JPWO2017090722A1 - Cell membrane permeable peptide and fish feed using cell membrane permeable peptide - Google Patents

Abstract

It is an object of the present invention to control the maturation of fish by producing seeds in the aquaculture industry by allowing them to function even in a small amount even when administering a gonad stimulating substance to fish. By administering a composition comprising a fish gonad stimulating substance and a cell membrane permeable peptide, sexual maturation is induced by efficient absorption into a living body. [Selection figure] None

Description

  The present invention relates to a cell membrane permeable peptide and a feed using the cell membrane permeable peptide.

  Several attempts have been made so far to orally administer physiologically active substances in fish and allow them to function as they are in the body of fish. Patent Document 1 describes that a polypeptide exhibiting fish growth hormone-like action functions even when orally administered. Patent Document 2 describes that a gonad stimulating substance functions even in oral administration in fish.

WO98-42748 WO2014-148603

  In seed production in the aquaculture industry, it is important to artificially control the maturity of fish by gonad stimulating substances. However, when the technique of Patent Document 2 is to be used, a relatively large amount of gonad stimulating substance has to be used, which is economically problematic.

  It is an object of the present invention to control the maturation of fish in the production of seedlings in the aquaculture industry by allowing them to function in small amounts even when orally administering a gonad stimulating substance to fish.

  As a result of various studies to solve the above problems, the present inventors have found that the effect can be enhanced by using a partial sequence of a cell membrane permeable peptide together with a reproductive stimulating substance, and the present invention has been completed. It came to do.

The present invention transfers a gonad stimulating substance of fish orally or enterally administered into the blood from the digestive tract, and contains the gonad stimulating substance and a cell membrane-permeable peptide as components. The gist is to provide.

The present invention has the following configuration.
(1) A composition for transferring a gonad stimulating substance of fish that has been orally or enterally administered into the blood from the digestive tract, comprising the gonad stimulating substance and a cell membrane-permeable peptide as components. .
(2) The composition according to (1), wherein the gonad stimulating substance is GnRH or GnRHa.
(3) The composition according to (1) or (2), wherein the cell membrane-permeable peptide contains the peptides (a) to (f).
(A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1.
(B) A peptide comprising a sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence of (a).
(C) A peptide consisting of the amino acid sequence of SEQ ID NO: 2.
(D) A peptide comprising a sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence of (c).
(E) A peptide consisting of the amino acid sequence of SEQ ID NO: 3.
(F) A peptide comprising a sequence in which 1 or 2 amino acids are deleted, substituted or added in the amino acid sequence of (e).
(4) The substitution in (b), (d), or (f) is performed from the hydrophobic index of the third amino acid from the N-terminus to 2 from the N-terminus in the amino acid sequence of (a), (c), or (e). The composition according to (3), wherein the substitution is such that the value obtained by subtracting the hydrophobic index of the amino acid is zero or a negative value.
(5) In the amino acid sequence of (a), (c), or (e), the substitution in (b), (d), or (f) is from the second amino acid to the fifth amino residue from the N-terminus. The composition according to (3) or (4), wherein the substitution is such that the standard deviation of the hydrophobic index is 4.0 or less.
(6) The composition according to (1) to (5), wherein the fish is a fish-eating fish.
(7) The composition according to (6), wherein the fish-eating fish is a mackerel fish.
(8) The composition of (7), wherein the mackerel subfamily is a mackerel fish.
(9) The composition according to any one of (6) to (8), wherein the fish-eating fish is a fish species having an adult fish length of 30 cm or more.
(10) A feed comprising the composition of (1) to (9).
(11) A method for inducing egg laying in fish characterized by oral or enteral administration of the composition or feed of (1) to (10).
(12) The egg-laying induction method according to (11), wherein the water temperature is adjusted.
(13) The method for inducing egg laying according to (11) or (12), wherein the sunshine is adjusted.
(14) A method for inducing spawning of fish by orally or enterally administering a gonad stimulating substance of 5 mg or less per kg of fish body weight.
(15) The method for inducing oviposition according to (14), wherein a cell-permeable peptide is administered together with a gonad stimulating substance when the gonad stimulating substance is orally or enterally administered.

  By using the composition or feed of the present invention, or by the method of the present invention, the gonad stimulating substance can be made to function in a smaller amount than when orally administered alone to fish. Oviposition by fish maturation can be controlled in the aquaculture industry using the composition, feed or method of the present invention.

  In the present invention, egg-laying induction means promoting egg-laying in fish. Egg laying is carried out in steps of ovulation and egg release. Ovulation is a phenomenon in which an oocyte is formed in the female ovary of an adult fish and detaches from the follicular tissue of the ovary. Ovulation is a phenomenon that discharges it to the outside. Oviposition that occurs spontaneously with mating or fertilization is called egg laying. In fish, eggs accumulated in the ovaries may be released to the body by some stimulus, but this phenomenon is also seen when eggs are not prepared for spawning. In such a case, it is distinguished from egg laying that fertilizes to obtain hatched larvae. In general, the maturation of eggs is promoted by the synthesis of hormones in the body stimulated by changes in water temperature and changes in sunshine duration. Egg maturation can also be determined by collecting ovarian eggs from a female body and measuring the egg diameter or observing the nuclei of eggs treated with sera.

