Anti-er recombinant rabbit monoclonal antibody and application thereof

Abstract

The application belongs to the technical field of immunochemistry, and particularly relates to an anti-ER recombinant rabbit monoclonal antibody and application thereof. The anti-ER 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 further relates to a nucleotide sequence encoding the anti-ER recombinant rabbit monoclonal antibody, a recombinant plasmid or an expression vector, a preparation method and application of the anti-ER recombinant rabbit monoclonal antibody in an ER protein detection method or device. The 658G3A2 clone ER recombinant rabbit monoclonal antibody of the application is good in specificity, strong in positive signal and the like, and is easier to score in IHC staining and more accurate in cancer detection and differentiation.

Description

Technical Field

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

[0002] The estrogen receptor (ER), a nuclear receptor protein with a molecular weight of approximately 66 kDa, is a core molecule mediating the biological effects of estrogen. It activates downstream signaling pathways by binding to estrogen, regulating various physiological processes such as cell proliferation, differentiation, apoptosis, and metabolism. It is widely involved in physiological activities including reproductive system development, skeletal homeostasis, and cardiovascular function regulation. Furthermore, ER dysfunction is closely related to the development and progression of various diseases, particularly tumors, metabolic diseases, and reproductive system diseases, where it holds significant pathological importance. Overactivation of the ER is a key driver of the development and progression of hormone-dependent tumors, typically breast cancer and endometrial cancer, and is also associated with ovarian cancer and prostate cancer.

[0003] However, immunohistochemistry (IHC) is commonly used in clinical practice for in situ detection of ER protein in tissues, observing its expression within cells under a microscope to assess whether abnormal ER expression exists. However, the anti-ER antibodies used in current detection techniques still have significant shortcomings in sensitivity and specificity, such as low sensitivity due to insufficient antibody affinity. This leads to problems such as high subjectivity and poor reproducibility in pathological evaluation of IHC staining results, affecting the accuracy of disease classification and treatment decisions based on ER status.

[0004] Therefore, the present invention provides an anti-ER recombinant rabbit monoclonal antibody and its application. Summary of the Invention

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a widely applicable and accurately identifiable anti-ER recombinant rabbit monoclonal antibody and its applications. The present invention also relates to the nucleotide sequence encoding the anti-ER recombinant rabbit monoclonal antibody, the recombinant plasmid or expression vector, the preparation method, and the application of the anti-ER recombinant rabbit monoclonal antibody in ER protein detection methods or devices.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides an anti-ER 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:8; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9.

[0008] This anti-ER recombinant rabbit monoclonal antibody (ER rabbit-derived antibody) can be used for immunohistochemical detection, and can identify and detect the expression of ER protein on tumor cells or immune cells with high specificity and high sensitivity.

[0009] The anti-ER monoclonal antibody was obtained through recombinant expression in mammalian cells. Specifically, the anti-ER 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-ER 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 ER protein.

[0010] The anti-ER monoclonal antibody can recognize recombinant ER antigen protein and ER molecules on tumor cells and immune cells; the anti-ER monoclonal antibody can also be used in immunohistochemical pathological diagnostic agents.

[0011] Secondly, the present invention provides a coding gene for encoding the above-mentioned anti-ER recombinant rabbit monoclonal antibody.

[0012] 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-ER 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-ER recombinant rabbit monoclonal antibody.

[0013] Thirdly, the present invention provides a nucleic acid molecule comprising a coding gene for encoding the aforementioned anti-ER recombinant rabbit monoclonal antibody.

[0014] Fourthly, the present invention provides an expression vector or recombinant plasmid comprising the above-described nucleic acid molecule.

[0015] Fifthly, the present invention provides a host cell that has been transformed or transfected with the above-mentioned expression vector or recombinant plasmid.

[0016] In a sixth aspect, the present invention provides a method for preparing an anti-ER 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-ER recombinant rabbit monoclonal antibody is obtained.

[0017] Seventhly, the application of the aforementioned anti-ER recombinant rabbit monoclonal antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid in the preparation of an ER protein molecule detection device. The detection device includes, but is not limited to, reagent kits, antibody chips, etc.

[0018] Eighthly, the present invention also provides an ER detection kit, which includes the above-mentioned anti-ER recombinant rabbit monoclonal antibody and immunohistochemical detection reagent.

[0019] Preferably, the ER detection kit comprises: anti-ER recombinant rabbit monoclonal antibody, horseradish peroxidase-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, 3,3'-diaminobenzidine concentrate, 3,3'-diaminobenzidine buffer, hematoxylin and blueing solution.

