JP5408802B2 - Separation method for immature fish germ cells using surface immature germ cell specific protein - Google Patents

Description

  The present invention relates to an antibody that can be used for separation of immature fish germ cells used in a method for growing fish by surrogate fish culture techniques, a method for separating immature fish germ cells using the antibody, and the like.

  With the recent increase in health awareness on a global scale, the consumption of Japanese food, especially fish containing high-quality lipids and proteins, is rising worldwide. As a result, large-scale high-quality fish such as bluefin tuna, which are particularly in high demand, have experienced a significant decrease in the number of survivors, and have come to a situation where they are threatened with extinction. As a means to protect large-scale high-quality fish resources such as bluefin tuna, so-called cultivated fisheries, in which artificial seedlings (fry) produced under artificial management are released into the natural sea area, are said to be effective. However, obtaining the eggs and sperm necessary for the production of artificial seedlings for the release of large fish has been a major technical and cost problem. In other words, in order to avoid the harmful effects of annihilation caused by specific diseases and the effects of crossing each other after the artificial seedlings have grown, the individual seedlings need to have genetic diversity. Considering that, it is desirable to collect eggs and sperm from a large number of parent fish, but it has been a long time for maturation of eggs and sperm such as fish species that reach several hundred kg, such as bluefin tuna. In fish species that require the moon, it has been extremely difficult technically and costly to grow a large number of parent fish under artificial control and collect eggs and sperm.

  In order to solve the above-mentioned problems related to the collection of eggs and sperm, the present inventors, for the first time in the world, produced surrogate parent fish for producing useful fish species such as eggs and sperm to related species that are quickly matured and easy to breed. Established technology. Specifically, primordial germ cells, which are germline stem cells such as eggs and sperm, are extracted from rainbow trout (donor) hatched fry, and pre- and hatched yamame trout (recipe) in which the immune system is immature and transplanted cells do not reject By transplanting the above-mentioned primordial germ cells into the ent) and nurturing and maturing the resulting host yamame trout, not only the eggs and sperm derived from the germ cells originally possessed by the yamame trout, but also the donor rainbow trout A technology for producing eggs and sperm derived from cells has been established (for example, see Patent Document 1). In this surrogate fish farming technique, in order to ensure sufficient transplantation efficiency for commercial mass production, only immature germ cells such as primordial germ cells are separated from the gonads and testis of donor fish and transplanted. Since it is necessary to use it, a recombinant rainbow trout in which a GFP gene was introduced into the regulatory region of the vasa gene that is expressed specifically in the primordial germ cell was used as a donor so that the GFP gene was expressed specifically in the primordial germ cell. However, eggs and sperm obtained by maturing primordial germ cells derived from this genetically modified rainbow trout are those into which GFP genes have been introduced, and juvenile fish produced using these eggs and sperm are Since it is a recombinant fish, it could not be used as an edible fish or a seedling for release, and there was an insufficient point in practical use.

  On the other hand, Patent Document 2 describes an antibody specific for a human primordial germ cell surface-specific antigen, and identifying a human primordial germ cell using this antibody. Non-Patent Document 1 describes that a monoclonal antibody that reacts with the gene product of Vasa gene that is specifically expressed in primordial germ cells at a specific developmental stage of Xenopus laevis is described.

  In addition, the present inventors aim to improve the above-mentioned insufficient points of the genetically modified fish in surrogate parent fish aquaculture technology, without introducing a marker gene such as a GFP gene, from a primordial germ cell from a donor fish. Attempts have been made to search for methods for isolating immature germ cells such as spermatozoa and spermatogonia. Specifically, rainbow trout has been used to search for genes that are specifically expressed in immature germ cells (see Non-Patent Document 2).

  An object of the present invention is to provide a gene, polynucleotide, polypeptide, an antibody that specifically binds to the polypeptide that is specifically expressed in immature germ cells of fish, and by using the antibody, An object of the present invention is to provide a method for separating immature germ cells from donor fish without introducing a marker gene such as a GFP gene, and a method for identifying immature germ cells derived from fish.

  In order to solve the above-mentioned problems, the present inventor has intensively studied, identified a cell surface antigen specifically expressed in rainbow trout spermatogonia, and prepared an antibody against this antigen. Specifically, by comprehensively analyzing mRNA expressed in rainbow trout spermatogonia (transcriptome analysis) and searching for genes encoding cell membrane surface proteins from the expressed genes A clone was obtained that was specifically expressed in immature germ cells such as rainbow trout spermatogonia and could be used as a marker in situ or the like. When cDNA was identified from this clone and homology search was performed, it was suggested that it was a CD205 gene encoding a complete transmembrane glycoprotein of 199 kDa. Compare the rainbow trout-derived CD205 gene with the CD205 gene of fish other than rainbow trout (zebrafish, bluefin tuna, soda tsutsuo, chub mackerel, yellowtail, flounder, red sea bream, mahata, nibe), and peptide antigens from regions conserved in various fish Then, an antibody against the peptide antigen was prepared. Using this antibody, immunostaining was performed on immature testis of rainbow trout immediately after sex differentiation. As a result, it was found that it specifically binds to immature germ cells, and the present invention was completed.

JP 2003-235558 A JP-T-2001-521380 Develop. Growth & Differ., 34 (2), 223-231 (1992) Abstracts of Annual Meeting of Marine Biotechnology Society Vol.9th, P148 (2006.5.27)

That is, the present invention relates to (1) a polynucleotide represented by the following (A), (B) or (C); (A) a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 79 to 5193 in SEQ ID NO: 1; B) A nucleotide sequence represented by nucleotide numbers 79 to 5193 in SEQ ID NO: 1, comprising a nucleotide sequence in which substitution, deletion, insertion, or addition of a number of nucleotides within the range of 1 to 20 and derived from fish of immature germ cells a polynucleotide encoding a polypeptide specifically expressed: (C) a nucleotide sequence or Ranaru polynucleotide shown in the nucleotide numbers 79-5193 of SEQ ID NO: 1 and 90% or more identity a, and encodes a polypeptide that is specifically expressed in immature germ cells from fish Polynucleotide: or, (2) the following (A) or (B) a polynucleotide encoding a polypeptide shown in; (A) a polypeptide consisting of the amino acid sequence represented by amino acid Nos 1-1705 in SEQ ID NO: 2 : (B) in the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2, consisting of an amino acid sequence in which substitution, deletion, insertion, or addition of the number of amino acids within the range of 1-20 , and A polypeptide specifically expressed in immature germ cells derived from fish; and (3) comprising a polynucleotide according to (1) or (2) above and specific to immature germ cells derived from fish The present invention relates to a recombinant vector comprising a gene encoding a polypeptide to be expressed and (4) the polynucleotide described in (1) or (2) above.

