Hybridoma cell lines, antibodies and their applications

Abstract

This invention proposes hybridoma cell lines, antibodies, and their applications. The antibodies comprise a CDR selected from at least one of the following: heavy chain variable region CDR: SEQ ID NO: 1–3 or its conserved modified amino acid sequence; light chain variable region CDR: SEQ ID NO: 4–6 or its conserved modified amino acid sequence. The antibody or antigen-binding fragment can efficiently and specifically recognize Cry4, effectively preventing or treating Cry4-mediated diseases, and detecting Cry4.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to hybridoma cell lines, antibodies, and their applications. Background Technology

[0002] Cryptochrome 4 (Cry4) is a newly discovered protein with a flavin prosthetic group found in non-mammalian vertebrates. Its photosensitizing properties in vitro suggest potential photo-induced rhythm regulation or photomagnetic effects. Animals carrying the cryptochrome 4 gene include birds (poultry), fish, amphibians, and other species. Key genes for rhythm regulation like cryptochromes are often highly correlated with the growth and reproduction rates of these animals; therefore, detecting the expression level of cryptochrome 4 protein has high value in scientific research and practical production.

[0003] Anti-cryptochrome 4 antibodies are primarily used to detect the presence and expression levels of cryptochrome 4 protein using immunological methods such as Western blotting, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). However, due to the relatively recent discovery of the cryptochrome 4 gene, there are currently no commercially available anti-cryptochrome 4 antibodies. Furthermore, existing anti-cryptochrome 4 antibodies in scientific research often suffer from insufficient specificity and limited application pathways. Therefore, the search for highly specific anti-cryptochrome 4 antibodies with multiple application pathways is crucial for detecting cryptochrome 4 expression, further understanding and mastering the physiological and biochemical changes in animals, developing high-yielding economic animal strains, and even detecting or treating potential cryptochrome 4-related diseases. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0005] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the invention, the antibody or antigen-binding fragment comprises a CDR selected from at least one of the following: a heavy chain variable region CDR: SEQ ID NO: 1-3 or an amino acid sequence thereof with conserved modifications; a light chain variable region CDR: SEQ ID NO: 4-6 or an amino acid sequence thereof with conserved modifications. The antibody or antigen-binding fragment according to an embodiment of the invention can efficiently and specifically recognize Cry4, effectively prevent or treat Cry4-mediated diseases, and detect Cry4.

[0006] In a second aspect, the present invention provides a hybridoma cell line that generates the antibody or antigen-binding fragment described in the first aspect. According to an embodiment of the present invention, the hybridoma cell line was deposited on March 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45831. The antibody or antigen-binding fragment generated by the hybridoma cell line according to the embodiment of the present invention can efficiently and specifically recognize Cry4, effectively preventing or treating Cry4-mediated diseases, and detecting Cry4.

[0007] In a third aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment. The antibody or antigen-binding fragment encoded by the nucleic acid molecule according to the embodiment of the invention can efficiently and specifically recognize Cry4, effectively prevent or treat Cry4-mediated diseases, and detect Cry4.

[0008] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. This effectively enables the expression of the aforementioned antibody or antigen-binding fragment, thereby achieving the large-scale in vitro production of the antibody or antigen-binding fragment.

[0009] In a fifth aspect, the present invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or expression vector or expressing the aforementioned antibody or antigen-binding fragment. Using this recombinant cell, under suitable conditions, the aforementioned antibody or antigen-binding fragment can be effectively expressed intracellularly.

[0010] In a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The pharmaceutical composition of the present invention can specifically recognize Cry4 and can effectively prevent or treat Cry4-mediated diseases.

[0011] In a seventh aspect, the present invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises: the aforementioned antibody or antigen-binding fragment; and a conjugation portion linked to the antibody or antigen-binding fragment. The conjugate of the present invention can specifically recognize Cry4 and can effectively prevent or treat Cry4-mediated diseases.

[0012] In an eighth aspect of the invention, the invention provides for the use of the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned pharmaceutical composition, or the aforementioned conjugate in the preparation of a medicament for the prevention and / or treatment of Cry4-mediated diseases.

[0013] In a ninth aspect, the present invention provides a kit. According to an embodiment of the invention, the kit comprises: the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cells. The kit of the present invention is capable of specifically binding to Cry4 and can effectively detect Cry4.

[0014] In a tenth aspect of the invention, the invention provides for the use of the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in the preparation of a kit for detecting Cry4.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This image shows the detection results of the anti-cryptochrome 4 antibody specifically recognizing exogenous cryptochrome 4 protein via immunoblotting according to an embodiment of the present invention. In the image, EGFP-GgCry1 in Cos7 represents Cos-7 cell lysates expressing chicken cryptochrome 1 fusion protein (EGFP-GgCry1) with an EGFP tag; EGFP-GgCry2 in Cos7 represents Cos-7 cell lysates expressing chicken cryptochrome 2 fusion protein (EGFP-GgCry2) with an EGFP tag; EGFP-GgCry4 in Cos7 represents Cos-7 cell lysates expressing chicken cryptochrome 4 fusion protein (EGFP-GgCry4) with an EGFP tag; Cos7 represents Cos7 cell lysates expressing EGFP-tagged proteins; and GgCry4 in E. coli represents E. coli lysates expressing GgGry4 protein with a His tag.

