Preparation and application of anti-cd38 rabbit recombinant monoclonal antibody

Abstract

The application relates to preparation and application of anti-CD38 rabbit recombinant monoclonal antibodies, the obtained antibodies have good specificity, most importantly, the antibodies have high affinity and can recognize natural cell surface CD38 proteins, and can be used for flow, ELISA and immunohistochemical detection applications.

Description

Technical Field

[0001] This invention belongs to the field of antibody preparation in biotechnology, and in particular relates to a rabbit recombinant monoclonal antibody that can specifically bind to the CD38 protein and its applications. Background Technology

[0002] CD38 is a single-chain transmembrane glycoprotein with a molecular weight of approximately 45 kDa. Because its N-terminus is intracellular and its C-terminus is extracellular, it belongs to the type II transmembrane protein category. CD38 can not only anchor to the cell membrane (45 kDa, mCD38) but can also be cleaved to form a soluble form (39 kDa, sCD38). CD38's ligand is CD31, and their interaction regulates cell migration, receptor-mediated adhesion, and signal transduction. Under normal circumstances, CD38 expression is relatively low in normal lymphoid cells, myeloid cells, and non-hematopoietic tissues, but it is widely expressed in hematopoietic cells, including morphological stem cells. CD38 is expressed in B cells, with particularly high expression in effector B cells (plasma cells). CD38 expression is associated with various diseases, including AIDS, autoimmune diseases such as systemic lupus erythematosus, type 2 diabetes, osteoporosis, and cancer. In addition, research has found that CD38 is highly expressed in a large number of malignant hematological cancers, especially in cancers such as multiple myeloma. Therefore, CD38 has become a popular target for the treatment of multiple myeloma.

[0003] Currently, antibodies targeting CD38 already exist in existing technologies. For example, Chinese patent application CN 2020106077816 discloses a nanobody that can bind to human CD38 and its application. Through the preparation of CD38 protein, immunization of alpacas, and the use of a phage library platform technology to display nanobodies, a nanobody VHH specifically binding to CD38 was screened, its CDR sequence was identified, and a humanized VHHhuFc1 was constructed. Patent applications CN2018800517512 and CN2018800790459 also prepared antibodies against CD38. Although the above antibodies have certain application prospects, there is still a need to develop monoclonal antibodies with stronger affinity and more epitopes. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant rabbit monoclonal antibody against CD38, selected from one or more of the following recombinant rabbit monoclonal antibodies:

[0005] The rabbit recombinant monoclonal antibody named 8A1 has the following amino acid sequences: CDR1, CDR2, and CDR3 of its heavy chain complementarity-determining regions are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 of its light chain complementarity-determining regions are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0006] The rabbit recombinant monoclonal antibody named 8H7 has the following amino acid sequences: CDR1, CDR2, and CDR3 of its heavy chain complementarity-determining regions are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 of its light chain complementarity-determining regions are shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively.

[0007] The rabbit recombinant monoclonal antibody named 9E10 has the following amino acid sequences: CDR1, CDR2, and CDR3 of the heavy chain complementarity-determining region (CDR1, CDR2, and CDR3) as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain complementarity-determining region (CDR1, CDR2, and CDR3) as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

[0008] Based on the above technical solution, a rabbit recombinant monoclonal antibody named 8A1 was developed, with the heavy chain variable region sequence being the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region sequence being the amino acid sequence shown in SEQ ID NO:8.

[0009] The rabbit recombinant monoclonal antibody named 8H7 has the heavy chain variable region sequence shown in SEQ ID NO:16 and the light chain variable region sequence shown in SEQ ID NO:17.

[0010] The rabbit recombinant monoclonal antibody named 9E10 has the following amino acid sequence: heavy chain variable region sequence as shown in SEQ ID NO:25 and light chain variable region sequence as shown in SEQ ID NO:26.

[0011] Based on the above technical solution, a rabbit recombinant monoclonal antibody named 8A1 was developed, and its SCFV sequence is the amino acid sequence shown in SEQ ID NO:9.

[0012] A rabbit recombinant monoclonal antibody named 8H7 has an SCFV sequence as shown in SEQ ID NO:18.

[0013] The rabbit recombinant monoclonal antibody named 9E10 has the SCFV sequence shown in SEQ ID NO:27.