The present invention also includes (5) a polypeptide represented by the following (A) or (B ) ; (A) a polypeptide comprising the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2, and (B) a SEQ ID NO: 1. An amino acid sequence represented by amino acid Nos. 1-1705 in No. 2, comprising an amino acid sequence in which a number of amino acids within the range of 1-20 are substituted, deleted, inserted, or added, and immature reproduction derived from fish A polypeptide specifically expressed in cells : (6) a polypeptide encoded by the gene described in (3) above and specifically expressed in immature germ cells derived from fish, (7) above The present invention relates to a fusion polypeptide in which the polypeptide according to (5) or (6) is bound to a marker polypeptide and / or a peptide tag.

  Furthermore, the present invention relates to (8) an antibody that specifically binds to the polypeptide described in (5) above, (9) an amino acid sequence represented by amino acid numbers 833 to 1107 in SEQ ID NO: 2, and amino acid numbers 1118 to 1395 The antibody according to (8), which specifically binds to a polypeptide comprising the amino acid sequence shown or the amino acid sequence shown by amino acid numbers 1407 to 1697.

  Furthermore, the present invention provides (10) a method for identifying immature germ cells derived from fish using the antibody according to (8) or (9) above, or (11) step (a): the labeled ( 8) or (9) a step of bringing the sample into contact with a sample; and a step (b): a step of confirming whether the antibody specifically binds to the sample; The present invention relates to a method for identifying immature germ cells derived therefrom.

  Furthermore, the present invention is (12) separating immature germ cells derived from fish from a sample containing immature germ cells derived from fish, using the antibody according to (8) or (9) above. A method for separating immature germ cells derived from fish, and (13) step (A): a sample containing immature germ cells derived from fish, and the labeled antibody according to (8) or (9) And a step (B) a step of separating immature germ cells derived from fish to which the antibody specifically binds; a method for separating immature germ cells derived from fish, (14) As a sample containing immature germ cells derived from fish, a sample containing cells selected from the cells of embryonic growth of hatched embryos of fish, cells of gonads of fish, and cells of testis of fish is used. As described in the above (12) or (13) It relates to a method of separating immature germ cells derived from fish.

The present invention (15) from the donor fish, the method of separation (12) to (14), separating the immature germ cells from donor fish, and the immature from separate donor fish A method for inducing differentiation of isolated immature germ cells, comprising a step of transplanting germ cells (isolated immature germ cells) into an early embryo of a recipient fish, and (16) a recipient fish as a recipient fish, (15) or (15), wherein the differentiation induction method according to (15) above, wherein (17) the recipient fish is a triploid fish. The differentiation induction method according to 16), or (18) transplantation of the recipient fish to the early embryo is transplantation of the recipient fish in the developmental stage before and after hatching to the back side of the peritoneal mesentery (15) to (18), wherein the differentiation induction method according to any one of (15) to (17), or (19) a primordial germ cell or a spermatogonia is used as the isolated immature germ cell. ) And / or the differentiation induction method for isolated immature germ cells according to any one of (15) to (19) above, and (20) eggs and / or sperm derived from donor fish. The present invention relates to a method for proliferating fish characterized by forming. In the present invention, the term “immature germ cell” refers to the lineage from primordial germ cells to spermatogonia, spermatocytes, sperm cells, sperm, primordial germ cells to oocytes, early oocytes, oocytes It means a cell selected from a primordial germ cell, a spermatogonia cell, an oocyte cell, and an early oocyte among germ cell lines consisting of a cell and an egg.

It is a figure which shows the result of the in situ hybridization with respect to the testis sample of a 3-month-old vasa-GFP gene introduction | transduction rainbow trout. Probes corresponding to the vasa gene, clone # 4017, and clone # 5206 were used. It is a figure which shows the result of in situ hybridization with respect to the testis sample of the vasa-GFP gene introduction | transduction rainbow trout early embryo which hold | maintains the primordial germ cell 35 days after fertilization. A probe corresponding to clone # 4017 was used. It is a figure which shows the result of in situ hybridization with respect to the testis sample of vasa-GFP gene introduction | transduction rainbow trout (12 months of age) which has an immature testis. A probe corresponding to clone # 4017 was used. It is a figure which shows the result of the in situ hybridization with respect to the testis sample of vasa-GFP gene introduction | transduction rainbow trout (18 months of age) which has a testis in a mature stage. A probe corresponding to clone # 4017 was used. It is a figure which shows the result of in situ hybridization with respect to the testis sample of vasa-GFP gene introduction | transduction rainbow trout which has a defecation testis. A probe corresponding to clone # 4017 was used. It is a figure which shows the result of in situ hybridization with respect to the ovary sample of vasa-GFP introduction | transduction rainbow trout which has an immature ovary. A probe corresponding to clone # 4017 was used. It is a figure which shows the alignment (amino acid sequence) of the preservation | save area | region of CD205 homologue derived from rainbow trout and other fish species. A: Alignment (nucleotide sequence) of the conserved region of CD205 homologue derived from rainbow trout and other fish species. B: Alignment (amino acid) of CD205 homologues of bluefin tuna, rainbow trout and zebrafish in another region. It is a figure which shows each area | region of rainbow trout CD205 cDNA. It is a figure which shows six extracellular regions which produced the recombinant about rainbow trout CD205. It is a figure which shows the result of SDS-PAGE about the recombinant polypeptide of rainbow trout CD205 obtained by refinement | purification. It is a figure which shows the result of the whole mount immunostaining performed with respect to the immature testis of vasa-GFP gene introduction | transduction rainbow trout immediately after sex differentiation. It is a figure which shows the result of the hematoxylin eosin dyeing | staining of the testis tissue of 2n yamame, non-transplanted 3n yamame, and transplanted 3n yamame. It is a figure which shows the result of PCR amplification of the GFP gene fragment from the semen DNA or somatic DNA extract | collected from the transplant 3n yamame trout (recipient). It is a figure which shows the result of the DNA fingerprint of the next-generation individual | organism | solid produced using the sperm extract | collected from the transplant 3n yamame trout. It is a figure which shows each gonad of the female individual | organism | solid of wild type 2n yamame (2N), non-transplanted 3n yamame (3N yamame), and transplanted 3n yamame (2N → 3N). The lowermost panel shows that the transplanted 3n yamame trout oocytes are GFP positive.