[0020] The immunohistochemical testing process includes dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, 3,3'-diaminobenzidine (DAB) staining, counterstaining, dehydration, mounting, and microscopic examination.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The recombinant rabbit monoclonal antibody against ER provided by this invention exhibits high specificity and sensitivity in binding to ER protein molecules. It can specifically recognize and detect ER protein expression on cells, showing high positive expression when detecting ER 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 fields, facilitating accurate assessment and detection results. The 658G3A2 clone of the recombinant rabbit monoclonal antibody against ER, 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

[0023] Figure 1These are immunohistochemical staining results of the 658G3A2 anti-ER monoclonal antibody prepared in this invention and a commercially available antibody in strongly positive breast cancer, weakly positive breast cancer, and cervical tissue. The concentration of the 658G3A2 anti-ER monoclonal antibody used was 1 μg / mL. Image a shows the immunohistochemical staining results of the 658G3A2 clone ER antibody in strongly positive breast cancer; image b shows the immunohistochemical staining results of the commercially available antibody in strongly positive breast cancer (images a and b are at the same magnification); image c shows the immunohistochemical staining results of the 658G3A2 clone ER antibody in weakly positive breast cancer; image d shows the immunohistochemical staining results of the commercially available antibody in weakly positive breast cancer (images c and d are at the same magnification); image e shows the immunohistochemical staining results of the 658G3A2 clone ER antibody in cervical tissue; image f shows the immunohistochemical staining results of the commercially available antibody in cervical tissue (images e and f are at the same magnification).

[0024] Figure 2 This is a statistical graph showing the titer of the 658G3A2 anti-ER monoclonal antibody of the present invention and commercially available antibodies at eight different concentration gradients.

[0025] Figure 3 The immunoblotting results of the 658G3A2 anti-ER monoclonal antibody of this invention as a primary antibody were used to verify its ability to recognize the ER protein. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] Example 1

[0029] This embodiment describes the preparation and screening of anti-ER recombinant rabbit monoclonal antibodies, including the following steps:

[0030] (1) Antigen preparation

[0031] The specific sequence of the ER antigen is shown in SEQ ID NO:1 below.

[0032] The sequence of SEQ ID NO: 1 is: YASGYHYGVWSCEGCK.

[0033] The aforementioned polypeptide sequence was selected based on the analysis of the ER 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 ER antigen to immunize rabbits.

[0034] (2) Immunity

[0035] The ER 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 ER 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.

[0036] (3) Cell fusion

[0037] 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.

[0038] (4) Screening and cloning

[0039] Seven to ten days after fusion, clonal cells were screened using an ELISA test with the ER 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 658G3A2.

[0040] (5) Perform antibody sequencing on the selected hybridoma cell lines.

[0041] Total RNA was isolated from 658G3A2 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. These sequences were then cloned into eukaryotic expression vectors (InvivoGen, pfuse-rchg, pfuse2-rclk1) for cell transfection. The specific primers used included VH-F and VH-R for the heavy chain variable region and VK-F and VK-R for the light chain variable region.

[0042] The sequence of the heavy chain variable region primer VH-F is shown in SEQ ID NO:2; the sequence of VH-R is shown in SEQ ID NO:3. The specific sequences of SEQ ID NO:2 and SEQ ID NO:3 are as follows:

[0043] The sequence of SEQ ID NO:2 is:

[0044] AGACTGGGCTGCGCTGGCTTC.

[0045] The sequence of SEQ ID NO:3 is:

[0046] GTGAGGGTGCCCGAG.

[0047] The sequence of the light chain variable region primer VK-F is shown in SEQ ID NO:4; the sequence of VK-R is shown in SEQ ID NO:5. The specific sequences of SEQ ID NO:4 and SEQ ID NO:5 are as follows:

[0048] The sequence of SEQ ID NO:4 is:

[0049] ATGGACAYGAGGGCCCCCACTC.

[0050] The sequence of SEQ ID NO:5 is:

[0051] GGTGGGAAGATGAGGACAGTAGG.

[0052] (6) Cell transfection and screening

[0053] 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.

[0054] 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.

[0055] (7) Preparation and purification of monoclonal antibodies on cells

[0056] 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.

[0057] Finally, the heavy chain variable region nucleotide sequence of the 658G3A2 anti-ER recombinant rabbit monoclonal antibody is shown in SEQ ID No. 6, and the light chain variable region nucleotide sequence of the anti-ER recombinant rabbit monoclonal antibody is shown in SEQ ID NO: 7.

[0058] The specific sequences of SEQ ID NO:6 and SEQ ID NO:7 are as follows:

[0059] The sequence of SEQ ID NO:6 is:

[0060] cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtgtctggattctccctcagcagctatgacatgaactgggtccgccaggctccagggaaggggctggaatggatcggagtcatttatgctaatggtgtc atagtctacgcgagctgggcaaaaggccgattcaccatctccagaacctcgaccacggtggatctgaaagtcaccagtccgacaaccgaggacacggccacctatttctgtgccagaacgggttttggcttgtggggccaaggcaccctggtcaccgtctcctca.

[0061] The sequence of SEQ ID NO:7 is:

[0062] gcgcaagtgctgacccagactgcatcccccgtgtctgcggctgtgggaggcacagtcaccatcagttgccagtccagtcagagtctttataataataaccgctttatactggttacagcagaaaccagggcagcctcccaagcaactgatctattctgcatccagt ctggcatctggggtcccatcgcggttcagcggcagtggatctgggacacagttcactctcaccatcagcgacctggagtgtgacgatgctgccacttactactgtgcaggcgcttatagtggtaatatttatgctttcggcggagggaccgaggtggtggtcaaa.