  In the present invention, the gonad stimulating substance has an effect of inducing spawning in fish, and includes, for example, reproductive hormones. Specifically, GnRH (Gonadotropin releasing hormone), luteinizing hormone LH (Luteinizing hormone), follicle stimulating hormone FSH (Follicle Stimulating Hormone), E2 (Estradiol9), DHP (17α, 20β-dihydroxy-4-) prognen-3-one), but not limited to these.

  In the present invention, as a reproductive hormone, a hypothalamic extract containing a high concentration thereof, a pituitary gland extract and a purified product thereof as well as those artificially synthesized by a chemical method can be used. Any method can be used for chemical synthesis as long as the target molecule can be synthesized. For example, in the case of peptide hormones, a peptide synthesizer can be used. Peptide hormone refers to a peptide that acts as a hormone. In order to use reproductive hormones, those chemically synthesized may be used, or those produced by bacterial cells or the like by genetic engineering techniques may be used. The genetic engineering technique refers to a technique for artificially manipulating genes, and refers to a technique for performing genetic manipulations on living organisms using techniques such as gene transfer and gene recombination.

  Moreover, you may use the microbial cell which contained the peptide hormone in the microbial cell as it is using the method of making a bacterium produce reproductive hormone. The bacteria to be used are not limited to these as long as they can produce peptide hormones in the cells by genetic engineering techniques, for example, Escherichia coli, Bacillus subtilis, lactic acid bacteria, yeast, actinomycetes, and cyanobacteria. When a method for producing cells is used, a culture, a culture solution, a cell collected after cultivation, a dried cell, a culture, or a culture solution obtained by drying the cell as long as peptide hormones are contained Any of a purified product obtained by purifying the product and a dried purified product obtained by drying the purified product may be used. It may be an alga body containing a reproductive hormone using a method of producing it in algae. In the case of such a method, the obtained culture, the culture solution, the algal bodies recovered after the culture, the dried algal bodies obtained by drying the algal bodies, the purified algal bodies obtained by purifying the culture or the culture liquid, Any dried alga body purified product obtained by drying the alga body purified product may be used. A cell containing a reproductive hormone may be used by a method of producing it in a cultured cell. Any cell can be used as long as it can produce the desired reproductive hormone, but cells derived from humans, mice, rats, guinea pigs, fish and insects are exemplified, but not limited thereto. In the case of such a method, any of the obtained cells, the culture solution, the cell purified product collected after the culture, and the dried cultured cell purified product obtained by drying the cell purified product may be used. In the present invention, as the reproductive hormone, one kind obtained by a specific origin and production method may be used alone, or two or more kinds having different origins and production methods may be combined.

  In the present invention, the peptide refers to a polymer of amino acid residues. All amino acid residues are preferably naturally occurring amino acid residues, but may include non-natural amino acid residues such as one or more amino acid analogs or amino acid mimetics. Also, all amino acid residues may be unnatural amino acid residues.

  In the present invention, amino acids refer to natural amino acids and synthetic amino acids, and amino acid analogs and amino acid mimetics that function in the same manner as natural amino acids. The amino acid may be either an L-amino acid or a D-amino acid. Natural amino acids refer to amino acids encoded by the genetic code and amino acids modified after translation in cells (eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). Amino acid analogs are compounds that have the same basic chemical structure as a natural amino acid (hydrogen, carboxy group, amino group, and α carbon bonded to the R group), but have a modified R group or a modified backbone (for example, Homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium). An amino acid mimetic refers to a compound having a structure different from that of a general amino acid but having a similar function. Amino acids are described herein in the generally known three-letter or one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. For each amino acid, the hydrophobicity index is used as an index of hydrophobicity. If the hydrophobicity index value has a second decimal place, it is rounded to the first decimal place. Specific amino acid hydrophobicity indices include arginine-4.5, lysine 3.9, aspartic acid-3.5, asparagine-3.5, glutamic acid-3.5, glutamine-3.5, histidine-3.2, proline-1.6, tyrosine-1.3, tryptophan-0.9 Serine-0.8, Threonine-0.7, Glycine-0.4, Alanine 1.8, Methionine 1.9, Cysteine 2.5, Phenylalanine 2.8, Leucine 3.8, Valine 4.2, Isoleucine 4.5 are used. (Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982))

Amino acids having basic side chains are composed of lysine, arginine and histidine, amino acids having acidic side chains are composed of aspartic acid and glutamic acid, and amino acids having uncharged polar side chains are glycine, asparagine, glutamine, serine, threonine and tyrosine. , Amino acids having a hydrophobic side chain, including cysteine, etc. include alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, etc., and amino acids having a branched side chain are composed of threonine, valine, leucine, isoleucine, and Amino acids having an aromatic side chain are tyrosine, tryptophan, phenylalanine, and histidine. It is known that substitution of these mutually similar amino acid residues within a peptide sequence can change the amino acid residues while retaining the properties of the original peptide. This is referred to as “conservative substitution” and is well known to those skilled in the art. The following eight groups are each known in the art as being conservative substitutions for each other. (Creighton, Proteins 1984).
1) Alanine (A), Glycine (G)
2) Aspartic acid (D), glutamic acid (E)
3) Asparagine (N), glutamine (Q)
4) Arginine (R), Lysine (K)
5) Isoleucine (I), leucine (L), methionine (M), valine (V);
6) Phenylalanine (F), tyrosine (Y), tryptophan (W);
7) serine (S), threonine (T); and 8) cysteine (C), methionine (M)