[0018] Figure 2This is a graph showing the detection results of the anti-cryptochrome 4 antibody specifically recognizing chicken endogenous cryptochrome 4 protein via immunoblotting, according to an embodiment of the present invention. Vinculin is the internal control used.

[0019] Figure 3 The image shows the detection results of the anti-cryptochrome 4 antibody specifically recognizing endogenous cryptochrome 4 protein by immunofluorescence according to an embodiment of the present invention.

[0020] Figure 4 This is a structural diagram of the pET21a-GgCry4 vector, which inserts the full-length coding region of GgCry4 between the NdeI and NotI sites of the pET21a vector according to an embodiment of the present invention.

[0021] Figure 5 This is a structural diagram of the pCAGGS-GgCry4 vector, which inserts the full-length coding region of GgCry4 between the KpnI and NheI sites of the pCAGGS vector according to an embodiment of the present invention.

[0022] Figure 6 This is a structural diagram of the pCAGGS-EGFP-GgCry4 vector, which inserts the full-length coding region of GgCry4 between the KpnI and NheI sites of the pCAGGS vector carrying a green fluorescent (EGFP) tag, according to an embodiment of the present invention.

[0023] Figure 7 A structural diagram of the pCAGGS-EGFP-GgCry1 vector, which inserts the full-length coding region of GgCry1 between the KpnI and NheI sites of the pCAGGS vector carrying a green fluorescent protein (EGFP) tag, according to an embodiment of the present invention; and

[0024] Figure 8 This is a structural diagram of the pCAGGS-EGFP-GgCry2 vector, which inserts the full-length coding region of GgCry2 between the KpnI and NheI sites of the pCAGGS vector carrying a green fluorescent protein (EGFP) tag, according to an embodiment of the present invention. Detailed Implementation

[0025] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0027] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0028] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0029] In this paper, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide that has been truncated at an N-terminus (N-terminus) or C-terminus (C-terminus) and / or internally deleted.

[0030] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0031] In this paper, the term "at least 90% identity" means at least 90% identity with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0032] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0033] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0034] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0035] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0036] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, pharmaceutical compositions, or conjugates of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the antibodies or antigen-binding fragments, pharmaceutical compositions, or conjugates of the present invention are administered via intravenous or subcutaneous injection.

[0037] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans and rodents, and also in birds, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to the administration of antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, or conjugates described herein to an individual in need.

[0038] An antibody or antigen-binding fragment

[0039] In some embodiments, the present invention provides an antibody or antigen-binding fragment comprising a CDR selected from at least one of the following: heavy chain variable region CDR: SEQ ID NO: 1-3 or its conserved modified amino acid sequence; light chain variable region CDR: SEQ ID NO: 4-6 or its conserved modified amino acid sequence. The antibody or antigen-binding fragments according to some specific embodiments of the present invention can efficiently and specifically recognize Cry4, effectively prevent or treat Cry4-mediated diseases, and detect Cry4.

[0040] GFNIKDTY (SEQ ID NO:1).

[0041] IDPVNGNS(SEQ ID NO:2).

[0042] SCGRPFVY (SEQ ID NO:3).

[0043] KSLLHSNGNIY(SEQ ID NO:4).

[0044] RLS (SEQ ID NO:5).

[0045] MQHLEYPLT (SEQ ID NO:6).

[0046] In this document, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, with no specific structural limitations, as long as they exhibit the desired biological activity. Antibody molecules typically consist of a lighter light chain and a heavier heavy chain linked by disulfide bonds. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl-terminus (C-terminus) is relatively stable and varies little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are referred to as VL and VH, respectively. As used herein, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of the antibody or its functional fragments to the antigens or epitopes it recognizes. In specific embodiments of this disclosure, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0047] In this document, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody's CDR. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments can be generated using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0048] In this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0049] According to some specific embodiments of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0050] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment includes a heavy chain framework region and / or a light chain framework region.

[0051] According to some specific embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, and mutants thereof.

[0052] According to some specific embodiments of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of mouse antibodies, chicken antibodies, duck antibodies, goose antibodies, red junglefowl antibodies, turkey antibodies, and fighting rooster antibodies.

[0053] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment includes a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 90% identity with it; and / or a light chain variable region of an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 90% identity with it.

[0054] EAQLQQSGAEFVKPGSSVRMSCTASGFNIKDTYIHWVRQRPEQGLEWIGRIDPVNG NSKYDPKFPGKATITSDTSSNTAYLQLSSLTSEDTAVYYCSCGRPFVYWGQGTLVTVST (SEQ ID NO: 7).

