Recombinate the preparation method and application of chorionic gonadotrophin rhCGTechnical field
The present invention relates to biological medicine and technical field of animal reproduction, specifically, being related to recombinating chorionic gonadotrophin rhCG'sPreparation method and application.
Background technology
Fish reproduction and residing ecological environment are closely related, under normal circumstances, ecological factor, such as nutrition, temperature, lightIt is the determinant of fish gonad development according to, flow etc..However under the conditions of artificial propagation, shortage rivers have comprehensive in pondEcological condition (flow velocity of such as water, the cataclysm of water level etc.) is closed, some chinese carp sexual glands cannot reach maturity, such as black carp parent population sexual gland oneAs be extremely difficult to for IV end of term；Even if the chinese carp of gonadal maturation can not voluntarily ovulate in pond, palpus manual injection hastens parturitionAgent can ovulate fertilization.The purpose of manual injection's ocyodinic is：On the one hand prevent certain sexual glands under artificial condition from voluntarily intoRipe fish (such as black carp) reach maturity with row's essence of laying eggs；On the other hand also promote the fish that can lay eggs smart concentration same period ovulationProduction essence is convenient for unified breeding management.
It hastens parturition in due course, reasonable employment ocyodinic is most important to fish reproduction.The upper common ocyodinic of production at present is mainThere are artificial synthetic analogues (LRH-A), fish brain hypophysis (PG), human chorionic gonadotrophin (chorionic gonadotrophin, hCG) and dioxone(DOM).Different types of parent population is very big to the sensitivity differences of different ocyodinics, needs selection quick to the parent population in practical applicationsSeveral ocyodinics are used cooperatively by the ocyodinic of sense, and effect is better than single ocyodinic.
HCG is a kind of glycoprotein hormones secreted by Human plactnta syncytiotrophoblast, including two non-covalent bonds of α and βWith reference to subunit.HCG promotes the formation reached maturity with corpus luteum of female egg mother cell, promotes the life of male creature spermInto.HCG is clinically usually used in assisted reproductive therapy in people doctor, and treatment is infertile.HCG is usually used in animal reproduction productionPig, ox, the estrus synchronization of sheep and superfecundation.The artificial repening that hCG is mainly used for fish in terms of fish reproduction hastens parturition, ovulatesProduction essence, is one of preferred ocyodinic of artificial propagation of fish.
The commercialization hCG mainly extraction purifications from urine of pregnant women at present, but large scale collection urine of pregnant women difficulty it is big, intoThis height, the hCG product batches differences purified are big, and there are the security risks that urine donor is brought.In addition hCG to light,The conditions such as temperature and acidity are extremely sensitive, if the urine of collection is extracted not in time, it is easy to lose biological activity, cause resourceWaste.Therefore, people have utilized technique for gene engineering exploitation novel human recombination hCG products, such as Merck Xue LannuoOvidrel (recombination physex parenteral solution), the injection chorionic gonadotrophin (beautiful pearl is currently urine source hCG) of beautiful pearl, recombinate hCGProduct quality is relatively uniform, purity is high, impurity is few, safety coefficient is big, gradually replaces people's urine source hCG products.
The common expression system of recombinant protein mainly has at present：Escherichia coli, yeast, insect cell and mammal are thinBorn of the same parents.Each expression system has respective advantage and deficiency, need to be according to the characteristics of destination protein and expression system, and it is suitable to selectExpression system.Escherichia expression system production cost is low, yield is high, expression flow is simple and quick, but is turned over due to a lack of proteinProcessing mechanism after translating, such as glycosylation, correct folding etc. are not suitable for expression complexity and most animals cell protein.Yeast tableThe advantages of having protokaryon and Higher eukaryotic expression system concurrently up to system, the albumen of harvest have certain post translational processing ability, butIt is that there are still deficiencies, such as easily forms protein multimers and excessive glycosylation, therefore be more suitable for expressing simple eucaryote eggIn vain.Insect expression system can express higher eucaryote albumen, and albumen can be carried out correctly to fold and modify, but protein contentIt is low, it is difficult to form seed lot.Mammalian cell expression system albumen can be carried out it is correct fold and complicated glycosylation modified,It is suitble to the complicated higher eucaryote albumen of expression；But expression flow is complicated, the period is long, and cost is higher.In practical applications, shouldAccording to different expression requirements, suitable expression system and strategy are selected.HCG is a kind of by Human plactnta syncytiotrophoblast pointThe glycoprotein hormones secreted, the subunit combined including two non-covalent bonds of α and β.Theoretically with mammalian cell come Expression productReproductive hormone albumen, space structure and the closer natural products of glycosylation, it is ensured that its physiological activity.