  When a peptide is modified by substitution, addition and / or insertion of an amino acid residue, as long as the modified peptide retains the properties of the original peptide, in addition to L-amino acids, D-amino acids, or amino acid analogs or amino acid mimetics Modifications may be made with non-natural amino acids such as the body. Introducing unnatural amino acids, such as D-amino acids, or amino acid analogs or amino acid mimetics, to increase the stability of the peptide in vivo is well known to those skilled in the art. If the modified peptide retains the properties of the original peptide, the peptide may contain D-amino acids and / or unnatural amino acid residues such as amino acid analogs or amino acid mimetics.

  Other substances may be linked to the peptide as long as the properties of the original peptide are retained. Other substances that can be linked to peptides include, but are not limited to, other peptides, lipids, sugars or sugar chains, acetyl groups, natural or synthetic polymers, and the like. Moreover, as long as the characteristics of the original peptide are maintained, modifications such as glycosylation, side chain oxidation, and phosphorylation may be performed on the peptide.

  The present invention can contain, as an active ingredient, a peptide in which the original peptide is modified by conservative substitution. As long as the modified peptide retains the properties of the original peptide, it includes non-conservative modifications. Here, the characteristic of a peptide refers to the expected physiological activity of the peptide. For example, when the peptide is GnRH, the type of modification and the number of amino acid residues to be modified are not limited as long as the modified peptide retains the egg-laying inducing activity of the original peptide.

  GnRH is a peptide hormone consisting of 10 amino acids. In addition to the amino acid sequence of SEQ ID NO: 4 which is a mammalian type, Thai type, salmon type, chicken type II and the like are known. In the present invention, any one may be used as long as it has physiological activity for stimulating the gonads. However, since it is generally used for promoting maturation of fish, it is preferable to use a mammal type. In addition, GnRH was replaced with D-alanine by replacing the sixth glycine of mammalian GnRH with D-type alanine, and the physiological activity was increased by replacing the 10th Gly with N-ethylamide. And modified GnRH such as those with both modifications. Any of them may be used as long as they have physiological activity, but it is particularly preferable to use GnRHa, which is a modified GnRH that has been modified with both modifications that are less susceptible to degradation from peptidases and have strong physiological activity.

  In the present invention, the cell membrane-permeable peptide refers to a peptide that passes through the cell membrane and moves into the cell when it coexists with the cell. The cell membrane permeable peptide may be used by being bound to a protein by a chemical method or a genetic engineering method, or may be used as a peptide. Examples include oligoarginine linked with arginine, peptide Tat derived from HIV-1 virus, penetratin derived from Drosophila, etc., but it is a peptide that has the property of translocating into the cell through the cell membrane when coexisting with the cell. There is no limitation to these.

In the present invention, penetratin means a peptide having the amino acid sequence of SEQ ID NO: 1 derived from Drosophila and having an activity of permeating a biological membrane by an energy-independent mechanism.
In the present invention, hydrophobic penetratin is a peptide having increased hydrophobicity by replacing one or two of the amino acid residues constituting penetratin with an amino acid residue having a higher hydrophobic index value. Say. Among the hydrophobic penetratins, N2 isoleucine-type penetratin refers to a peptide having the amino acid sequence of SEQ ID NO: 2 in which the second amino acid residue glutamine from the N-terminus of penetratin is replaced with isoleucine to increase the hydrophobicity. In the present invention, hydrophilic penetratin is a peptide having increased hydrophilicity by replacing one or two of the amino acid residues constituting penetratin with an amino acid residue having a lower hydrophobicity index. Say. Among the hydrophilic penetratins, N3,5 alanine-type penetratin is SEQ ID NO: 3 in which isoleucine at both the 3rd and 5th amino acid residues from the penetratin N-terminus is substituted with alanine to increase hydrophilicity. A peptide having an amino acid sequence.

  When penetratin or modified penetratin is used as the cell membrane permeable peptide, a peptide having the same amino acid sequence as penetratin or modified penetratin can be used, and in each amino acid sequence, 1 or 2 amino acids are deleted, substituted, or A peptide consisting of an added sequence can be used. The substitution is preferably a conservative substitution. In particular, in order to act on fish, the substitution is zero or negative in each amino acid sequence by subtracting the hydrophobic index of the third amino acid from the N-terminal from the hydrophobic index of the second amino acid from the N-terminal. It is preferable that the substitution is such that the standard deviation of the hydrophobicity index from the second amino acid to the fifth amino residue from the N-terminus is 4.0 or less in each amino acid sequence.

  Since the life cycle of fish is approximately 24 hours per day, the dose of gonad stimulating substance is basically 24 hours a day. The action of gonad stimulants is usually proportional to blood concentration, and blood volume is proportional to fish weight. Therefore, the dose of gonad stimulating substance is calculated as a numerical value per kg of fish weight (BW). In order to promote egg laying in fish according to the present invention, the dosage of gonad stimulating substance is preferably 0.01 mg / kg BW / day or more, and in order to obtain a good egg quality egg, 0.1 mg / kg BW / day or more is preferable, In order to induce egg laying more strongly, 1 mg / kg BW / day or more is particularly preferable. The upper limit is not particularly limited as long as the fish does not cause side effects, but for economic reasons, it is usually 100 mg / kg BW / day or less, and 10 mg / kg BW / day or less is particularly preferable.