[0014] Based on the above technical solution, the light chain constant region of the rabbit recombinant monoclonal antibody is the κ chain, and the heavy chain constant region is of the IgG type.

[0015] The present invention also provides a nucleic acid molecule comprising a nucleic acid sequence of a heavy chain complementarity-determining region or a light chain complementarity-determining region capable of encoding a rabbit recombinant monoclonal antibody that binds to the CD38 protein.

[0016] The present invention also provides a carrier containing the above-mentioned nucleic acid molecules.

[0017] The present invention also provides a host cell containing the above-mentioned rabbit recombinant monoclonal antibody that binds to CD38 protein, the above-mentioned nucleic acid molecule, or the above-mentioned vector.

[0018] The present invention also provides a conjugate containing the above-mentioned antibody.

[0019] The present invention also provides a pharmaceutical composition comprising a main component and an excipient, wherein: the main component is one or more of the above-mentioned rabbit recombinant monoclonal antibody binding to CD38 protein, the above-mentioned nucleic acid molecule, the above-mentioned carrier, the above-mentioned host cell, and the above-mentioned conjugate; and the excipient is selected from pharmaceutically acceptable carriers or excipients, and optionally other bioactive substances.

[0020] The present invention also provides the use of the above-mentioned rabbit recombinant monoclonal antibody that binds to CD38 protein, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned host cell, and the above-mentioned conjugate in the preparation of drugs for treating diseases or detection reagents.

[0021] The present invention also provides a kit comprising the above-described rabbit recombinant monoclonal antibody that binds to the CD38 protein.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] Currently, CD38 monoclonal antibodies are mainly derived from mice and humans. Because the immunization process in rabbits is more complex than in mice, antibodies with broader epitope coverage can be obtained. Therefore, this invention utilizes the CD38 extracellular protein to immunize rabbits, and then prepares an anti-CD38 rabbit recombinant monoclonal antibody using B-cell cloning technology. Importantly, the obtained anti-CD38 rabbit recombinant monoclonal antibody exhibits high affinity and good specificity, capable of recognizing native cell surface CD38 protein, and can be used for flow cytometry, ELISA, and immunohistochemical detection applications. Furthermore, future development of other diagnostic and therapeutic reagents targeting CD38 is possible. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The image shows an SDS-PAGE image of the CD38 protein.

[0026] Figure 2 The results of PCR amplification of the heavy and light chains of the anti-CD38 rabbit recombinant monoclonal antibody are shown.

[0027] Figure 3 The image shows an SDS-PAGE image of the purified CD38 rabbit recombinant monoclonal antibody.

[0028] Figures 4 - 7 This demonstrates the specificity of FACs in detecting CD38 rabbit recombinant monoclonal antibodies.

[0029] Figure 8 The binding affinity of FACs to CD38 rabbit recombinant monoclonal antibody was demonstrated.

[0030] Figure 9 The results demonstrate the effectiveness of IHC detection of recombinant rabbit monoclonal antibody against CD38-8A1 in human T-cell malignant lymphoma and human tonsils. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The present invention will be further described below: Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Additionally, to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0033] The term "antibody" as used herein includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding moieties") or single chains thereof. An "antibody" is a glycoprotein, or its antigen-binding moieties, comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. The proteins or fragments thereof involved in this invention may be naturally purified products, chemically synthesized products, or products generated from a eukaryotic host (e.g., mammalian cells) using recombinant technology. All raw materials and reagents used in this invention are commercially available.

[0034] The technical solution of the present invention will be further described below with reference to the embodiments.

[0035] Example:

[0036] (1) Preparation of CD38 protein: In this invention, a CD38 FC fusion tag protein eukaryotic expression plasmid was constructed. After transfecting 293F cells for 5 days, the cell culture supernatant was collected, purified using Protein A resin, and the protein concentration was identified. The obtained CD38 protein was analyzed by SDS-PAGE, and the protein with a concentration of approximately 70-72 kDa was identified as CD38 protein. The protein expression, purification, and identification results are shown in […]. Figure 1 .

[0037] (2) Obtaining peripheral blood mononuclear cells (PBMCs) from immunized animals: New Zealand white rabbits were selected as immunized animals. For the first immunization, 250 μg of CD38 protein was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple points on the back of the New Zealand white rabbits. A second immunization was performed 21 days later, with 120 μg of protein emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously into the back of the New Zealand white rabbits. A third immunization was performed 21 days later, with 120 μg of protein emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously into the back of the New Zealand white rabbits. Peripheral blood was aseptically collected one week after the third immunization.

