Anti-pr recombinant rabbit monoclonal antibody and application thereof

Abstract

The application belongs to the technical field of immunochemistry, and particularly relates to an anti-PR recombinant rabbit monoclonal antibody and application thereof. The anti-PR 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: 4. The amino acid sequence of the light chain variable region is shown as SEQ ID NO: 5. The application further relates to a nucleotide sequence encoding the anti-PR recombinant rabbit monoclonal antibody, a recombinant plasmid or an expression vector, a preparation method and application of the anti-PR recombinant rabbit monoclonal antibody in a PR protein detection method or device. The anti-PR recombinant rabbit monoclonal antibody has the characteristics of good specificity and strong positive signal, and is easier to score in IHC staining and more accurate in distinguishing cancer.

Description

Technical Field

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

[0002] PR protein, or progesterone receptor (PR), is a core transcription factor mediating the biological effects of progesterone (P4). This protein has two isoforms: PR-A (approximately 94 kDa) and PR-B (approximately 110 kDa). PR is a dual-function protein, acting as a ligand-dependent transcription factor directly interacting with nuclear DNA to regulate transcription, and also as a non-transcription factor regulating extranuclear signaling pathways. By binding to progesterone, PR protein regulates downstream gene expression and is widely involved in physiological processes such as female reproductive system development (e.g., uterus and breast), pregnancy maintenance, and embryo implantation; it plays a crucial role in mediating and regulating the function of the ovary, uterus, and breast, as well as reproductive activities. Studies have shown that PR dysfunction is closely related to the occurrence and development of various reproductive system diseases and hormone-dependent tumors, especially breast cancer and endometrial diseases, where it has key pathological significance and serves as an important molecular marker for disease diagnosis, classification, and treatment strategy development.

[0003] Currently, immunohistochemistry (IHC) is commonly used in clinical practice for in situ detection of PR proteins in tissues. Microscopic observation of their intracellular expression allows for assessment of any abnormal PR expression. However, existing anti-PR antibodies have significant limitations in sensitivity and specificity, particularly in differentiating between PR-A and PR-B subtypes, identifying low-expression samples, and reducing non-specific staining. This leads to high subjectivity and poor reproducibility in IHC staining results during pathological evaluation, impacting the accuracy of disease classification and treatment decisions based on PR status.

[0004] Therefore, developing an anti-PR antibody with high sensitivity, strong specificity, and suitability for pathological testing scenarios such as IHC is of great practical significance for achieving accurate identification of PR proteins, improving the reliability of related disease diagnosis, and promoting the formulation of personalized treatment strategies. 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-PR recombinant rabbit monoclonal antibody and its application. The present invention also relates to the nucleotide sequence encoding the anti-PR recombinant rabbit monoclonal antibody, the recombinant plasmid or expression vector, the preparation method, and the application of the anti-PR recombinant rabbit monoclonal antibody in PR 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 an anti-PR recombinant rabbit monoclonal antibody, 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:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

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

[0011] The anti-PR monoclonal antibody was obtained through recombinant expression in mammalian cells. Specifically, the anti-PR 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-PR 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 PR protein.

[0012] The anti-PR monoclonal antibody can recognize recombinant PR antigen protein and PR molecules on tumor cells and immune cells; the anti-PR 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-PR recombinant rabbit monoclonal antibody.

[0014] Preferably, the encoding gene includes a DNA sequence as shown in SEQ ID NO:2 or a complementary sequence thereto, for encoding the heavy chain variable region of the anti-PR recombinant rabbit monoclonal antibody; and a DNA sequence as shown in SEQ ID NO:3 or a complementary sequence thereto, for encoding the light chain variable region of the anti-PR recombinant rabbit monoclonal antibody.

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

[0019] Seventhly, the application of the aforementioned anti-PR recombinant rabbit monoclonal antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid in the preparation of a PR 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 PR detection kit, which includes the above-mentioned anti-PR recombinant rabbit monoclonal antibody and immunohistochemical detection reagent.

[0021] Preferably, the PR detection kit includes: anti-PR 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.