  The polynucleotide of the present invention includes one or several nucleotides in the polynucleotide consisting of the nucleotide sequence represented by nucleotide numbers 79-5193 in SEQ ID NO: 1, or the nucleotide sequence represented by nucleotide numbers 79-5193 in SEQ ID NO: 1 Or a polynucleotide encoding a polypeptide specifically expressed in fish-derived immature germ cells, or nucleotide numbers 79-5193 in SEQ ID NO: 1 A polynucleotide that encodes a polypeptide that hybridizes under stringent conditions with a polynucleotide comprising a sequence complementary to the nucleotide sequence shown in, and that is specifically expressed in immature germ cells derived from fish; At number 2 Substitution, deletion or insertion of one or several amino acids in a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in amino acid Nos. 1-1705, or in the amino acid sequence shown in amino acid Nos. 1-1705 in SEQ ID No. 2. Or a polynucleotide that encodes a polypeptide that consists of an added amino acid sequence and that is specifically expressed in immature germ cells derived from fish, and at least the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2 The polynucleotide is not particularly limited as long as it is a polynucleotide that encodes a polypeptide consisting of an amino acid sequence having a homology of 85% or more and that is specifically expressed in immature germ cells derived from fish. The gene of the present invention includes the polynucleotide of the present invention, and fish The gene is not particularly limited as long as it is a gene encoding a polypeptide specifically expressed in a conventional immature germ cell. The polypeptide of the present invention is represented by amino acid numbers 1-1705 in SEQ ID NO: 2. Consisting of an amino acid sequence in which one or several amino acid substitutions, deletions, insertions, or additions are made in the polypeptide containing the amino acid sequence or the amino acid sequence shown in amino acid numbers 1-1705 in SEQ ID NO: 2, and A polypeptide specifically expressed in immature germ cells derived from fish, or an amino acid sequence having at least 85% homology with the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2, and derived from fish A polypeptide that is specifically expressed in immature germ cells, or the gene of the present invention, The polypeptide is not particularly limited as long as it is a polypeptide that is specifically expressed in immature germ cells derived from fish. In the present invention, the polypeptide that is specifically expressed in immature germ cells derived from fish is any of A polypeptide that is specifically expressed in immature germ cells derived from fish and is located at least partially outside or on the surface of the immature germ cells.

  The above “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 5. It means an amino acid sequence in which any number of amino acids are deleted, substituted or added. The “nucleotide sequence in which one or several nucleotides are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and further preferably 1 Means a nucleotide sequence in which any number of ~ 5 nucleotides has been deleted, substituted or added.

  For example, a polynucleotide consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted, or added (mutant polynucleotide) can be any of those known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis. It can also be produced by this method. Specifically, a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 79-5193 in SEQ ID NO: 1 or a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 79-5193 in SEQ ID NO: 1 (for example, shown in SEQ ID NO: 1) Nucleotide sequence) is obtained by introducing a mutation into these polynucleotides using a method of contacting with a mutagen agent, a method of irradiating ultraviolet rays, a genetic engineering technique, etc. can do. Site-directed mutagenesis, which is one of genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 2001. (hereinafter abbreviated as "Molecular Cloning 3rd Edition"), Current Protocols in Molecular Biology, Supplements 1-38, John Wiley & Sons (1987-1997), etc. be able to. By expressing this mutant polynucleotide using an appropriate expression system, a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added can be obtained.

  The above-mentioned “polynucleotide hybridizing under stringent conditions” uses a polynucleotide such as DNA or RNA as a probe, and uses a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Specifically, using a filter on which a colony or plaque-derived polynucleotide or a fragment thereof is immobilized, a high concentration is obtained at 65 ° C. in the presence of 0.7 to 1.0 M NaCl. After hybridization, the filter is washed under conditions of 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Identifiable polynu Reochido can be mentioned. Hybridization can be performed according to the method described in Molecular Cloning 3rd edition and the like.

  That is, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Specifically, polynucleotides having a homology of 50 to 70% or more (particularly, DNA) are hybridized and polynucleotides having lower homology (especially DNA) are not hybridized with each other, or are washing conditions for normal Southern hybridization at 65 ° C., 1 × SSC, 0.1% SDS Or conditions for hybridizing at a salt concentration corresponding to 0.1 × SSC, 0.1% SDS. For example, the polynucleotide that can hybridize under stringent conditions can include a polynucleotide having a certain homology with the base sequence of the polynucleotide used as a probe, for example, 60% or more, preferably Is preferably a polynucleotide having a homology of 70% or more, more preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. It can be illustrated.

  The method for obtaining and preparing the polynucleotide of the present invention, such as DNA, is not particularly limited. The nucleotide sequence information in the polynucleotide sequence shown in SEQ ID NO: 1 disclosed in the present specification and the sequence shown in SEQ ID NO: 2 are shown. Prepare appropriate probes and primers based on the amino acid sequence information in the amino acid sequence, and use them to isolate fish CD205 genes by screening fish DNA libraries, or by chemical synthesis according to conventional methods can do. In addition, as a kind of polynucleotide of this invention, DNA can be illustrated suitably.

  The gene of the present invention is a gene that contains the polynucleotide of the present invention and encodes a polypeptide that is specifically expressed in immature germ cells derived from fish. Specifically, the nucleotide number in SEQ ID NO: 1 Preferable examples include a polynucleotide consisting of the nucleotide sequence shown in 79-5349 and a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in amino acid number 1-1757 in SEQ ID NO: 2.

  The fish in the present invention is not particularly limited as long as the immature germ cell is a fish that specifically expresses the polypeptide of the present invention, bluefin tuna, mahata, yellowtail, nibe, Japanese flounder, Japanese mackerel, red sea bream, eel, Examples include saltwater fish such as coelacanth and freshwater fish such as rainbow trout.

  The method for obtaining and preparing the polypeptide of the present invention is not particularly limited, and may be a naturally occurring polypeptide, a chemically synthesized polypeptide, or a recombinant polypeptide produced by a gene recombination technique. When a naturally occurring polypeptide is obtained, the polypeptide of the present invention can be obtained by appropriately combining the methods for isolating and purifying the polypeptide from cells or tissues expressing such a polypeptide. When the polypeptide is prepared by chemical synthesis, for example, the polypeptide of the present invention is synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). be able to. Moreover, the polypeptide of the present invention can be synthesized using various commercially available peptide synthesizers. When a polypeptide is prepared by a gene recombination technique, the polypeptide of the present invention can be prepared by introducing a polynucleotide such as DNA consisting of a base sequence encoding the polypeptide into a suitable expression system. . Among these, preparation by a gene recombination technique that can be prepared in a large amount by a relatively easy operation is preferable.

