Antibody or antigen-binding fragment thereof that specifically binds to porcine y sperm and use thereof

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to porcine Y sperm, a composition comprising same, and use thereof. It was confirmed that treatment with the antibody or an antigen-binding fragment thereof, of the present invention, induced an aggregation reaction of Y chromosome sperm, thereby enabling easy distinction between X chromosome sperm and the Y chromosome sperm. Thus, pigs having a specific sex can be mass-produced, and pigs having a desired sex can be selectively produced, thereby contributing to planned breeding, breeding improvement, and management efficiency.

Description

Antibody or antigen-binding fragment thereof that specifically binds to porcine Y sperm and uses thereof

[0001] The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to porcine Y sperm, a composition comprising the same, and the use thereof.

[0002] Sperm sexing technology for livestock is a technology that artificially separates only sperm of the desired sex to induce fertilization and pregnancy in order to produce offspring of the desired sex, and it is a very important technology in terms of production efficiency and commercial aspects.

[0003] Looking at the livestock industry, each species has specific sexes desired. For instance, in the case of dairy cows, farms seeking breeding improvements must continuously breed female calves capable of milk production, and they must also produce female calves with superior genetic traits through successive generations. Conversely, for beef cattle, there is a desire to produce male calves with rapid growth rates and high weight gain. In the case of pigs, breeding farms that produce purebred pigs for breeding improvement are called Great Grand Parents (GGP) farms, while farms that raise these purebreds to produce candidate pigs are called Grand Parents (GP) farms. The system consists of fattening farms (PS) that produce fattening pigs through artificial insemination using sows distributed from GP farms. The system is structured so that GGP farms produce sows and supply them to GP farms, while GP farms produce sows and supply them to PS farms. Therefore, all pig farms, from the primary breeding farm to the final fattening farm, absolutely require female pigs capable of producing piglets, and these animals play a crucial role. In the case of pigs produced at fattening farms, sows undergo rearing and fattening processes to be supplied as livestock products, while boars are castrated before one week of age to remove a characteristic odor (boar taint). Therefore, GGP and GP farms have the advantage of generating enormous economic benefits, such as reduced management costs, feed costs, and labor costs, by mass-producing sows and supplying them to downstream pig farms.

[0004] In addition, with the implementation of animal ethics laws starting in Europe, general anesthesia is required for male castration, which incurs significant costs. Therefore, there is an urgent need for the development of new technologies to replace this, and as the most critical solution to this, there is an urgent need for methods to increase the efficiency of sow production through sex control technologies and methods.

[0005] Against this backdrop, the present invention was completed by developing a technology that can effectively control sex from animal sperm.

[0006] One aspect provides an antibody for sperm binding or an antigen-binding fragment thereof, comprising: a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence of SEQ ID NO. 1, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence of SEQ ID NO. 3; and a light chain complementarity determining region 1 (LCR1) comprising an amino acid sequence of SEQ ID NO. 4, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence of SEQ ID NO. 5, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence of SEQ ID NO. 6.

[0007] Another aspect is to provide an isolated nucleic acid encoding the antibody or its antigen-binding fragment.

[0008] Another aspect is to provide a vector containing the isolated nucleic acid.

[0009] Another aspect is to provide isolated host cells transformed with the above vector.

[0010] Another aspect is to provide a cell line that expresses or produces the above-mentioned antibody for sperm binding or its antigen binding.

[0011] Another aspect provides a method for producing an antibody for sperm binding or an antigen-binding fragment thereof, comprising the step of culturing the host cell or the cell line to express an antibody.

[0012] Another aspect is to provide a composition for sperm sex determination comprising the above antibody or an antigen-binding fragment thereof.

[0013] Another aspect is to provide a sperm sex determination kit comprising the above antibody or an antigen-binding fragment thereof.

[0014] Another aspect provides a method for sex determination of mammalian sperm other than humans, comprising: 1) mixing semen separated from an individual with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4; and 2) separating sperm specifically bound to the antibody or unbound sperm from the mixture.

[0015] Another aspect provides a method for producing mammals other than humans having a specific sex, comprising: 1) mixing semen separated from an individual with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4; 2) separating sperm specifically bound to the antibody or unbound sperm from the mixture; 3) fertilizing using the separated sperm; and 4) producing offspring of a desired sex.

[0016] One aspect provides an antibody for sperm binding or an antigen-binding fragment thereof, comprising: a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence of SEQ ID NO. 1, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence of SEQ ID NO. 3; and a light chain complementarity determining region 1 (LCR1) comprising an amino acid sequence of SEQ ID NO. 4, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence of SEQ ID NO. 5, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence of SEQ ID NO. 6.

