Preparation method and application of zebrafish cd4-1 and cd8 alpha monoclonal antibodies based on joint immunization and plasma cell enrichment

Abstract

The application discloses a zebrafish CD4-1 and CD8 alpha monoclonal antibody preparation method and application based on joint immunity and plasma cell enrichment. The hybridoma cell strains 2G8C5 and 1A12F2 are preserved in the China Center for Type Culture Collection (CCTCC), and the preservation numbers are CCTCC NO: C202622 and CCTCC NO: C202623 respectively. The application further discloses a specific monoclonal antibody against zebrafish CD4-1 and CD8 alpha. The application adopts a joint immunity mode of 'cell immunity + recombinant protein immunity', and innovatively introduces CD138 + plasma cell sorting technology, and realizes efficient immunity and cell fusion. The zebrafish CD4-1 and CD8 alpha monoclonal antibodies have specificity, and provide an important tool for researching the role and mechanism of lymphocytes in adaptive immune response of bony fishes.

Description

Technical Field

[0001] This invention belongs to the field of fish immunology and relates to a method for preparing and applying zebrafish CD4-1 and CD8α monoclonal antibodies based on combined immunization and plasma cell enrichment. Specifically, it relates to a zebrafish T lymphocyte surface co-receptor CD4-1 and CD8α monoclonal antibody, its preparation method and application, and especially to a hybridoma cell line that secretes zebrafish T lymphocyte surface co-receptor CD4-1 and CD8α monoclonal antibodies, the monoclonal antibody, its preparation method and application. Background Technology

[0002] T lymphocytes are a core component of the adaptive immune response in vertebrates, playing a crucial role in pathogen clearance, maintaining immune homeostasis, and the formation of immune memory. Based on differences in their surface co-receptors, T lymphocytes are mainly divided into CD4+ and CD5+. + T cells and CD8 + T cells have two subsets. CD4 and CD8 molecules, as co-receptors of the T cell receptor (TCR), play a crucial synergistic role in T cell antigen recognition and activation. They bind to major histocompatibility complex class II (MHC-II) and class I (MHC-I) molecules on the surface of antigen-presenting cells, respectively, thereby enhancing the interaction between the TCR and the peptide-MHC complex and mediating downstream signal transduction, determining the functional differentiation direction of T cells.

[0003] CD4 + T cells primarily function as helper immune cells. In peripheral immune tissues, they can differentiate into multiple functional subsets, including Th1, Th2, Th17, Treg, and Tfh, regulating immune responses by secreting specific cytokines. CD8 + T cells, also known as cytotoxic T lymphocytes, primarily eliminate virus-infected or tumor cells by secreting perforin and granzymes, or by inducing apoptosis in target cells through the Fas-FasL signaling pathway. In bony fishes, an adaptive immune system has gradually developed, exhibiting high conservation in its overall framework compared to mammals, but significant differences remain in molecular composition and regulatory mechanisms. Previous studies have shown that bony fish CD4 molecules typically contain two subtypes, CD4-1 and CD4-2, whose structure and function differ somewhat from mammalian CD4. The main functional form of the CD8 molecule is the CD8αβ heterodimer, composed of CD8α and CD8β chains, with CD8α being a key component for its immune function. However, systematic and in-depth research on the specific functions of CD4-1 and CD8α in T cell development, differentiation, and immune responses in bony fish remains lacking.

[0004] Zebrafish (Danio rerio), as an important vertebrate model organism, is widely used in developmental biology, toxicology, and disease model research due to its advantages such as transparent embryos, ease of genetic manipulation, rapid development, and complete genomic information. In recent years, the value of zebrafish in immunological research has also become increasingly prominent, with its innate and adaptive immune systems holding significant comparative evolutionary value. However, limited by the lack of specific immunological research tools, current research on zebrafish T cells mainly focuses on gene expression levels or transcriptome analysis, making it difficult to analyze CD4 at the protein or cellular level. + T cells and CD8 + Accurate identification, sorting, and functional analysis of T cells are crucial. Therefore, preparing and obtaining specific monoclonal antibodies against zebrafish CD4-1 and CD8α is of great significance for accurately identifying zebrafish T cell subsets and elucidating their functions in infection, inflammation, and immune regulation. This will not only help to improve the functional atlas of adaptive immunity in bony fish T cells but also provide key technical support for immunological research in zebrafish disease models, vaccine evaluation, and disease-resistant breeding. Furthermore, it has significant scientific value and practical implications for elucidating the evolutionary patterns of the adaptive immune system in vertebrates. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a hybridoma cell line that secretes monoclonal antibodies against zebrafish CD4-1 and CD8α, its preparation method, and its applications. Specifically, it proposes a monoclonal antibody against zebrafish T lymphocyte surface co-receptors CD4-1 and CD8α, its preparation method, and its applications. In particular, it proposes a hybridoma cell line that secretes monoclonal antibodies against zebrafish T lymphocyte surface co-receptors CD4-1 and CD8α, the monoclonal antibody, its preparation method, and its applications.

[0006] This invention addresses the shortcomings of existing technologies by providing more efficient immunization and fusion methods. It provides hybridoma cell lines 2G8C5 and 1A12F2 that can stably secrete monoclonal antibodies against zebrafish T lymphocyte surface coreceptors CD4-1 and CD8α, as well as the monoclonal antibodies, their preparation methods, and applications. These methods enable the specific identification of zebrafish T lymphocyte CD4-1 and CD8α subsets, providing an important immunological tool for the study of adaptive immune mechanisms in bony fish.

[0007] This invention prepares and obtains specific monoclonal antibodies against zebrafish CD4-1 and CD8α, which is of great significance for accurately identifying zebrafish T cell subsets and elucidating their functions in infection, inflammation, and immune regulation. This not only helps to improve the functional atlas of adaptive immunity in bony fish T cells, but also provides key technical support for immunological research on zebrafish disease models, vaccine evaluation, and disease-resistant breeding. Furthermore, it has significant scientific value and practical implications for elucidating the evolutionary patterns of the adaptive immune system in vertebrates.

[0008] The innovation of this invention lies in the use of a combined immunization strategy of "cellular immunity + recombinant protein immunity", while simultaneously introducing CD138. + Plasma cell sorting enriched highly secretory B cells for fusion, significantly improving the efficiency of obtaining antibodies against natural membrane protein conformation epitopes. Furthermore, the genes of the variable regions of the heavy and light chains of the monoclonal antibody were cloned and sequenced to confirm the antibody at the molecular sequence level.

[0009] In this invention, the hybridoma cell lines 2G8C5 and 1A12F2 are deposited at the China Center for Type Culture Collection (CCTCC) with accession numbers CCTCC NO: C202622 and CCTCC NO: C202623, respectively.

[0010] This invention proposes a specific monoclonal antibody against zebrafish CD4-1, which can specifically bind to zebrafish CD4-1.

[0011] In one specific embodiment, the antibody is produced by hybridoma cell line 2G8C5, which was deposited on January 16, 2026, at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202622.

[0012] The present invention also proposes a specific monoclonal antibody against zebrafish CD8α, which can specifically bind to zebrafish CD8α.

[0013] In one specific embodiment, the antibody is produced by hybridoma cell line 1A12F2, which was deposited on January 16, 2026, at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202623.

[0014] The present invention also proposes a specific monoclonal antibody against zebrafish CD4-1 and / or its antigen-binding fragment, wherein the specific monoclonal antibody and / or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, etc.; wherein the heavy chain variable region comprises CDRH1 as shown in SEQ ID NO:11, CDRH2 as shown in SEQ ID NO:12, CDRH3 as shown in SEQ ID NO:13, etc.; and the light chain variable region comprises CDRL1 as shown in SEQ ID NO:14, CDRL2 as shown in SEQ ID NO:15, CDRL3 as shown in SEQ ID NO:16, etc.

[0015] In one specific embodiment, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 85% sequence identity with it.

[0016] In one specific embodiment, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 85% sequence identity with it.

[0017] This invention also proposes a specific monoclonal antibody against zebrafish CD8α and / or its antigen-binding fragment, wherein the specific monoclonal antibody and / or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, etc.; wherein the heavy chain variable region comprises CDRH1 as shown in SEQ ID NO: 19, CDRH2 as shown in SEQ ID NO: 20, CDRH3 as shown in SEQ ID NO: 21, etc.; the light chain variable region comprises CDRL1 as shown in SEQ ID NO: 22, CDRL2 with the sequence GAS, CDRL3 as shown in SEQ ID NO: 23, etc.