  For example, when preparing the polypeptide of the present invention by genetic recombination techniques, to recover and purify the polypeptide from cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose Known methods including chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, preferably high performance liquid chromatography are used. In particular, as a column used for affinity chromatography, for example, a column in which an antibody such as a monoclonal antibody against the polypeptide of the present invention is bound, or when a normal peptide tag is added to the polypeptide of the present invention, this peptide tag A purified product of these polypeptides can be obtained by using a column to which a substance having an affinity for is bound. In addition, when the polypeptide of the present invention is expressed on the cell membrane, a purified sample can be obtained by carrying out the above purification treatment after allowing the cell membrane degrading enzyme to act.

  Furthermore, the immature germ cell derived from fish, comprising an amino acid sequence in which one or several amino acid substitutions, deletions, insertions, or additions are made in the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2. Or an amino acid sequence having at least 85% homology with the amino acid sequence represented by amino acid Nos. 1-1705 in SEQ ID NO: 2 and expressed in fish-derived immature germ cells A polypeptide that is specifically expressed can be obtained by those skilled in the art based on the nucleotide sequence information represented by nucleotide numbers 79 to 5193 in SEQ ID NO: 1 encoding the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2. It can be prepared or obtained as appropriate.

  The recombinant vector of the present invention is a recombinant vector comprising the polynucleotide of the present invention and capable of expressing a polypeptide specifically expressed in immature germ cells derived from fish in vivo. The recombinant vector of the present invention can be constructed by appropriately integrating the polynucleotide of the present invention into an expression vector. The expression vector is preferably one that can replicate autonomously in the host cell, or one that can be integrated into the chromosome of the host cell. In addition, a promoter, enhancer, terminator, etc. can be placed at a position where the polynucleotide of the present invention can be expressed. Those containing a control sequence can be preferably used. The expression vector is preferably a recombinant vector using an expression vector for animal cells, particularly an expression vector for fish cells, and the procedure and method for constructing an expression vector that can be used in the present invention are commonly used in the field of genetic engineering. Can be used.

  Examples of the control sequence that can be used in the present invention include a constitutive promoter, an inducible or regulatory promoter, a tissue-specific promoter, or a promoter derived from a gene of an expressed antigen. As long as it can be expressed in, it is not particularly limited thereto. Examples of the constitutive promoter include a promoter sequence derived from cytomegalovirus (CMV), or rous sarcoma virus (RSV), simian virus-40 (SV-40), muscle β-actin promoter, or herpes simplex virus (HSV). Examples include strong promoters. Examples of the tissue-specific promoter include a thymidine kinase promoter. Examples of inducible or regulatable promoters include growth hormone regulatable promoters, promoters under the control of the lac operon sequence, or zinc inducible metallothionein promoters. The transcriptional regulatory sequence can be operably linked to a nucleotide sequence encoding an immunogenic polypeptide (ie, so that expression of the nucleotide sequence can be regulated).

  The control sequence can include an expression control sequence including a promoter (eg, the inducible or constitutive promoter) DNA sequence, optionally further comprising an enhancer element, transcription or polyadenylation signal [eg, simian virus-40. One or more copies of an intron sequence for splicing (derived from (SV-40) or bovine growth hormone), or an immunostimulatory DNA sequence known as a CpG motif can be included.

  In addition, the expression vector may be selected, for example, from a bacterial replication origin sequence, or an antibiotic resistance (eg, kanamycin) gene or a non-antibiotic resistance gene (eg, β-galactosidase gene) for selection. Sex markers can be included.

  The fusion polypeptide of the present invention comprises a fusion polypeptide in which the polypeptide of the present invention is bound to a marker polypeptide and / or a peptide tag. Examples of the marker polypeptide include GFP, luciferase protein, and LacZ protein. Examples of the peptide tag include Myc tag, His tag, FLAG tag, and GST tag. The fusion peptide of the present invention can be prepared by using known genetic engineering using the polypeptide of the present invention, a marker polypeptide, or a peptide tag.

  The antibody that specifically binds to the polypeptide of the present invention is not particularly limited as long as it is an antibody that specifically binds to the polypeptide of the present invention, but the amino acid sequence represented by amino acid numbers 833 to 1107 in SEQ ID NO: 2, It is preferably an antibody that specifically binds to an amino acid sequence represented by amino acid numbers 1118 to 1395 or a polypeptide comprising the amino acid sequence represented by amino acid numbers 1407 to 1697. Specific examples of the antibodies of the present invention include immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and the like. Can be prepared by a conventional method using an antibody as an antigen. Among them, a monoclonal antibody is more preferable in terms of its specificity. Such an antibody that specifically binds to the polypeptide of the present invention, such as a monoclonal antibody, is useful for, for example, separating and quantifying the polypeptide of the present invention and clarifying the molecular mechanism of the polypeptide of the present invention.

  The antibody against the polypeptide of the present invention can be obtained by administering a fragment containing the polypeptide or an epitope thereof or a cell expressing the polypeptide on the membrane surface to an animal (preferably other than human) using a conventional protocol. For example, for the preparation of monoclonal antibodies, the hybridoma method (Nature 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (Immunology Today 4), which results in antibodies produced by continuous cell line cultures. , 72, 1983) and EBV-hybridoma method (Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).

  Examples of the monoclonal antibody of the present invention include fluorescent substances such as FITC (fluorescein isocyanate) or tetramethylrhodamine isocyanate, radioisotopes such as 125I, 32P, 14C, 35S or 3H, alkaline phosphatase, peroxidase, By using a fusion polypeptide in which a fluorescent protein such as green fluorescent protein (GFP) is fused with an antibody labeled with an enzyme such as β-galactosidase or phycoerythrin, or the antibody of the present invention, Functional analysis can be performed. Examples of the immunological measurement method include RIA method, ELISA method, fluorescent antibody method, plaque method, spot method, hemagglutination method, and octalony method.

The method for identifying immature germ cells derived from fish of the present invention is not particularly limited as long as it is a method using the antibody of the present invention, but a method having the following steps (a) and (b) is preferably exemplified. be able to.
Step (a): contacting the labeled antibody of the present invention with a sample;
Step (b): confirming whether the antibody specifically binds to the sample;

  As the antibody label in the above step (a), the aforementioned fluorescent substances and enzymes can be used. The contact between the antibody and the sample in the step (a) can be performed in an appropriate solvent. Whether or not the antibody and the sample are specifically bound in the step (b) can be confirmed by a known immunoassay method such as ELISA or RIA.