[0038] (3) Obtaining CD38-specific B lymphocytes: PBMC cells were separated from the collected peripheral blood using lymphocyte separation solution; CD38 protein was coupled to the magnetic beads according to the instructions for using immunomagnetic beads; the mixture of CD38 protein-coupled magnetic beads and separated PBMC cells was incubated at room temperature for 50 min and then placed in a magnetic rack. After 5 min, all the magnetic beads sank to the bottom. The supernatant was discarded, and sterile PBS was added to wash the cells. The washing was repeated 3 times. The cells obtained in the end were CD38-specific B lymphocytes.

[0039] (4) Identification of B lymphocytes: The isolated B lymphocytes were diluted several times and placed in 96-well cell culture plates. 1640 medium containing 10% fetal bovine serum (FBS) and 2 μg / ml human IL2 was added, and the plates were cultured at 37℃ and 5% CO2 for 6 days. The supernatant of the medium was collected for antibody identification.

[0040] Indirect ELISA identification:

[0041] The plate was coated with 1 μg / ml CD38 protein, 100 μl / well, and incubated at 4℃ for 16 h. The next day, after discarding the coating solution, the plate was blocked with PBS containing 1% bovine serum albumin (BSA), 150 μl / well, and incubated at 37℃ for 1 h. The blocking solution was discarded, and B cell supernatant was added, 50 μl / well, and incubated at 37℃ for 1 h. The B cell supernatant was discarded, and the plate was washed 5 times with PBS containing 0.5 wt.% Tween-20, 2 min each time. Finally, goat anti-rabbit IgG-HRP secondary antibody diluted 5000 times was added, and the plate was incubated at 37℃ for 1 h. The secondary antibody was discarded, and the plate was washed 5 times with phosphate-Tween buffer (PBST), 2 min each time. The washing solution was discarded, the plate was patted dry, and substrate was added for color development. B cell clones labeled 8A1, 8H7, and 9E10 were identified as positive by ELISA. The detection results are shown in Table 1.

[0042] Clone number OD450 8A1 2.23 8H7 1.78 9E10 1.83

[0043] (5) Cloning of antibody genes

[0044] B cells that tested positive were collected, and RNA was extracted using standard RNA extraction methods. The RNA was then reverse transcribed into cDNA. The antibody heavy chain gene primers were used for amplification: upstream primer 5'-CAGTCGCTGGAGGAGTCCGG-3' and downstream primer 5'-CCATTGGTGAGGGTGCCCGAG-3'. The antibody light chain gene primers were used for amplification: upstream primer 5'-GACATTGTGATGACCCAGAC-3' and downstream primer 5'-CCACCTCGGTCCCTTCGCCG-3'. The amplification conditions were: denaturation at 94℃ for 3 min, followed by 30 cycles of (95℃ for 1 min, 56℃ for 30 s, and 72℃ for 1 min), and a final extension at 72℃ for 10 min. (PCR amplification results are shown in [link to PCR results]). Figure 2 The PCR products were recovered using a DNA gel purification and recovery kit. The heavy and light chain genes of the rabbit recombinant monoclonal antibody were cloned into an expression vector and transformed. Single colonies were verified using colony PCR, and gene sequencing was performed on positive colonies to obtain the gene sequence of the specific antibody. The amino acid sequences of each region of the specific antibody were obtained by translating the gene sequence of the specific antibody according to the codons.

[0045] The amino acid sequences of the specific antibodies ultimately obtained from B lymphocytes labeled 8A1, 8H7, 9E10 are shown in Table 2.