The dynamic hCG for protecting sector application is still mainly extracted from pregnant woman urine at present, there is no the related report of recombination hCG productsRoad.
Invention content
The object of the present invention is to provide the preparation methods of recombination chorionic gonadotrophin rhCG.
It is a further object of the present invention to provide the applications of recombination chorionic gonadotrophin rhCG.
Another object of the present invention is to provide a kind of method that fish are hastened parturition.
The design of the present invention is as follows：Recombination hCG products, and quality phase can be obtained on a large scale using gene recombination technologyTo it is uniform, purity is high, impurity is few, safety coefficient is big.Mammalian cell expression system, can compared with other expression systemsIt obtains in terms of molecular structure, physicochemical property and biological function closest to natural higher organism protein molecular.
In order to realize the object of the invention, the present invention provides the preparation method of recombination chorionic gonadotrophin rhCG, utilizes mammalEukaryotic expression system expression recombination chorionic gonadotrophin rhCG；The recombination chorionic gonadotrophin rhCG includes α and β subunits, and the two passes through modelDe Huali connections.
Wherein, the α subunits：
I) by SEQ ID NO:The albumen that amino acid sequence shown in 1 is formed；Or
ii)SEQ ID NO:Amino acid sequence shown in 1 is substituted, lacks and ors add one or several amino acid and sameEtc. functions the albumen as derived from i)；Or
Iii) with SEQ ID NO:Amino acid sequence homology shown in 1 is more than 90%, and the ammonia with equal functionThe albumen that base acid sequence is formed.
The β subunits：
I) by SEQ ID NO:The albumen that amino acid sequence shown in 3 is formed；Or
ii)SEQ ID NO:Amino acid sequence shown in 3 is substituted, lacks and ors add one or several amino acid and sameEtc. functions the albumen as derived from i)；Or
Iii) with SEQ ID NO:Amino acid sequence homology shown in 3 is more than 90%, and the ammonia with equal functionThe albumen that base acid sequence is formed.
Aforementioned method according to the gene order of NCBI hCG α and the β subunits announced, is had a preference for according to mammalian codonsProperty carry out gene order optimization, the gene order of the α and β subunits after optimization is implemented in different expression cassettes, then connects respectivelyOnto same expression vector, host cell is converted, and expressed in host cell, isolate and purify target protein；Alternatively,
The gene order of α and β subunits after optimization is building up on expression vector respectively, gained recombinant vector cotransformation placeChief cell, and expressed in host cell, isolate and purify target protein.
In the present invention, the gene order of the α and β subunits after optimization is respectively such as SEQ ID NO:Shown in 2 and 4.
Aforementioned method, the expression vector are selected from pCG, pcDNA3.1, pMS2-GFP, pCMV5, preferably pcDNA3.1.
Aforementioned method, the host cell CHO or 293 cells.Wherein, Chinese hamster ovary cell (CHO) is by the U.S.FDA is confirmed as safe genetic engineering recipient cell, is one of large-scale production most common expression cell of Medicines, preferablyChinese hamster ovary celI.Chinese hamster ovary celI genetic background understands that physiological metabolism is stablized, and expression is relatively high, shearing force is resistant to, convenient for advising greatlyMould culture is one of most common expression system of current recombinant protein.
The recombinant vector of above-mentioned hCG α and the β subunits containing recombination is gone in host expression system, mammalian expression systemsAlbumen can be instructed correctly to fold, complicated N-type glycosylation and accurately a variety of post translational processing work(such as O-shaped glycosylation are providedCan, it can obtain in terms of molecular structure, physicochemical property and biological function closest to natural protein molecular.
The present invention also provides prepare according to the method described above recombination chorionic gonadotrophin rhCG (and its derived protein or by modificationAlbumen) following any application：
1) promote the application in animal reproduction；
2) application in the medicine preparation for the treatment of animals' reproduction relevant disease；
3) application in ocyodinic is prepared.