[0017] The term "antibody" in this specification refers to a specific immunoglobulin directed toward an antigenic site. The forms of the antibody include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies, such as ScFv fragments, diabodies, short-chain antibodies, etc., and include all immunoglobulin antibodies. The antibody may include not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule that does not have the structure of a complete antibody having two light chains and two heavy chains, but possesses an antigen-binding function by having a specific antigen-binding site, i.e., a binding domain, directed toward an antigenic site. The heavy chains are of five types: γ, δ, α, μ, and ε, and the heavy chain can determine the type of antibody. α and γ consist of 450 amino acids, while μ and ε consist of 550 amino acids. The heavy chain has two regions, namely a variable region and a constant region. The light chain has two types, λ and κ, and can consist of approximately 211 to 217 amino acids. In each human antibody, there is exactly only one type of chain. The light chain consists of a constant region and a variable region that are continuous.

[0018] The term "antigen-binding fragment" as used herein refers to a fragment thereof relative to the entire structure of an immunoglobulin, which is a part of a polypeptide comprising a portion capable of binding to an antigen. Such fragment comprises a functional fragment of an antibody molecule that is not a complete antibody having two light chains and two heavy chains. For example, the antigen-binding fragment may be scFv, (scFv)2, Fv, Fab, Fab', F(ab')2, or a combination thereof. For example, it may be F(ab')2, Fab', Fab, Fv, or scFv. Among the antigen-binding fragments, Fab comprises a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region of the heavy chain (C H1 It has a structure containing ) and possesses one antigen-binding site. Fab' is heavy chain C H1 It differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain. F(ab')2 antibodies are generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments, which are minimal antibody fragments possessing only heavy chain variable regions and light chain variable regions, are widely known in the art. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by non-covalent bonds, whereas in single-chain Fv, the heavy chain variable region and the single chain variable region are generally connected by covalent bonds via a peptide linker or directly at the C-terminus, allowing them to form a dimer-like structure similar to that of two-chain Fv. The above antigen-binding fragment can be obtained using a proteolytic enzyme (for example, if the whole antibody is restricted to papain, Fab can be obtained, and if it is restricted to pepsin, F(ab')2 fragment can be obtained). In addition, the above antigen-binding fragment can be produced through genetic recombination technology.

[0019] In this specification, the term "Complementarity determining region" (CDR) refers to a region where three exist in each of the light and heavy chains of the variable region, and which is a region with particularly high variability in amino acid sequences among the variable regions, and due to this high variability, specific antibodies against various antigens can be detected. The three complementarity determining regions of the heavy chain are designated as HCDR1, HCDR2, and HCDR3 in order from the amino terminus to the carboxyl terminus, and the three complementarity determining regions of the light chain are designated as LCDR1, LCDR2, and LCDR3 in order from the amino terminus to the carboxyl terminus. In a single antibody, the above six complementarity determining regions come together to form an antigen binding site.

[0020] The "% of sequence homology" for the above amino acid sequence can be confirmed by comparing the comparison region with two optimally arranged sequences.

[0021] Furthermore, functional variants of the sperm-binding antibody of the present invention comprise biological equivalents of the sperm-binding antibody sequences described herein, within a range capable of specifically recognizing sperm. For example, additional modifications may be made to the amino acid or polynucleotide sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include deletion, insertion, and / or substitution of amino acid sequence residues of the antibody and are based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0022] The above antibody or its antigen-binding fragment comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity therewith; a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence of SEQ ID NO. 2 or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity therewith; and a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 3 or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity therewith; It may include a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO. 4 or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity therewith, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO. 5 or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity therewith, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO. 6 or an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity therewith.

[0023] In one embodiment, the antibody or the antigen-binding fragment thereof may comprise a heavy chain complementarity determining region 1 (HCDR1) consisting of an amino acid sequence represented by SEQ ID NO. 1, a heavy chain complementarity determining region 2 (HCDR2) consisting of an amino acid sequence represented by SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (HCDR3) consisting of an amino acid sequence represented by SEQ ID NO. 3; and a light chain complementarity determining region 1 (LCR1) consisting of an amino acid sequence represented by SEQ ID NO. 4, a light chain complementarity determining region 2 (LCDR2) consisting of an amino acid sequence represented by SEQ ID NO. 5, and a light chain complementarity determining region 3 (LCDR3) consisting of an amino acid sequence represented by SEQ ID NO. 6.

[0024] The above antibody or its antigen-binding fragment may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising an amino acid sequence of SEQ ID NO. 8. Specifically, it may comprise a heavy chain variable region comprising an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 7; and a light chain variable region comprising an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 8.

[0025] In one embodiment, the antibody or its antigen-binding fragment may comprise a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO. 7 and a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO. 8.