[0046] SEQ ID NO. Designation Format / Source Type Sequence 1 8A1 VH CDR1 aa GFSFSTSYW 2 VH CDR2 aa IYTGSGST 3 VH CDR3 aa ARDPGVHSGTNL 4 VL CDR1 aa EDINTL 5 VL CDR2 aa LAS 6 VL CDR3 aa QCTYYGSTYGGT 7 VH aa QSLEESGGDLVKPAGSLTLTCTASGFSFSTSYWICWVRQAPGKGLEWIACIYTGSGSTPYASWAKGRFTISKTSSTTVTLQVTSLTAADTATYFCARDPGVHSGTNLWGQGTLVSVSS 8 VL aa DVVMTQTPASVSAAVGGTVTIKCQASEDINTLLAWYQQKPGQPPKLLIYLASNLASGVSSRFKGSRSGTEFTLTISDLECADAATYYCQCTYYGSTYGGTFGGGTEVVVK 9 scfv aa QSLEESGGDLVKPAGSLTLTCTASGFSFSTSYWICWVRQAPGKGLEWIACIYTGSGSTPYASWAKGRFTISKTSSTTVTLQVTSLTAADTATYFCARDPGVHSGTNLWGQGTLVSVSSDVVMTQTPASVSAAVGGTVTIKCQASEDINTLLAWYQQKPGQPPKLLIYLASNLASGVSSRFKGSRSGTEFTLTISDLECADAATYYCQCTYYGSTYGGTFGGGTEVVVK 10 8H7 VH CDR1 aa GFSLSSYG 11 VH CDR2 aa VAVGGTT 12 VH CDR3 aa VRNGVSGSSDI 13 VL CDR1 aa QSIYSY 14 VL CDR2 aa SAS 15 VL CDR3 aa QQDYSGSNVDNT 16 VH aa QSVKESEGGLFKPTDTLTLTCTVSGFSLSSYGVIWVRQAPGKGLEYIGFVAVGGTTYYANWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCVRNGVSGSSDIWGPGTLVTVSS 17 VL aa AYDMTQTPASVSEPVGGTVTIKCQASQSIYSYLSWYQQKLGQPPKLLIYSASTLASGVSSRFKGSGSGTEYTLTISGVECADAATYYCQQDYSGSNVDNTFGGGTEVVVK 18 scfv aa QSVKESEGGLFKPTDTLTLTCTVSGFSLSSYGVIWVRQAPGKGLEYIGFVAVGGTTYYANWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCVRNGVSGSSDIWGPGTLVTVSSAYDMTQTPASVSEPVGGTVTIKCQASQSIYSYLSWYQQKLGQPPKLLIYSASTLASGVSSRFKGSGSGTEYTLTISGVECADAATYYCQQDYSGSNVDNTFGGGTEVVVK 19 9E10 VH CDR1 aa GFSFSTSYW 20 VH CDR2 aa IYTGSGST 21 VH CDR3 aa ARDPGVHSGTNL 22 VL CDR1 aa QSIYSY 23 VL CDR2 aa YAS 24 VL CDR3 aa QQDYSGYNVDNT 25 VH aa QSLEESGGDLVKPAGSLTLTCTASGFSFSTSYWICWVRQAPGKGLEWIACIYTGSGSTPYASWAKGRFTISKTSSTTVTLQVTSLTAADTATYFCARDPGVHSGTNLWGQGTLVSVSS 26 VL aa AYDMTQTPASVEVAVGGTVTIKCQASQSIYSYCSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQQDYSGYNVDNTFGGGTEVVVK 27 scfv aa QSLEESGGDLVKPAGSLTLTCTASGFSFSTSYWICWVRQAPGKGLEWIACIYTGSGSTPYASWAKGRFTISKTSSTTVTLQVTSLTAADTATYFCARDPGVHSGTNLWGQGTLVSVSSAYDMTQTPASVEVAVGGTVTIKCQASQSIYSYCSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQQDYSGYNVDNTFGGGTEVVVK 11A7 VH CDR1 aa GFDFTTYYY VH CDR2 aa IYTGSSDSI VH CDR3 aa ARDRPSGNDDYIYDGGMDL VL CDR1 aa QTINSW VL CDR2 aa QAS VL CDR3 aa QQGYSFGNVDNA VH aa QSLEESGGDLVKPGASLTLTCTASGFDFTTYYYMCWVRQAPGKGLEWIGCIYTGSSDSIYYASWAKGRFTISKTSSTTVTLQMSSLTAADTATYFCARDRPSGNDDYIYDGGMDLWGPGTLVTVFS VL aa AYDMTQTPASVEVAVGGTVSIKCQASQTINSWLSWYQQKPGQPPKLLIYQASKLAASGVSSRFKGSGSGTEYTLTISGVECADAATYYCQQGYSFGNVDNAFGGGTEVVVK scfv aa QSLEESGGDLVKPGASLTLTCTASGFDFTTYYYMCWVRQAPGKGLEWIGCIYTGSSDSIYYASWAKGRFTISKTSSTTVTLQMSSLTAADTATYFCARDRPSGNDDYIYDGGMDLWGPGTLVTVFS AYDMTQTPASVEVAVGGTVSIKCQASQTINSWLSWYQQKPGQPPKLLIYQASKLAASGVSSRFKGSGSGTEYTLTISGVECADAATYYCQQGYSFGNVDNAFGGGTEVVVK