By the albumen of modification, combined including the rhCG through glycosylation, Pegylation, acetylation or with BSA,It belongs to the scope of protection of the present invention.
By the albumen of transformation, do not change the rhCG protein actives including what the rhCG and other protein fusions were formedFusion protein, all belong to the scope of protection of the present invention.
Aforementioned application, the animal are mammal or fish；Wherein, the mammal is the economy such as pig, ox, sheepAnimal, family's pack animal or dog.
In the present invention, the fish include fresh water or marine fishes, preferably freshwater fish, including four large Chinese carps (black carp, grassFish, silver carp and bighead) and the economic fish such as Cypriniformes, Perciformes, Silurformes, salmon shape mesh, Gadiformes, Clupeiformes, Gadiformes, moreIt is preferred that Cypriniformes, Perciformes, Silurformes economic fish.For example, the fresh-water fishes such as silver carp, snakehead, Pelteobagrus fulvidraco, black carp.
The promotion animal reproduction refers to promote oocyte maturation, promotion estrus synchronization, superfecundation, induced breedingDeng.It hastens parturition especially suitable for fish.
The present invention also provides a kind of fish ocyodinic, at least contain the rhCG in active constituent.In addition, also it may includeLRH-A (such as LRH-A2、LRH-A3), fish brain hypophysis (PG), the ingredients such as dioxone (DOM).
The present invention also provides the methods that fish are hastened parturition, and are hastened parturition by injecting above-mentioned ocyodinic to fish.
Aforementioned method, the injection dosage of rhCG is 200-2000IU/kg weight.For the injection of freshwater fish rhCGIt measures as 200-2000IU/kg weight.
Preferably, it is hastened parturition using two needle injections, the ocyodinic injected for the first time is LRH-A, and injection dosage is 1-2 μ g/kgWeight；It injects for the first time and carries out second after 10-12h and inject, the ocyodinic of second of injection is LRH-A2-10 μ g/kg weight,DOM 5-6 μ g/kg weight and rhCG 400-600IU/kg weight.
For the silver carp parent population of 5kg or so, induced spawning method is as follows：LRH-A is injected for the first time2, dosage is 1 μ g/kg weight；It is firstSecond is carried out after secondary injection 12h to inject, and injects LRH-A for the second time25 μ g/kg weight, 5 μ g/kg weight of DOM and rhCG500IU/kg weight.
For the snakehead parent population of 1.5kg or so, induced spawning method is as follows：LRH-A is injected for the first time3, dosage is 2 μ g/kg weight；Second of injection is carried out after injecting 12h for the first time, injects LRH-A for the second time32 μ g/kg weight, 5 μ g/kg weight of DOM and rhCG400IU/kg weight.
For the Pelteobagrus fulvidraco parent population of raun 100g or so, milter 200g or so, induced spawning method is as follows：LRH- is injected for the first timeA3, dosage is 1 μ g/kg weight；Second of injection is carried out after injecting 10h for the first time, injects LRH-A for the second time310 μ g/kg weight,5 μ g/kg weight of DOM and rhCG 600IU/kg weight.
By above-mentioned technical proposal, the present invention at least has following advantages and advantageous effect：
(1) fish provided by the invention recombination hCG and its derived protein or the albumen by modification is thin in mammalHigh efficient expression in born of the same parents-Chinese hamster ovary celI, quality is relatively uniform, and rhCG purity of protein after purification is up to 93%, and purity is high, safety coefficientGreatly；It is active high, than living up to 14300IU/mg.
(2) fish provided by the invention recombination hCG and its derived protein or the albumen by modification, can effectively improveSpawning effect to the silver carp for urinating source hCG sensitivities, with reference to ocyodinic LRH-A2With DOM use, the spawning rate of silver carp parent population and bySmart rate is up to 95% and 96.7%, higher than the 80% and 85.6% of urine source hCG products.
(3) fish provided by the invention recombination hCG and its derived protein or the albumen by modification, to urine source hCG notSensitive perciform fishes have good spawning effect, with reference to ocyodinic LRH-A3It is used with DOM, improves the oviposition of snakehead parent populationRate and rate of fertilization, reduce Effect time, and spawning rate and rate of fertilization (being respectively 95% and 96.3%) are above urine source hCG products(being respectively 75% and 87.3%).