[0026] The above antibody or its antigen-binding fragment includes all of the following: monoclonal antibodies, polyclonal antibodies, mouse antibodies, goat antibodies, sheep antibodies, guinea pig antibodies, rat antibodies, rabbit antibodies, human antibodies, humanized antibodies, chimeric antibodies (e.g., humanized murine antibodies), and heterojunction antibodies (e.g., sheep-specific antibodies). Additionally, the above antibody may include diabodies, triabodidies, and tetrabodies. The above monoclonal antibody may be of the IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type.

[0027] The above antibody or its antigen-binding fragment may bind to sperm containing a Y chromosome derived from a mammal. The sperm containing a Y chromosome may be Y chromosome sperm.

[0028] The above mammal may include, but is not limited to, one or more selected from the group consisting of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle, dogs, and pigs. Specifically, the above mammal may be a pig. Additionally, the above mammal may be one excluding humans.

[0029] In one embodiment, the pig may include one or more breeds selected from the group consisting of Duroc, Landrace, Yorkshire, Berkshire, Yucatan Mini, Hampshire, Poland China, Korean native breeds and crossbreeds thereof, but is not limited thereto. Specifically, the pig may include one or more breeds selected from the group consisting of Duroc, Landrace, and Yorkshire.

[0030] The above antibody or its antigen-binding fragment may not bind to sperm containing an X chromosome derived from a mammal. The sperm containing an X chromosome may be X chromosome sperm.

[0031] In one embodiment, the antibody or its antigen-binding fragment may induce an agglutination reaction of sperm containing a Y chromosome derived from a mammal; specifically, the antibody or its antigen-binding fragment may specifically bind to a cell membrane protein of the tail portion of a Y chromosome sperm to induce an agglutination reaction of the Y chromosome sperm. Additionally, since the antibody or its antigen-binding fragment does not specifically bind to X chromosome sperm, it does not induce an agglutination reaction of X chromosome sperm. Therefore, by utilizing these properties, Y chromosome sperm and X chromosome sperm can be easily distinguished or separated.

[0032]

[0033] Another aspect is to provide an isolated nucleic acid encoding the antibody or its antigen-binding fragment. The same parts described above apply equally to the nucleic acid.

[0034] In this specification, the term "nucleic acid" comprehensively includes DNA and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues in which sugar or base sites are modified. The sequence of nucleic acid encoding a variable region of a heavy chain and light chain of one aspect may be modified. Such modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0035] The above nucleic acid is interpreted to include a nucleotide sequence that exhibits substantial identity with respect to the nucleotide sequence of the above nucleic acid. Substantial identity means a nucleotide sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology when one aspect of the nucleotide sequence is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0036]

[0037] Another aspect is to provide a vector containing the isolated nucleic acid. The same parts described above apply equally to the vector.

[0038] The above vector may be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing a target antibody) for the expression of an antibody or its antibody fragment in a suitable host cell, with DNA encoding partial or full-length light and heavy chains, and may include essential regulatory elements operably linked to enable the expression of a DNA (gene) insert. "Operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function, and that the gene is linked to the expression regulatory sequence so that the gene can be expressed.

[0039] The term "expression control sequence" above refers to a DNA sequence that regulates the expression of a linked DNA sequence operable in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence regulating the termination of transcription and translation, a start codon, a stop codon, a polyadenylation signal, and an enhancer. Those skilled in the art may recognize that the design of the expression vector may vary by selecting a different control sequence depending on factors such as the selection of the host cell to be transformed and the expression level of the protein.

[0040] The above vector is not particularly limited in type as long as it is a vector commonly used in the fields of cloning and antibody production; examples include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. The above plasmids include Escherichia coli-derived plasmids (pBR322, pBR325, pUC118, and pUC119, pET-21b(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, and YCp50), and the above viruses may include animal viruses such as retroviruses, adenoviruses, or vaccinia viruses, and insect viruses such as baculoviruses. Vectores of the pComb3 family, commonly used for phage labeling, may be used, and vectors commonly used for expressing proteins in mammalian cells to express antibodies in mammalian cells, such as pcDNA or pVITRO, may be used.

[0041]

[0042] Another aspect is to provide isolated host cells transformed with the above-mentioned vector. The same parts described above apply equally to the said host cells.

[0043] In this specification, the term "transformation" refers to a molecular biological technique in which a fragment of DNA chain or a plasmid containing a type of foreign gene different from that of the original cell infiltrates between cells and binds to the DNA present in the original cell, thereby altering the genetic traits of the cell. The vector is transfected or transfected into a host cell. Various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for transfection or transfection may be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. The transformation may be performed under in vitro conditions.