  As a result of the step (b), the sample specifically bound to the antibody or the substance in the sample can be identified as a fish-derived immature germ cell, while the antibody did not bind specifically. The sample or material in the sample can be identified as not being immature germ cells from fish.

As long as the method for separating immature germ cells derived from fish of the present invention is a method for separating immature germ cells derived from fish from a sample containing immature germ cells derived from fish using the antibody of the present invention. Although it does not restrict | limit in particular, The method which has the following processes (A) and (B) can be illustrated suitably.
Step (A): contacting the sample containing immature germ cells derived from fish with the labeled antibody of the present invention;
Step (B) separating immature germ cells derived from fish to which the antibody specifically binds;

The sample used in the above step (A) is not particularly limited as long as it is a sample containing immature fish cells of fish, but a sample containing reproductive ridges of fish embryos and fish gonads and testis cells is preferably used. A sample obtained by dissociating the above-mentioned genital ridges, gonads and testes until they are separated into cells by proteolytic enzymes and dispersing them in a suitable solvent can be more preferably exemplified.
In addition, the aforementioned fluorescent substances and enzymes can be used for labeling the antibody in the step (A). Furthermore, the contact between the antibody and the sample in the step (A) can be performed in an appropriate solvent. Separation in the step (B) is performed by a method using a cell beader or the like, a method using a secondary antibody recognizing the antibody of the present invention labeled with magnetic beads (magnetic bead labeled secondary antibody), and the like. Only immature germ cells bound with the antibody of the present invention can be easily separated. The use of a magnetic bead-labeled secondary antibody is preferable in that the separation in the step (B) can be performed quickly and easily by using a magnet.

  In the method for inducing differentiation of isolated immature germ cells of the present invention, immature germ cells (isolated immature germ cells) derived from the donor fish separated by the above-described separation method of the present invention are transplanted into the early embryos of the recipient fish. Made up of. Transplantation of the isolated immature germ cells into the early embryo of the recipient fish can be performed by transplanting the recipient fish into the peritoneal lining of the peritoneal mesentery at an early developmental stage. When the isolated immature germ cells are transplanted into the peritoneal cavity of a hatched embryo of a recipient fish, for example, by a method such as a microinjection method according to the differentiation induction method of the present invention, the transplanted immature germ cells are spontaneously reciped. It moves into the gonads of ent fish, where it can induce differentiation into oocytes or spermatocytes, and further induce differentiation into eggs or sperm. As the isolated immature germ cells, immature germ cells isolated from embryos before and after hatching, preferably primordial germ cells and spermatogonia can be used, and as an early embryo of the recipient fish to be transplanted It is preferable to use recipient fish in the developmental stage before and after hatching. In addition, when a fish whose chromosome is triploid is used as a recipient fish, eggs and / or sperm derived from the recipient fish are hardly formed, and in eggs and / or sperm derived from the donor fish, particularly in females. Most preferred is that eggs derived from donors are specifically formed.

  Recipient fish with triploid chromosomes can be easily prepared by the method described in JP-A-2006-101845. Specifically, artificial insemination is performed in a normal manner using eggs from a diploid fish female parent and sperm from a normal diploid fish male parent, and under flowing water at 10 ° C. In addition to the method of starting development and immersing in warm water at 28 ° C. after 15 minutes after fertilization and then becoming normal eggs with flowing water at 10 ° C., eggs from normal females of diploid fish After normal insemination using spermatozoa from diploid fish male parents, the egg chromosomes were triploidized by blocking the second polar body by pressure treatment. A method of producing triploid juveniles through fertilized eggs, or letting sperm inactivated by ultraviolet rays or γ rays to occur on the eggs and generating females, and the second polar body release inhibition method (development process) In this method, the second polar body is prevented from being released by pressure stimulation or temperature stimulation to make a diploid) or first Splitting method (When the second polar body is released at the stage of development and the development begins only in the female pronucleus, a physicochemical stimulus is applied, and the first cleavage is blocked to produce a diploid. And the method of doubling chromosomes by a male hormone (testosterone) to cause sex change and maleification, and then mating this pseudomale with a normal female. . As triploid recipient fish, females in which almost all donor-derived eggs are specifically formed are preferred.

  Moreover, although the same kind may be sufficient as the kind of recipient fish and donor fish in the differentiation-inducing method of this invention, a different kind of fish can also be used. Examples of the different species of fish include the same species of different species and different species of fish.For example, when the donor fish is a rainbow trout, the same species of different species of recipient fish are yamame trout, the different species of recipient fish are brown trout, The char can be specifically exemplified, and when the donor fish is bluefin tuna, as the recipient fish of the same genus and different species, bigeye tuna, albacore tuna, yellowfin tuna, bigeye tuna and the like are specifically exemplified as the different species recipient fish In the case where the donor fish is yellowtail or amberjack, examples of the recipient fish of different genera include horse mackerel and nibs. In addition, when the donor fish are groupers or eels, it is possible to use dissimilar fish that are easily reared. When heterogeneous fish are used, for example, immature germ cells derived from donor fish use immature germ cells of fish that are relatively expensive and labor-intensive to grow up to adult fish (for example, larger fish than recipient fish). When using fish that are relatively low cost and low labor to grow up to adult fish (for example, smaller fish than donor fish), the recipient fish eggs are grown by growing the recipient fish after transplantation. It is possible to induce differentiation of sperm and sperm at a relatively low cost and with low labor, for example, using a relatively small water tank. Examples of preferred combinations of donor fish and recipient fish to enjoy this merit include a combination of bluefin tuna (donor) and chub mackerel (recipient), and a combination of rainbow trout (donor) and yamame trout (recipient). it can.

  The method for proliferating fish of the present invention comprises forming eggs and / or sperm derived from donor fish by the method for inducing differentiation of isolated immature germ cells of the present invention. More specifically, for example, donor-derived eggs and sperm are collected from recipient fish after transplantation, fertilized eggs are produced using these eggs and sperm, and hatched to obtain donor fry, This can be bred to create a new individual of the donor fish and to grow the donor fish in large quantities.