(4) fish provided by the invention recombination hCG and its derived protein or the albumen by modification, to there is no at presentThe Silurformes fish that effect ocyodinic is hastened parturition also have good spawning effect, with reference to ocyodinic LRH-A3It is used with DOM, yellow forehead can be madeThe spawning rate and rate of fertilization of fish are improved to 90% and 85.7%, and being above urine source hCG products, (spawning rate and rate of fertilization are respectively65% and 71.2%).
(5) fish provided by the invention recombination hCG and its derived protein or the albumen by modification, to freshwater fishSpawning effect is higher than urine source hCG products, can replace application of the urine source hCG products in freshwater fish is hastened parturition.
Description of the drawings
Fig. 1 is the SDS-PAGE electrophoresis that fish recombinates hCG in the embodiment of the present invention 2.Wherein left figure is non-reduced electrophoresisFigure, right figure are reduction electrophoretogram；MK：Albumen Marker；1：Clarified broth；2：CM FF collection liquids；3：Phenyl collection liquids；4：QFF collection liquids.
Fig. 2 is the non-reduced electrophoretograms of SDS-PAGE that fish recombinates hCG and uhCG in the embodiment of the present invention 2.MK：AlbumenMarker；1：Recombinate hCG；2：UhCG.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodimentAccording to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,Molecular Cloning:A Laboratory Manual, 2001) or the condition according to manufacturer's specification suggestion.
Embodiment 4-6 chooses silver carp, snakehead, Pelteobagrus fulvidraco parent population and carries out experiment of hastening parturition respectively, in the first tenday period of a month in May in Guangdong fishing groundIt carries out.
Embodiment 1 recombinates the screening of hCG protein expression systems
HCG is investigated in Escherichia coli (Escherichia coli)-BL21 (DE3), P. pastoris cellThe expression of (Pichia pastoris)-GS115 and mammalian cell-Chinese hamster ovary cell (CHO).In geneThe nucleotide sequence of hCG α (GenBank NM_000735.3) and hCG β (GenBank NM_000737.3) subunit is retrieved in libraryIt is and artificial synthesized.The nucleotide sequence of hCG α subunits and β subunits is transferred to respectively expression vector pET-28a, pPIC9K andIn pcDNA3.1.The recombinant vector containing α subunits and β subunits is transferred in BL21 (DE3), GS115 and Chinese hamster ovary celI respectively.BL21 (DE3) does not obtain soluble recombination hCG albumen；Recombination hCG polymers are obtained in GS115 cells, and activity is very low；CHOActive recombination hCG is obtained in cell, but protein content is low, need to optimize.Final choice Chinese hamster ovary celI is as recombination hCGExpression system, and the expression system is optimized.
Embodiment 2 recombinates expression and purifying of the hCG albumen in Chinese hamster ovary celI
The nucleotide sequence of hCG α and β subunits that will be retrieved in embodiment 1, according to mammalian codons preferences intoRow codon optimization, α subunits nucleotide sequence such as SEQ ID NO after optimization:Shown in 2, β subunits nucleotide sequence such as SEQ IDNO:Shown in 4.
The nucleotide sequence of artificial synthesized hCG α subunits and β subunits is transferred to respectively in expression vector pcDNA3.1, it willExpression recombination hCG in Chinese hamster ovary celI is transferred to after recombinant vector linearisation together.
The steady cell that turns is subjected to fermented and cultured in fermentation tank, zymotic fluid is by two-stage in-depth filtration film packet removal cell and carefullyBorn of the same parents' fragment then with 0.22 μm of membrane filtration, obtains clear zymotic fluid.Clear zymotic fluid uses weak cation exchange layer firstAnalyse (such as CM FF, GE Healthcare) purifying：Loading is balanced with equilibrium liquid (20mM ammonium acetates, pH5.0), then uses eluentEluent is collected in (50Mm PB, 3M sodium chloride, pH8.5) elution.Secondly weak cation exchange is chromatographed into collection liquid hydrophobic layerAnalyse (such as Phenyl, GE Healthcare) purifying：Loading is balanced with equilibrium liquid (50mM PB, 3M sodium chloride, pH8.5), thenIt eluted with eluent (10mM PB, pH8.5), collect eluent.Finally hydrophobic chromatography collection liquid is exchanged (such as with strong anionQFF, GE Healthcare) chromatography be further purified：Loading is balanced with equilibrium liquid (50mM PB, pH8.5), then uses eluent(50Mm PB, 3M sodium chloride, pH8.5) is eluted, and is collected.PAGE gel electrophoresis is carried out to the recombination hCG (rhCG) of purifying(Fig. 1)；RhCG and urine source hCG carries out PAGE gel electrophoresis (Fig. 2) after purification.