[0055] DIVMTQAAPSLPVTPGESVSISCRSTKSLLHSNGNIYLYWFLQRPGQSPQLLIYRLSNL ASGVPDRFSGSGSGTVFTLRINRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK (SEQ ID NO: 8).

[0056] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment further includes a constant region.

[0057] According to some specific embodiments of the present invention, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0058] According to some specific embodiments of the present invention, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, red junglefowl antibodies, turkey antibodies, fighting rooster antibodies, and mutants thereof.

[0059] According to some specific embodiments of the present invention, at least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, chicken antibodies, duck antibodies, goose antibodies, wild chicken antibodies, turkey antibodies, and fighting rooster antibodies.

[0060] According to some specific embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ type or λ type.

[0061] According to some specific embodiments of the present invention, the heavy chain constant region and / or light chain constant region are derived from mouse antibodies, chicken antibodies, duck antibodies, goose antibodies, wild chicken antibodies, turkey antibodies, fighting rooster antibodies or mutants thereof.

[0062] According to some specific embodiments of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region; and / or the N end of the light chain constant region is connected to the N end of the light chain variable region.

[0063] According to some specific embodiments of the present invention, the heavy chain constant region includes: an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it; and / or the light chain constant region includes: an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it.

[0064] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSCGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKFVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTA QTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEDLHNHHTEKSLSHSPGK(SEQ ID NO:9).

[0065] RADAAPTVSIFPPSSEEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT DQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 10).

[0066] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain of an amino acid sequence as shown in SEQ ID NO:11 or an amino acid sequence having at least 90% identity with it; and / or a light chain of an amino acid sequence as shown in SEQ ID NO:12 or an amino acid sequence having at least 90% identity with it.

[0067] EAQLQQSGAEFVKPGSSVRMSCTASGFNIKDTYIHWVRQRPEQGLEWIGRIDPVNGNSKYDPKFPGKATITSDTSSNTAYLQLSSLTSEDTAVYYCSCGRPFVYWGQGTLVTVSTAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSCGVHTFPAVLQSDLY TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKFVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKE FKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEDLHNHHTEKSLSHSPGK(SEQ IDNO:11).

[0068] DIVMTQAAPSLPVTPGESVSISCRSTKSLLHSNGNIYLYWFLQRPGQSPQLLIYRLSNLASGVPDRFSGSGSGTVFTLRINRVEAEDVGVYYCMQHLEYPLTFGAGTKLEL KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:12).

[0069] According to some specific embodiments of the present invention, the antibody comprises at least one selected from full-length monoclonal antibodies, Fab antibodies, Fab' antibodies, F(ab')2 antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and minimal recognition units; or, the antigen-binding fragment comprises at least one selected from F(ab')2 fragments, Fab' fragments, Fab fragments, F(ab)2 fragments, Fv fragments, scFv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, and minimal recognition units.

[0070] In this article, the terms “full-length antibody”, “full-length monoclonal antibody” or “full-length monoclonal antibody” refer to antibodies composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0071] In this paper, the terms “single-domain antibody,” “nanobody,” and “VHH antibody” are used interchangeably. The antibody was originally described as an antigen-binding immunoglobulin (variable) domain of a “heavy chain antibody” (i.e., “antibody lacking light chains”) containing a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically binds to antigen proteins (e.g., Cry4) through the heavy chain variable region.

[0072] In this article, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0073] In this paper, the term “F(ab')2 antibody” or “F(ab')2 fragment” has two antigen-binding F(ab') parts linked together by disulfide bonds.

[0074] In this article, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0075] In this paper, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by linking the variable regions of the antibody heavy chain and light chain through short peptides.

[0076] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, with a very small molecular weight, accounting for only about 1% of a complete antibody.

[0077] Hybridoma cell lines, nucleic acid molecules, expression vectors, and recombinant cells

[0078] In some embodiments, the present invention provides a hybridoma cell line that produces the aforementioned antibody or antigen-binding fragment. According to an embodiment of the present invention, the hybridoma cell line was deposited on March 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45831. The antibody or antigen-binding fragment produced by the hybridoma cell line according to the embodiment of the present invention can efficiently and specifically recognize Cry4, effectively preventing or treating Cry4-mediated diseases, and enabling the detection of Cry4.

[0079] In some embodiments, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment. The antibody or antigen-binding fragment encoded by the nucleic acid molecule according to embodiments of the present invention can efficiently and specifically recognize Cry4, effectively preventing or treating Cry4-mediated diseases, and detecting Cry4.

[0080] According to some specific embodiments of the present invention, the nucleic acid molecule is DNA.

[0081] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0082] In some embodiments, the present invention provides an expression vector. According to embodiments of the present invention, the expression vector carries the aforementioned nucleic acid molecule. When the nucleic acid molecule is ligated to the vector, it can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. These control elements can be directly derived from the vector itself or are exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and control elements need to be operably linked. In this context, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended function of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids, bacteriophages, etc. After the expression vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned antibody or antigen-binding fragment can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro acquisition of the antibody or antigen-binding fragment.