[0063] 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 658G3A2 anti-ER recombinant rabbit monoclonal antibody was shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region of the anti-ER recombinant rabbit monoclonal antibody was shown in SEQ ID NO:9.

[0064] The specific sequences of SEQ ID NO:8 and SEQ ID NO:9 are as follows:

[0065] The sequence of SEQ ID NO:8 is:

[0066] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYDMNWVRQAPGKGLEWIGVIYANGVIVYASWAKGRFTISRTSTTVDLKVTSPTTEDTATYFCARTGFGLWGQGTLVTVSS.

[0067] The sequence of SEQ ID NO:9 is:

[0068] AQVLTQTASPVSAAVGGTVTISCQSSQSLYNNNRLYWLQQKPGQPPKQLIYSASSLASGVPSRFSGSGSGTQFTLTISDLECDDAATYYCAGAYSGNIYAFGGGTEVVVK.

[0069] Example 2

[0070] This example demonstrates immunohistochemical detection using an anti-ER recombinant rabbit monoclonal antibody as the primary antibody. The method is as follows:

[0071] (1) Sample preparation: The strong positive breast cancer, weak positive breast cancer, and cervical tissue sections fixed in formalin and embedded in paraffin were baked in a 60℃ constant temperature oven for 1-2 hours and stored for later use.

[0072] (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 min each time;

[0073] (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.

[0074] (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.

[0075] (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);

[0076] (6) Primary antibody incubation: Add 100 μL of 1 μg / mL 658G3A2 anti-ER 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;

[0077] (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.

[0078] (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.

[0079] (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;

[0080] (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;

[0081] (11) Mounting: Mount the sample with neutral resin.

[0082] Depend on Figure 1The results showed that ER protein specifically stained the nuclei of strongly positive breast cancer, weakly positive breast cancer, and cervical tissue. Furthermore, the 658G3A2 clone of the recombinant rabbit ER monoclonal antibody exhibited better staining performance and a deeper staining color than commercially available ER antibodies. This indicates that the 658G3A2 clone of the recombinant rabbit ER monoclonal antibody of this invention, due to its high specificity and strong positive signal, is easier to assess and has higher identification accuracy in IHC staining, making it more accurate for detecting and differentiating cancers.

[0083] Example 3

[0084] This example describes the determination of the affinity of the 658G3A2 anti-ER recombinant rabbit monoclonal antibody. The determination method is as follows:

[0085] (1) Remove the labeled ER 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℃.

[0086] (2) The ER recombinant rabbit monoclonal antibody of 658G3A2 clone 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;

[0087] (3) Add the diluted anti-ER 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.

[0088] (4) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0089] (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.

[0090] (6) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0091] (7) Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution at 100 μL / well and react at room temperature for 6 minutes;

[0092] (8) After the reaction is complete, add 2M H2SO4 at a rate of 50μL / well to stop the color development;

[0093] (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.

[0094] The results showed that, in eight concentration gradient experiments, the anti-ER recombinant rabbit monoclonal antibody of the 658G3A2 clone of the present invention had a strong affinity and high sensitivity for ER protein molecules, and could still achieve a high OD value under low antibody concentration conditions, which can save experimental and detection costs.

[0095] Example 4

[0096] This example demonstrates the detection using Western blotting with the anti-ER recombinant rabbit monoclonal antibody 658G3A2 as the primary antibody. The method is as follows:

[0097] (1) Activate the polyvinylidene fluoride (PVDF) membrane using MCF-7, T47D, and ZR-75-1 cell lysates by activating it with methanol for 1 min, washing it 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.

[0098] (2) Primary antibody incubation: Dilute the 658G3A2 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.

[0099] (3) Remove the membrane and wash it in TBST solution 3 times (2×5min+1×10min).

[0100] (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;

[0101] (5) Remove the membrane strip and wash it in TBST solution 4 times (3×5min+1×8min).

[0102] (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;

[0103] (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.

[0104] The theoretical molecular weight of ER protein is around 66 kDa. Figure 3In the original text, MCF-7 represents human breast cancer cell lysis, T47D represents human breast ductal carcinoma cell lysis, and ZR-75-1 represents human breast cancer cell lysis. Figure 3 The results showed that ER-positive bands appeared in all three lysis buffers, with the bands located at approximately 66 kDa, i.e., in the swimming lanes. The 658G3A2 anti-ER recombinant rabbit monoclonal antibody could specifically recognize ER protein in MCF-7, T47D, and ZR-75-1 cells, indicating that the 658G3A2 cloned recombinant rabbit monoclonal antibody of the present invention can recognize ER protein with high specificity.

[0105] 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. An anti-ER recombinant rabbit monoclonal antibody, 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-ER 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-ER 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-ER 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 an anti-ER recombinant rabbit monoclonal antibody, 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-ER recombinant rabbit monoclonal antibody.

8. The application of the anti-ER 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 an ER detection device.

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

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

Images