[0018] In one specific embodiment, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 85% sequence identity with it.

[0019] In one specific embodiment, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence having at least 85% sequence identity with it.

[0020] The present invention also proposes a recombinant protein comprising one or more of the specific monoclonal antibody as described above, or the specific monoclonal antibody as described above and / or its antigen-binding fragment.

[0021] The present invention also proposes an antibody conjugate comprising:

[0022] (a) One or more of the following: a specific monoclonal antibody as described above, or a specific monoclonal antibody and / or its antigen-binding fragment as described above, or a recombinant protein as described above; and

[0023] (b) Conjugates selected from the following groups: detectable markers, enzymes, cytokines, radionuclides, nanoparticles / nanoparticles

[0024] One or more of the following: rod, etc.

[0025] In one specific embodiment, the antibody conjugate comprises a specific monoclonal antibody against zebrafish CD4-1 and a biotinylate marker, as described above.

[0026] In one specific embodiment, the antibody conjugate comprises a specific monoclonal antibody against zebrafish CD8α as described above and a FITC fluorescent marker, etc.

[0027] The present invention also proposes a hybridoma cell line, which is capable of producing specific monoclonal antibodies against zebrafish CD4-1 as described above.

[0028] In one specific embodiment, the hybridoma cell line includes hybridoma cell line 2G8C5, etc., wherein hybridoma cell line 2G8C5 was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C202622.

[0029] The present invention also proposes a hybridoma cell line, which is capable of producing specific monoclonal antibodies against zebrafish CD8α as described above.

[0030] In one specific embodiment, the hybridoma cell line includes hybridoma cell line 1A12F2, etc., wherein hybridoma cell line 1A12F2 was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C202623.

[0031] The present invention also proposes a reagent / kit comprising one or more of the following: a specific monoclonal antibody as described above, or a recombinant protein as described above, or a specific monoclonal antibody and / or an antigen-binding fragment as described above, or an antibody-drug conjugate as described above, or a hybridoma cell line as described above.

[0032] The present invention also proposes a detection system comprising one or more of the following: a specific monoclonal antibody as described above, or a recombinant protein as described above, or a specific monoclonal antibody and / or antigen-binding fragment as described above, or an antibody-drug conjugate as described above, a reagent / kit as described above, or a hybridoma cell line as described above.

[0033] This invention also proposes a combined immunization method, which includes primary cellular immunization and protein-enhanced immunization. The core of this method is: firstly, primary immunization is performed by delivering natural conformational antigens using live cells that stably express membrane proteins to activate an immune response against conformational epitopes; subsequently, enhanced immunization is performed using high-purity recombinant proteins to synergistically enhance antibody titers and affinity against both linear and conformational epitopes.

[0034] In one specific embodiment, the combined immunization method includes: performing cell-based immunization using live cells stably expressing the target antigen as immunogens; and performing protein-enhanced immunization using recombinant extracellular domain proteins of the target antigen after the cell-based immunization.

[0035] In one specific embodiment, the combined immunization method includes: ① cellular immunization: using live cells stably expressing the target antigen as immunogens to immunize experimental animals; ② protein immunization: after the cellular immunization, using the extracellular domain recombinant protein of the target antigen in combination with an adjuvant to perform multiple booster immunizations on the same experimental animal.

[0036] Specifically, the method includes the following steps:

[0037] The first step involves constructing mammalian cells stably expressing zebrafish CD4-1 or CD8α, and then using these live cells to perform primary immunization on experimental animals, i.e., cell-mediated immunization. The target antigen is a transmembrane protein or membrane-anchored protein with a native membrane conformation, preferably including, but not limited to, zebrafish CD4-1 or CD8α proteins and their homologs or variants. The experimental animals are preferably mice, rats, or rabbits, animals suitable for monoclonal antibody preparation. This step, through the presentation of the antigen's native membrane conformation, can efficiently induce an immune response against conformation-dependent epitopes.

[0038] The cell-mediated immunization is performed 2-3 times, with an interval of 1-2 weeks; preferably, the cell-mediated immunization is performed 2 times, with an interval of 2 weeks.

[0039] The cell dose range for each cellular immunization was 5 × 10⁻⁶. 8 Up to 5×10 9 Cells / kg, preferably 5×10⁻⁶ 8 Cells / kg

[0040] The second step involves preparing the extracellular recombinant protein of the target antigen using a eukaryotic expression system. The eukaryotic expression system is preferably a mammalian cell expression system (such as CHO, HEK293, etc.). Expression in this system ensures that the recombinant protein has correct folding and post-translational modifications. Subsequently, the expression product is purified to obtain a high-purity recombinant protein immunogen, which will be used for the booster immunization in the third step, i.e., protein immunization.

[0041] Preferably, the purification method includes affinity chromatography and the like.

[0042] The third step involves administering booster immunizations to the same group of experimental animals using the high-purity recombinant protein and adjuvant prepared in the second step, following the completion of the cellular immunization procedure described in the first step. This adjuvant is preferably one or more of Freund's incomplete adjuvant, aluminum adjuvant, or TiterMax. This combined immunization strategy synergistically activates immune responses against both native and linear epitopes of the antigen, significantly increasing the titer and affinity of polyclonal antibodies, ultimately laying the foundation for the preparation of highly efficient hybridoma cells.

[0043] In this process, a recombinant protein solution with a concentration of 1.0-2.0 mg / mL is mixed with Freund's adjuvant suspension at a volume ratio ranging from 1:(0.5-1.0). Preferably, the volume ratio of 2.0 mg / mL recombinant protein to adjuvant is 1:1.

[0044] The booster immunization is performed 2-4 times, with an interval of 1-2 weeks between each booster immunization, and the recombinant protein dose for each booster immunization ranges from 5-10 mg / kg; preferably, the booster immunization is performed 2 times, with an interval of 1 week between each booster immunization, and the recombinant protein dose for each booster immunization is 5 mg / kg.

[0045] In one specific embodiment, the method includes the following steps:

[0046] The first step involved constructing mammalian cells stably expressing zebrafish CD4-1 or CD8α, and administering two intraperitoneal immunizations (cell-mediated immunity) to mice in live cell form, with a two-week interval. This allowed the antigen to be presented in its native membrane conformation, thereby inducing an immune response against conformation-dependent epitopes. The cell dose for each immunization ranged from 1 × 10⁻⁶ cells / mL. 6 Up to 1×10 7 5 × 10 cells, preferably 5 × 10 6 Each cell.

[0047] The second step involves using a eukaryotic expression system to prepare recombinant CD4-1 or CD8α extracellular domain proteins with correct folding and post-translational modifications, and then obtaining high-purity immunogens through affinity purification.

[0048] The third step involves two additional cellular immunizations, followed by two more recombinant protein-adjuvant immunizations (protein immunizations) to enhance antibody titer and affinity, enabling synergistic recognition of natural and linear epitopes, ultimately for subsequent high-efficiency hybridoma preparation. The volume ratio of recombinant protein to adjuvant is 1:1. The protein immunizations are performed twice, one week apart, with each immunization using 100 μg of recombinant protein.

[0049] This invention also proposes a targeted and efficient hybridoma fusion method, which includes enrichment of highly secretory plasma cells and conformational antigen-induced targeted fusion of B cells. Based on traditional whole-cell fusion of spleen cells, this method innovatively introduces CD138 from the spleen. + Plasma cell flow cytometry sorting step, CD138 obtained by sorting + The number of plasma cells was 5 × 10 4 Up to 5×10 5 The purity of the cells is not less than 85%. During the terminal phase of immunization, a population of B cells in a high antibody-secreting state is preferentially isolated to improve the positive fusion rate of specific antibodies from a cellular perspective. This is combined with the conformational epitope-dominant response induced by previous "cell-based immunization + recombinant protein immunization" (the aforementioned combined immunization methods). Compared with existing technologies that rely on random spleen cell fusion, this method represents a substantial improvement in cell selection strategy, pre-fusion cell state control, and antibody source directionality, overcoming technical challenges such as low positive clone ratios, high screening workload, and difficulty in obtaining antibodies that recognize natural conformational epitopes during antigen immunization.