[0044] An antibody or its antigen-binding fragment according to one aspect may be expressed in eukaryotic cells, preferably mammalian host cells, considering the applicability to microorganisms such as bacteria (E. coli) or yeast, or to mammalian cells. The mammalian host cell may be, for example, any one selected from the group consisting of Chinese hamster ovary (CHO) cells, NSO myeloma cells, COS cells, SP2 cells, F2N cells, HEK293 cells, and antibody-producing hybridoma cells, but is not limited thereto.

[0045]

[0046] Another aspect is to provide a cell line that expresses and / or produces the above-mentioned antibody for sperm binding or its antigen-binding fragment. The same parts described above apply equally to said cell line.

[0047]

[0048] Another aspect provides a method for producing an antibody for sperm binding or an antigen-binding fragment thereof, comprising the step of culturing the isolated host cell or cell line to express an antibody or an antigen-binding fragment thereof. The same parts as described above apply equally to the method.

[0049] The above method may include the step of transforming a host cell for producing one type of antibody or its antigen-binding fragment into a vector operably linked to DNA encoding said antibody or its antigen-binding fragment. The type of selected host cell and recombinant expression vector is as described above, and this step may be carried out by selecting an appropriate transformation method. When the recombinant expression vector encoding said antibody gene is introduced into a mammalian host cell, the antibody may be produced by culturing the host cell for a period sufficient to cause the antibody to be expressed in the host cell, or more preferably, for a period sufficient to cause the antibody to be secreted into the culture medium in which the host cell is cultured.

[0050] In addition, the above method may further include the step of culturing the transformed isolated host cells to produce a polypeptide of an antibody or an antigen-binding fragment thereof according to one aspect from a recombinant expression vector introduced into the host cells. The composition of the culture medium, culture conditions, and culture time for culturing the selected host cells may be appropriately selected, and the antibody molecules produced in the host cells may be accumulated in the cytoplasm of the cells, secreted outside the cells or into the culture medium by an appropriate signal sequence, or targeted to periplasms, etc. In addition, the protein may be refolded and given a functional structure using methods known in the art so that the antibody according to one aspect maintains binding specificity to mesothelin. In addition, when producing an IgG-type antibody, the heavy chain and light chain may be expressed in separate cells and brought into contact at a separate stage to form a complete antibody, or the heavy chain and light chain may be expressed in the same cell to form a complete antibody inside the cell.

[0051] Additionally, the above method may further include a step of obtaining an antibody or its antigen-binding fragment produced in isolated host cells. The method of obtaining can be appropriately selected and controlled by considering the characteristics of the polypeptide of the antibody or its antigen-binding fragment produced in the host cells, the characteristics of the host cells, the mode of expression, or whether the polypeptide is targeted. For example, the antibody or its antigen-binding fragment secreted into the culture medium can be recovered by obtaining the medium in which the host cells were cultured and removing impurities through centrifugation, and if necessary, the cells may be lysed to a extent that does not affect the functional structure of the antibody or its antigen-binding fragment in order to release and recover the antibody present in specific organelles or cytoplasm within the cell to the outside.

[0052] The obtained antibody may undergo an additional process of further removing impurities and concentrating it through methods such as chromatography, filtration using a filter, or dialysis. The separation or purification of the obtained antibody may be performed by separation and purification methods commonly used for proteins, for example, by chromatography. The chromatography may include, for example, affinity chromatography including a protein A column, a protein G column, and a protein L column, ion exchange chromatography, or hydrophobic chromatography. In addition to the chromatography, the antibody may be separated and purified by combining filtration, ultrafiltration, salting out, dialysis, etc.

[0053]

[0054] Another aspect is to provide a composition for sperm sex determination or a use for sperm sex determination comprising the above-mentioned antibody for sperm binding or its antigen binding. The same parts as described above apply equally to the composition or the use.

[0055] The term "sperm sex determination" in this specification refers to the separation or differentiation of X chromosome sperm and Y chromosome sperm. Since it is difficult to distinguish these sperm based solely on motility or morphological differences, the present invention aims to separate or differentiate them using a sperm separation method involving binding with antibodies and a sperm agglutination reaction mediated by antibodies and antibody fragments.

[0056] Generally, mammalian eggs have only an X chromosome as a sex chromosome, whereas sperm in semen contain X or Y chromosomes in a ratio of about 50:50, and sperm play a role in determining the sex of the fertilized egg when they meet an egg inside the female's body and fertilize it. Therefore, by identifying or separating the sex of sperm, it is possible to produce offspring of a specific sex.

[0057] The above composition may be for sex determination of sperm derived from, isolated from, and / or obtained from mammals.