Other specific examples of the use of the differentiation induction method of the present invention for fish propagation methods include the following.
(1) Use for preservation of genetic resources of fish: If immature germ cells of rare and endangered species are cryopreserved and transplanted to recipients of related species that are easy to breed when needed, these recipes The ents can produce eggs and sperm from rare or endangered species (sometimes already extinct). In addition, when various useful strains and varieties are produced by techniques such as gene transfer in the future, it is possible to maintain these without the need for breeding, and transplantation to recipient embryos when necessary. It becomes possible to change to an individual.
(2) Use of isolated immature germ cells for molecular biological and biochemical analysis: By extracting and analyzing proteins and nucleic acids from separated immature germ cells, Protein identification becomes possible. In particular, it is useful as an analysis system for stem cells, which is attracting attention in regenerative medicine.
(3) Use for fish breeding (breeding improvement): after immature germ cells that have been isolated are genetically modified in vitro, transplanted into recipient individuals, differentiated into eggs and sperm, and fertilized Thus, an individual with the desired genetic modification can be produced efficiently. For genetic modification of immature germ cells, as described above, known gene introduction methods and gene modification methods already used in fish can be used. Examples of the method include a microinjection method and an electroporation method that are widely used as a method for gene transfer into fish.
(4) Use for analysis of gene function in fish: If immature germ cells can be cultured in vitro, after targeting the cells to the gene, By converting into an individual, for example, a knockout fish in which the function of the gene is destroyed or a knock-in fish in which the sequence of the gene is modified can be produced. That is, it can be used as a substitute for embryonic stem cells (ES cells) used in mice, and this makes it possible to clarify the role of a novel gene whose function is unknown.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Search for polypeptides specifically expressed in fish immature germ cells]
The rainbow trout introduced by the present inventors in Japanese Patent Application Laid-Open No. 2003-235558 ([0040]) into which a GFP gene controlled by the transcriptional regulatory region of the vasa gene is introduced (hereinafter referred to as “vasa-GFP gene introduced rainbow trout”). ), 1 × 10 ⁷ type A spermatogonia were isolated by flow cytometry using GFP fluorescence as an index. MRNA was extracted from the isolated type A spermatogonia, and a cDNA library was prepared according to a conventional method. Subsequently, 5206 clones were randomly selected from the cDNAs contained in this library, and the 5 ′ terminal nucleotide sequence was determined. Furthermore, using a membrane protein prediction program in silico, 82 clones having characteristics as membrane proteins were identified from these 5206 clones. Although these 82 clones are clearly expressed in type A spermatogonia, their expression is specific to type A spermatogonia (immature germ cells) or not germ cells It was unknown whether it was also expressed in gonadal somatic cells. Therefore, in order to identify clones that are specifically expressed in immature germ cells among the obtained 82 clones, primers for each clone were designed and RT-PCR analysis was performed according to a conventional method. In addition, as template cDNA in this RT-PCR, cDNA derived from the above-mentioned type A spermatogonia isolated by flow cytometry and cDNA derived from somatic cells of the gonad were used. As a result, 20 clones out of 82 clones were found to be expressed specifically or at a particularly high level in immature germ cells.
The expression level of a polypeptide that is specifically expressed in immature fish germ cells is advantageous when the polypeptide is used as a target for the fractionation of fish immature germ cells using an antibody. desired. Here, since the above RT-PCR has extremely high sensitivity, it cannot be said to be optimal for screening those 20 clones having higher expression levels. Therefore, using each of these 20 clones as a probe, in situ hybridization was performed on a testis sample of a 3-month-old vasa-GFP transgenic rainbow trout according to a conventional method, and analysis of the signal intensity was attempted. did. As a result, 2 clones (clone # 4017 and # 5206) out of 20 clones showed a signal specific to type A spermatogonia of the 3-month-old testis and a stronger signal (FIG. 1). Therefore, it was decided to perform further detailed expression analysis using clone # 4017 having a stronger signal among these two clones.

[Expression analysis of clone # 4017]
When an antibody that specifically binds to a polypeptide that is specifically expressed in immature germ cells is prepared, in order to increase the versatility of the antibody, the polypeptide was used for the above in situ hybridization and the like 3 It is required to specifically recognize immature germ cells such as primordial germ cells and spermatogonia in not only A-type spermatogonia of the month but also in all stages of the gonad. Therefore, instead of the sample used in the above in situ hybridization, a testis sample of a rainbow trout early embryo introduced with vasa-GFP gene that retains primordial germ cells 35 days after fertilization (FIG. 2), a vasa with an immature testis -Testis sample of GFP transgenic rainbow trout (12 months of age) (Fig. 3), vasa-GFP transgenic rainbow trout (18 months of age) testis sample (Fig. 4), vasa- with sperm testis The same in situ hybridization was performed using a testis sample of a GFP-transfected rainbow trout (FIG. 5) or a vasa-GFP-introduced rainbow trout ovary sample (FIG. 6) having an immature ovary. In addition, as a probe, the antisense probe (sequence number 3) with respect to clone # 4017 used in the said Example 1 was used. The results are shown in FIGS. As a result, in all samples, it was revealed that clone # 4017 was specifically expressed in primordial germ cells, spermatogonia, oocytes and early oocytes.
From the above experiments, it was shown that clone # 4017 is suitable as a marker molecule for immature germ cells such as primordial germ cells, spermatogonia, oocytes and early oocytes.

[Determination of the polynucleotide sequence of clone # 4017]
The experiment of Example 2 above showed that clone # 4017 was expressed specifically in immature germ cells and was suitable as its marker molecule. Therefore, the full length of clone # 4017 was isolated by the conventional RACE-PCR method, and its entire base sequence was determined. As a result, it was revealed that this clone was a cDNA of 6541 bp in total (SEQ ID NO: 1) containing a 5274 bp open coding region encoding a 199 kD polypeptide (SEQ ID NO: 2). When homology analysis was performed on the polypeptide of this clone, it was suggested that this clone may be a homologue of CD205. Therefore, in order to confirm whether CD205 homologues exist in fish other than rainbow trout, we investigated zebrafish, bluefin tuna, soda tsutsuo, chub mackerel, yellowtail, Japanese flounder, red sea bream, red sea bream, mahata, nibe. Was isolated. By analyzing these CD205 homologues and searching for regions conserved among the respective fish species from the deduced amino acid sequences, the amino acid sequence shown in FIG. 7 (amino acid numbers 1222-1260 in SEQ ID NO: 2 and the CD205 homologue in Comparison with the corresponding amino acid sequence) and in the region shown in FIG. 8A (comparison between the nucleotide sequence shown in nucleotide numbers 3742-3858 in SEQ ID NO: 1 and the corresponding nucleotide sequence in the CD205 homolog) in rainbow trout CD205 It was found that the homology was extremely high. In another region shown in FIG. 8B, the amino acid sequences of CD205 homologues of bluefin tuna, rainbow trout and zebrafish were found to be quite homologous.