[0083] According to some specific embodiments of the present invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0084] According to some specific embodiments of the present invention, the expression vector is a plasmid expression vector.

[0085] In some embodiments, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or expression vector or expressing the aforementioned antibody or antigen-binding fragment. Using this recombinant cell, under suitable conditions, the aforementioned antibody or antigen-binding fragment can be efficiently expressed intracellularly.

[0086] It should be noted that the "suitable conditions" mentioned in this application specification refer to conditions suitable for the expression of the antibody or antigen-binding fragment described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the antibody or antigen-binding fragment include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the antibody or antigen-binding fragment according to the specific environment of their laboratory.

[0087] According to some specific embodiments of the present invention, the recombinant cells are eukaryotic cells.

[0088] According to some specific embodiments of the present invention, the recombinant cells are mammalian cells.

[0089] Pharmaceutical compositions, conjugates and kits

[0090] In some embodiments, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The pharmaceutical composition of the present invention can specifically recognize Cry4 and can effectively prevent or treat Cry4-mediated diseases.

[0091] According to some specific embodiments of the invention, pharmaceutically acceptable excipients are further included.

[0092] The pharmaceutical compositions of this invention can be administered by any acceptable method of administration. The pharmaceutical compositions of this invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, and current methods for preparing these dosage forms are known or readily apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a subject.

[0093] In some embodiments, the present invention provides a conjugate. According to embodiments of the present invention, the conjugate comprises: the aforementioned antibody or antigen-binding fragment; and a conjugation portion linked to the antibody or antigen-binding fragment. The conjugate of the present invention can specifically recognize Cry4 and can effectively prevent or treat Cry4-mediated diseases.

[0094] According to some specific embodiments of the present invention, the coupling portion includes at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, and chemotherapeutic agents.

[0095] In this document, the carrier can be a substance capable of suspension or dispersion in a liquid phase (e.g., solid-phase carriers such as particles and magnetic beads), or a solid phase capable of containing or carrying a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic paths, glass capillaries, nanopillars, and monolithic columns); it can also be a labeling carrier for labeling antibody or antigen-binding fragments, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), etc. Examples of fluorescent substances include: luciferin (e.g., streptavidin and biotin, or complementary sense and antisense nucleic acids), fluorescent substances (e.g., luciferin, luciferin isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., insect luciferin, aequorin, acridine ester, tris(2,2'-bipyridine)ruthenium, luminol), radioactive isotopes (e.g., 3H, 14C, 32P, 35S, 125I), and gold colloids.

[0096] According to some specific embodiments of the present invention, the drug is a small molecule drug that can bind to an antibody or antigen-binding fragment.

[0097] According to some specific embodiments of the present invention, the protein tag includes, but is not limited to, His tag, Flag tag, GST tag, MBP tag, SUMO tag and C-Myc tag.

[0098] According to some specific embodiments of the present invention, the term "modifier" should be interpreted broadly and may refer to substances used to modify proteins. Exemplarily, it may be polyethylene glycol or a derivative thereof.

[0099] According to some specific embodiments of the present invention, the chemotherapeutic agents include, but are not limited to, albumin-bound paclitaxel, cyclophosphamide, ifosfamide, phenylalanine nitrogen mustard, methotrexate, fluorouracil, actinomycin D, and vincristine.

[0100] It should be noted that the binding method between the conjugated portion and the antibody or antigen-binding fragment can use methods known in the art. For example, methods such as physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin), and ion binding can be used.

[0101] In some embodiments, the present invention provides a kit. According to embodiments of the present invention, the kit comprises: the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cells. As is known prior, the aforementioned antibody or antigen-binding fragment can bind to Cry4, and the aforementioned antibody or antigen-binding fragment can specifically bind to Cry4. Furthermore, under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cells can all express the antibody or antigen-binding fragment. Further, the kit containing the above substances can effectively bind to Cry4 and can be used for the effective detection of Cry4. The kit can be used in scientific research, such as for the qualitative or quantitative detection of Cry4 in biological samples, and can also be used to determine the state of an object, such as determining whether the Cry4 level of the object is too high or too low than normal after obtaining the Cry4 level. The biological sample can be cells, tissues, blood, etc.

[0102] use

[0103] In some embodiments, the present invention provides for the use of the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned pharmaceutical composition, or the aforementioned conjugate in the preparation of a medicament for the prevention and / or treatment of Cry4-mediated diseases.

[0104] In some embodiments, the present invention provides for the use of the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in the preparation of a kit for detecting Cry4.

[0105] method

[0106] In some embodiments, the present invention provides a method for treating and / or preventing Cry4-mediated diseases. According to embodiments of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned antibody or antigen-binding fragment, the aforementioned pharmaceutical composition, or the aforementioned conjugate. The method of the present invention can effectively treat or prevent Cry4-mediated diseases.

