Anti-bcl2 recombinant rabbit monoclonal antibody and application thereof

Abstract

The application provides an anti-BCL2 recombinant rabbit monoclonal antibody and application thereof, the anti-BCL2 recombinant rabbit monoclonal antibody includes heavy chain variable region and light chain variable region, the heavy chain variable region has the CDR of heavy chain that is composed of CDR-H1, CDR-H2, CDR-H3, the amino acid sequence of CDR-H1, CDR-H2, CDR-H3 is respectively as shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8;The light chain variable region has the CDR of light chain that is composed of CDR-L1, CDR-L2, CDR-L3;The amino acid sequence of CDR-L1, CDR-L3 is respectively as shown in SEQ ID NO.9 and SEQ ID NO.10, the amino acid sequence of CDR-L2 is GAS.The BCL2 recombinant rabbit monoclonal antibody of the application is good in specificity, is easier to score in IHC dyeing, and is more accurate for detecting and distinguishing cancer.

Description

Technical Field

[0001] This invention belongs to the field of immunomedical technology, and particularly relates to a recombinant rabbit monoclonal antibody against BCL2 and its applications, especially in immunohistochemical detection. Background Technology

[0002] BCL2 protein is a core anti-apoptotic protein regulating apoptosis. Its encoding gene is structurally overexpressed in follicular lymphoma due to chromosomal translocation, making it one of the earliest discovered apoptosis-related oncogenic proteins in humans. The protein has a molecular weight of approximately 26 kDa. It inhibits the mitochondrial apoptosis pathway and blocks the release of pro-apoptotic factors, allowing cells to survive abnormally and evade physiological apoptosis, thereby promoting tumorigenesis and progression. Its abnormally high expression is widespread in lymphoma, leukemia, and various solid tumors, and is also closely related to tumor chemotherapy resistance and poor patient prognosis, making it a key molecular target for tumor pathology research and targeted therapy. Because BCL-2 has highly similar sequences to homology family proteins such as BCL-XL and MCL-1, cross-reactivity can easily occur, leading to false positives. Therefore, antibodies with higher specificity are needed to recognize the BCL2 protein. Summary of the Invention

[0003] (a) Technical problems to be solved

[0004] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a widely applicable recombinant rabbit monoclonal antibody against BCL2 that can accurately identify BCL2 expression, and its application. The present invention also relates to the nucleotide sequence encoding the recombinant rabbit monoclonal antibody against BCL2, the recombinant plasmid or expression vector, the preparation method, and the application of the recombinant rabbit monoclonal antibody against BCL2 in BCL2 protein detection methods or devices.

[0005] (II) Technical Solution

[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 a recombinant rabbit monoclonal antibody against BCL2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a heavy chain CDR composed of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively;

[0008] The light chain variable region has a light chain CDR composed of CDR-L1, CDR-L2, and CDR-L3; the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively, and the amino acid sequence of CDR-L2 is GAS.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5.

[0010] This recombinant rabbit monoclonal antibody against BCL2 (rabbit-derived BCL2 antibody) can be used for immunohistochemical detection, and can identify and detect the expression of BCL2 protein on tumor cells or immune cells with high specificity and high sensitivity.

[0011] The anti-BCL2 monoclonal antibody was obtained through recombinant expression in mammalian cells. Specifically, the anti-BCL2 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-BCL2 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 BCL2 protein.

[0012] The anti-BCL2 monoclonal antibody can recognize recombinant BCL2 antigen protein and BCL2 molecules on tumor cells and immune cells; the anti-BCL2 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-BCL2 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-BCL2 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-BCL2 recombinant rabbit monoclonal antibody.

[0015] Thirdly, the present invention provides a nucleic acid molecule comprising a coding gene for encoding the aforementioned anti-BCL2 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] Sixthly, the present invention provides a method for preparing a recombinant rabbit monoclonal antibody against BCL2, wherein the host cells are transformed or transfected using the above-mentioned expression vector or recombinant plasmid, the transformed or transfected cells are cultured, the cell supernatant is collected and purified, and the recombinant rabbit monoclonal antibody against BCL2 is obtained.