[0058] In one embodiment, the antibody for sperm binding or the antigen binding thereof; or a composition containing the same may bind to sperm containing a mammalian Y chromosome, and specifically may bind to the caudal cell membrane of a Y chromosome sperm to induce a sperm agglutination reaction. Accordingly, based on the sperm agglutination reaction, Y chromosome sperm and X chromosome sperm can be distinguished / differentiated or separated and obtained.

[0059] In one embodiment, the sperm agglutinated by binding to or agglutinating the antibody for sperm binding or the antigen binding thereof; or a composition containing the same, can be determined to be sperm containing a Y chromosome.

[0060] In one embodiment, sperm that is not bound to or not aggregated by the antibody for sperm binding or the antigen binding thereof; or a composition containing the same, can be determined to be sperm containing an X chromosome.

[0061]

[0062] Another aspect is to provide a sperm sex determination kit comprising the above-mentioned antibody for sperm binding or the antigen binding thereof. The same parts as described above also apply to the above-mentioned composition.

[0063] The above kit may be for sex determination of sperm derived from, isolated from, and / or obtained from mammals.

[0064] In one embodiment, the kit may further include reagents and / or apparatus for isolating, purifying, and / or obtaining Y-chromosome sperm in which an agglutination reaction is induced by the antibody for sperm binding or by the antigen binding thereof, or X-chromosome sperm to which the antibody is not bound.

[0065]

[0066] Another aspect provides a method for sex determination of sperm of mammals other than humans, comprising: 1) mixing a sample containing semen, sperm, or sperm isolated from an individual with an antibody for sperm binding or an antigen binding thereof; and 2) separating sperm specifically bound to the antibody or unbound sperm from the mixture. The same parts as described above apply equally to the method.

[0067] The term "individual" in this specification refers to a subject from which semen or sperm is to be collected, and may be a mammal. The mammal may include, but is not limited to, one or more selected from the group consisting of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle, dogs, and pigs. Specifically, the mammal may be a pig. Additionally, the mammal may be any animal other than a human.

[0068] The semen, sperm, or sperm separated from the above-mentioned individual may refer to in vitro semen, sperm, or sperm. Accordingly, the above method may be performed under in vitro conditions.

[0069] In the above method, the above '1) step' may include inducing the binding of the Y chromosome sperm contained in a sample containing semen, sperm, or sperm separated from an individual with the antibody for sperm binding or the antigen binding thereof, and specifically, may include inducing an agglutination reaction of the Y chromosome sperm.

[0070] In the above method, 'step 2)' is for separating sperm specifically bound to or unbound from the antibody or its antigen-binding fragment, and may use one or more methods selected from the group consisting of flow cytometry separation techniques, magnetic separation techniques, filter separation techniques, panning separation techniques, separation methods using nanomaterials, and direct administration techniques, but is not limited thereto.

[0071] The above method may further include the step of 3) determining that sperm specifically bound to the antibody are sperm containing a Y chromosome, and unbound sperm are sperm containing an X chromosome. Specifically, sperm that agglutinated due to the sperm agglutination reaction may be Y chromosome sperm, and sperm that did not agglutinate may be X chromosome sperm.

[0072]

[0073] Another aspect provides a method for producing mammals other than humans of a specific sex, comprising: 1) mixing semen separated from an individual with the antibody for sperm binding or the antigen binding thereof; 2) separating sperm specifically bound to the antibody or unbound sperm from the mixture; 3) fertilizing using the separated sperm; and 4) producing offspring of a desired sex. The same parts as described above apply equally to the method.

[0074] In the above method, the above '1) step' may include inducing the binding of the Y chromosome sperm contained in a sample containing semen, sperm, or sperm separated from an individual with the antibody for sperm binding or the antigen binding thereof, and specifically, may include inducing an agglutination reaction of the Y chromosome sperm.

[0075] In the above method, 'step 2)' is for separating sperm specifically bound to or unbound from the antibody or its antigen-binding fragment, and may use one or more methods selected from the group consisting of flow cytometry separation techniques, magnetic separation techniques, filter separation techniques, panning separation techniques, separation methods using nanomaterials, and direct administration techniques, but is not limited thereto.

[0076] In the above method, the above '3) step' may involve fertilizing using the separated sperm and egg, and specifically may include in vitro fertilization in a test tube or artificial fertilization in the body by directly injecting into the uterus.

[0077] The term "in vitro fertilization" in this specification refers to obtaining a fertilized egg by culturing eggs collected from slaughterhouses or superovulation-treated females with sperm in a test tube. For the purposes of the present invention, it refers to a sex-separated fertilized egg obtained by performing fertilization in a test tube using sperm that has already been sex-separated or separated in a test tube, and using this, offspring of the desired sex can be produced by injecting them into the body of a female.