[0050] The present invention also proposes a method for detecting zebrafish CD4-1 and / or CD8α, the method comprising detecting zebrafish CD4-1 and / or CD8α by one or more of the following: a specific monoclonal antibody as described above, or a recombinant protein as described above, or a specific monoclonal antibody and / or antigen-binding fragment as described above, or an antibody-drug conjugate as described above, a reagent / kit as described above, or a detection system as described above, or a hybridoma cell line as described above.

[0051] In one specific embodiment, the detection includes one or more of the following: flow cytometry, immunofluorescence assay, ELISA, Western blot (WB) assay, and semi-quantitative assay.

[0052] In one specific embodiment, the method includes in vivo methods and in vitro methods, etc.

[0053] In one specific embodiment, the method includes diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic methods, etc.

[0054] In one specific embodiment, the detection includes qualitative detection and quantitative detection, etc.

[0055] The present invention also proposes a method for detecting or identifying positive hybridoma cell lines. The method includes using one or more of the following as detection tools: a specific monoclonal antibody as described above, or a recombinant protein as described above, or a specific monoclonal antibody and / or antigen-binding fragment as described above, or an antibody-drug conjugate as described above, a reagent / kit as described above, or a detection system as described above, to detect and screen specific antibodies expressed in the supernatant or on the cell surface of hybridoma cells, thereby efficiently and accurately identifying positive hybridoma cell lines that can secrete monoclonal antibodies that recognize the natural conformational epitopes of target antigens.

[0056] The present invention also proposes a method for preparing the hybridoma cell line as described above, wherein the preparation method obtains the hybridoma cell line by means of the combined immunization method and the directed and efficient hybridoma fusion method as described above.

[0057] This invention also proposes the following methods for preparing detection reagents / kits for detecting zebrafish CD4-1 and / or CD8α: specific monoclonal antibodies as described above, recombinant proteins as described above, specific monoclonal antibodies and / or antigen-binding fragments as described above, antibody conjugates as described above, reagents / kits as described above, detection systems as described above, hybridoma cell lines as described above, or methods as described above. These methods include: preparing detection plates for detecting zebrafish CD4-1 and / or CD8α; preparing specific monoclonal antibodies and / or antigen-binding fragments against zebrafish CD4-1 and / or CD8α; screening for specific monoclonal antibodies and / or antigen-binding fragments against zebrafish CD4-1 and / or CD8α; preparing fusion cells; detecting zebrafish CD4-1 and / or CD8α; and improving the T cell line of bony fish. Applications include functional mapping of cellular adaptive immunity, immunological research on zebrafish disease models / vaccine evaluation / disease-resistant breeding, elucidating the evolutionary laws of the adaptive immune system in vertebrates, and research on the role and mechanism of lymphocytes in the adaptive immune response of bony fish.

[0058] In the application, the detection includes one or more of the following: flow cytometry, immunofluorescence assay, ELISA, Western blot (WB) assay, and semi-quantitative assay.

[0059] In the application described, the method includes diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic methods, etc.

[0060] In one specific embodiment, the detection includes qualitative detection and quantitative detection, etc.

[0061] In one specific embodiment, the method includes in vivo methods and in vitro methods, etc.

[0062] In one specific embodiment of the present invention, the nucleotide sequence described above includes a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence; or a nucleic acid sequence having the same function as the nucleotide sequence formed by substitution, deletion or addition of one or more nucleic acid bases; or a nucleotide sequence that hybridizes with the nucleotide sequence or its full-length complement under stringent conditions; or a nucleotide sequence that is distinct from the nucleotide sequence due to the degeneracy of the genetic codon.

[0063] In one specific embodiment of the present invention, the amino acid sequence described above includes: an amino acid sequence having at least 85% sequence identity with the amino acid sequence; or an amino acid sequence having the same function but with one or more amino acid residues substituted, deleted, or added; or an amino acid fragment expressed having the same function after substitution, deletion, or addition of one or more nucleic acid bases based on the coding nucleotide sequence of the amino acid sequence; or an amino acid sequence having the same function that can hybridize with the coding nucleotide sequence of the amino acid sequence under moderately stringent conditions and encode the same function.

[0064] Compared with existing technologies, the beneficial effects of this invention include: this invention adopts a combined immunization method of "cell-based immunity + recombinant protein immunity", and innovatively introduces CD138. + Plasma cell sorting technology enables highly efficient immunization and cell fusion. Simultaneously, this invention establishes a screening system combining flow cytometry, immunofluorescence, and semi-quantitative methods. Through complete sequencing of the heavy chain variable region and light chain variable region genes, antibody sequences are confirmed at the molecular level. This successfully prepared zebrafish CD4-1 and CD8α monoclonal antibodies, ensuring sufficient specificity and providing an important tool for subsequent research on the role and mechanism of lymphocytes in the adaptive immune response of bony fish. Attached Figure Description

[0065] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0066] Figure 1 This image shows the transfection effect of BOSC cells as detected by flow cytometry.

[0067] Figure 2Image showing the transfection effect of BOSC cells under a fluorescence microscope;

[0068] Figure 3 The infection effect of NIH / 3T3 cells as detected by flow cytometry is shown in the figure.

[0069] Figure 4 Flow cytometry results for detecting the specific binding of fusion cell culture supernatant in wells 2G8C5 and 1A12F2 to zebrafish spleen leukocytes;

[0070] Figure 5 This image shows the results of specific binding of monoclonal antibodies to CD4-1 and CD8α in zebrafish under a fluorescence microscope.

[0071] Figure 6 CD4-1 + Cell population, CD4-1 - Cell population and CD8 + Cell population, CD8 - Agarose gel electrophoresis results of cell-specific gene expression in various cell populations;

[0072] Figure 7 CD4-1 in zebrafish before and after Aeromonas hydrophila infection + and CD8α + Flow cytometry analysis results of T lymphocytes. Detailed Implementation

[0073] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0074] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0075] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0076] This invention discloses a hybridoma cell line secreting monoclonal antibodies against zebrafish CD4-1 and / or CD8α, its preparation method, and its applications. The hybridoma cell lines 2G8C5 and 1A12F2 are deposited at the China Center for Type Culture Collection (CCTCC), with accession numbers CCTCC NO: C202622 and CCTCC NO: C202623, respectively. This invention also discloses a specific monoclonal antibody against zebrafish CD4-1 and / or CD8α, which specifically binds to zebrafish CD4-1 and / or CD8α. This invention employs a combined immunization approach of "cell-based immunity + recombinant protein immunity" and innovatively introduces CD138. + Plasma cell sorting technology enables efficient immunization and cell fusion. The zebrafish CD4-1 and / or CD8α monoclonal antibodies described in this invention are specific, providing an important tool for subsequent research on the role and mechanism of lymphocytes in the adaptive immune response of bony fish.

[0077] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0078] In this embodiment of the invention, the experimental materials used are: DMEM medium (Gibco), RPMI 1640 medium (Gibco), PEG (MERCK), FITC powder (MCE), APC-Streptavidin (BioLegend), Biotin (ThermoFisher), Hoechst-33342 (Beyotime), Protein G (Beyotime), fetal bovine serum FBS (Gibco), penicillin-streptomycin (Thermo Fisher), Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Abcam), and Percoll (Cytiva).

[0079] Example 1:

[0080] This embodiment provides two methods for preparing zebrafish immunogens, namely cell-based immunogens and protein-based immunogens.

[0081] 1. Furthermore, the above-mentioned method for preparing cell-mediated immunity includes the following steps:

[0082] Zebrafish CD4-1 and CD8α gene cloning:

[0083] Total RNA was extracted from zebrafish leukocytes, and qualified RNA was selected for reverse transcription to synthesize cDNA templates for later use. Primer pairs for PCR amplification were designed, including a forward primer F1 and a reverse primer R1. Specifically, the forward primer F1 sequence for CD4-1 is CCGCTCGAGGCCACCATGCTCGGTTTGATTCTAAT (SEQ ID NO: 1), and the reverse primer R1 sequence is CGGAATTCTCAGGTCTTGTAGAATCCCT (SEQ ID NO: 2); the forward primer F2 sequence for CD8α is GAAGATCTGCCACCATGTATCAAATATCCACTGG (SEQ ID NO: 3), and the reverse primer R2 sequence is CGGAATTCCTAGAAGTGGCTGTTGGGCA (SEQ ID NO: 4). Subsequently, using the cDNA as a template, a PCR amplification reaction system was prepared and amplification was performed. After amplification, the PCR products were detected by agarose gel electrophoresis, and the target bands were recovered. The recovered products are the isolated zebrafish CD4-1 gene and zebrafish CD8α gene (hereinafter collectively referred to as "CD4-1 and CD8α genes"). "Isolated" means that the genes have been separated from the DNA sequences flanking them in their natural state and have been isolated from the nucleic acid and protein components that accompany them in their natural state.