[Preparation of antibody against polypeptide of clone # 4017]
According to the sequence predicted to be more antigenic within the conserved region of CD205 found in Example 3 (amino acid sequence shown by amino acid numbers 1222-1260 in SEQ ID NO: 2), two types derived from rainbow trout CD205 Peptide antigens were synthesized, and each type of antiserum was prepared by immunizing two (two in total) rabbits with each antigen. However, when Western blotting or immunohistochemical staining was performed using these four types of antisera, none of these antisera specifically recognized CD205. Therefore, next, the extracellular region of the polypeptide of SEQ ID NO: 2 (rainbow trout CD205) (FIG. 9) was divided into six regions (FIG. 10), and the corresponding cDNA sequences were expressed in the expression vector pCold I (TaKaRa). And the obtained vector was transformed into Escherichia coli BL21 Star, and the transformant was cultured to produce the aforementioned 6 regions of the polypeptide. Affinity purification using a His tag was attempted for the polypeptides in these 6 regions, and the obtained sample was confirmed by SDS-PAGE. As a result, 4 regions (region 2, region 4, region 5, region 6) of 6 regions were identified. Only the polypeptide could be purified (FIG. 11). These four regions of polypeptide were immunized to different rabbits, and antiserum was collected from each rabbit. Whole mount immunostaining was performed on immature testis of vasa-GFP gene-introduced rainbow trout immediately after sex differentiation using each of the obtained four types of antisera. FIG. 12 shows the results of whole mount immunostaining of one type of antiserum (hereinafter also referred to as “# 4017 antibody”) among the four types of antisera obtained. The upper row is an image detected with the # 4017 antibody, the middle row is an image of detecting GFP produced by germ cells, and the lower row is an image obtained by superimposing the upper and middle images. From this result, it was shown that # 4017 antibody specifically recognizes immature germ cells (spermatogonia). Similar images were obtained when the other three types of antisera were used.

[Reference Example] Induction of donor rainbow trout germ cell differentiation in yamame trout transplanted with rainbow trout immature germ cells, and proliferation of donor rainbow trout using the yamame trout immature reproduction from donor fish isolated from donor fish using GFP as an indicator By transplanting the cells into the early embryo of the recipient fish and growing the resulting recipient recipient fish, donor-derived germ cells can be obtained, or the donor fish-derived reproduction obtained from the transplant recipient fish In order to confirm whether the fry of donor fish can be obtained using cells, the following experiment was performed.

  First, as a recipient fish, a hatched fry of a triploid yamame trout (hereinafter also referred to as “triploid yamame” or “3n yamame”) is prepared according to the method described in JP-A-2006-101845. did. This method utilizes the fact that salmon fish such as yamame trout can easily and reliably prevent the release of the second polar body when a fertilized egg that has passed 15 minutes after fertilization is immersed in warm water at 27 ° C. for 15 minutes. Since triploid individuals become infertile, when transplanted with donor immature germ cells, recipients can produce only donor-derived eggs and sperm without mixing with recipient's own eggs and sperm. Preferred as fish.

  Transplanting type A spermatogonia of diploid vasa-GFP transgenic rainbow trout (donor) having fertility into the peritoneal cavity of a triploid yamame hatchling (recipient) obtained by the above method did. Thereafter, the recipient individual after transplantation was bred continuously, and testis was collected from a male individual (transplanted 3n yamame trout) immediately before the spawning season. The collected testicular tissue was sectioned and the tissue morphology was observed by hematoxylin and eosin staining. In addition, as a control, the testicular tissue of a male individual immediately before the spawning period of a triploid yamame trout that had not been transplanted with donor cells (non-transplanted 3n yamame trout) or a male individual immediately before the spawning period of a wild type diploid yamame trout (2n The testicular tissue of yamame trout was also observed. These observation results are shown in FIG. As can be seen from the results of FIG. 13, the non-transplanted 3n yamame had only spermatogonia in the somatic division phase, whereas immature germ cells of diploid donor rainbow trout (type A spermatozoa In the transplanted 3n yamame trout transplanted with the cells), the testicular tissue filled with mature sperm could be confirmed as in the wild type 2n yamame trout.

  When the above transplanted 3n yamame trout was further bred, a part of male individuals matured within one year after transplantation, and DNA was extracted from the semen of 10 individuals. PCR was performed using this DNA and a GFP primer specific for donor rainbow trout cells. As a result of electrophoresis of the obtained PCR products (“Serious” of # 1 to # 10) on an agarose gel, amplification of the GFP gene fragment was observed in all of these 10 individuals (# 1 to # 10) (FIG. 14). ). In addition, instead of these 10 semen-derived DNAs, the same PCR was performed using somatic cell-derived DNA, and the obtained PCR products ("body" of # 1 to # 10) were similarly electrophoresed. In the case, amplification of the GFP gene fragment was not observed. Furthermore, donor rainbow trout (vasa-GFP transgenic rainbow trout) (positive control individual), wild type yamame trout (negative control individual) and wild type rainbow trout (negative control individual) are similarly used using DNA extracted from semen and somatic cells, respectively. As a result of PCR analysis, GFP gene expression was confirmed in semen and somatic cells of donor rainbow trout, whereas in the case of wild yamame trout and wild rainbow trout, expression of GFP gene from both semen and somatic cells. Was not confirmed. Further, β-actin was used as an internal standard.

Next, in order to examine whether semen obtained from mature transplanted 3n yamame trout actually functions as donor-derived semen, the following experiment was conducted.
When the semen collected from the above-mentioned mature transplanted 3n yamame trout was fertilized into an unfertilized egg collected from a wild-type rainbow trout, a rainbow trout fry (hereinafter referred to as “rainbow trout F1 fry”) hatched from the obtained egg. . Moreover, the timing of this hatching was exactly the same as the case of fertilized eggs derived from wild-type rainbow trout (32-33 days after fertilization). It has been confirmed that the fertilized eggs of hybrids produced by fertilization of rainbow trout eggs and yamame spermatozoa are greatly delayed in hatching, and the fry after hatching is completely annihilated by the start of feeding.
The above-mentioned rainbow trout F1 juvenile holds the GFP gene, which is a marker for donor rainbow trout, and it was confirmed that germ cells are labeled with green fluorescence. Furthermore, as a result of DNA identification by the known RAPD (Random Amplification DNA Fingerprinting) method, all of the 50 rainbow trout F1 juveniles examined showed a completely rainbow trout type DNA pattern, and no DNA fragment peculiar to yamame trout was observed at all. It was. FIG. 15 shows the results of the RAPD method for 20 individuals among 50 rainbow trout F1 juveniles. In addition, as a control, the results of rainbow trout, yamame trout, and a hybrid of rainbow trout and yamame trout (rainbow trout) are also shown.
From the above results, it was shown that semen obtained from mature transplanted 3n yamame trout actually functions as semen of rainbow trout as a donor.