  According to the present invention, it is possible to provide a method for inducing egg laying of fish by orally or enterally administering a gonad stimulating substance of 5 mg or less per kg of fish body weight. In order to bring the gonad stimulating substance to 5 mg or less per 1 kg of fish body weight, the cell membrane-permeable peptide of the present invention may be used. The cell membrane permeable peptide can be administered together with the gonad stimulating substance during oral or enteral administration of the gonad stimulating substance.

  When administering the gonad stimulating substance and the cell membrane permeable peptide according to the present invention, a predetermined dose may be administered once a day, or may be divided into several times a day. Since the fish meal cycle is once a day after sunrise or before sunset, or twice a day after sunrise and before sunset, it is preferably performed once or twice a day. If administration is performed for 1 day, egg-laying can be induced, but it may be performed continuously for 2 days. If performed for 3 consecutive days, so much egg laying is induced.

  For oral administration, the gonad stimulating substance and the cell membrane permeable peptide may be directly administered as an aqueous solution, or may be mixed with feed and administered. In the case of direct administration, water may be used as a solvent. However, depending on the substance to be administered, it is desirable to use a buffer because it is unstable in pH, and it is particularly preferable to use a phosphate buffer because it is harmless to a living body. Further, in order to prevent the administered substance from flowing out of the mouth, a thickener such as chitansan gum, carrageenan, guar gum and the like can be used at the same time, but guar gum is preferred for economic reasons. When peptide hormones are mixed with feed, it is preferable to mix them with feed having a low peptidase activity in order to prevent peptide degradation. The activity of peptidase in feed can be reduced by heat treatment, acid treatment, alkali treatment, drying treatment, pressure treatment and the like. In preparing solutions or feeds for administration, peptides are preferably prepared at the time of use because of their stability problems, but those stored at low temperatures can also be used. The storage temperature is preferably 10 ° C. or lower, more preferably −4 ° C. or lower, still more preferably −20 ° C. or lower, and most preferably −80 ° C. or lower. When a cryopreserved solution is used for administration, it is preferably administered immediately after dissolution in order to suppress degradation as much as possible.

  Oral administration may be performed directly from the mouth of the fish using a syringe. The tip of the syringe release part may be inserted directly into the mouth or esophagus using a syringe, but the cannula is connected to the syringe release part so that the tip reaches the fish esophagus or stomach. May be administered directly to the esophagus or stomach. It is preferable to insert a cannula connected to a syringe into the stomach for administration because a fixed amount can be reliably administered to fish. The material of the cannula may be anything as long as durability and flexibility are maintained, but polyethylene is preferable from the viewpoint of durability, flexibility and economy.

  When an oral administration is performed using a syringe, use of anesthesia makes it possible to reliably give the fish a dose. Anesthesia can be anything as long as the fish is temporarily sedated and then restored to normal. Any drug, needle, electric shock, carbon dioxide gas or the like can be used as long as it can temporarily sedate the fish. For example, 2-phenoxyethanol is preferably used for anesthesia using the drug. Prepare a fish tank containing 2-phenoloxyethanol 500ppm water or seawater, put the fish into it, and take it out of the tank as soon as it calms down. Return to an aquarium containing seawater or water immediately after administration to confirm recovery from anesthesia. In order not to burden the fish as much as possible, it is desirable that the time for removing the fish from the water is as short as possible, preferably within 30 seconds.

  Enteral administration may be performed directly from the anal part of the fish. A cannula or syringe connected to a syringe is intubated directly from the fish anal part and the administration is inserted directly into the fish intestine part. In this case, anesthesia may be used at the time of administration. The digestive tract is the passage of food from the mouth to the anus. This includes the oral cavity, esophagus, stomach, and intestinal tract. Anesthesia can be anything as long as the fish can be temporarily sedated, but drugs, needles, electricity, carbon dioxide, etc. can be used.

  When a gonad stimulating substance and a cell membrane permeable peptide are mixed with a feed and orally administered, the nutrition of the feed is provided without excess or deficiency. Preferably, a feed having a protein content of 5% by weight or more, more preferably 10% by weight or more, and particularly preferably 15% by weight or more may be given. Particularly, in the case of raw bait, it is preferable to give a bait having a protein content of 5% by weight or more, more preferably 10% by weight or more, and particularly preferably 15% by weight or more as a raw material. In addition, in the mixed feed, it is preferable to feed the whole feed with a protein content of 5% by weight or more, more preferably 15% by weight or more, particularly preferably 25% by weight or more, and most preferably 35% by weight or more. If the diet contains too little protein, orally administered gonad stimulating substances are susceptible to degradation by peptidases contained in the gastrointestinal tract, affecting the induction rate of spawning. Also, it causes poor growth and no egg-laying behavior is observed. On the other hand, when the protein content in the food is too high, the protein in the food induces peptidases in the digestive tract, and orally administered gonad stimulating substances are easily degraded, affecting the induction rate of egg laying. Moreover, calorie deficiency is relatively short, and egg-laying behavior is not seen. In order to avoid this, it is preferable to give a feed having a protein content of 90% by weight or less, preferably 80% by weight or less, more preferably 70% by weight or less, and particularly preferably 60% by weight or less. Fish growth may be controlled by administering both gonad stimulating substance and cell membrane permeable peptide mixed feed and non-mixed feed. In this case, it is preferable that each nutrient component such as protein, lipid, vitamin, etc. is given without excess or deficiency in the total of both feeds.