[0019] Seventhly, the application of the aforementioned anti-BCL2 recombinant rabbit monoclonal antibody, encoding gene, nucleic acid molecule, expression vector, or recombinant plasmid in the preparation of a BCL2 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 BCL2 detection kit, which includes the above-mentioned anti-BCL2 recombinant rabbit monoclonal antibody and immunohistochemical detection reagent.

[0021] Preferably, the BCL2 detection kit includes: recombinant rabbit monoclonal antibody against BCL2, 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 BCL2 provided by this invention exhibits high specificity and sensitivity in binding to the BCL2 protein molecule. It can specifically recognize and detect the expression of BCL2 protein on cells, showing a high positive expression when detecting BCL2 protein. Immunohistochemical assays in various tissues have shown 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 361F0D1 clone of the recombinant rabbit monoclonal antibody against BCL2 in 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 The images show the immunohistochemical results of the 361F0D1 anti-BCL2 monoclonal antibody prepared in this invention and a commercially available antibody (purchased from Abcam, catalog number: ab182858) in tonsil, appendix, and placental tissues. The concentration of the 361F0D1 anti-BCL2 monoclonal antibody used was 1 μg / mL. Figure 1 Image 'a' in the figure shows the immunohistochemical detection results of the BCL2 antibody of clone 361F0D1 in the tonsils. Figure 1 Figure b in the image shows the immunohistochemical detection results of commercially available antibodies in the tonsils. Figure 1 (The magnification of 'a' and 'b' is the same). Figure 1 In the image, c represents the immunohistochemical detection result of the BCL2 antibody of clone 361F0D1 in the appendix. Figure 1 In the figure, d represents the immunohistochemical detection result of commercially available antibodies in the appendix. Figure 1 (The magnification of c and d is the same). Figure 1 The image shows the immunohistochemical detection results of the BCL2 antibody of clone 361F0D1 in placental tissue, represented by the number 'e'. Figure 1 f in the figure represents the immunohistochemical detection results of commercially available antibodies in placental tissue. Figure 1 (The magnification factors of e and f are the same).

[0026] Figure 2 This is a statistical chart showing the titer of the 361F0D1 anti-BCL2 monoclonal antibody of this invention and commercially available antibodies at eight different concentration gradients.

[0027] Figure 3 The immunoblotting results of the 361F0D1 anti-BCL2 monoclonal antibody of this invention as a primary antibody were used to verify its ability to recognize the BCL2 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 BCL2. The steps include:

[0032] (1) Antigen preparation

[0033] The specific sequence of the BCL2 antigen is shown in SEQ ID NO.1 below.

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

[0035] The aforementioned polypeptide sequence was selected based on the analysis of the BCL2 molecular sequence, considering the structure, antigenicity, hydrophilicity / hydrophobicity of the constituent amino acids, and secondary structure of the BCL2 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 polypeptide with the sequence shown in SEQ ID NO.1 was conjugated with hemocyanin (KLH) and used as a BCL2 antigen to immunize rabbits.

[0036] (2) Immunity

[0037] The BCL2 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 BCL2 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 BCL2 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 361F0D1.

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

[0043] Total RNA was isolated from 361F0D1 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 (heavy chain variable region primers, VH-F: AGACTGGGCTGCGCTGGCTTC (SEQ ID NO.11), VH-R: GTGAGGGTGCCCGAG (SEQ ID NO.12); light chain variable region primers: VK-F ATGGACAYGAGGGCCCCCACTC (SEQ ID NO.13), VK-R: GGTGGGAAGATGAGGACAGTAGG (SEQ ID NO.14)). These nucleotide sequences 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 361F0D1 anti-BCL2 recombinant rabbit monoclonal antibody is shown in SEQ ID NO.2, and the light chain variable region nucleotide sequence of the anti-BCL2 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] SEQ ID NO.2 is:

[0052] cagtcgctggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggattctccctcagtaactactatatgagctgggtccgccaggctccagggaaggggctggaatggatcggaggcattagtattagtgctat tatatactacgcgagctgggcgaaaggccgattcaccatctccaaaacctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagagggataaacttgtggggccaaggcaccctggtcaccgtctcctca.