Next, in order to confirm whether or not an egg obtained from a transplanted 3n yamame female actually functions as a donor rainbow trout egg, the following experiment was performed.
A 9-month-old transplanted 3n yamame trout (2N → 3N) female individual was dissected and observed, and a filamentous ovary holding only small oocytes typical of non-transplanted 3n yamame trout (3N) females. A colony composed of large oocytes that can be retained by wild-type 2n yamame (2N) yamame trout could be observed in the center of each of the above. Furthermore, these large oocytes emitted clear GFP fluorescence that is a marker for donor rainbow trout (FIG. 16). Therefore, as a result of continuous breeding of these female individuals, ovulation was observed in 5 out of 50 female parent fish. Therefore, the eggs obtained from these five female individuals were slaughtered with semen obtained from the above-mentioned transplanted 3n yamame male individuals. As a result, as shown in FIG. 16, all fertilized eggs were generated normally, and hatching was completed at exactly the same timing as normal rainbow trout. DNA analysis of the chromosomal genome of each hatched fry and the mitochondrial genome known to be maternally inherited proved that all fry individuals were completely rainbow trout. These larvae also grew normally thereafter.
From the above, it was shown that by using a male individual and a female individual of transplanted 3n yamame trout, it is possible to produce fry only of rainbow trout as a donor.

By using the polynucleotide, gene or polypeptide of the present invention as an indicator, immature germ cells derived from fish can be easily separated or identified.
In addition, if the present invention is used, only immature germ cells can be isolated from donor fish without using genetic recombination techniques such as GFP, so that they can be used as food fish or seedlings for release. Parent fish farming techniques can be realized with high efficiency.
In addition, in this surrogate parent fish culture technique, fish that are relatively expensive and labor-intensive to grow up to adult fish are donor fish, and fish that is relatively inexpensive and labor-intensive to grow up to adult fish are recipient fish. By transplanting immature germ cells derived from donor fish into recipient fish and growing them to adult fish, differentiation can be induced into donor fish germ cells at a relatively low cost and effort. By using cells, it is possible to grow donor fish with relatively low cost and low labor.

Claims (20)

The polynucleotide shown in the following (A), (B) or (C).
(A) a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 79 to 5193 in SEQ ID NO: 1:
(B) a nucleotide sequence represented by nucleotide numbers 79 to 5193 in SEQ ID NO: 1, comprising a nucleotide sequence of substitution, deletion, insertion, or addition of a number of nucleotides within the range of 1 to 20; and fish A polynucleotide encoding a polypeptide that is specifically expressed in immature germ cells of origin:
(C) a polypeptide having 90% or more identity with a polynucleotide comprising the nucleotide sequence represented by nucleotide numbers 79 to 5193 in SEQ ID NO: 1 and specifically expressed in immature germ cells derived from fish Encoding polynucleotide: A polynucleotide encoding a polypeptide represented by the following (A) or (B).
(A) A polypeptide consisting of the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2:
(B) The amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2, consisting of an amino acid sequence in which substitution, deletion, insertion, or addition of the number of amino acids in the range of 1-20 is made, and fish Polypeptides specifically expressed in immature germ cells of origin: A gene encoding the polypeptide comprising the polynucleotide of claim 1 or 2 and specifically expressed in immature germ cells derived from fish. A recombinant vector comprising the polynucleotide according to claim 1 or 2. The polypeptide shown in the following (A) or (B).
(A) a polypeptide comprising the amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2:
(B) The amino acid sequence represented by amino acid numbers 1-1705 in SEQ ID NO: 2, consisting of an amino acid sequence in which substitution, deletion, insertion, or addition of the number of amino acids in the range of 1-20 is made, and fish Polypeptides specifically expressed in immature germ cells of origin: A polypeptide encoded by the gene according to claim 3 and specifically expressed in immature germ cells derived from fish. A fusion polypeptide in which the polypeptide according to claim 5 is bound to a marker polypeptide and / or a peptide tag. An antibody that specifically binds to the polypeptide of claim 5. The amino acid sequence represented by amino acid numbers 833 to 1107 in SEQ ID NO: 2, the amino acid sequence represented by amino acid numbers 1118 to 1395, or the polypeptide comprising the amino acid sequence represented by amino acid numbers 1407 to 1697, which specifically binds. 9. The antibody according to 8. A method for identifying immature germ cells derived from fish using the antibody according to claim 8 or 9. Step (a): contacting the labeled antibody of claim 8 or 9 with a sample; and step (b): confirming whether the antibody specifically binds to the sample;
A method for identifying immature germ cells derived from fish, characterized by comprising: A method for separating immature germ cells derived from fish, comprising separating immature germ cells derived from fish from a sample containing immature germ cells derived from fish using the antibody according to claim 8 or 9. . Step (A): contacting a sample containing immature germ cells derived from fish with the labeled antibody according to claim 8 or 9; and step (B) derived from fish to which the antibody is specifically bound. Isolating immature germ cells of
A method for separating immature germ cells derived from fish, characterized by comprising: As a sample containing immature germ cells derived from fish, it is characterized by using a sample containing cells selected from cells of embryonic growth of fish hatched embryos, cells of fish gonads, and cells of fish testis The method for separating immature germ cells derived from fish according to claim 12 or 13. From the donor fish, the method of separation according to claim 12 to 14, separating the immature germ cells from donor fish, and the immature germ cells from isolated donor fish (the separation immature germ cells), A method for inducing differentiation of isolated immature germ cells, comprising a step of transplanting to an early embryo of a recipient fish. The differentiation induction method according to claim 15, wherein a fish different from the donor fish is used as the recipient fish. The differentiation induction method according to claim 15 or 16, wherein a fish having a triploid chromosome is used as the recipient fish. The differentiation induction according to any one of claims 15 to 17, wherein the transplantation of the recipient fish to the early embryo is a transplantation of the recipient fish in the developmental stage before and after hatching to the back side of the intraperitoneal mesentery. Method. The differentiation induction method according to any one of claims 15 to 18, wherein a primordial germ cell or a spermatogonia is used as the isolated immature germ cell. A method for growing fish, characterized in that eggs and / or sperm derived from donor fish are formed by the method for inducing differentiation of isolated immature germ cells according to any one of claims 15 to 19.

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