  In the present invention, the form of feed is not limited. For example, there are raw feeds that are used by cutting or chopping fish, moist pellets that are formed by mixing powdered feed containing raw foods and binders, and dry pellets that are solid feeds using dry ingredients. It is not limited as long as the fish has palatability and is recognized as food and taken into the body. The fish used for the feed includes sardines, horse mackerel, mackerel, squid, and krill, but is not limited to these as long as the fish taste can be obtained. Even if it does not contain fish as a raw material, it may be anything that can be recognized as food and taken into the body, and it may be a capsule or solid containing the composition to be administered. Capsules and solids are preferably those that disintegrate in the body. It may be in the form of a gel produced by heating vegetable or animal protein or starch. Examples of vegetable protein include soy protein, wheat protein, animal protein includes fish, chicken, pork, beef, mutton, gelatin, and starch includes wheat starch, corn starch, potato starch, and potato starch. However, the present invention is not limited to these. Moreover, as long as it has the property to gelatinize, the mixture with another substance may be sufficient.

  As a method of mixing the gonad stimulating substance and the cell membrane permeable peptide with the feed, the feed may be produced by mixing the feed with the feed raw material at the time of production. Further, a liquid containing a gonad stimulating substance and a cell membrane permeable peptide may be soaked in the feed or injected into the feed. In the case of a feed having a multilayer structure, it may be injected into the inner layer, or the feed may be produced by adding only to the raw material of the inner layer at the time of producing the feed. If the gonad stimulating substance and the cell membrane permeable peptide are contained only in the inner layer, the gonad stimulating substance can be reliably incorporated into the living body.

  The gonad stimulating substance and the cell membrane permeable peptide may be dissolved in the same solution and administered, or separate solutions may be prepared and administered. As long as the gonad stimulating substance and the cell membrane permeable peptide are simultaneously present in the same gastrointestinal tract, they may be administered separately or simultaneously. Moreover, when administering with a feed, a feed may be created so that it may be contained in the same feed, and a feed may be created so that it may be contained in a separate feed. When administered, as long as the gonad stimulating substance and the cell membrane permeable peptide are simultaneously present in the same gastrointestinal tract, they may be administered simultaneously or separately. It is necessary to administer the optimal amount of the gonad stimulating substance that can exert its effect, but for economic reasons it is desired to use as little as possible. For example, in the case of GnRHa, less than 5 mg per kg fish weight Is preferable, 3 mg or less is more preferable, and 2 mg or less is most preferable. It is necessary to administer a sufficient amount of the cell membrane permeable peptide to promote absorption of the gonad stimulant into the cell, but it is necessary to suppress the amount of the peptide used for economic reasons. For example, when penetratin is administered with a gonad stimulating substance, it is preferable to use 5 times the mass of the gonad stimulating substance, more preferably 4 times the most, and most preferably 3 times the mass.

  When performing egg-laying induction, it is preferable to adjust the growth temperature as appropriate. The growth temperature varies depending on the fish species, but egg-laying is not easily induced by stress near the upper or lower limit of the temperature range in which it can grow. For this reason, it is preferable to induce around the median temperature at which growth is possible. The water temperature is adjusted at the same time as oral administration of gonad stimulating substance and cell membrane-permeable peptide, or prior to oral administration of gonad-stimulating substance and cell membrane-permeable peptide, in the case of mackerel fish species such as sesame mackerel and chub mackerel. It is preferable to induce at a temperature of 21 to 21 ° C., 24 to 27 ° C. for mackerel fish species such as suma, bonito and tuna, and 18 to 22 ° C. for summer swordfish and marlin species such as swordfish. The water temperature is preferably adjusted 1 day to 200 days before the spawning induction, and more preferably 3 days to 90 days. The water temperature can be adjusted by using a heater, a cooler, or a combination thereof. In the adjustment, the temperature may be adjusted within a range of ± 2 ° C from the set temperature, preferably within a range of ± 1 ° C, and more preferably within a range of ± 0.5 ° C. The set temperature can be changed around the median temperature for growth, but in order to reduce environmental irritation to fish, breeding is continued at the same temperature during oral administration of gonad stimulating substances and cell membrane-permeable peptides. It is preferable to do.

  According to the present invention, egg-laying can be induced even when reared in an onshore water tank. The onshore water tank can be of any type such as a breeding water type or a flush type. By the present invention, examples of fish species that can induce spawning in captive land tanks include suma, sesame mackerel, bonito, skipjack, bluefin tuna, yellowfin and cocinaga.

  The fish of the fish-eating fish of the present invention refers to fish mainly containing fish, and is represented by mackerel fish. Fish-eating fish need to break down the protein of many fish that serve as food, and their digestion is regulated by enzyme activity, food gastrointestinal residence time, intestinal bacteria, and the like. Usually, even when a peptide is administered orally, it is difficult to predict a sufficient physiological activity of the peptide because it is digested and decomposed in the same manner as a protein in this system. However, in the present invention, when a peptide physiologically active substance was orally administered in the presence of a cell membrane permeable peptide, surprisingly, a higher effect was recognized as compared with the case where it was administered alone.