[0047] (6) Production and identification of CD38 rabbit recombinant monoclonal antibody

[0048] The expression plasmids of the heavy and light chain genes of the antibody were co-transfected into 293 cells and cultured at 37℃ with 5% CO2 for 72 h. Cell supernatant was collected, and the antibody was purified using Protein A resin. The purified antibody was then identified by SDS-PAGE. The results are shown below. Figure 3 .

[0049] The purified antibody underwent functional identification, as detailed below:

[0050] a) ELISA reaction

[0051] The CD38 protein at a concentration of 1 μg / ml was coated at 100 μl / well and incubated at 4℃ for 16 h. The next day, the coating solution was discarded, and the plate was blocked with PBS containing 1% BSA at 150 μl / well and incubated at 37℃ for 1 h. The blocking solution was discarded, and antibodies diluted at different fractions were added at 50 μl / well and incubated at 37℃ for 1 h. The antibodies were discarded, and the plate was washed 5 times with PBS containing 0.5% Tween-20 for 2 min each time. Finally, goat anti-rabbit IgG-HRP secondary antibody diluted 5000 times was added and incubated at 37℃ for 1 h. The secondary antibody was discarded, and the plate was washed 5 times with PBST for 2 min each time. The washing solution was discarded, the plate was patted dry, and substrate was added for color development. The ELISA results are shown in the figure. Figure 4 .

[0052] b) Flow cytometry-based specific assay method

[0053] 293 cells were transfected with the full-length CD38 protein particle for testing. Transfected cells were collected in centrifuge tubes, washed twice with sterile PBS, and Fc receptor blocking buffer was used to block the Fc receptors on the cell surface. The cells were incubated at 4°C for 30 min. Cells were collected by centrifugation, washed twice with PBS containing 0.5 wt.% BSA, and then incubated at 4°C for 30 min with different concentrations of antibody. Cells were then washed twice more with PBS containing 0.5 wt.% BSA, and finally, goat anti-rabbit IgG-488 fluorescent secondary antibody was added and incubated at 4°C for 30 min. After washing twice with PBS containing 0.5 wt.% BSA, the cells were resuspended in 200 μl of PBS containing 0.5 wt.% BSA and analyzed by flow cytometry. Specificity results of the flow cytometry assay are shown below. Figures 5-7 The analysis results show that the screened antibodies can specifically recognize CD38 on the cell membrane.

[0054] (c) Flow cytometry-based method for determining binding force

[0055] Raji cells were used for testing. Cells were collected in centrifuge tubes, washed twice with sterile PBS, and Fc receptors on the cell surface were blocked using Fc receptor blocking solution, incubated at 4°C for 30 min. Cells were collected by centrifugation, washed twice with PBS containing 0.5 wt.% BSA, and then incubated at 4°C for 30 min with different concentrations of antibody. Cells were then washed twice more with PBS containing 0.5 wt.% BSA, and finally, goat anti-rabbit IgG-488 fluorescent secondary antibody was added and incubated at 4°C for 30 min. After washing twice with PBS containing 0.5 wt.% BSA, cells were resuspended in 200 μl of PBS containing 0.5 wt.% BSA and tested using a flow cytometer. The results of the flow cytometry binding affinity are shown below. Figure 8 Flow cytometry binding affinity analysis showed that the screened antibodies had different binding affinity.

[0056] d) Immunohistochemical (IHC) detection method

[0057] 1) Tissue fixation: Before dewaxing, place the sections in a constant temperature oven and bake at 60℃ for 60 minutes.

[0058] 2) Dewaxing: Soak the sections in xylene for 15 minutes, then replace with new xylene and soak for another 15 minutes.