[0053] SEQ ID NO.3 is:

[0054] gatgttgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtgagagcatttatagtttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatggtgcatccgctctg gaatctggggtcccatcgcggttcaaaggcagtagatctgggacagagtacactctcagtatcagcgacctggagtgtgccgatgctgccacttactactgtcaaaactatgattctgataatgcttggcctttcggcggagggaccgaggtggtggtcaaa.

[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 361F0D1 anti-BCL2 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-BCL2 recombinant rabbit monoclonal antibody was shown in SEQ ID NO.5.

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

[0057] SEQ ID NO.4 is:

[0058] QSLEESGGRLVTPGTPLTLTCTVSGFSLSNYYMSWVRQAPGKGLEWIGGISISAIIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGINLWGQGTLVTVSS.

[0059] The sequence of CDR-H1 is: GFSLSNYY (SEQ ID NO.6);

[0060] The sequence of CDR-H2 is: ISISAII (SEQ ID NO.7);

[0061] The sequence of CDR-H3 is: ARGINL (SEQ ID NO.8).

[0062] SEQ ID NO.5 is:

[0063] DVVMTQTPASVSEPVGGTVTIKCQASESIYSSLAWYQQKPGQPPKLLIYGASALESGVPSRFKGSRSGTEYTLSISDLECADAATYYCQNYDSDNAWPFGGGTEVVVK.

[0064] The sequence of CDR-L1 is: ESIYSS (SEQ ID NO.9);

[0065] CDR-L2 sequence: GAS;

[0066] The sequence of CDR-L3 is: QNYDSDNAWP (SEQ ID NO.10).

[0067] Example 2

[0068] This example demonstrates immunohistochemical detection using a recombinant rabbit monoclonal antibody against BCL2 as the primary antibody. The method is as follows:

[0069] (1) Sample preparation: The tonsil, appendix and placental 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;

[0070] (2) Dewaxing of sections: Paraffin sections are first placed in fresh xylene for dewaxing, soaked twice, 10 min each time;

[0071] (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.

[0072] (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.

[0073] (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).

[0074] (6) Primary antibody incubation: Add 100 μL of 1 μg / mL 361F0D1 anti-BCL2 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;

[0075] (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.

[0076] (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.

[0077] (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;

[0078] (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;

[0079] (11) Mounting: Mount the sample with neutral resin.

[0080] Depend on Figure 1 The results showed that BCL2 protein specifically stained the nuclear cells of human tonsils, appendix, and placental tissues. Furthermore, the staining effect of the 361F0D1 cloned BCL2 recombinant rabbit monoclonal antibody was better than that of the commercially available BCL2 antibody (purchased from Abcam, catalog number: ab182858), with a deeper staining color. This indicates that the 361F0D1 cloned BCL2 recombinant rabbit monoclonal antibody of this invention, due to its high specificity and strong positive signal, is easier to evaluate in IHC staining, has high identification accuracy, and is more accurate in detecting and differentiating cancers.

[0081] Example 3

[0082] This example describes the determination of the affinity of the 361F0D1 anti-BCL2 recombinant rabbit monoclonal antibody for BCL2. The determination method is as follows:

[0083] (1) Remove the labeled BCL2 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℃.

[0084] (2) The BCL2 recombinant rabbit monoclonal antibody cloned by 361F0D1 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;

[0085] (3) Add the diluted anti-BCL2 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.

[0086] (4) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0087] (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.

[0088] (6) After the reaction is complete, remove the microplate, discard the liquid, rinse with purified water 5 times, and pat dry.

[0089] (7) Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution at 100 μL / well and react at room temperature for 6 minutes;

[0090] (8) After the reaction is complete, add 2M H2SO4 at a rate of 50μL / well to stop the color development;

[0091] (9) Read the OD value at 450 nm on the microplate reader, organize the data, and analyze the results as follows: Figure 2 The figure shows a comparison of the potency of 361F0D1 antibody and commercially available BCL2 antibody (purchased from abcam, catalog number: ab182858).