  As the fish belonging to the subfamily Sabaidae of the present invention, fishes belonging to the marsh family, the swordfish family, and the licorice family are known in addition to the mackerel fish, and examples thereof include marlin, swordfish, and prickly fish. Examples of the fish belonging to the mackerel family include tuna such as chub mackerel, sesame mackerel, guru bear, suma, sawara, hagatsuo, bonito, soda bonito, and bluefin tuna, Atlantic bluefin tuna, southern bluefin tuna, yellowfin, binnaga, bigeye, cochinaga, etc. For use in the present invention, mackerel sub-fishes with high fish-eating properties are preferred, and mackerel fishes with high migratory properties are more preferred. Examples of mackerel fish with high migratory activity include chub mackerel, sesame mackerel, skipjack, bluefin tuna, Atlantic bluefin tuna, and southern bluefin tuna.

  In the present invention, an adult fish means a fish that has grown reproductively. The body length is 25 cm or more for chub mackerel or sesame, 40 cm or more for suma, 42 cm or more for skipjack, and 115 cm or more for bluefin tuna. In the case of swordfish, it means an individual of 140cm or more. In terms of body weight, it is 300g or more for masaba and sesame, 1,400g or more for suma, 1,500g or more for skipjack, 30kg or more for bluefin tuna, 70kg or more for swordfish .

  In the present invention, the body length refers to the length from the tip of the head to the rear end of the lower tailbone (bone that supports the tail fin). For the caudal side, bend the caudal fin to the left and right, and measure the distance from the bending point to the tip of the head to obtain the body length.

  Egg collection may be performed by squeezing the abdomen from a mature female, or may be performed by opening the abdomen. Moreover, you may collect what spawned naturally. Sperm can be used for artificial insemination that is obtained by squeezing the abdomen from a mature male. Egg maturation can be confirmed by sampling from adult female fish that have been induced to lay eggs and observing under a microscope. In the case of natural egg laying, an egg collection net is installed in the drainage part, and the eggs laid using the net are collected. As long as the net can collect eggs, the size and material of the mesh may be anything, but if the mesh is too fine, drainage will be hindered, and if it is too coarse, the egg cannot be collected. Therefore, the mesh is 1 mm or less, preferably 800 μm or less, more preferably 500 μm or less.

  In the present invention, the water temperature is adjusted by heating and cooling the water temperature with a cooler, heater, boiler, sunshine, solar system, etc., but is not limited to this as long as the water temperature can be adjusted appropriately. . It also includes moving the ginger to an environment with an appropriate water temperature.

  In the present invention, the sunshine duration can be adjusted by raising the fish outdoors or indoors and adjusting the illuminance of the lamp with a timer or manually, or by shading with a sheet. Adjustment of daylight hours is repeated 24 hours a day and night, but at the time of induction, the daytime and nighttime ratios are adjusted to the daylight hours corresponding to the spawning season of each fish species and reared to an appropriate daytime and nighttime ratio. . The egg-laying season is, for example, from 5 to July for bluefin tuna and from 1 to April for sesame mackerel, so it is preferable to adjust to the sunshine hours in the area corresponding to each period.

  In the present invention, egg quality refers to the ability of an egg to fertilize and hatch. Egg quality can be evaluated by floating egg rate, fertilized egg rate and hatching rate. The floating egg rate, fertilized egg rate, and hatching rate are obtained as follows. Suspend the egg treated with sperm in 1L seawater, transfer 2ml of it to a 12-well plate and count the number of eggs. The total number of eggs is obtained by multiplying the average value measured three times by 500. Next, among the eggs in 2 ml used for measuring the total number of eggs, the number of eggs that floated on the surface of the water within 5 minutes is the number of floating eggs, and the eggs that have been confirmed to be broken by microscopic observation among the floating eggs Is the number of fertilized eggs, and the number of larvae hatched until 2 days later is the number of hatched larvae. Furthermore, the floating egg rate (= floating egg number / total egg number x 100), fertilized egg rate (= fertilized egg number / total egg number x 100), and hatching rate (= hatched larvae number / total egg number) Calculate to find. It can be evaluated that the higher the floating egg rate, fertilized egg rate, and hatching rate, the better the egg quality.

Particular embodiments of the present invention will now be described in greater detail through the following examples. The examples are intended to more fully support the description of selected embodiments of the invention and should not be construed as limiting the scope of the invention in any way.