[0059] 3) Hydration: Soak in anhydrous ethanol for 5 minutes, then replace with new anhydrous ethanol and soak for another 5 minutes, then soak in 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes.

[0060] 4) Washing: Soak in dd H2O for 5 minutes, then wash 3 times.

[0061] 5) Antigen retrieval (microwave irradiation method): Treat with 0.3% H2O2 methanol for 10 min, rinse with tap water and then with distilled water. Place the slides in a dedicated retrieval box containing pH 6.0 citrate buffer and heat to boiling for 10 min. After the retrieval solution cools to room temperature, wash three times with PBS.

[0062] 6) Washing: Soak in dd H2O for 5 min, wash twice; soak in PBST for 5 min, wash twice.

[0063] 7) Inactivating enzyme: Soak the slices in 20ml of inactivating enzyme reagent, protect from light, and incubate at room temperature for 15min.

[0064] 8) Washing: Soak in PBST for 5 minutes and wash 3 times.

[0065] 9) Sealing: Add an appropriate amount of sealing solution and let it sit in a humidified chamber at room temperature for 60 minutes.

[0066] 10) Primary antibody reaction: Draw a circle with neutral resin to form a trough, add diluted primary antibody to each tissue, and incubate overnight in a humidified chamber at 4°C.

[0067] 11) Rewarming: After being removed from 4℃, incubate at room temperature for 60 min.

[0068] 12) Washing: Rinse gently with PBST and soak for 5 minutes. Repeat 4 times.

[0069] 13) Enzyme-labeled secondary antibody reaction: Add diluted HRP-labeled secondary antibody to each tissue and incubate at room temperature for 60 min.

[0070] 14) Washing: Rinse gently with PBST and soak for 5 minutes. Repeat 4 times.

[0071] 15) DAB color development method: Prepare DAB color development solution, react in the dark for 10-15 min, add it to the slide, and develop color for 1-5 min.

[0072] 16) Termination of color development: Distilled water terminates the color development reaction.

[0073] 17) Resuscitation: Add an appropriate amount of hematoxylin staining solution to each tissue, stain for 5-10 minutes, and rinse thoroughly with distilled water.

[0074] 18) Decolorization and Blue Inversion: Place the slice in 1% hydrochloric acid-ethanol for 2-3 seconds to decolorize, then quickly remove it and place it in distilled water to stop the decolorization. Then place it in PBST for blue inversion for 5-10 minutes.

[0075] 19) Mounting: Immerse in 75% ethanol for 5 min; 85% ethanol for 5 min; 95% ethanol for 5 min; and anhydrous ethanol for 5 min respectively. Immerse in xylene for 15 min, replace the xylene and immerse for another 15 min, then cover with a coverslip.

[0076] 20) Microscopic observation and recording of results. 8A1 was selected for testing; the results are shown below. Figure 8 This antibody can specifically bind to human T-cell malignant lymphoma and tonsils.