[0078] The term "artificial insemination" in this specification refers to the process of inducing conception by artificially injecting the semen of a male animal into the reproductive organs of a female animal, rather than through natural mating, to combine an egg and sperm. For the purposes of the present invention, by injecting the semen of a male animal into the reproductive organs of a female animal, sperm selected for sex as described above enter the cytoplasm of the egg, thereby enabling the production of offspring of the desired sex.

[0079] In one embodiment, if the separated sperm is a sperm specifically bound to an antibody, it may produce a male individual (offspring).

[0080] In one embodiment, if the separated sperm is sperm unbound to an antibody, it may produce a female individual (offspring).

[0081] It has been confirmed that by treating with the antibody of the present invention or its antigen-binding fragment, an agglutination reaction of Y chromosome sperm can be induced, thereby allowing for easy differentiation between X chromosome sperm and Y chromosome sperm. This enables the mass production of pigs of a specific sex and, by selectively producing pigs of a desired sex, contributes to planned breeding, breeding improvement, and management efficiency.

[0082] Figure 1 is the result of confirming whether the sperm bound to the antibody protein of the present invention using sperm of different pig breeds.

[0083] Figure 2 is the result of confirming the binding site between the antibody protein of the present invention and sperm using a fluorescence microscope.

[0084] Figure 3 is the result of confirming, using an electron microscope, whether the sperm agglutination reaction of a pig breed is induced by the antibody protein treatment of the present invention.

[0085] Figure 4 is the result of confirming the chromosomes of the antibody-positive sperm and antibody-negative sperm of the present invention, separated by a flow cytometer, using PCR.

[0086] Figure 5 is the result of visually confirming whether sperm agglutination occurred following the treatment of the antibody protein of the present invention using porcine liquid sperm.

[0087] Figure 6 shows the results of verifying the efficiency of X chromosome sperm separation using a mesh filter after inducing a sperm agglutination reaction by treating with the antibody protein of the present invention.

[0088] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0089]

[0090] Example 1: Production and Sequence Analysis of Antibodies Capable of Specifically Binding to Porcine Y Sperm

[0091] The inventors produced an antibody capable of specifically binding to porcine Y sperm (Y chromosome sperm) having the sequences of Table 1 and Table 2 below.

[0092] CDR Region Amino Acid Sequence Sequence Number Heavy chain CDR1GYTITSGYD1CDR2ISSSGRT2CDR3SSLGAY3Light chain CDR1KSVSTSGYSC4CDR2LVS5CDR3QHIRELT6

[0093] CDR regionAmino acid sequenceSequence numberHeavy chain variable regionVal Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Thr Ile Thr Ser Gly Tyr Asp Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met Gly His Ile Ser Ser Ser Gly Arg Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ala Lys Asn Gln Phe Phe Leu Gln Leu Thr Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ser Ser Leu Gly Ala Tyr Trp Gly Gln7 Light chain variable region Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Cys Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly8

[0094]

[0095] Example 2: Confirmation of the binding ability of porcine Y sperm-specific binding antibodies to sperm

[0096] To confirm the binding ability of the antibody produced and sequenced in Example 1 above with pig sperm, the following experiment was performed.

[0097] Specifically, liquid semen from commercially available pig breeds was separated using a centrifuge, and a sperm dilution solution diluted to 1,000,000 cells / 1 ml was used as the sperm for analysis. To verify the binding of the antibody protein prepared and sequenced in Example 1, 100,000 sperm cells were each treated with the antibody protein, and a primary binding reaction was carried out for approximately 20 minutes. Unbound primary antigen proteins were then removed by centrifugation. Next, the sperm cells to which the primary antibody was bound were stained with PE fluorescent substance-conjugated anti-rat antibody protein (secondary antibody) at room temperature for approximately 20 minutes, and these were used as samples for analysis. To exclude dead sperm before flow cytometry analysis, PI fluorescent substance was added, and only live sperm were selected. Additionally, rat isotype IgG protein was added as a control for the sperm, and the samples were stained with the same secondary antibody. After selecting only the sperm portion using Forward Scatter (FSC) and Side Scatter (SSC) of a flow cytometer, the binding of sex-identifying antibody proteins in sperm of each breed was determined on only the sperm population, and 10,000 sperm data points per sperm were stored and analyzed.

[0098] As a result of the experiment above, as shown in Fig. 1, only unbound sperm were detected in the control group without antibody protein treatment (Fig. 1A), whereas in the group to which the sex determination antibody protein of the present invention was added to sperm of pig breeds Duroc, Landrace, and Yorkshire, antibody-negative sperm (antibody protein unbound sperm) and antibody-positive sperm (antibody protein bound sperm) were observed separated in a 50:50 ratio (Figs. 1B, C, and D).