[0092] The results showed that, in eight concentration gradient experiments, the 361F0D1 clone of the present invention exhibited strong affinity and high sensitivity for the BCL2 protein molecule, and could still achieve a high OD value under low antibody concentration conditions, thus saving experimental and detection costs.

[0093] Example 4

[0094] This example demonstrates the detection using Western blotting with the anti-BCL2 recombinant rabbit monoclonal antibody 361F0D1 as the primary antibody. The method is as follows:

[0095] (1) Activate the polyvinylidene fluoride (PVDF) membrane using H. tonsil, H. thymus, Jurkat, U-937, THP-1, and HeLa cell lysates. Activate with methanol for 1 min, wash twice with pure water, and then wash three times with TBST. Block: 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 surfactant Tween-20.

[0096] (2) Primary antibody incubation: Dilute the 361F0D1 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.

[0097] (3) Remove the membrane and wash it in TBST solution 3 times (2×5min+1×10min).

[0098] (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;

[0099] (5) Remove the membrane strip and wash it in TBST solution 4 times (3×5min+1×8min).

[0100] (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;

[0101] (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.

[0102] The theoretical molecular weight of BCL2 protein is around 26 kDa. Figure 3 In this context, H. tonsil represents human tonsil cell lysis; H. thymus represents human thymocyte lysis; Jurkat represents human T-lymphocytic leukemia cell lysis; U-937 represents human histiocytic lymphoma cell lysis; THP-1 represents human acute monocytic leukemia cell lysis; and Hela represents human cervical cancer cell lysis. Figure 3 The results showed that BCL2 positive bands appeared in the lysates of H. tonsil, H. thymus, Jurkat, U-937, THP-1, and Hela cells. That is, in the swimming lanes, the 361F0D1 anti-BCL2 recombinant rabbit monoclonal antibody can specifically recognize BCL2 protein in H. tonsil, H. thymus, Jurkat, U-937, THP-1, and Hela cells, indicating that the 361F0D1 cloned BCL2 recombinant rabbit monoclonal antibody of the present invention can recognize BCL2 protein with high specificity.

[0103] 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 BCL2, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a heavy chain CDR composed of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively; The light chain variable region has a light chain CDR composed of CDR-L1, CDR-L2, and CDR-L3; the amino acid sequences of CDR-L1 and CDR-L3 are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively, and the amino acid sequence of CDR-L2 is GAS.

2. The anti-BCL2 recombinant rabbit monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

5.

3. A gene encoding a gene, characterized in that, Used to encode the anti-BCL2 recombinant rabbit monoclonal antibody as described in claim 1 or 2.

4. The encoding gene according to claim 3, 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 anti-BCL2 recombinant rabbit monoclonal antibody, and DNA sequences as shown in SEQ ID NO.3 are used to encode the light chain variable region of the anti-BCL2 recombinant rabbit monoclonal antibody.

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

6. An expression vector or recombinant plasmid, characterized in that, It contains the nucleic acid molecule as described in claim 5.

7. Transform or transfect the host cell of the expression vector or recombinant plasmid as described in claim 6.

8. A method for preparing a recombinant rabbit monoclonal antibody against BCL2, characterized in that, The expression vector or recombinant plasmid described in claim 6 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-BCL2 recombinant rabbit monoclonal antibody.

9. The application of the anti-BCL2 recombinant rabbit monoclonal antibody according to claim 1 or 2, the encoding gene according to claim 3 or 4, the nucleic acid molecule according to claim 5, the expression vector or recombinant plasmid according to claim 6, and the host cell according to claim 7 in the preparation of a BCL2 detection device.

10. A BCL2 detection kit, characterized in that, It includes the anti-BCL2 recombinant rabbit monoclonal antibody and immunohistochemical detection reagent as described in claim 1 or 2.

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