[0107] The effective amount of the antibody or antigen-binding fragment, pharmaceutical composition, or conjugate described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the subject's weight, the subject's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0108] The antibodies or antigen-binding fragments, pharmaceutical compositions, or conjugates of the present invention can be incorporated into medicaments suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These medicaments can be prepared in various forms, such as liquids, semi-solids, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical medicaments are in the form of injection solutions or infusion solutions. The aforementioned antibodies or antigen-binding fragments, pharmaceutical compositions, or conjugates can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0109] According to embodiments of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0110] In some embodiments, the present invention provides a method for detecting Cry4, the method comprising: contacting the sample to be tested with the aforementioned antibody or antigen-binding fragment or the aforementioned kit to form an immune complex.

[0111] According to some specific embodiments of the present invention, the presence of Cry4 or the content of Cry4 in the sample to be tested is determined based on the signal of the immune complex.

[0112] According to some specific embodiments of the present invention, the immune complex further includes a second antibody, which binds to the antibody or a functional fragment thereof.

[0113] According to some specific embodiments of the present invention, the immune complex further includes a second antibody that binds to Cry4.

[0114] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0115] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994) and "Current Protocols in Immunology" (JEColigan et al., ed., 2011), each of which is explicitly incorporated herein by reference.

[0116] In this embodiment of the invention, the nucleotide sequence used to prepare the expression vector can be obtained from its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0117] Example 1: Preparation of anti-cryptochrome 4 monoclonal antibody

[0118] (1) Expression and purification of antigen

[0119] The full-length coding region of red junglefowl cryptochrome 4 (GgCry4) was synthesized (NCBI accession No: NM_001039596.1) and subcloned into the pET21a vector between the NdeI and NotI restriction sites to construct the pET21a-GgCry4 vector. The vector structure is shown below. Figure 4It encodes a GgCry4-His fusion protein with a C-terminal His tag. pET21a-GgCry4 was transformed into BL21(DE3) strain. After plating, single colonies were inoculated into 5 mL of LB broth containing ampicillin and cultured overnight at 37°C with shaking at 220 rpm. After incubation, all colonies were inoculated into 1 L of LB broth containing ampicillin and cultured at 37°C with shaking at 220 rpm to promote rapid cell growth. When the OD600 was in the range of 0.8–1.0, IPTG was added to the medium to a final concentration of 1 mM, the temperature was lowered to 22°C, and the culture was continued for 16 h. The cells were then collected by centrifugation at 4000 rpm for 30 min. Add 30 mL of lysis buffer (50 mM NaH2PO4, 200 mM NaCl, 10 mM imidazole, pH 8.0) and lysozyme to a final concentration of 0.2 mg / mL, along with 0.1 mM protease inhibitor AEBSF, to the bacterial cells. Incubate on ice for 30 min to induce initial lysis. After sonication, centrifuge the lysate. Mix the supernatant with 1 mL of Ni-NTA resin (Qiagen) at 4°C for 1 h using continuous vortexing to ensure the target protein binds fully to Ni-NTA. After mixing, pour the mixture into a chromatography column to allow unbound protein to elute. Wash the column once with 30 mL of wash buffer (50 mM NaH2PO4, 200 mM NaCl, pH 8.0) containing 40 mM imidazole, followed by a second wash with wash buffer containing 60 mM imidazole. Then, add 2 mL of elution buffer (50 mM NaH2PO4, 200 mM NaCl, 300 mM imidazole, pH 8.0), mix thoroughly for 30 min, and collect the eluent containing GgCry4 protein, which will be used as the crude antigen in subsequent experiments.

[0120] (2) Animal Immunization

[0121] The purified antigen obtained in step (1) above was dialyzed at 4°C to 0.01M phosphate buffer (pH 7.2) and used as the immunogenic antigen. Four SPF-grade BALB / c female mice were subcutaneously immunized with the antigen for a total of five immunizations, with each immunization spaced two weeks apart. The first immunization used Freund's complete adjuvant for emulsification, and the second to fifth immunizations used incomplete Freund's adjuvant for booster immunizations. Each immunization used 30 μg of antigen. Blood was collected 10 days after the fourth immunization for ELISA testing.

[0122] The specific procedure for ELISA detection is as follows: First, dilute the dialyzed antigen shown in Part (1) above to a concentration of 2 μg / mL using NaHCO3 buffer. Add 100 μL of the diluted antigen to each well of a 96-well plate and incubate overnight at 4°C. Block with 5% skim milk powder for 2 hours. Serially dilute the serum at ratios of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, and add 100 μL of serum at each dilution to each well, incubate for 1 hour. After washing three times with 5% skim milk powder, add HRP-labeled goat anti-rabbit IgG antibody (dilution: 1:50000) and incubate for 1 hour. After adding TMB for 20 minutes of color development, add stop solution (0.5M sulfuric acid) to terminate the reaction and read the values. The results showed an antibody titer of 1:128000, indicating a highly positive result. Therefore, two weeks after the fifth immunization, blood was collected from the animals, and all serum was separated and preserved. The mice with the highest ELISA titer were used for the next step of the cell fusion experiment.