[0099] Considering that sperm in semen have X or Y chromosomes in a ratio of about 50:50, the above results indicate that the antibody protein developed in the present invention can specifically bind to X chromosome sperm or Y chromosome sperm of pig breeds.

[0100]

[0101] Example 3: Confirmation of sperm binding site and sperm agglutination reaction of porcine Y sperm-specific binding antibody

[0102]

[0103] 3.1: Confirmation of the binding site with sperm

[0104] The structure of sperm is broadly divided into the head, which contains the nucleus and acrosome; the neck, which contains the apex and the terminal end. Accordingly, to identify the specific part of the sperm to which the antibody prepared in Example 1 binds, an experiment was conducted as follows.

[0105] Specifically, immunohistochemistry was performed to examine the binding site of the sperm, and the antibody protein of the present invention bound to the pig sperm was stained in the same manner as in Example 2 above, and the binding site was confirmed using a fluorescence microscope.

[0106] As a result of the above experiment, as shown in Figure 2, it was confirmed that the antibody protein of the present invention binds to the caudal cell membrane of pig sperm.

[0107] Based on the above results, it can be seen that the antibody protein of the present invention specifically binds to the cell membrane protein of the tail portion of an X chromosome sperm or a Y chromosome sperm.

[0108]

[0109] 3.2: Confirmation of Sperm Agglutination Reaction

[0110] The structure of the antibody protein consists of one structural protein FC portion and two antigen-binding sites, and when the antibody protein binds to an antigen, an agglutination reaction is induced. Accordingly, the following experiment was performed to determine whether the antibody prepared in Example 1 above causes a sperm agglutination reaction upon binding to sperm.

[0111] Specifically, liquid semen from commercially available pig breeds was separated using a centrifuge, antibody proteins were added, and the mixture was reacted by gently shaking at 37°C for 14 hours, after which it was observed using a CASA sperm analyzer. As a control group, a group without the addition of the antibody of the present invention was used.

[0112] As a result of the experiment above, as shown in Fig. 3, no sperm agglutination reaction was observed in the control group without the addition of antibody protein, whereas in the group with the addition of antibody protein, it was confirmed that the antibody protein bound to the tail protein of the sperm, inducing a sperm agglutination reaction in which the tails of the sperm bound to each other (Fig. 3).

[0113] Based on the above results, it can be seen that the antibody protein of the present invention specifically binds to the cell membrane protein of the Y chromosome sperm tail and promotes binding between sperm tails, thereby inducing a sperm agglutination reaction.

[0114]

[0115] Example 4: Evaluation of the Efficacy of Y Chromosome Sperm Separation by Porcine Y Sperm-Specific Binding Antibodies

[0116] In order to determine whether the unbound sperm (negative sperm) and bound sperm (positive sperm) with the antibody prepared in Example 1, as distinguished in Example 2 above, were X chromosome sperm or Y chromosome sperm, respectively, the following experiment was performed.

[0117] Specifically, a gene amplification test (PCR) was performed using Y chromosome-specific primers (PY Primers), and the sequence information is shown in Table 3 below.

[0118] Primer Nucleotide Sequence Gene Size PY forward 5'- AAT CCA CCA TAC CTC ATG GAC C -3' (Sequence No. 9) 377 bp PY reverse 5'- TTT CTC CTG TAT CCT CCT GC- 3' (Sequence No. 10)

[0119] Porcine sperm stained with the antibody prepared in Example 1 were separated into 100,000 antibody-positive sperm and 100,000 antibody-negative sperm from Example 2 using a flow cytometer, placed in a 1.5 ml tube, and the supernatant was removed using a centrifuge. After adding 50 μl of distilled water (DW) to the precipitated sperm tube, the sperm nuclei were isolated by heating in a PCR machine at 99°C for 10 minutes. After adding 2 μl of Y chromosome-specific primer (PY primer) to 2 μl of sperm DNA in a PCR premixture tube containing PCR enzymes, a PCR genetic test was performed. After performing the PCR reaction for 5 minutes at 95°C as the initial denaturation step, a total of 30 cycles were performed at 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds, and an extension reaction was performed at 72°C for 5 minutes to investigate whether the gene amplified was an X chromosome sperm or a Y chromosome sperm.

[0120] As a result of the experiment above, as shown in Figure 4, the antibody-positive sperm of the present invention was amplified by a Y chromosome-specific primer (PY primer), confirming that it was a Y chromosome sperm, and the antibody-negative sperm of the present invention was confirmed to be an X chromosome sperm.

[0121] Based on the above results, it can be seen that the antibody of the present invention specifically binds to the cell membrane protein of the tail portion of the Y chromosome sperm and induces a Y chromosome sperm agglutination reaction.