[0084] In this step, the total volume of the PCR amplification reaction system is 50 μL, which, by volume percentage, includes: 5% forward primer F1 or F2, 5% reverse primer R1 or R2, 50% 2× PrimerSTAR DNAPolymerase, 5% cDNA template, and the remaining volume is made up with dH2O.

[0085] The amplification program was as follows: 98 °C pre-denaturation for 10 s, 55 °C annealing for 15 s, 75 °C extension for 10 s, for 35 cycles; then 0.5 μL rTaq was added to the reaction system and the reaction was extended at 72 °C for 20 min.

[0086] The amplified zebrafish CD4-1 and CD8α gene fragment sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Comparative analysis of their intron and exon structures revealed that these gene fragments exhibit high structural similarity to other species, particularly other bony fishes.

[0087] Construction of zebrafish CD4-1 and CD8α expression vectors:

[0088] 4.5 μL of the recovered CD4-1 or CD8α gene fragment was mixed with the vector ligation system (including 0.5 μL LPMD19T vector and 5 μL Solution I) and ligated at 16 ℃ for 15 h to obtain the first ligation system. 10 μL of the first ligation system was added to 50 μL of Trans10 competent cells, placed on ice for 30 min, and then heat-shocked in a 42 ℃ water bath for 45 s, followed immediately by placing on ice for 2 min.

[0089] Add 400 μL of antibiotic-free LB liquid medium to the transformation system and incubate at 37 ℃ with shaking for 1 h. Then, take 200 μL of bacterial culture and spread it on LB solid medium containing ampicillin (1:1000) and incubate overnight at 37 ℃. Select positive monoclonal strains for sequencing identification. After confirmation, extract the PMD19T recombinant plasmid containing the CD4-1 or CD8α gene to obtain CD4-1-PMD19T plasmid and CD8α-PMD19T plasmid, respectively.

[0090] Take 1 μg each of the recombinant plasmid and the pLenti-CMV-GFP empty vector, add them to a double digestion system (including 1 μL BglⅡ, 1 μL EcoRⅠ, 4 μL 3.1 10× FastDigest Green Buffer, and bring the volume to 40 μL with dH2O), and digest at 37 ℃ for 4 h to obtain the corresponding digestion products. Take 4.5 μL of the digestion products and mix them with a DNA ligation system (including 1 μL T4 DNA Ligase and 1 μL T4 DNA Ligase Buffer), and ligate at 16 ℃ for 18 h to obtain a second ligation system. Transform the second ligation system into 100 μL of Trans10 competent cells, place on ice for 30 min, heat shock at 42 ℃ for 40 s, then incubate on ice for 2 min, add 500 μL of fresh LB medium, and shake and culture for 45 min. Take 100 μL of the bacterial culture and spread it on LB solid medium containing kanamycin, and incubate at 37 ℃. The cultures were incubated overnight at ℃, and positive monoclonal strains were selected the next day. The positive monoclonal strains were then expanded and plasmids were extracted to obtain expression vectors containing the CD4-1 or CD8α gene, namely pLenti-CMV-GFP-CD4-1 plasmid and pLenti-CMV-GFP-CD8α plasmid.

[0091] Retrovirus preparation:

[0092] Frozen BOSC cells were removed from liquid nitrogen, thawed, revived, and passaged to obtain passaged BOSC cells, which were passaged twice. 500 μL of trypsin was used to digest the passaged BOSC cells at 37 ℃, followed by resuspending the cells in 5 mL of DMEM medium. The cells were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded to obtain a cell pellet. The cell pellet was resuspended and counted, and then seeded into each 60 mm culture dish with 2 × 10⁶ cells / mL. 6 Each cell.

[0093] When the confluence of BOSC cells reached 85%, the culture supernatant was discarded, and 3 mL of DMEM medium containing 25 μM chloroquine (containing 1% penicillin and 10% FBS) was slowly added, and the cells were incubated at 37 ℃. In an EP tube, 10 μg of retroviral expression vector (pLenti-CMV-GFP-CD4-1 or pLenti-CMV-GFP-CD8α), 5 μg of Helper plasmid, and 50 μL of 2.5 M CaCl2 were added, and the volume was brought up to 500 μL with sterile water to form a mixture. Bubbles were then created by pipetting the mixture into a glass tube, and 500 μL of 2× HEBS buffer was slowly added dropwise during the pipetting process to prepare the transfection system. 1 mL of the transfection system was mixed thoroughly and added dropwise to a cell culture dish, gently shaken, and transfected. Eight hours after transfection, the culture supernatant was discarded, and 3 mL of fresh DMEM medium (containing 1% penicillin and 10% FBS) was added. The culture medium was replaced 1-2 times during subsequent culture with double antibiotics and 10% FBS. The cell culture supernatant was collected 12 h after the last culture medium replacement, centrifuged at 1200 rpm for 5-10 min, and the supernatant containing retroviruses was collected, aliquoted and stored at -80℃.

[0094] As shown in Figures 1-2, the transfection effect of cells after transfection was detected by fluorescence microscopy and flow cytometry, respectively. The transfected cells showed a green positive reaction under the fluorescence microscope.

[0095] NIH / 3T3 cell infection:

[0096] Frozen NIH / 3T3 cells were removed from liquid nitrogen, thawed, and then passaged to obtain NIH / 3T3 cells passaged 1-3 times. 500 μL of trypsin was used to digest the passaged NIH / 3T3 cells at 37 ℃. The cells were resuspended in 5 mL of DMEM medium, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended and counted. Then, 6 × 10⁶ cells were seeded in each 100 mm culture dish. 5 Each cell.

[0097] When the confluence of NIH / 3T3 cells reaches 50%, discard the culture supernatant and slowly add 5-10 mL of DMEM medium containing 5 μM polybrene (containing 1% antibiotics and 10% FBS). Add 550-650 μL of retrovirus suspension to each culture dish, mix gently, and incubate at 37 ℃ to complete cell infection. After 6-8 h of infection, add 1-2 mL of DMEM medium without polybrene to terminate the infection, and replace the medium with DMEM medium without polybrene 1-2 times during subsequent culture. 24 h after the last medium replacement, collect the cells, centrifuge, digest with 500 μL trypsin at 37 ℃ for 1 min, resuspend the cells in 5 mL of DMEM medium, and centrifuge. Discard the supernatant, resuspend in 10 mL of PBS and centrifuge twice. Finally, resuspend the cells in 200 μL of PBS to obtain transfected NIH / 3T3 cells.

[0098] As shown in Figure 3, flow cytometry analysis revealed that infected NIH / 3T3 cells exhibited a significant green fluorescent positive signal, indicating that the cells stably expressed the zebrafish T lymphocyte surface co-receptors CD4-1 and CD8α proteins on their cell surface.