[0123] (3) Cell fusion

[0124] Take the thymus from the mouse and crush it. Pour the crushed thymus cells into a 15mL centrifuge tube, add 2mL of HAT and 1mL of HT, and place it in an incubator for later use.

[0125] Healthy sp2 / 0 cells were gently pipetted from the culture flask wall and transferred to a 50 mL centrifuge tube. Blood was collected from the eyeballs of mice, and the mice were then sacrificed by neck retraction. The spleen was harvested, ground, and the cells were transferred to the sp2 / 0 cell tube. The cells were centrifuged at 1500 rpm for 5 min. The cells were then reconstituted with serum-free IMDM and centrifuged again. The centrifuge tube, after removing the supernatant, was placed in 37°C warm water and incubated for approximately 1 minute. 1 mL of PEG was slowly added, and the tube was incubated for another 1 min. Then, 2 mL of serum-free IMDM was slowly added over 2 min, followed by 8 mL of serum-free IMDM over another 2 min. The tube was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 10 mL of serum was added to the pellet, and the cells were carefully reconstituted. This pellet was then transferred to the previously prepared thymocyte culture medium, and the volume was adjusted to 50 mL with sterile semi-solid culture medium. The mixture was thoroughly mixed. Then pour it evenly into 30 cell culture dishes, and place the cell culture dishes in a humidified box for incubation.

[0126] (4) Clone screening

[0127] Ten × 93 cell clones were selected and cultured in 96-well cell culture plates (pre-coated with 100 μL / well of thymocytes). After 2 days of culture, the supernatant of the single-clonal cells in the 96-well plates was discarded, and 200 μL / well of 20% newborn calf IMDM medium (containing HT) was added for the first selection. The plates were coated with GgCry4 protein, and the selected clones were screened a second time using ELISA, with the experimental procedure similar to the previous ELISA detection, resulting in 24 positive hybridoma cell lines. The plates were then coated again with GgCry4 protein and 6×His peptide, and a third selection was performed using ELISA to obtain the target positive hybridoma cell lines.

[0128] (5) Identification of monoclonal cell subclasses

[0129] The subclass coating antibody (Goat Anti-Mouse Ig, Human ads-UNLB) was diluted with 100 μL / well of coating buffer to a final antibody concentration of 2 μg / mL. The cells were incubated overnight at 4°C, and washed three times with washing buffer after incubation. Blocking was performed with 200 μL / well of blocking buffer, incubated at 37°C for 2 h, and then washed three times with washing buffer. 100 μL / well of fusion cell culture supernatant and negative control (SP2 / 0 culture supernatant) were added, and the cells were incubated at 37°C for 1 h. After staining, the cells were washed three times with washing buffer. The subclass secondary antibody was diluted with 100 μL / well of PBS and added to appropriate wells. The cells were incubated at 37°C for 1 h, and then washed three times with washing buffer. 100 μL / well of TMB chromogenic solution was added, and the chromogenic time was approximately 10 min. 50 μL of stop solution was added to each well to terminate the staining. The absorbance was measured at dual wavelengths (450 nm and 630 nm) to obtain IgG-positive hybridoma cell lines.

[0130] (6) Specific screening of hybridoma cells

[0131] The GgCry4 coding region obtained in Part (1) was subcloned into the space between the KpnI and NheI sites of the pCAGGS-Flag vector to construct the pCAGGS-GgCry4 vector. For the specific structure, please refer to [link to relevant documentation]. Figure 5The protein encodes the Flag-GgCry4 fusion protein with an N-terminal Flag tag. pCAGGS-GgCry4 was transfected into LMH cells using Lipofectamin 3000 (Thermo). Forty-eight hours after transfection, cells were lysed with RIPA (50 mM Tris-HCl, pH 8.0, with 150 mM NaCl, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS). The lysate was used as a positive sample, and the lysate of pCAGGS-Flag empty vector 293T cells was used as a negative sample for Western blotting experiments. The procedures were standard in the art. Hybridoma cell supernatant was used as the primary antibody (diluted 1:5), and goat anti-mouse antibody (Jackson) was used as the secondary antibody. The supernatant with the best specificity and shortest exposure time was selected, and the corresponding hybridoma cells (Cry4-AB22) were expanded and cultured using standard procedures in the art.

[0132] (7) Preparation of ascites

[0133] The hybridoma cells obtained in part (6) were cultured to the logarithmic growth phase, washed with PBS, and the cell count was quantified to 5 × 10⁶ cells / day. 5 ~9×10 5 Cells / mL. Paraffin oil was injected intraperitoneally, with 500 μL injected into each Balb / c mouse. One week later, 1 mL of the above-mentioned cells was injected intraperitoneally into each mouse. Seven days after cell injection, ascites fluid was collected from the mice, centrifuged at 5000 rpm for 10 min, and the supernatant was collected and stored at -20℃.