[0077] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. sequence list <110> Suzhou Dima Biotechnology Co., Ltd. <120> Preparation and application of anti-CD38 rabbit recombinant monoclonal antibody <160> 36 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <400> 1 Gly Phe Ser Phe Ser Thr Ser Tyr Trp 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Ile Tyr Thr Gly Ser Gly Ser Thr 1 5 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <400> 3 Ala Arg Asp Pro Gly Val His Ser Gly Thr Asn Leu 1 5 10 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <400> 4 Glu Asp Ile Asn Thr Leu 1 5 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <400> 5 Leu Ala Ser 1 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <400> 6 Gln Cys Thr Tyr Tyr Gly Ser Thr Tyr Gly Gly Thr 1 5 10 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <400> 7 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Thr Ser Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Thr Gly Ser Gly Ser Thr Pro Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Val Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Asp Pro Gly Val His Ser Gly Thr Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Ser Val Ser Ser 115 <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <400> 8 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Asp Ile Asn Thr Leu 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Tyr Tyr Gly Ser Thr 85 90 95 Tyr Gly Gly Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 9 <211> 228 <212> PRT <213> Artificial Sequence <400> 9 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Thr Ser Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Thr Gly Ser Gly Ser Thr Pro Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Val Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Asp Pro Gly Val His Ser Gly Thr Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Ser Val Ser Ser Asp Val Val Met Thr Gln Thr Pro Ala Ser 115 120 125 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 130 135 140 Glu Asp Ile Asn Thr Leu Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 145 150 155 160 Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Ala Ser Gly Val 165 170 175 Ser Ser Arg Phe Lys Gly Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr 180 185 190 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys 195 200 205 Thr Tyr Tyr Gly Ser Thr Tyr Gly Gly Thr Phe Gly Gly Gly Thr Glu 210 215 220 Val Val Val Lys 225 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <400> 10 Gly Phe Ser Leu Ser Ser Tyr Gly 1 5 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <400> 11 Val Ala Val Gly Gly Thr Thr 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <400> 12 Val Arg Asn Gly Val Ser Gly Ser Ser Asp Ile 1 5 10 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <400> 13 Gln Ser Ile Tyr Ser Tyr 1 5 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <400> 14 Ser Ala Ser 1 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <400> 15 Gln Gln Asp Tyr Ser Gly Ser Asn Val Asp Asn Thr 1 5 10 <210> 16 <211> 116 <212> PRT <213> Artificial Sequence <400> 16 Gln Ser Val Lys Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Gly 20 25 30 Val Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Phe Val Ala Val Gly Gly Thr Thr Tyr Tyr Ala Asn Trp Ala Lys Ser 50 55 60 Arg Ser Thr Ile Thr Arg Asn Thr Asn Glu Asn Thr Val Thr Leu Lys 65 70 75 80 Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Val Arg 85 90 95 Asn Gly Val Ser Gly Ser Ser Asp Ile Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 17 <211> 110 <212> PRT <213> Artificial Sequence <400> 17 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Tyr Ser Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Gly Ser Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 18 <211> 226 <212> PRT <213> Artificial Sequence <400> 18 Gln Ser Val Lys Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Gly 20 25 30 Val Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Phe Val Ala Val Gly Gly Thr Thr Tyr Tyr Ala Asn Trp Ala Lys Ser 50 55 60 Arg Ser Thr Ile Thr Arg Asn Thr Asn Glu Asn Thr Val Thr Leu Lys 65 70 75 80 Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Val Arg 85 90 95 Asn Gly Val Ser Gly Ser Ser Asp Ile Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser 115 120 125 Glu Pro Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser 130 135 140 Ile Tyr Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro 145 150 155 160 Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Ser Ser 165 170 175 Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser 180 185 190 Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr 195 200 205 Ser Gly Ser Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val 210 215 220 Val Lys 225 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <400> 19 Gly Phe Ser Phe Ser Thr Ser Tyr Trp 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <400> 20 Ile Tyr Thr Gly Ser Gly Ser Thr 1 5 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <400> twenty one Ala Arg Asp Pro Gly Val His Ser Gly Thr Asn Leu 1 5 10 <210> twenty two <211> 6 <212> PRT <213> Artificial Sequence <400> twenty two Gln Ser Ile Tyr Ser Tyr 1 5 <210> twenty three <211> 3 <212> PRT <213> Artificial Sequence <400> twenty three Tyr Ala Ser 1 <210> twenty four <211> 12 <212> PRT <213> Artificial Sequence <400> twenty four Gln Gln Asp Tyr Ser Gly Tyr Asn Val Asp Asn Thr 1 5 10 <210> 25 <211> 118 <212> PRT <213> Artificial Sequence <400> 25 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Thr Ser Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Thr Gly Ser Gly Ser Thr Pro Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Val Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Asp Pro Gly Val His Ser Gly Thr Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Ser Val Ser Ser 115 <210> 26 <211> 110 <212> PRT <213> Artificial Sequence <400> 26 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Tyr Ser Tyr 20 25 30 Cys Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Gly Tyr Asn 85 90 95 Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 27 <211> 228 <212> PRT <213> Artificial Sequence <400> 27 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Ala Gly Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Thr Ser Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Thr Gly Ser Gly Ser Thr Pro Tyr Ala Ser Trp Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr Leu 65 70 75 80 Gln Val Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Asp Pro Gly Val His Ser Gly Thr Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Ser Val Ser Ser Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 115 120 125 Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 130 135 140 Gln Ser Ile Tyr Ser Tyr Cys Ser Trp Tyr Gln Gln Lys Pro Gly Gln 145 150 155 160 Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser Gly Val 165 170 175 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr 180 185 190 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 195 200 205 Asp Tyr Ser Gly Tyr Asn Val Asp Asn Thr Phe Gly Gly Gly Thr Glu 210 215 220 Val Val Val Lys 225 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <400> 28 Gly Phe Asp Phe Thr Thr Tyr Tyr Tyr 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <400> 29 Ile Tyr Thr Gly Ser Ser Asp Ser Ile 1 5 <210> 30 <211> 19 <212> PRT <213> Artificial Sequence <400> 30 Ala Arg Asp Arg Pro Ser Gly Asn Asp Asp Tyr Ile Tyr Asp Gly Gly 1 5 10 15 Met Asp Leu <210> 31 <211> 6 <212> PRT <213> Artificial Sequence <400> 31 Gln Thr Ile Asn Ser Trp 1 5 <210> 32 <211> 3 <212> PRT <213> Artificial Sequence <400> 32 Gln Ala Ser 1 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <400> 33 Gln Gln Gly Tyr Ser Phe Gly Asn Val Asp Asn Ala 1 5 10 <210> 34 <211> 126 <212> PRT <213> Artificial Sequence <400> 34 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe Thr Thr Tyr Tyr 20 25 30 Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile Tyr Thr Gly Ser Ser Asp Ser Ile Tyr Tyr Ala Ser Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Asp Arg Pro Ser Gly Asn Asp Asp Tyr Ile Tyr Asp Gly Gly 100 105 110 Met Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Phe Ser 115 120 125 <210> 35 <211> 111 <212> PRT <213> Artificial Sequence <400> 35 Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val Ser Ile Lys Cys Gln Ala Ser Gln Thr Ile Asn Ser Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Lys Leu Ala Ala Ser Gly Val Ser Ser Arg Phe Lys 50 55 60 Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu 65 70 75 80 Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Gly 85 90 95 Asn Val Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <210> 36 <211> 237 <212> PRT <213> Artificial Sequence <400> 36 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe Thr Thr Tyr Tyr 20 25 30 Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile Tyr Thr Gly Ser Ser Asp Ser Ile Tyr Tyr Ala Ser Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Asp Arg Pro Ser Gly Asn Asp Asp Tyr Ile Tyr Asp Gly Gly 100 105 110 Met Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Phe Ser Ala Tyr 115 120 125 Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly Gly Thr 130 135 140 Val Ser Ile Lys Cys Gln Ala Ser Gln Thr Ile Asn Ser Trp Leu Ser 145 150 155 160 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gln 165 170 175 Ala Ser Lys Leu Ala Ala Ser Gly Val Ser Ser Arg Phe Lys Gly Ser 180 185 190 Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu Cys Ala 195 200 205 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Phe Gly Asn Val 210 215 220 Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 225 230 235