[0122]

[0123] Example 5: Verification of sex determination efficacy using porcine Y sperm-specific binding antibodies

[0124] In order to confirm whether the porcine Y sperm-specific binding antibody produced in Example 1 above can be utilized in actual artificial insemination, the following experiment was conducted to verify the sex determination efficacy using liquid semen sperm used in actual artificial insemination.

[0125] Specifically, after semen was separated from porcine liquid semen using a centrifuge, a porcine Y sperm-specific binding antibody (sex determination antibody protein) prepared in Example 1 at a concentration of 150 μg was added to a sperm dilution solution diluted to 1,400,000,000 sperm / 10 ml, and the reaction was carried out by gently shaking at 37 ℃ for 14 hours. Subsequently, sperm were separated using an 18 μM mesh, and 100,000 sperm samples—both the separated and the original sperm—were stained with PE fluorescent conjugated anti-rat antibody (secondary antibody) at room temperature for approximately 20 minutes, after which they were used as analytical samples. As a control for the sperm, rat isotype IgG protein was added and stained with the same secondary antibody. After selecting only the sperm portion using Forward Scatter (FSC) and Side Scatter (SSC) of a flow cytometer, the binding efficiency of sex-determining antibody proteins in the sperm of each experimental group was determined on only the sperm population, and 10,000 sperm data points per sperm were stored and analyzed.

[0126] As a result, as shown in Fig. 5, it was confirmed that in the control group without the addition of the antibody protein of the present invention, no sperm agglutination reaction occurred, whereas in the group with the addition of the antibody protein of the present invention, the sperm agglutination reaction proceeded and settled at the bottom of the tube (Fig. 5). In addition, as shown in Fig. 6, when X chromosome sperm and Y chromosome sperm, which existed in a 50:50 ratio before separation, were separated using a filter to separate only X chromosome sperm through the treatment with the antibody protein of the present invention, a high separation efficiency of over 90% was observed.

[0127] Based on the above results, it can be seen that only sperm with a Y chromosome or sperm with an X chromosome can be isolated and obtained from pig sperm using the antibody of the present invention, and that pigs of the desired sex can be obtained by proceeding with artificial insemination using the isolated and obtained sperm.

[0128]

[0129] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence of SEQ ID NO. 1, a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence of SEQ ID NO. 3; and A light chain variable region comprising a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence of SEQ ID NO. 4, a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence of SEQ ID NO. 5, and a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence of SEQ ID NO. 6, Antibody for sperm binding or antigen-binding fragment thereof.

2. The antibody or the antigen-binding fragment of claim 1, wherein the antibody or the antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising an amino acid sequence of SEQ ID NO.

8.

3. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof binds to sperm containing a Y chromosome.

4. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof induces an agglutination reaction of sperm containing a Y chromosome.

5. Isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A vector comprising the isolated nucleic acid of claim 5.

7. Isolated host cells transformed with the vector of claim 6.

8. A cell line that expresses or produces an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

9. A method for producing an antibody for sperm binding or an antigen-binding fragment thereof, comprising the step of culturing the host cells of claim 7 to express an antibody.

10. A composition for sperm sex determination comprising an antibody according to any one of claims 1 to 4 or an antigen-binding fragment thereof.

11. A composition according to claim 10, wherein the antibody or its antigen-binding fragment binds to sperm containing the Y chromosome of a mammal to induce a sperm agglutination reaction.

12. A kit for sperm sex determination comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4. 13.1) A step of mixing a sample comprising semen, sperm, or sperm separated from an individual with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4; and 2) A step of separating sperm specifically bound to or unbound from the antibody or its antigen-binding fragment from the above mixture. A method for sex determination of mammalian sperm other than humans, including 14. The method of claim 13, wherein the method further comprises the step of 3) determining that sperm specifically bound to the antibody is sperm containing a Y chromosome, and unbound sperm is sperm containing an X chromosome. 15.1) A step of mixing a sample containing semen, sperm, or sperm separated from an individual with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4; 2) A step of separating sperm specifically bound to the antibody or unbound sperm from the above mixture; 3) A step of fertilizing using the separated sperm; and 4) The stage of producing offspring of the desired sex A method of producing mammals other than humans having a specific sex, including 16. A method according to claim 15, wherein the separated sperm in step '3)' is sperm specifically bound to an antibody, thereby producing a male individual.

17. A method according to claim 15, wherein the separated sperm in step '3)' is sperm unbound to the antibody, thereby producing a female individual.

18. A method for producing an antibody for sperm binding or an antigen-binding fragment thereof, comprising the step of culturing the cell line of claim 8 to express an antibody.

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