[0134] (8) Antigen affinity chromatography to purify antibodies

[0135] Take 10 mg of the crude antigen described in step (1) of this embodiment and dialyze it into a 20 mM NaHCO3 coupling buffer. Centrifuge at 10,000 rpm for 10 min at 4 °C and collect the supernatant. Add 1.5 mL of pure water and 1.5 mL of hydrogen bromide to a 10 mL empty chromatography column to activate the column material. After it settles naturally, wash it sequentially with 10 mL of water and 10 mL of 20 mM NaHCO3. After draining, add the dialyzed antigen and incubate the resulting mixture at 4 °C overnight. Drain the column material the next morning. Wash the column bed with 20 mM NaHCO3 until the flow-through level is colorless according to the G250 test. Wash the column bed with 50 mL of Tris blocking buffer. After draining, add 5 mL of Tris blocking buffer, gently resuspend the column material by pipetting, and mix at 4 °C overnight. Drain the chromatography column the next day and wash it with 50 mL of water. Block the chromatography column with 10 mL of NaCl-sodium azide buffer. At this point, the affinity chromatography column construction is complete.

[0136] Take the constructed affinity chromatography column, wash with 10 column volumes of water, and then wash with 100 mM sodium acetate buffer for another 10 column volumes. Centrifuge serum at 12,000 rpm for 10 min at 4 °C, collect the supernatant, and filter through a 0.45 μm filter membrane. Mix one part of ascites fluid with four parts of sodium acetate buffer and load the sample at a rate of 0.5 mL / min. After loading, continue washing with sodium acetate buffer until the G250 test is colorless. After eluting the antibody with glacial acetic acid elution buffer, quickly adjust the pH to neutral with saturated Na2CO3. After ultrafiltration and concentration of the eluted antibody, dialyze it into 0.1 M phosphate buffer containing 50% glycerol at pH 7.2. Centrifuge the dialyzed antibody at 12,000 rpm for 10 min at 4 °C, collect the supernatant, and store the centrifuged antibody at -20 °C.

[0137] Example 2: Application evaluation of anti-cryptochrome 4 antibody in immunoblotting

[0138] The coding regions for chicken cryptochrome 4 and two proteins that may have cross-immunogenic reactions, namely chicken cryptochrome 1 (GgCry1) and chicken cryptochrome 2 (GgCry2), were subcloned into the KpnⅠ and NheⅠ regions of the pCAGGS-EGFP vector with a green fluorescent protein (EGFP) tag, thus constructing the pCAGGS-EGFP-GgCry4 vector (see [link to pCAGGS-EGFP-GgCry4 vector]). Figure 6 ), pCAGGS-EGFP-GgCry1 vector (see pCAGGS-EGFP-GgCry1 vector) Figure 7 ), pCAGGS-EGFP-GgCry2 vector (see pCAGGS-EGFP-GgCry2 vector) Figure 8 The three vectors encode EGFP-Cry4 (EGFP-GgCry4), EGFP-Cry1 (EGFP-GgCry1), and EGFP-Cry2 (EGFP-GgCry2) proteins, respectively. Cos-7 cells (Chinese Academy of Sciences Cell Bank) were transfected with Lipofectamine 3000 (Thermo) to express the target proteins. BL21(DE3) strains transformed with the pET21a-GgCry4 vector (similar to those in Example 1) were induced to express GgCry4 protein using IPTG. The bacteria and transfected cells were lysed using RIPA lysis buffer, and Cos-7 cells transfected with the pCAGGS-EGFP empty vector were lysed as a negative control. The lysis buffer was centrifuged at 12000×g for 5 min, and the supernatant was collected. Using the above five samples, the feasibility and specificity of the antibody produced in this invention for detecting exogenous cryptochrome 4 protein were assessed using Western blotting (antibody dilution ratio 1:1000). The results are as follows: Figure 1As shown, although cryptochrome 4 has high homology with cryptochrome 1 and cryptochrome 2, the antibody described in this invention can still specifically recognize cryptochrome 4 by immunoblotting, but not cryptochrome 1 or cryptochrome 2, and does not have cross-immunoreactivity with other proteins in the cell.

[0139] One-day-old chicks were euthanized, and their heart tissue was added to RIPA lysis buffer and ground. The mixture was then centrifuged at 12000×g for 10 min, and the supernatant was collected. Using the above heart lysis buffer sample, the feasibility and specificity of identifying endogenous cryptochrome 4 protein with the antibody produced in this invention using Western blotting were assessed. The results are as follows: Figure 2 As shown, the antibody prepared by this invention can be used to detect endogenous cryptochrome 4 in chickens by immunoblotting (antibody dilution ratio 1:500), and it hardly undergoes cross-immunoreactivity with other proteins in the body.