[0099] 2. Furthermore, the above-mentioned method for preparing eukaryotic protein immunoassay includes the following steps:

[0100] To obtain correctly folded zebrafish CD4-1 and CD8α functional proteins with post-translational modifications, this invention employs a mammalian cell expression system for secretory expression. First, expression vectors are constructed via molecular cloning. Using previously obtained zebrafish CD4-1 and CD8α gene fragments as templates, specific primers are designed and synthesized for PCR amplification of their extracellular domain coding sequences. For the CD4-1 extracellular domain, forward primer F3: ctagctagcGCCACCATGGATTACAAGGATGACGATGACAAGGAAGATCTGCCACCATGATGTTTTG (SEQ ID NO: 5) and reverse primer R3: ccgctcgagTCACTTATCGTCGTCATCCTTGTAATCGTCCTTGTAGAATCCC (SEQ ID NO: 6) were used; for the CD8α extracellular domain, forward primer F4: ctagctagcGCCACCATGGATTACAAGGATGACGATGACAAGCCGCTCGAGGCCACCATGTATCAAATATAC (SEQ ID NO: 7) and reverse primer R4: ccgctcgagTCACTTATCGTCGTCATCCTTGTAATCAAAAGTGGTTACTGGGCA (SEQ ID NO: 8). The PCR reaction volume was 50 μL, containing 25 μL of 2×Phanta Max Master Mix, approximately 100 ng of template cDNA, 2 μL each of 10 μM forward and reverse primers, with the remainder supplemented with nuclease-free water. The reaction program was as follows: 95℃ pre-denaturation for 3 minutes; followed by 35 cycles of 98℃ for 15 seconds, 60℃ for 15 seconds, and 72℃ for 30 seconds; and a final extension at 72℃ for 5 minutes. The amplified product was ligated to the pFUSE-hIgG1-Fc2 vector, which had been linearized by double digestion with Nhe I and Xho I, at an insert-to-vector molar ratio of 3:1. The linearized vector was 100 ng, corresponding to approximately 100 ng of the purified PCR product. Ligation was performed using a homologous recombination kit (ClonExpress Ultra). The ligation products were transformed into Stbl3 competent cells, and positive clones were screened on ampicillin-resistant plates. After sequencing verification, the recombinant expression plasmids pFUSE-zCD4-1-ECD-Fc and pFUSE-zCD8α-ECD-Fc were obtained.

[0101] Protein expression and collection:

[0102] Subsequently, protein expression was performed using HEK293F cells in suspension culture. When the cell density reached 1.5 × 10⁻⁶ cells... 6At cell / mL, the PEI MAX transfection method was used, with 30 μg of recombinant plasmid and 60 μL of PEI MAX (1 mg / mL) diluted separately in 1.5 mL of Opti-MEM. TM In step I, after mixing and allowing to stand for 20 minutes to form a complex, 30 mL of cell suspension was added. Six hours after transfection, 1.5 mL of 10×Feed Solution was added, and the mixture was incubated at 37°C, 8% CO2, and 130 rpm for 120 hours. The culture was collected, centrifuged at 3000 ×g for 30 minutes at 4°C, and the supernatant was filtered to obtain a clear solution containing the target protein.

[0103] Protein purification: Protein purification was performed using Protein A affinity chromatography. 1 mL of Protein A Sepharose was used. TM Fast Flow packing material was used. After equilibration with binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4), cell culture supernatant was loaded to allow the Fc fusion protein to bind to the packing material. After washing with 10 μL of CV binding buffer, elution was performed with low-pH elution buffer (0.1 M glycine-HCl, pH 3.0), and the fraction was immediately neutralized with 1 M Tris-HCl (pH 8.5) and collected. The purified proteins were pooled, concentrated using 10 kDa ultrafiltration centrifuge tubes at 4°C, and then transferred to PBS buffer (pH 7.4). The final recombinant protein was aliquoted and stored at -80°C.

[0104] Example 2:

[0105] This embodiment provides a combined immunization approach of "cellular immunity + recombinant protein immunity". Specifically, cellular immunity is first used to ensure that the antigen is presented in its natural conformation, thereby improving the ability to recognize conformational epitopes; then, high-dose protein immunization is used to significantly enhance the immune response.

[0106] 1. First immunization (cell-based immunity)

[0107] NIH / 3T3 cells stably expressing zebrafish CD4-1 or CD8α were used as antigens and immunized by intraperitoneal injection, with each mouse receiving 1 × 10⁻⁶ cells. 7 1 cell, injection volume of 200 μL, without adjuvant.

[0108] 2. Secondary immunization (cellular immunity)

[0109] A second immunization was administered 14 days after the first immunization, using the same method, cell type, and dosage as the first immunization, and was still administered via intraperitoneal injection.

[0110] By administering two consecutive cellular immunizations, the mouse immune system was able to fully recognize the extracellular domains of zebrafish CD4-1 or CD8α under native conformational conditions, thereby enhancing the immune response to conformational epitopes.

[0111] 3. Third immunization (protein immunity)

[0112] Seven days after the second cellular immunization, a third immunization was performed. The purified and refolded zebrafish CD4-1 or CD8α recombinant protein (5 μg / μL) obtained in Example 1 of this invention was fully emulsified with Freund's complete adjuvant at a ratio of 1:1 (volume ratio). 100 μg of recombinant protein antigen was injected intraperitoneally into each mouse, with an injection volume of 200 μL.

[0113] 4. Fourth immunization (protein immunity)

[0114] Seven days after the third immunization, a fourth immunization was performed. The recombinant protein obtained in Example 1 of this invention was emulsified with Freund's incomplete adjuvant at a 1:1 (volume ratio), and 100 μg of recombinant protein antigen was injected intraperitoneally into each mouse. After four immunizations, cell fusion was performed.

[0115] Example 3:

[0116] In this embodiment, CD138 is innovatively introduced in the cell fusion process. + Plasma cell sorting technology. Through antigen-specific enrichment, plasma cell populations that have successfully recognized the target and differentiated into the terminal stage of antibody secretion are screened out, and then subjected to efficient cell fusion. This combined strategy of "functional screening + targeted fusion" significantly improves cell fusion efficiency.

[0117] Cell fusion:

[0118] On the third day after the end of immunization in mice, the animals were euthanized by cervical dislocation, and the spleen was removed under sterile conditions to prepare a spleen cell suspension.

[0119] To significantly improve the positive rate of subsequent hybridoma screening, this invention makes a key improvement to the traditional process: Before cell fusion, CD138 (a plasma cell-specific surface marker) antibody is used to specifically sort plasma cells from the prepared spleen cell suspension using immunomagnetic bead sorting technology. These plasma cells are terminally differentiated B cells that can secrete target antibodies after immune stimulation. Fusion with this population can enrich antigen-specific B cells, thereby greatly improving the efficiency of obtaining positive hybridoma clones.

[0120] Meanwhile, SP2 / 0 myeloma cells were resuscitated one week prior to the cell fusion procedure and passaged the day before fusion to ensure optimal cell growth. On the day of fusion, SP2 / 0 cells in logarithmic growth phase were collected for later use.

[0121] The cell fusion procedure was as follows: The sorted plasma cells were fused with the SP2 / 0 cells described above. First, the SP2 / 0 cells were gently pipetted from the culture flask wall using 10 mL of additive-free RPMI-1640 basal medium to prepare a single-cell suspension. Then, the plasma cell suspension and SP2 / 0 cell suspension were added to a centrifuge tube at a volume ratio of 10:1 and mixed thoroughly. The mixture was centrifuged at 1200 rpm for 8 minutes, the supernatant was discarded, and the two cell pellets were carefully resuspended and mixed until a paste was formed. The bottom of the centrifuge tube containing the mixed cell pellet was placed in a beaker preheated to 37°C in a water bath. Using a pipette, 1 mL of preheated 37°C polyethylene glycol (PEG) solution was added dropwise to the centrifuge tube slowly and continuously over 90 seconds, gently agitating the bottom of the tube while adding. After the addition was complete, the centrifuge tube was left to stand in the water bath for another 90 seconds. Subsequently, PEG dilution and inactivation were performed: 3 mL of preheated RPMI-1640(-) culture medium, approximately 30 seconds apart, was slowly added to the centrifuge tube in five portions to allow the cells to gradually adapt to the osmotic pressure change, until a total of 15 mL was added. Then, RPMI-1640(-) culture medium was added to bring the total volume to 50 mL, and the tube was centrifuged at 800 rpm for 8 minutes. The supernatant was discarded to obtain the confluent cell pellet. The cell pellet was resuspended in 3 mL of GIT selective culture medium containing HAT or HT to obtain the confluent cell resuspension. For safety, a portion of this resuspension can be cryopreserved as a backup. A certain amount of plasma cell suspension or feeder cells was added to the remaining cell resuspension, mixed thoroughly, and then seeded at 100 µL per well in a 96-well cell culture plate.