Example 1 Induction of egg laying of sesame mackerel For sesame mackerel, individuals (555 ± 51 g) caught in Tateyama Bay, Chiba Prefecture were used. A total of 8 males and 4 males were housed in a 1-ton FRP water tank. GnRHa1.2 group administered 1.2 mg GnRHa per kg body weight of fish, GnRHa1.2 + P group administered with 1.2 mg GnRHa and fishnetine 4.6 mg per kg body weight, 1 kg of fish body weight A total of 4 sections were prepared: GnRHa1.2 + SP group administered with 1.2 mg of GnRHa and 4.6 mg of N2 isoleucine penetratin, and GnRHa6.0 group administered with 6.0 mg of GnRHa per kg of fish body weight.
During administration, 2 ml of phosphate buffered buffer (NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM, KH ₂ ) from the mouth of sesame mackerel anesthetized in 200 to 500 ppm of 2-phenoxyethanol (Wako Pure Chemicals) A solution in which guar gum (final concentration 1%) and various hormones were mixed in PO ₄ 1.76 mM) was injected with a syringe using a cannula.
In order to collect the eggs laid, a drain pipe adjusted to the height of the breeding water surface was provided on the wall surface of the aquarium, and drainage from the drain pipe was received by an egg collection net. The recovered eggs were suspended in 1 L seawater, 2 ml of which was transferred to a 12-well plate, and the number of eggs was counted. The number obtained by multiplying the average value measured three times by 500 was defined as the number of eggs laid. Egg collection and measurement were performed daily during the experiment. Next, among the eggs in 2 ml used for measuring the number of eggs laid, the number of eggs that floated on the surface of the water within 5 minutes was counted as the number of floating eggs, and the number of larvae hatched by 2 days later was counted as the number of hatched larvae. Table 1 shows the number of eggs laid, the number of floating eggs, and the number of hatching during the test period.

Example 2 GnRHa blood kinetics of sesame mackerel Experiments were conducted from June 15 to 18, 2014. The sesame mackerel used six individuals (429 ± 42.7 g) caught in Tateyama Bay, Chiba Prefecture, and was housed in a 1-ton FRP tank. GnRHa1.2 group administered 1.2 mg GnRHa / kg fish weight, GnRHa1.2 + P group administered 1.2 mg GnRHa and penetratin 4.6 mg / kg fish weight, 1 kg fish weight GnRHa1.2 + HP with 1.2 mg GnRHa and 4.6 mg N3,5 alanine penetratin, GnRHa1.2 + SP with 1.2 mg GnRHa and 4.6 mg N2 isoleucine penetratin per 1 kg fish weight, fish A total of 6 groups were prepared, GnRHa 6.0 group administered with 6.0 mg of GnRHa per kg body weight, and non-administered group administered nothing.

At the time of administration, guar gum (final concentration 1%) and various hormones are mixed in 2 ml of phosphate buffered buffer PBS (NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM, KH ₂ PO ₄ 1.76 mM) The syringe was filled and injected with a cannula from the mouth of sesame. Blood was collected after 0.5, 1, 2, and 3 hours after the injection, and the GnRHa concentration in the blood was measured using time-resolved fluorescence measurement (DELFIA). Blood was collected from the tail with a heparinized syringe and centrifuged at 16,000 × g for 10 minutes at 4 ° C. to obtain plasma. Contaminants were removed from the plasma using a reverse phase extraction column strata-X (Shimadzu GLC Co., Ltd.), and the product eluted in an 80% (v / v) acetonitrile aqueous solution was used for the measurement of GnRHa.

細胞膜はリン脂質2重膜により構成されているので、細胞膜を透過するためにはその分子の疎水性が重要であると考えられる。ペネトラチンよりも疎水性ペネトラチンの方が他の共存するタンパク質を細胞膜を通過させ細胞内に移行させる活性が高く、また親水性ペネトラチンはペネトラチンよりも活性が低いことが予想された。確かに疎水性ペネトラチンはペネトラチンよりも高い活性が確認されたが、驚くべきことに親水性ペネトラチンであっても活性は維持され、投与後3時間の共存させた生殖ホルモンの血中濃度にあっては親水性ペネトラチンの効果がペネトラチンの効果を上回る効果が見られた。N2イソロイシン型ペネトラチンおよびN3,5アラニン型ペネトラチンはいずれもペネトラチンから改変したものであるが、特に活性の増強に関係のあると考えられる部位は改変しているＮ末端部分である。Ｎ末端から2番目と3番目のアミノ酸の疎水性インデックスを比較すると、元のペネトラチンがGln-3.5、Ile4.5に対し、N2イソロイシン型ペネトラチンGln-3.5、Ala1.8、N3,5アラニン型ペネトラチンIle4.5、Ile4.5となり、その2番目と3番目の疎水性インデックスの差が持続的な効果に関係があると考えられる。またペネトラチンの活性に関係が強いと考えられるＮ端から2番目のアミノ酸から5番目のアミノ残基までの疎水性インデックスについて、標準偏差を計算したところ、ペネトラチンは4.1に対し、N3,5アラニン型ペネトラチン2.75、N2イソロイシン型ペネトラチン3.64となった。このことからＮ端から2番目のアミノ酸から5番目のアミノ残基までの疎水性インデックスの値の標準偏差が小さくなることが持続的な効果に影響すると考えられた。The measurement of GnRHa was carried out by the following procedure. Add 100 μl of 0.1 M sodium carbonate buffer (pH 9.3) in which GnRHa is dissolved at a concentration of 50 ng / ml to each well of Nunc fluoronunc plate C96 clear maxi soap (Thermo Fisher Scientific Co., Ltd.) at room temperature. Allowed to stand for 24 hours. Thereafter, 200 μl of PBST (NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 10 mM, KH ₂ PO ₄ 1.76 mM, 0.05% Tween 20) was added to each well and washed. After washing 3 times, add 200 μl of blocking buffer (BSA 0.1%, Na ₂ HPO ₄ 0.05 M, sucrose 3.0%, NaN ₃ 0.05%) and block at 37 ° C for 1 hour. went. After washing GnRHa immobilized and blocked plate 3 times with PBST, DELIFIA buffer (Tris 6.055 g / L, NaCl 6 g / L, BSA 1 g / L, NaN ₃ 0.5 g / L, Tween-40 0.1 50 μl of standard solution diluted with g / L, DTPA 7.867 mg / L, pH 7.75) and plasma-derived sample were added to each well. Subsequently, 50 μl of anti-GnRHa goat polyclonal antibody (Agrisera) diluted 40,000 times with DELFIA buffer was added to each well and reacted at 37 ° C. for 2 hours. After washing 3 times with PBST, 50 μl of europium-labeled antibody rabbit IgG goat polyclonal antibody (PerkinElmer) dissolved in DELFIA buffer at a concentration of 500 ng / ml was added as a secondary antibody to each well for 1 hour at 37 ° C. Reacted. After washing 3 times with PBST, 100 μl of enhancer reagent (PerkinElmer) was added to each well and shaken at room temperature for 30 minutes to amplify europium fluorescence intensity. The fluorescence intensity of each well was measured using a microplate reader Infinite 200F Pro (Tecan), and the GnRHa concentration in the sample was calculated from the obtained value.