[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 PR provided by this invention exhibits high specificity and sensitivity in binding to PR protein molecules. It can specifically recognize and detect PR protein expression on cells, showing high positive expression when detecting PR protein. Immunohistochemical assays in various tissues have revealed that this antibody can be applied to immunohistochemistry (IHC), indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody chip preparation, flow cytometry, and other detection and screening methods, facilitating accurate assessment and detection results. The PR recombinant rabbit monoclonal antibody of the 278G8D6 clone 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 278G8D6 anti-PR monoclonal antibody prepared in this invention and a commercially available antibody in ovarian cancer, breast cancer, and uterine tissues. The concentration of the 278G8D6 anti-PR monoclonal antibody used was 1 μg / mL. Image a shows the immunohistochemical staining results of the 278G8D6 clone PR antibody in ovarian cancer; image b shows the immunohistochemical staining results of the commercially available antibody in ovarian cancer (images a and b are at the same magnification); image c shows the immunohistochemical staining results of the 278G8D6 clone PR 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 278G8D6 clone PR antibody in uterine tissue; image f shows the immunohistochemical staining results of the commercially available antibody in uterine tissue (images e and f are at the same magnification).

[0026] Figure 2 This is a statistical graph showing the titer of the 278G8D6 anti-PR monoclonal antibody of the present invention and commercially available antibodies at eight different concentration gradients.

[0027] Figure 3 The Western blotting results of the 278G8D6 anti-PR monoclonal antibody of this invention as a primary antibody were used to verify its ability to recognize the PR 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 anti-PR recombinant rabbit monoclonal antibodies, including the following steps:

[0032] (1) Antigen preparation

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

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

[0035] The aforementioned polypeptide sequence was selected based on the analysis of the PR molecule sequence, considering its structure, antigenicity, hydrophilicity / hydrophobicity of constituent amino acids, and secondary structure. 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 polypeptide with the sequence shown in SEQ ID NO:1 was conjugated with hemocyanin (KLH) and used as the PR antigen to immunize rabbits.

[0036] (2) Immunity

[0037] The PR 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 PR antigen containing the above sequence (the 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 PR antigen (SEQ ID NO:1). The corresponding cell line numbers were labeled, and the cells in the positive wells were subjected to limiting dilution until the entire 96-well plate showed a positive result for the ELISA test. Stable monoclonal lines with high positive values ​​were selected to obtain hybridoma cell lines secreting specific monoclonal antibodies, recorded as 278G8D6.

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

[0043] Total RNA was isolated from 278G8D6 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:6), VH-R GTGAGGGTGCCCGAG (SEQ ID NO:7); and VK-F ATGGACAYGAGGGCCCCCACTC (SEQ ID NO:8), VK-R GGTGGGAAGATGAGGACAGTAGG (SEQ ID NO:9). The nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region were then cloned into eukaryotic expression vectors (InvivoGen, pfuse-rchg, pfuse2-rclk1) 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 278G8D6 anti-PR recombinant rabbit monoclonal antibody is shown in SEQ ID No. 2, and the light chain variable region nucleotide sequence of the anti-PR recombinant rabbit monoclonal antibody is shown in SEQ ID NO: 3.

[0050] The specific sequences of SEQ ID NO:2 and SEQ ID NO:3 are as follows:

[0051] The sequence of SEQ ID NO:2 is:

[0052] cagtcgctggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcagtagctactacatgagctgggtccgccaggctccaggggaggggctggaatggatcggaaccattagtgatggtgggcgc acatggtacgcgagctgggcgaaaggccgattcaccatctccgaaacctcgaccacggtggatctgaaaatgtccagtctgacaaccgaggacacggccacctatttctgtgtcagaagtgaaggttcattgtggggccaaggcaccctggtcaccgtctcctca.

[0053] The sequence of SEQ ID NO:3 is:

[0054] gatgttgtgatgacccagactccagcctccgtggaggcagctgtgggaggcacagtcaccatcaagtgccaggccagtcagagcattagcagttggttatcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatagggcatccactctggaatct ggggtcccatcgcggttcaaaggcagtggatctgggacagagttcactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaatgcacttatggtagtagtagtagtagtaatggtggagttttcggcggagggaccgaggtggtggtcaaa.

[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 278G8D6 anti-PR recombinant rabbit monoclonal antibody was shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the anti-PR recombinant rabbit monoclonal antibody was shown in SEQ ID NO:5.

[0056] The specific sequences of SEQ ID NO:4 and SEQ ID NO:5 are as follows:

[0057] The sequence of SEQ ID NO:4 is:

[0058] QSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMSWVRQAPGEGLEWIGTISDGGRTWYASWAKGRFTISETSTTVDLKMSSLTTEDTATYFCVRSEGSLWGQGTLVTVSS.

[0059] The sequence of SEQ ID NO:5 is:

[0060] DVVMTQTPASVEAAVGGTVTIKCQASQSISSWLSWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGSSSSSNGGVFGGGTEVVVK.