[0122] The culture plates were placed in a constant temperature incubator at 37℃ and 5% CO2. During culture, the cells were observed periodically under an inverted microscope. When the fused cells (i.e., hybridoma cells) grew to approximately 1 / 3 of the well bottom area, the culture supernatant was collected by centrifugation for screening. Under the microscope, the hybridoma cells showed good growth: active cell division, plump and rounded morphology, strong refractive index, uniform size, adherent growth, and exhibiting the expected continuous and unlimited proliferative potential.

[0123] Fusion cell screening and cloning:

[0124] Flow cytometry was used to detect the cell culture supernatant from hybridoma-positive wells. Specifically, spleen tissue from healthy zebrafish was collected, washed and ground under aseptic conditions, filtered through a 200-mesh sieve, and centrifuged at 2000 rpm for 5 min. The supernatant was discarded, and the cell pellet was collected. The cell pellet was resuspended in L-15 medium and added to Percoll separation medium. The mixture was centrifuged at 23 ℃ and 500 g for 35 min, and the resulting white ring-shaped cell layer was aspirated. This cell layer was washed with L-15 medium to obtain zebrafish peripheral leukocytes.

[0125] 100 μL of the supernatant from the hybridoma-positive wells to be tested was added to the resulting leukocyte pellet as the primary antibody, while SP2 cell culture supernatant was added to the control wells. The mixture was incubated at room temperature for 30 min. After incubation, the cells were centrifuged at 2500 rpm for 3 min to remove the supernatant, and the cell pellet was washed with FACS Buffer. Then, 100 μL of Alexa Fluor 488-labeled fluorescent secondary antibody (1:2000 dilution) was added to each well, and the mixture was incubated on ice in the dark for 30 min. After incubation, the cells were centrifuged again to remove the supernatant, and the cell pellet was thoroughly washed with FACS Buffer. Finally, the cells were resuspended in 200 μL of FACS Buffer for flow cytometry analysis to screen for positive wells that specifically bind to target cells. Flow cytometry results showed that the cell population distribution in wells 2G8 and 1A12 was significantly different from the control, suggesting that the fusion cells in these two wells can secrete specific antibodies and recognize specific proteins on the surface of zebrafish lymphocytes.

[0126] The fusion cells obtained from the positive wells 2G8 and 1A12 were further subcloned:

[0127] The specific procedure was as follows: After euthanizing the mice, the thymus tissue was removed under aseptic conditions, ground through a 200-mesh sieve, and repeatedly pipetted with RPMI-1640 culture medium to prepare a thymus cell suspension. The suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the suspension was resuspended in preheated GIT culture medium for later use.

[0128] After counting the fusion cells in the positive wells, the cells were serially diluted 10-fold with culture medium. Approximately 100 fusion cells were mixed with a thymocyte suspension, thoroughly mixed, and seeded at a volume of 100 μL / well into 96-well cell culture plates. The plates were then incubated at 37 ℃ and 5% CO2. Once clones had formed and grown to approximately one-third of the well area, the culture supernatant from each well was collected and screened again by flow cytometry. This yielded two stable hybridoma cell lines, 2G8C5 and 1A12F2, that secrete specific antibodies.

[0129] As shown in Figure 4, the monoclonal antibody secreted by the hybridoma cell line can specifically recognize zebrafish T lymphocytes, among which CD4-1 + T lymphocytes account for approximately 14.4% of the total lymphocyte count, CD8α + T lymphocytes accounted for approximately 8.3%, indicating that the obtained hybridoma cell lines have good specificity and application value. Subsequently, the hybridoma cell lines 2G8C5 and 1A12F2 were cryopreserved for subsequent experiments and applications.

[0130] Example 4:

[0131] This embodiment relates to the preparation and labeling method of anti-zebrafish T lymphocyte surface co-receptor monoclonal antibody secreted by hybridoma cell lines 2G8C5 and 1A12F2 obtained in Embodiment 3 of the present invention.

[0132] Ascites preparation: Ten-week-old BALB / c mice were selected, and 500 μL of sterile paraffin oil was injected intraperitoneally to induce ascites formation. After 10 days of treatment, hybridoma cell lines 1H10G6 and 1C11B7 in good growth condition were collected, washed with PBS to remove the culture medium, and resuspended in 200 μL of sterile PBS. Each mouse was then injected intraperitoneally with 2 × 10⁶ cells / mL of the solution. 5 The mice were inoculated with cells. Their physiological condition was observed daily after inoculation. When significant abdominal distension and limited movement occurred, the mice were euthanized by cervical dislocation, and ascites fluid was collected. The ascites fluid was centrifuged at 2000 rpm for 5 min, and the pale yellow supernatant in the middle layer was collected, aliquoted, and stored at -80 ℃ for later use.

[0133] Antibody purification: 200 μL of rProtein G agarose was added to a 15 mL centrifuge tube, washed three times with PBS, and then 500 μL of ascites fluid sample was added. The mixture was diluted with PBS to a total volume of 8 mL and incubated overnight at 4 °C to allow the antibody to bind to Protein G. After incubation, the agarose beads were washed multiple times with PBS to remove non-specific binding components. Elution was then performed with 500 μL of 0.1 mol / L Glycine-HCl solution (pH 2.8) to obtain the target monoclonal antibody. Immediately after elution, 1 / 10 of the volume of 1 mol / L Tris-HCl (pH 8.5) was added to neutralize the acidic environment. The resulting antibody was aliquoted and stored at -80 °C.

[0134] Biotin labeling: The purified CD4-1 antibody was placed in NaHCO3 buffer at pH 8.0 and dialyzed at 4 °C for 8 h. After dialysis, 120 μL of biotin solution at a concentration of 1 mg / mL was added per 1 mg of antibody, and the reaction was continued at room temperature for 3 h. After the reaction was completed, 8% of the system volume of 1 mol / L NH4Cl solution was added to terminate the labeling reaction, and the antibody was dialyzed for 10 min. Subsequently, the antibody was placed in a dialysis bag and dialyzed repeatedly with PBS at 4 °C to remove free biotin. Finally, 0.5 mg / mL sodium azide and 1 mg / mL BSA were added to the antibody solution to obtain the biotin-labeled CD4-1 monoclonal antibody, namely CD4-1-biotin, which was stored at low temperature for later use.

[0135] FITC fluorescent labeling: The purified CD8α antibody was first placed in PBS and dialyzed at 4 °C for 8 h. Then, it was transferred to Na₂CO₃ / NaHCO₃ buffer (25 mmol / L) and dialyzed for another 8 h to meet the conditions for fluorescent labeling. After labeling, the antibody solution was repeatedly dialyzed in PBS until no absorption signal was observed at 480 nm. Finally, 0.5 mg / mL sodium azide was added to the antibody to obtain the FITC-labeled CD8α monoclonal antibody, i.e., CD8α-FITC, which was stored at low temperature and protected from light.

[0136] Example 5:

[0137] This embodiment describes the determination of the nucleic acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody obtained in Embodiment 3 of the present invention.

[0138] Hybridoma cells in logarithmic growth phase were used to extract total RNA using the Trizol (TAKARA) method. cDNA was generated by reverse transcription using oligo(dT)20 (Invitrogen) as primers. Then, using cDNA as a template, the heavy chain variable region and light chain variable region genes were amplified using specific PCR primers. After the PCR products were purified by agarose gel electrophoresis, they were cloned into the pMD-19T vector via TA cloning, followed by sequencing and sequence analysis.

[0139] ① The amino acid sequence of the variable region of the CD4-1 heavy chain in zebrafish is: DVQLQESGPGLVKPSQTLSLTCTVS GYSFTSY W IEWIRQHPGKGLEWIG YINPYNDGTKYNEKFKG RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR DYYGSSYAMD Y WGQGTTVTVSS. (SEQ ID NO: 9). The underscore indicates the CDR area.

[0140] The amino acid sequence of the zebrafish CD4-1 light chain variable region is: DIVMTQSPSSLSASVGDRVTITC RASQSISDYLH WYQQKPGKAPKLLIY YASQSIS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPYT FGQGTKVEIK (SEQ ID NO: 10). The underscore indicates the CDR area.

[0141] Furthermore, the CDR regions (CDRH1, CDRH2, CDRH3) of the heavy chain variable region of zebrafish CD4-1 respectively contain the sequences shown in SEQ ID NO: 11-13, and the CDR regions (CDRL1, CDRL2, CDRL3) of the light chain variable region respectively contain the sequences shown in SEQ ID NO: 14-16.