Since the cell membrane is composed of a phospholipid bilayer membrane, the hydrophobicity of the molecule is thought to be important in order to permeate the cell membrane. It was predicted that hydrophobic penetratin is more active than penetratin in transferring other coexisting proteins through the cell membrane and moving into the cell, and hydrophilic penetratin is less active than penetratin. Certainly, hydrophobic penetratin was confirmed to have higher activity than penetratin, but surprisingly, even hydrophilic penetratin maintained its activity and was in the blood concentration of reproductive hormones coexisting for 3 hours after administration. The effect of hydrophilic penetratin exceeded that of penetratin. N2 isoleucine type penetratin and N3,5 alanine type penetratin are both modified from penetratin, but the site considered to be particularly related to the enhancement of activity is the modified N-terminal part. Comparing the hydrophobic index of the 2nd and 3rd amino acids from the N-terminal, the original penetratin is Gln-3.5, Ile4.5, N2 isoleucine penetratin Gln-3.5, Ala1.8, N3,5 alanine penetratin Ile4.5 and Ile4.5, and the difference between the second and third hydrophobicity indices is thought to be related to the sustained effect. The standard deviation of the hydrophobic index from the 2nd amino acid to the 5th amino acid residue from the N-terminus, which is thought to be strongly related to the activity of penetratin, was N3,5 alanine compared to 4.1 for penetratin. It became penetratin 2.75 and N2 isoleucine penetratin 3.64. From this, it was considered that the small standard deviation of the value of the hydrophobic index from the second amino acid to the fifth amino residue from the N-terminal affects the sustained effect.

Claims (15)

A composition for transferring an orally or enterally administered gonad stimulating substance of fish from the digestive tract into the blood, comprising the gonad stimulating substance and a cell membrane-permeable peptide as components. The composition of claim 1, wherein the gonad stimulating substance is GnRH or GnRHa. The composition according to claim 1 or 2, wherein the cell membrane permeable peptide contains the peptides (a) to (f).
(A) A peptide consisting of the amino acid sequence of SEQ ID NO: 1.
(B) A peptide comprising a sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence of (a).
(C) A peptide consisting of the amino acid sequence of SEQ ID NO: 2.
(D) A peptide comprising a sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence of (c).
(E) A peptide consisting of the amino acid sequence of SEQ ID NO: 3.
(F) A peptide comprising a sequence in which 1 or 2 amino acids are deleted, substituted or added in the amino acid sequence of (e). The substitution in (b) (d) or (f) is the second amino acid from the N-terminal to the hydrophobic index of the third amino acid from the N-terminal in the amino acid sequence of (a), (c) or (e) The composition according to claim 3, wherein the substitution is such that the value obtained by subtracting the hydrophobic index is zero or a negative value. The substitution in (b) (d) or (f) is a hydrophobic index from the second amino acid to the fifth amino residue from the N-terminus in the amino acid sequence of (a), (c) or (e). The composition according to claim 3 or 4, wherein the substitution is such that the standard deviation of is 4.0 or less. The composition according to any one of claims 1 to 5, wherein the fish is a fish-eating fish. 7. The composition of claim 6, wherein the fish-eating fish is a mackerel subfamily. 8. The composition of claim 7, wherein the mackerel subfamily is a mackerel fish. The composition according to any one of claims 6 to 8, wherein the fish-eating fish is a fish species having an adult fish length of 30 cm or more. A feed comprising the composition of claims 1-9. A method for inducing spawning of fish, characterized by oral or enteral administration of the composition or feed of claim 1. The egg-laying induction method according to claim 11, wherein the water temperature is adjusted. The egg-laying induction method according to claim 11 or 12, wherein the method adjusts sunshine. A method for inducing egg laying in fish by orally or enterally administering a gonad stimulating substance of 5 mg or less per kg of fish body weight. The method of inducing egg laying according to claim 14, wherein the cell-penetrating peptide is administered together with the gonad stimulating substance at the time of oral or enteral administration of the gonad stimulating substance.