6B8 clone GATA3 antibody in kidney tissue; image f shows the immunohistochemical staining results of the commercially available antibody in kidney tissue (images e and f are at the same magnification).

[0026] Figure 2 This is a statistical graph showing the titer of the 255B6B8 anti-GATA3 monoclonal antibody of the present invention and commercially available antibodies at eight different concentration gradients.

[0027] Figure 3 The Western blotting results of the 255B6B8 anti-GATA3 monoclonal antibody of this invention as a primary antibody were used to verify its ability to recognize the GATA3 protein. Detailed Implementation

[0028] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Human tissue samples are formalin-fixed and paraffin-embedded human tissue samples, all of which have been pathologically verified, and informed consent has been obtained from the patients.

[0030] Example 1

[0031] This embodiment describes the preparation and screening of recombinant rabbit monoclonal antibodies against GATA3. The steps include:

[0032] (1) Antigen preparation

[0033] The specific sequence of the GATA3 antigen is shown in SEQ ID NO:1 below.

[0034] SEQ ID NO:1 is: LHSYMDAAQ.

[0035] The aforementioned polypeptide sequence was selected based on the analysis of the GATA3 molecular sequence, considering the structure, antigenicity, hydrophilicity / hydrophobicity of the constituent amino acids, and secondary structure of the GATA3 protein molecule. The polypeptide with the sequence shown in SEQ ID NO:1 was artificially synthesized and used as an antigen for immunizing rabbits. During immunization, the GATA3 antigen polypeptide with the sequence shown in SEQ ID NO:1 was conjugated with hemocyanin (KLH) and used as the GATA3 antigen to immunize rabbits.

[0036] (2) Immunity

[0037] The GATA3 antigen obtained in step (1) was mixed with complete Freund's adjuvant (volume ratio 1:1) and emulsified. Multiple New Zealand white rabbits were immunized by subcutaneous injection. Two weeks later, the GATA3 antigen containing the above sequence (GATA3 antigen polypeptide shown in SEQ ID NO:1) was emulsified with incomplete Freund's adjuvant (volume ratio 1:1) for a second and third immunization. After the three immunizations, blood was collected and serum titers were determined by serial dilution using ELISA. The rabbit with the highest antibody titer against the SEQ ID NO:1 antigen was selected for the next step of cell fusion.

[0038] (3) Cell fusion

[0039] Prepare mouse-derived sp2 / 0 myeloma cells in advance, ensuring they are in the logarithmic growth phase at the time of fusion. Immunized rabbit spleens were used to prepare a single-cell suspension of lymphocytes. The rabbit spleen lymphocytes were mixed with the myeloma cells, and 50% PEG1500 was added. IMDM medium was added, and after centrifugation and discarding the supernatant, the cells were gently resuspended and mixed in HAT medium. The volume was adjusted to 800 mL, and the mixture was aliquoted into 96-well plates and incubated at 37°C with 5% CO2. The fused cell status in the 96-well plates was observed 6-9 days after fusion. The medium was changed with HT, and the plates were continued to be incubated at 37°C with 5% CO2.

[0040] (4) Screening and cloning

[0041] Seven to ten days after fusion, clonal cells were screened using an ELISA test with the GATA3 antigen (SEQ ID NO:1). The corresponding cell line numbers were labeled, and the positive wells were subjected to limiting dilution until the entire 96-well plate showed a positive ELISA result. Stable monoclonal lines with high positive values ​​were selected to obtain hybridoma cell lines secreting specific monoclonal antibodies, recorded as 255B6B8.

[0042] (5) Perform antibody sequencing on the selected hybridoma cell lines.

[0043] Total RNA was isolated from 255B6B8 hybridoma cells according to the TriZol RNA extraction reagent instructions. Following the instructions of the TIANScript first-strand cDNA synthesis kit, the total RNA was reverse transcribed into cDNA. The nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were amplified using specific primers. The primers for the heavy chain variable region were: VH-F AGACTGGGCTGCGCTGGCTTC (SEQ ID NO:2), VH-R GTGAGGGTGCCCGAG (SEQ ID NO:3); and VK-F ATGGACAYGAGGGCCCCCACTC (SEQ ID NO:4), VK-R GGTGGGAAGATGAGGACAGTAGG (SEQ ID NO:4). NO:5) Amplify the nucleotide sequences of the antibody heavy chain variable region and the antibody light chain variable region, and then clone the nucleotide sequences of the antibody heavy chain variable region and the antibody light chain variable region into the eukaryotic expression vector (InvivoGen, pfuse-rchg, pfuse2-rclk1) in preparation for cell transfection.