[0142] Heavy chain CDR1 (VHCDR1): GYSFTSYW (SEQ ID NO: 11)

[0143] Heavy chain CDR2 (VHCDR2): YINPYNDGTKYNEKFKG (SEQ ID NO: 12)

[0144] Heavy chain CDR3 (VHCDR3): DYYGSSYAMDY (SEQ ID NO: 13)

[0145] Light chain CDR1 (VLCDR1): RASQSISDYLH (SEQ ID NO: 14)

[0146] Light chain CDR2 (VLCDR2): YASQSIS (SEQ ID NO: 15)

[0147] Light chain CDR3 (VLCDR3): QQSYSTPYT (SEQ ID NO: 16)

[0148] ②The amino acid sequence of the CD8α heavy chain variable region of zebrafish is: QVQLKQSGPGLVQPSQTLSLTCTVS GGSISSS YGYTMH WVKQSHGKSLEWIG YIYPYNGGTGYNQKFKG NKVSNRPSGNSRQTISKDTSKNQVFFKMNSLQSDDTAIYYCAR SYYYGMDV WGQGTTVTVSS (SEQ ID NO: 17). The underscore indicates the CDR area.

[0149] The amino acid sequence of the CD8α light chain variable region of zebrafish is: MDGGKMDTVQQSPSVLVAPSQSLSITCTVS KSVS TSGYNY VSWYQQHPGKAPKLMIY GAS NLASGVPSRFSGSGSGTSYSLTISSVEAEDAATYYC QQGYSYPYT FGGGTKLEIK

[0150] (SEQ ID NO: 18). The underlined area represents the CDR area.

[0151] Furthermore, the CDR regions (CDRH1, CDRH2, CDRH3) of the zebrafish CD8α heavy chain variable region contain the sequences shown in SEQ ID NO: 19-21, respectively, and the CDR regions (CDRL1, CDRL2, CDRL3) of the light chain variable region contain the sequences shown in SEQ ID NO: 22, GAS, and SEQ ID NO: 23, respectively.

[0152] Heavy chain CDR1 (VHCDR1): GGSISSSYGYTMH (SEQ ID NO: 19)

[0153] Heavy chain CDR2 (VHCDR2): YIYPYNGGTGYNQKFKG (SEQ ID NO: 20)

[0154] Heavy chain CDR3 (VHCDR3): SYYYGMDV (SEQ ID NO: 21)

[0155] Light chain CDR1 (VLCDR1): KSVSTSGYNY (SEQ ID NO: 22)

[0156] Light chain CDR2 (VLCDR2): GAS

[0157] Light chain CDR3 (VLCDR3): QQGYSYPYT (SEQ ID NO: 23)

[0158] Example 6:

[0159] This embodiment provides an application of the monoclonal antibody described in Embodiment 4 of the present invention in the study of adaptive immune response in bony fish.

[0160] The indirect immunofluorescence assay for the identification of monoclonal antibodies includes the following steps:

[0161] Leukocytes were isolated from the spleen of healthy grass carp and resuspended in 5 mL PBS. A suitable density of resuspended leukocytes was prepared into cell drops using a cell smear machine and fixed with methanol for 5 min. 100 μL of 1% BSA was added, and the plate was blocked at 37 ℃ for 1 h. The plate was washed twice with PBST and once with PBS, 5 min each time. 100 μL of the CD4-1-biotin antibody (1:200) labeled in Example 4 of this invention was added to the well plate, and the plate was incubated at 37 ℃ for 1 h. The plate was washed twice with PBST and once with PBS, 5 min each time. Then, APC-labeled streptavidin (BioLegend) and the CD8α-FITC antibody (both diluted 1:200 in PBS) labeled in Example 4 of this invention were added, and the plate was incubated at 37 ℃ for 1 h. The plate was washed twice with PBST and once with PBS, 5 min each time. One drop of Hoechst-33342 dye was added as a mounting medium, and the plate was examined under a fluorescence microscope.

[0162] like Figure 5 As shown, CD4-1 + T cells are highly expressed in lymphocytes, and CD4-1 + With CD8α + The fact that T cells do not co-express the antibodies indicates that the two antibodies have good specificity and do not cross-express.

[0163] The semi-quantitative identification of monoclonal antibodies includes the following steps:

[0164] Leukocytes were isolated from the spleen of healthy zebrafish and resuspended in 5 mL L-15 medium. 100 μL of the leukocyte suspension was added to a 96V plate and centrifuged at 2500 rpm for 3 min. The supernatant was discarded, and the supernatant of hybridoma cells from the positive wells in Example 3 of this invention was added as primary antibody. The plate was incubated on ice for 30 min. After centrifugation at 2500 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 200 μL FACS Buffer (L-15, 2% FBS). The plate was centrifuged again, and the resulting precipitate was washed once more. 100 μL of Goat Anti-Mouse IgG H&L (Alexa Fluor 647) (1:1000) fluorescent secondary antibody was added to each well, and the plate was incubated on ice in the dark for 30 min. After centrifugation at 2500 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 200 μL FACS Buffer (L-15, 2% FBS). Resuspend cells in %FBS, centrifuge again, and wash the resulting precipitate once more. Separate positive and negative cell populations, centrifuge at 2500 rpm for 5 min to obtain positive and negative cells respectively, extract total RNA from each, and reverse transcribe it into cDNA templates. Take quantitative amounts of cDNA templates from positive and negative cells for PCR reactions, and perform agarose gel electrophoresis on the PCR products to ensure consistency of β-actin between positive and negative cells.

[0165] like Figure 6 The electrophoresis results show that the positive clusters (i.e., CD4-1) obtained by 2G8C5 sorting + The expression levels of CD3ε and CD4-1 genes in the negative group were extremely high, while CD8 and IgM were not expressed, whereas the expression levels of CD4-1 were extremely high. - No CD4-1 gene expression was observed, but a certain amount of CD8 and IgM gene expression was observed; simultaneously, the positive group obtained from 1A12F2 sorting (i.e., CD8) + The expression levels of CD3ε and CD8 genes in the negative group were extremely high, while CD4-1 and IgM were not expressed, whereas the negative group (i.e., CD8) showed extremely high expression levels. - The absence of CD8 gene expression but the presence of CD4-1 and IgM gene expression indicates that the monoclonal antibody can specifically recognize the proteins of CD4-1 and CD8α co-receptors on the surface of zebrafish T lymphocytes, accurately locate T lymphocyte subsets, and accurately sort out CD4-1 and CD8α positive cell populations in zebrafish.

[0166] The experiment on changes in T lymphocyte populations after Aeromonas hydrophila infection included the following steps:

[0167] Healthy zebrafish were injected intraperitoneally with 7 × 10 5Aeromonas hydrophila at CFU / mL was used as the infection group, and grass with the same health status was injected with the same dose of PBS as the control group. On day 5 after infection, leukocytes were isolated from the spleens of both the infection and control groups. 2 mL of FACS Buffer (PBS, 2% FBS) was used to resuspend the leukocytes from the spleens of both groups to obtain leukocyte suspensions. 100 μL of each leukocyte suspension was added to the wells of a plate, and the plates were centrifuged at 2500 rpm for 3 min. The supernatant was discarded, and 100 μL of CD4-1-biotin (1:400) collected in Example 4 of this invention was added to each well as the primary antibody. The plates were incubated on ice in the dark for 30 min. After centrifugation at 2500 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 200 μL of FACS Buffer (L-15, 2% FBS), centrifuged again, and the resulting cell pellet was washed once more. The resulting cell suspension was then mixed with APC-Streptavidin (1:400). The CD8α-FITC (1:400) collected in Example 4 of this invention were incubated on ice for 30 min, and then incubated on ice in the dark for 30 min. The cells were centrifuged at 2500 rpm for 3 min, the supernatant was discarded, the cells were resuspended in 200 μL FACS Buffer, centrifuged again, and the resulting precipitate was washed once more. The cells were resuspended in 200 μL FACS Buffer / well and flow cytometry was performed.