Claims

1. A rabbit recombinant monoclonal antibody that binds to the CD38 protein, characterized in that: The rabbit recombinant monoclonal antibody that binds to the CD38 protein is named 8A1: the amino acid sequences of its heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of its light chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

2. The rabbit recombinant monoclonal antibody binding to CD38 protein according to claim 1, characterized in that: The rabbit recombinant monoclonal antibody named 8A1 has a heavy chain variable region sequence as shown in SEQ ID NO:7 and a light chain variable region sequence as shown in SEQ ID NO:

8.

3. The rabbit recombinant monoclonal antibody binding to CD38 protein according to claim 1 or 2, characterized in that: The rabbit recombinant monoclonal antibody named 8A1 has an SCFV sequence that is the amino acid sequence shown in SEQ ID NO:

9.

4. The rabbit recombinant monoclonal antibody binding to CD38 protein according to claim 1, characterized in that: The rabbit recombinant monoclonal antibody has a light chain constant region of κ chain and a heavy chain constant region of IgG type.

5. A nucleic acid molecule, characterized in that: It encodes the rabbit recombinant monoclonal antibody that binds to the CD38 protein as described in any one of claims 1-4.

6. A carrier, characterized in that: It contains the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that: The host cell contains the rabbit recombinant monoclonal antibody that binds to CD38 protein as described in any one of claims 1 to 4, the nucleic acid molecule as described in claim 5, or the vector as described in claim 6.

8. The use of the rabbit recombinant monoclonal antibody binding CD38 protein as described in any one of claims 1 to 4, the nucleic acid molecule as described in claim 5, the vector as described in claim 6, and the host cell as described in claim 7 in the preparation of a reagent for detecting CD38 protein.

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