[0044] (6) Cell transfection and screening

[0045] Prepare 293 cells for transfection in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 24-well plates at a density of 1.5 ml per well (3 × 10⁻⁶ cells / well).6 per ml.

[0046] The eukaryotic expression vector was mixed with polyethyleneimine (PEI) at a mass ratio of 1:6 and added to the prepared 293 cells. The cells were then cultured in a shaker at 37°C and 5% CO2. After 3-5 days of culture, the transfected cell supernatant was subjected to ELISA to screen for positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunohistochemical detection. A positive immunohistochemical test confirmed the correct antibody sequence.

[0047] (7) Preparation and purification of monoclonal antibodies on cells

[0048] The confirmed positive expression vector was used to transfect a large number of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography with protein A to obtain antibodies with a purity >95%. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4-8°C.

[0049] Finally, the heavy chain variable region nucleotide sequence of the 255B6B8 anti-GATA3 recombinant rabbit monoclonal antibody is shown in SEQ ID NO:6, and the light chain variable region nucleotide sequence of the anti-GATA3 recombinant rabbit monoclonal antibody is shown in SEQ ID NO:7.

[0050] The specific sequences of SEQ ID NO:6 and SEQ ID NO:7 are as follows:

[0051] The sequence of SEQ ID NO:6 is:

[0052] cagtcgctggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggaatcgacctcagtagctatgcaatgagctgggtccgccaggctccagggaaggggctggaatggctcggaatcattggtggtagtggtacc acatactacgcgagctgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagactctggaatatctggggcccaggcaccctggtcaccgtctcctca.

[0053] The sequence of SEQ ID NO:7 is:

[0054] gcccaagtgctgacccagactgcatcgtccgtgtctgcagctgtggggggcacagtcaccatcagttgccagtccagtcagagtgttgttgataacaaccgcttatcctggtatcagcagaaaccagggcagcctcccaaaggcctgatctacgatgcatccaaa ctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcgacgtgcagtatgacgatgctgccacttactactgtctaggcggtgttagtggtaatatttatcttttcggcggagggaccgaggtggtggtcaaa.

[0055] The obtained base sequence was translated into an amino acid sequence and analyzed. The amino acid sequence of the heavy chain variable region of the 255B6B8 anti-GATA3 recombinant rabbit monoclonal antibody is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region of the anti-GATA3 recombinant rabbit monoclonal antibody is shown in SEQ ID NO:9.

[0056] The specific sequences of SEQ ID NO:8 and SEQ ID NO:9 are as follows:

[0057] The sequence of SEQ ID NO:8 is:

[0058] QSLEESGGRLVTPGTPLTLTCTASGIDLSSYAMSWVRQAPGKGLEWLGIIIGGSGTTYYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARLWNIWGPGTLVTVSS.

[0059] The sequence of SEQ ID NO:9 is:

[0060] AQVLTQTASSVSAAVGGTVTISCQSSQSVVDNNRLSWYQQKPGQPPKGLIYDASKLASGVPSRFKGSGSGTQFTLTISDVQYDDAATYYCLGGVSGNIYLFGGGTEVVVK.

[0061] Example 2

[0062] This example demonstrates immunohistochemical detection using recombinant rabbit monoclonal antibody against GATA3 as the primary antibody. The method is as follows:

[0063] (1) Sample preparation: The formalin-fixed paraffin-embedded tonsil, breast cancer and kidney tissue sections were baked in a 60℃ constant temperature oven for 1-2 hours and stored for later use;

[0064] (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 minutes each time;

[0065] (3) Hydration of sections: The sections were hydrated by soaking in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes in sequence, and then rinsed twice with purified water for 3 minutes each time.

[0066] (4) Antigen retrieval: It is recommended to use the high temperature heat retrieval method for 3 min (if using an automatic retrieval instrument, the high temperature retrieval at 98℃ for 20 min can be set). After the slides are naturally cooled to room temperature, the tissue to be tested is circled with an immunohistochemical pen and rinsed twice with purified water for 3 min each time.