[0061] Example 2

[0062] This example demonstrates immunohistochemical detection using anti-PR recombinant rabbit monoclonal antibody as the primary antibody, and the method is as follows:

[0063] (1) Sample preparation: The ovarian cancer, breast and uterine tissue sections fixed in formalin and embedded in paraffin were baked in a constant temperature oven at 60℃ 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 minutes (if using an automatic retrieval instrument, you can set it to 98℃ for 20 minutes). After the slides have cooled to room temperature, circle the tissue to be tested with an immunohistochemical pen and rinse twice with purified water for 3 minutes 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 278G8D6 anti-PR 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] The PR antibody prepared in this application and a commercially available PR antibody were used to perform the above-mentioned immunohistochemical detection in human ovarian cancer, breast, and uterine tissues, respectively. The results are shown in the figure. Figure 1 .

[0075] Depend on Figure 1 The results showed that PR protein exhibited specific nuclear staining in human ovarian cancer, breast, and uterine tissues. Furthermore, the PR recombinant rabbit monoclonal antibody of the 278G8D6 clone showed better staining results and a deeper staining color than commercially available PR antibodies. This indicates that the PR recombinant rabbit monoclonal antibody of the 278G8D6 clone 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.

[0076] Example 3

[0077] This example describes the determination of the affinity of the 278G8D6 anti-PR recombinant rabbit monoclonal antibody. The determination method is as follows:

[0078] (1) Remove the labeled PR 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 and incubate overnight at 4℃. Then block with 2% bovine serum albumin (BSA) overnight at 4℃.

[0079] (2) The PR recombinant rabbit monoclonal antibody of clone 278G8D6 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;

[0080] (3) Add the diluted anti-PR 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.

[0081] (4) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0082] (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.

[0083] (6) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0084] (7) Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution at 100 μL / well and react at room temperature for 6 minutes;

[0085] (8) After the reaction is complete, add 2M H2SO4 at a rate of 50μL / well to stop the color development;

[0086] (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.

[0087] Figure 2 The results showed that, in eight concentration gradient experiments, the anti-PR recombinant rabbit monoclonal antibody of the 278G8D6 clone of the present invention had a strong affinity and high sensitivity for PR protein molecules, and could still achieve a high OD value under low antibody concentration conditions, which can save experimental and detection costs.

[0088] Example 4

[0089] This example demonstrates the detection using Western blotting with anti-PR recombinant rabbit monoclonal antibody 278G8D6 as the primary antibody. The method is as follows:

[0090] (1) Activation was performed using a polyvinylidene fluoride (PVDF) membrane containing T47D cell lysate. The membrane was activated with methanol for 1 min, washed twice with pure water, and then washed three times with TBST. Blocking: The membrane was placed in a blocking solution prepared with 5% bovine serum albumin (BSA) and shaken 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.

[0091] (2) Primary antibody incubation: Dilute the 278G8D6 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.

[0092] (3) Remove the membrane and wash it in TBST solution 3 times (2×5min+1×10min).

[0093] (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;

[0094] (5) Remove the membrane strip and wash it in TBST solution 4 times (3×5min+1×8min).

[0095] (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;

[0096] (7) Exposure: Place the negative film in a cassette and expose the X-ray film for different periods according to the fluorescence intensity; then perform operations in the order of 1 minute of development, washing, 1 minute of fixation, and finally wash and dry; The results are as Figure 3 shown.

[0097] There are two subtypes of PR proteins, PR-A and PR-B, and their theoretical molecular weights are around 94 kDa and 110 kDa. Figure 3 In

[0097] , T47D represents the lysate of human breast ductal carcinoma cells. From Figure 3 the results, it can be seen that there are PR positive bands in the T47D lysate. That is, in the lane, the 278G8D6 anti-PR recombinant rabbit monoclonal antibody can specifically recognize the PR protein in T47D cells, indicating that the 278G8D6 cloned PR recombinant rabbit monoclonal antibody of the present invention can highly specifically recognize the PR protein.

[0098] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant rabbit monoclonal antibody against PR protein, 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:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

2. A gene encoding a gene, characterized in that, Used to encode the recombinant rabbit monoclonal antibody against the anti-PR protein as described in claim 1.

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

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 PR protein, 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 recombinant rabbit monoclonal antibody against PR protein.

8. The application of the anti-PR protein 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 PR protein detection device.

9. A PR protein detection kit, characterized in that, It includes the recombinant rabbit monoclonal antibody against PR protein as described in claim 1.

10. The PR protein detection kit according to claim 9, characterized in that, The test kit also includes: horseradish peroxidase-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, 3,3'-diaminophenylhydrazine concentrate, 3,3'-diaminophenylhydrazine buffer, hematoxylin, and blueing solution.

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