[0140] Example 3: Application evaluation of anti-cryptochrome 4 antibody in immunofluorescence

[0141] The pCAGGS-GgCry4 vector from Part (6) of Example 1 was transfected into HeLa cells lacking background Cry4 expression using Lipofectamine 3000 (Thermo) to express the Flag-GgCry4 fusion protein; pCAGGS-Flag vector was also transfected as a negative control. After transfection, cells were cultured for 48 hours, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. Subsequently, the antibody prepared in this invention was used as the primary antibody (antibody dilution ratio 1:500), and Goat anti-rabbit Alexa 594 (Jackson) was used as the secondary antibody for immunofluorescence labeling of GgCry4; the Flag tag was immunofluorescently labeled using the highly specific Anti-Flag M2-FITC antibody (Sigma); cell nuclei were stained with DAPI. Results are as follows: Figure 3 As shown, the signal originating from the Flag tag is presented in green (Anti-Flag), the signal originating from cryptochrome 4 is presented in red (Anti-Cry4), and the cell nucleus is shown in blue; the green and red signals highly overlap in the superimposed signals, indicating that the antibody produced by this invention specifically recognizes cryptochrome 4 without cross-immunizing with other proteins in the cell.

[0142] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "embodiment," or "specific embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0143] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, characterized in that, Including the following variable region CDRs for heavy chains and variable region CDRs for light chains: The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:1; The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:2; The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:3; The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO:4; The amino acid sequence of the light chain variable region CDR2 is shown in SEQ ID NO:5; The amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that, Includes heavy chain framework region and / or light chain framework region.

3. The antibody or antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, and goose antibodies.

4. The antibody or antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, chicken antibodies, duck antibodies, and goose antibodies.

5. The antibody or antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of human antibodies, chicken antibodies, turkey antibodies, and fighting rooster antibodies.

6. The antibody or antigen-binding fragment according to claim 1, characterized in that, include: Heavy chain variable region of the amino acid sequence shown in SEQ ID NO:7; and The light chain variable region of the amino acid sequence shown in SEQ ID NO:

8.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, characterized in that, The antibody or antigen-binding fragment further includes a constant region.

8. The antibody or antigen-binding fragment according to claim 7, characterized in that, The constant region includes the heavy chain constant region and / or the light chain constant region.

9. The antibody or antigen-binding fragment according to claim 8, characterized in that, At least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, and goose antibodies.

10. The antibody or antigen-binding fragment according to claim 8, characterized in that, At least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of mouse antibodies, chicken antibodies, duck antibodies, and goose antibodies.

11. The antibody or antigen-binding fragment according to claim 8, characterized in that, At least a portion of the heavy chain constant region and / or light chain constant region is derived from at least one of human antibodies, chicken antibodies, turkey antibodies, and fighting rooster antibodies.

12. The antibody or antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; or The light chain constant region includes light chain constant regions selected from κ-type or λ-type.

13. The antibody or antigen-binding fragment according to claim 8, characterized in that, The N-end of the heavy chain constant region is connected to the C-end of the heavy chain variable region; and / or the N-end of the light chain constant region is connected to the N-end of the light chain variable region.

14. The antibody or antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region includes: an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% identity with it; and / or The light chain constant region includes an amino acid sequence such as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity with it.

15. The antibody or antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment comprises: a heavy chain with the amino acid sequence shown in SEQ ID NO:11; and The light chain of the amino acid sequence shown in SEQ ID NO:

12.

16. A hybridoma cell line that produces the antibody or antigen-binding fragment according to any one of claims 1 to 14, characterized in that, The hybridoma cell line was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 26, 2024, with accession number CGMCC No. 45831.

17. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment as described in any one of claims 1 to 15.

18. An expression carrier, characterized in that, Contains the nucleic acid molecule as described in claim 17.

19. The expression vector according to claim 18, characterized in that, The expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

20. The expression vector according to claim 18, characterized in that, The expression vector is a plasmid expression vector.

21. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 17 or express the antibody or antigen-binding fragment of any one of claims 1 to 15.

22. The recombinant cell according to claim 21, characterized in that, The recombinant cells are obtained by introducing the expression vector according to any one of claims 18 to 20 into host cells.

23. The recombinant cell according to claim 21, characterized in that, The recombinant cells are eukaryotic cells.

24. The recombinant cell according to claim 21, characterized in that, The recombinant cells are mammalian cells.

25. A pharmaceutical composition, characterized in that, Includes at least one of the following: The antibody or antigen-binding fragment according to any one of claims 1 to 15; The nucleic acid molecule of claim 17; The expression vector according to any one of claims 18-20; or The recombinant cells according to any one of claims 21 to 24.

26. The pharmaceutical composition according to claim 25, characterized in that, The pharmaceutical composition further includes pharmaceutically acceptable excipients.

27. A coupling, characterized in that, Include: The antibody or antigen-binding fragment according to any one of claims 1 to 15; and The coupling portion is linked to the antibody or antigen-binding fragment, and the coupling portion includes a protein tag.

28. A reagent kit, characterized in that, include: The antibody or antigen-binding fragment according to any one of claims 1 to 15; The nucleic acid molecule of claim 17; The expression vector according to any one of claims 18-20; or The recombinant cells according to any one of claims 21 to 24.

29. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 15, the nucleic acid molecule according to claim 17, the expression vector according to any one of claims 18 to 20, or the recombinant cells according to any one of claims 21 to 24 in the preparation of a kit for detecting Cry4.

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