[0067] (5) Inactivation of endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocking agent to completely cover the tissue, incubate at room temperature for 10 min, rinse twice with purified water for 3 min each time, and rinse once with phosphate buffer (PBST);

[0068] (6) Primary antibody incubation: Add 100 μL of 1 μg / mL 255B6B8 anti-GATA3 recombinant rabbit monoclonal antibody to completely cover the tissue, incubate in a 37℃ incubator for 1 h, and wash with PBST 3 times for 5 min each time.

[0069] (7) Secondary antibody incubation: Perform secondary antibody incubation according to the instructions of the DAB staining solution kit of the secondary antibody staining system used. After incubation, rinse the slides with PBST 3 times for 5 minutes each time, and rinse with purified water once.

[0070] (8) DAB staining: Prepare DAB staining solution according to the instructions of the DAB staining solution kit. Drop an appropriate amount of the prepared DAB staining solution to completely cover the tissue. Stop staining when the color does not deepen. Rinse 3 times with purified water.

[0071] (9) Hematoxylin counterstaining: Counterstain the sections according to the operating steps and suggestions in the instructions of the hematoxylin manufacturer, and rinse with PBST or tap water to return to blue;

[0072] (10) Dehydration and clearing: Soak in 70%, 85%, 95%, 100%, and 100% graded alcohols sequentially for 3 minutes each time; clear with xylene twice for 5 minutes each time;

[0073] (11) Mounting: Mount the sample with neutral resin.

[0074] Depend on Figure 1The results showed that GATA3 protein exhibited specific nuclear staining in human tonsil, breast cancer, and kidney tissues. Furthermore, the 255B6B8 clone of GATA3 recombinant rabbit monoclonal antibody showed better staining performance and a deeper color than commercially available GATA3 antibodies. This indicates that the 255B6B8 clone of GATA3 recombinant rabbit monoclonal antibody of this invention, due to its high specificity and strong positive signal, is easier to evaluate and has higher identification accuracy in IHC staining, making it more accurate for detecting and differentiating cancers.

[0075] Example 3

[0076] This example describes the determination of the affinity of the 255B6B8 anti-GATA3 recombinant rabbit monoclonal antibody. The determination method is as follows:

[0077] (1) Remove the labeled GATA3 peptide (SEQ ID NO:1) from 4℃ and allow it to return to room temperature. Dilute to a concentration of 1 μg / ml and add 100 μL / well to a 96-well microplate. Incubate overnight at 4℃, then block overnight at 4℃ with 2% bovine serum albumin (BSA).

[0078] (2) The GATA3 recombinant rabbit monoclonal antibody of clone 255B6B8 was diluted to an initial concentration of 0.5 μg / mL, and then serially diluted 2-fold, with a total of 8 concentration gradients for comparison.

[0079] (3) Add the diluted anti-GATA3 recombinant rabbit monoclonal antibody at 100 μL / well to a 96-well microplate containing peptides, cover with a sealing film, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium.

[0080] (4) After the reaction is complete, remove the enzyme-labeled plate, discard the liquid, rinse 5 times with purified water, and pat dry.

[0081] (5) Dilute horseradish peroxidase (HRP) labeled goat anti-rabbit IgG according to the instructions for use of the secondary antibody, add 100 μL / well to the microplate, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium.

[0082] (6) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0083] (7) Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution at 100 μL / well and react at room temperature for 6 minutes;

[0084] (8) After the reaction is complete, add 2M H2SO4 at a rate of 50μL / well to stop the color development;

[0085] (9) Read the OD value at 450 nm on the microplate reader, organize the data, and analyze the results as follows. Figure 2 As shown.

[0086] The results showed that, in eight concentration gradient experiments, the anti-GATA3 recombinant rabbit monoclonal antibody of the 255B6B8 clone of the present invention had a strong affinity for GATA3 protein molecules and high sensitivity. It could still achieve a high OD value under low antibody concentration conditions, which could save experimental and detection costs.

[0087] Example 4

[0088] This example demonstrates the detection using Western blotting with recombinant rabbit monoclonal antibody 255B6B8 against GATA3 as the primary antibody. The method is as follows:

[0089] (1) Activate the polyvinylidene fluoride (PVDF) membrane with CCRF-CEM and SH-SY5Y cell lysis buffer by activating it with methanol for 1 min, washing the membrane twice with pure water, and then washing it three times with TBST; Blocking: Place the membrane in a blocking solution prepared with 5% bovine serum albumin (BSA) and shake at room temperature for 2 h; TBST is a commonly used washing buffer suitable for experiments such as immunoblotting. It contains three basic components: Tris buffer, salt (usually sodium chloride), and the surfactant Tween-20.