[0168] like Figure 7 As shown, flow cytometry analysis revealed that 5 days after Aeromonas hydrophila infection, CD4-1... + The proportion of T cells in total lymphocytes increased from 9.15% before infection to 22.5% after infection; CD8α + The proportion of T cells in total lymphocytes increased from 3.06% before infection to 16.5% after infection. The proportions of both types of T cells significantly increased after infection, indicating that during infection, the monoclonal antibody in Example 4 of this invention recognized CD4-1... + and CD8α + T lymphocytes participate in adaptive immune responses and play an important role in antibacterial immunity.

[0169] In summary, this invention employs a combined immunization approach of "cell-based immunity + recombinant protein immunity" and innovatively introduces CD138. +Plasma cell sorting technology enables highly efficient immunization and cell fusion. Simultaneously, this invention establishes a screening system combining flow cytometry, immunofluorescence, and semi-quantitative methods, thereby successfully preparing zebrafish CD4-1 and CD8α monoclonal antibodies while fully ensuring the specificity of the monoclonal antibodies. This provides an important tool for subsequent research on the role and mechanism of lymphocytes in the adaptive immune response of bony fish.

[0170] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0171] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0172] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0173] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that, The monoclonal clone includes one or more of the following: (1) A specific monoclonal antibody against zebrafish CD4-1, wherein the antibody can specifically bind to zebrafish CD4-1, and the antibody is produced by hybridoma cell line 2G8C5; The hybridoma cell line 2G8C5 was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202622. (2) A specific monoclonal antibody against zebrafish CD8α, said antibody being specifically bound to zebrafish CD8α, said antibody being produced by hybridoma cell line 1A12F2; The hybridoma cell line 1A12F2 was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C202623.

2. A monoclonal antibody and / or its antigen-binding fragment, characterized in that, The monoclonal antibody and / or its antigen-binding fragment includes one or more of the following: (1) A specific monoclonal antibody against zebrafish CD4-1 and / or its antigen-binding fragment, wherein the specific monoclonal antibody and / or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises CDRH1 as shown in SEQ ID NO:11, CDRH2 as shown in SEQ ID NO:12, and CDRH3 as shown in SEQ ID NO:13; and / or, the light chain variable region comprises CDRL1 as shown in SEQ ID NO:14, CDRL2 as shown in SEQ ID NO:15, and CDRL3 as shown in SEQ ID NO:16; (2) A specific monoclonal antibody against zebrafish CD8α and / or its antigen-binding fragment, wherein the specific monoclonal antibody and / or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises CDRH1 as shown in SEQ ID NO: 19, CDRH2 as shown in SEQ ID NO: 20, and CDRH3 as shown in SEQ ID NO: 21; and / or, the light chain variable region comprises CDRL1 as shown in SEQ ID NO: 22, CDRL2 having the sequence GAS, and CDRL3 as shown in SEQ ID NO:

23.

3. The specific monoclonal antibody and / or its antigen-binding fragment as described in claim 2, characterized in that, In (1), the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:10, or an amino acid sequence having at least 85% sequence identity with it; and / or, In (2), in (1), the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:17, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:18, or an amino acid sequence having at least 85% sequence identity with it.

4. A recombinant protein, characterized in that, The recombinant protein comprises one or more of the specific monoclonal antibody as described in claim 1, or the specific monoclonal antibody as described in claim 2 or 3 and / or its antigen-binding fragment.

5. An antibody conjugate, characterized in that, The antibody conjugate comprises: (a) One or more of the specific monoclonal antibody as claimed in claim 1, or the specific monoclonal antibody and / or its antigen-binding fragment as claimed in claim 2 or 3, or the recombinant protein as claimed in claim 4; and (b) Conjugates selected from the following groups: detectable markers, enzymes, cytokines, radionuclides, nanoparticles / nanoparticles One or more of the following: rods.

6. A hybrid cell tumor cell line, characterized in that, The hybrid cell line includes one or two of the following: (1) The hybridoma cell line is capable of producing the specific monoclonal antibody against zebrafish CD4-1 as described in claim 1; the hybridoma cell line includes the hybridoma cell line with accession number CCTCC NO: C202622; (2) The hybridoma cell line is capable of producing the specific monoclonal antibody against zebrafish CD8α as described in claim 1; the hybridoma cell line includes the hybridoma cell line with accession number CCTCC NO: C202623.

7. A substance characterized in that, The substance includes one or more of the following: (1) A reagent / kit comprising one or more of the following: the specific monoclonal antibody as described in claim 1, or the specific monoclonal antibody and / or antigen-binding fragment as described in claim 2 or 3, or the recombinant protein as described in claim 4, or the antibody conjugate as described in claim 5, or the hybridoma cell line as described in claim 6; (2) A detection system comprising one or more of the following: a specific monoclonal antibody as described in claim 1, or a specific monoclonal antibody and / or antigen-binding fragment as described in claim 2 or 3, or a recombinant protein as described in claim 4, or an antibody-drug conjugate as described in claim 5, or a hybrid cell tumor cell line as described in claim 6, or a reagent / kit as described in (1).

8. A method, characterized in that, The method includes one or more of the following: (1) A combined immunization method, the method comprising primary cellular immunization and protein-enhanced immunization; (2) A method for targeted and efficient hybridoma fusion, the method comprising enrichment of highly secretory plasma cells and induced B cell targeted fusion by conformation antigen; (3) A method for detecting zebrafish CD4-1 and / or CD8α, the method comprising detecting zebrafish CD4-1 and / or CD8α by one or more of the following: the specific monoclonal antibody as described in claim 1, or the specific monoclonal antibody and / or antigen-binding fragment as described in claim 2 or 3, or the recombinant protein as described in claim 4, or the antibody-drug conjugate as described in claim 5, or the hybrid cell tumor cell line as described in claim 6, or the substance as described in claim 7; (4) A method for detecting or identifying positive hybridoma cell lines, the method comprising using one or more of the following as detection tools: the specific monoclonal antibody as described in claim 1, or the specific monoclonal antibody and / or antigen-binding fragment as described in claim 2 or 3, or the recombinant protein as described in claim 4, or the antibody conjugate as described in claim 5, or the hybridoma cell line as described in claim 6, or the substance as described in claim 7, to detect and screen specific antibodies expressed in the supernatant or on the cell surface of hybridoma cells, thereby efficiently and accurately identifying positive hybridoma cell lines that can secrete monoclonal antibodies that recognize the natural conformational epitopes of target antigens; (5) A method for preparing the hybridoma cell line as described in claim 6, wherein the preparation method is to prepare the hybridoma cell line by the combined immunization method as described in (1) and the directed high-efficiency hybridoma fusion method as described in (2).

9. The method as described in claim 8, characterized in that, In (1), the method includes: performing cell-mediated immunization using live cells stably expressing the target antigen as an immunogen; performing protein-enhanced immunization using a recombinant extracellular domain protein of the target antigen after the cell-mediated immunization; and / or, In (2), the method includes: introducing CD138 based on the overall fusion of spleen cells. + The plasma cell sorting step prioritizes the separation of B cell populations in a high antibody secretion state at the end of the immunization phase, thereby increasing the positive fusion rate of specific antibodies from the cell source, while combining the conformational epitope dominant response induced by the combined immunization method in (1).

10. The specific monoclonal antibody as described in claim 1, or the specific monoclonal antibody and / or antigen-binding fragment as described in claim 2 or 3, or the recombinant protein as described in claim 4, or the antibody-drug conjugate as described in claim 5, or the hybridoma cell line as described in claim 6, or the substance as described in claim 7, or the method as described in claim 8 or 9, for the preparation of a detection reagent / kit for detecting zebrafish CD4-1 and / or CD8α, the preparation of a detection plate for detecting zebrafish CD4-1 and / or CD8α, the preparation of specific monoclonal antibodies and / or antigen-binding fragments against zebrafish CD4-1 and / or CD8α, the screening of specific monoclonal antibodies and / or antigen-binding fragments against zebrafish CD4-1 and / or CD8α, the preparation of fusion cells, the detection of zebrafish CD4-1 and / or CD8α, and the improvement of bony fish T cells. Applications of research in functional mapping of cellular adaptive immunity, immunological studies of zebrafish disease models / vaccine evaluation / disease-resistant breeding, elucidating the evolutionary laws of the adaptive immune system in vertebrates, and the role and mechanism of lymphocytes in adaptive immune responses in bony fish.

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