[0090] (2) Primary antibody incubation: Dilute the 255B6B8 antibody to a concentration of 0.5 μg / mL, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking.

[0091] (3) Remove the membrane and wash it in TBST solution 3 times (2×5min+1×10min).

[0092] (4) Secondary antibody incubation: HRP-anti-rabbit IgG was diluted with FG solution at a ratio of 1:5000, mixed well and added to the membrane strip, and shaken at room temperature for 1 hour;

[0093] (5) Remove the membrane strip and wash it in TBST solution 4 times (3×5min+1×8min).

[0094] (6) Substrate: Mix equal volumes of luminol / enhancer solution and hydrogen peroxide solution diluted 5 times with pure water in the same container, add the membrane strip, and incubate for 2 min;

[0095] (7) Exposure: Place the film in the dark box and expose the X-ray film for different time periods according to the fluorescence intensity; then perform the operation in the order of 1 min development, 1 min cleaning, and 1 min fixing, and finally clean and dry; the results are as follows Figure 3 As shown.

[0096] The theoretical molecular weight of GATA3 protein is around 48 kDa. Figure 3 CCRF-CEM represents human T-lymphocytic leukemia cell lysate, and SH-SY5Y represents human neuroblastoma cell lysate. Figure 3 The results showed that GATA3 positive bands appeared in the lysates of CCRF-CEM and SH-SY5Y cells. That is, in the swimming lanes, the 255B6B8 anti-GATA3 recombinant rabbit monoclonal antibody could specifically recognize GATA3 protein in CCRF-CEM and SH-SY5Y cells, indicating that the 255B6B8 clone of the present invention can recognize GATA3 recombinant rabbit monoclonal antibody with high specificity.

[0097] The anti-GATA3 recombinant rabbit monoclonal antibody proposed in this invention can be prepared into a kit for GATA3 detection. The GATA3 detection kit includes: anti-GATA3 recombinant rabbit monoclonal antibody, horseradish peroxidase-labeled secondary antibody, EDTA3 retrieval solution, catalase blocking solution, 3,3'-diaminophenylhydrazine concentrate, 3,3'-diaminophenylhydrazine buffer, hematoxylin and blueing solution.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant rabbit monoclonal antibody against GATA3, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

2. A gene encoding a gene, characterized in that, Used to encode the anti-GATA3 recombinant rabbit monoclonal antibody of claim 1.

3. The encoding gene according to claim 2, characterized in that, It includes: DNA sequences as shown in SEQ ID NO:6 are used to encode the heavy chain variable region of the anti-GATA3 recombinant rabbit monoclonal antibody, and DNA sequences as shown in SEQ ID NO:7 are used to encode the light chain variable region of the anti-GATA3 recombinant rabbit monoclonal antibody.

4. A nucleic acid molecule, characterized in that, It contains the coding gene as described in claim 2 or 3.

5. An expression carrier, characterized in that, It contains the nucleic acid molecule as described in claim 4.

6. Transform or transfect the host cells of the expression vector according to claim 5.

7. A method for preparing a recombinant rabbit monoclonal antibody against GATA3, characterized in that, The expression vector described in claim 5 is used to transform or transfect host cells, the transformed or transfected cells are cultured, the cell supernatant is collected and purified to obtain the anti-GATA3 recombinant rabbit monoclonal antibody.

8. The application of the anti-GATA3 recombinant rabbit monoclonal antibody of claim 1, the encoding gene of claim 2 or 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, and the host cell of claim 6 in the preparation of a GATA3 detection device.

9. A GATA3 detection kit, characterized in that, It includes the anti-GATA3 recombinant rabbit monoclonal antibody and immunohistochemical detection reagent as described in claim 1.

10. The GATA3 detection kit according to claim 9, characterized in that, It includes: The anti-GATA3 recombinant rabbit monoclonal antibody, horseradish peroxidase-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, 3,3'-diaminophenylhydrazine concentrate, 3,3'-diaminophenylhydrazine buffer, hematoxylin and blueing solution as described in claim 1.

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