Embodiment 3 recombinates the Activity determination of hCG albumen
By measuring effects of the rhCG to underage mouse uterus weight, the activity of recombination hCG is determined.With reference to Chinese veterinary drugPharmacopeia " chorionic gonadotrophin biologic assay ", method is as follows：By hCG standard items and recombination hCG samples, (estimating activity is respectively15000IU/mg) it is configured to high, medium and low three dosage of 0.625IU/ml, 0.375IU/ml, 0.225IU/ml.Health is chosen to closeLattice, birth 17-23 day, weight 9-13g, same source female immature mouse, be randomly divided into 6 groups, every group 15 by weight.DailyGive the standard items or recombination hCG 0.2ml per sub-cutaneous injections respective concentration respectively in the roughly the same time, once a day, continuouslyAnimal in last time injection is put to death, weighed, dissected, extracted uterus, remove the tissue of attachment, go by injection 3 times afterwards for 24 hoursExcept ovary, intrauterine liquid is pressed dry, the uterus weight of every 10g weight is directly weighed and be converted into, according to the quantitative response in biologic assayIt is 14300IU/mg that parallel line assay method, which calculates recombination hCG potency, is urine source hCG (uhCG) potency as defined in veterinary drug allusion quotation3.2 times of 4500IU/mg.
Embodiment 4 recombinates application of the hCG albumen in silver carp is promoted to hasten parturition
Silver carp, also known as Hypophthalmichthys molitrix, Cypriniformes Cyprinidae silver carp category fish are one of four large Chinese carps of China, the excellent fish more preferably cultivatedOne of kind.Property it is active, be apt to jump, the zooplankters such as phytoplanktons and wheel animalcule such as staple food diatom, green algae.
Weight 5kg or so is selected, the silver carp parent population physical strong, disease-free and injury-free, constitution is plentiful is randomly divided into 4 groups：It is negativeControl group, uhCG (uhCG) group, rhCG-1 groups and rhCG-2 groups, every group of 10 tail raun, 5 tail milter.Nurturing period adds throwing appropriateFine fodder, increases bath number before hastening parturition, during 25 DEG C of water temperature, hastened parturition using two needle injections, phase is injected according to following table time and dosageDrug is answered, wherein milter injection halves, and after second is injected, records Effect time.The male and female parent population of heat is manually doneMethod is inseminated, and method is as follows：Fish body is dried, the ovum for waiting to produce female parent population is squeezed into basin, squeezes into milter sperm at once, it is light with handLight stirring makes smart ovum fully combine and improves rate of fertilization.Being denoted as not laying eggs in raun 6h is not produced, and oviposition is denoted as half on a small quantity in 6hProduction.Parent population after hastening parturition cleans injury with 20ppm potassium permanganate, injects the penicillin anti-inflammatory of 30,000/tail.Fertilized eggs are placed in hatchingArtificial incubation is carried out in circuit, rate of fertilization is calculated after 12h.
The results are shown in Table 1, and silver carp individually injects the spawning rate of rhCG silver carp parent populations and rate of fertilization difference to hCG sensitivitiesIt for 65% and 77.3%, but is shared with LRH-A and DOM, spawning effect is more excellent, wherein the female fish parent fish Effect time of rhCG-1 groups(7h) is equal with uhCG groups (7h) raun, is below negative control group (8.5h) and rhCG-2 groups (8h).9 tail raun of rhCG-1 groupsFull production, 1 tail half produce, and spawning rate reaches 95%, rate of fertilization 96.7%, be above negative control group (55% and 67.2%),UhCG groups (80% and 85.6%) and rhCG-2 groups.
Embodiment 5 recombinates application of the hCG albumen in snakehead is promoted to hasten parturition
Snakehead is commonly called as snakeheaded fish, and Perciformes murrel section Ophiocephalus fish, meat is fine and smooth, meat flavour is delicious, and growth is fast, yield is high, cultivationDensity is high, high efficiency, is a kind of higher rare economic fish of economic value.Property it is violent, be food with fish, shrimp etc..
Select 1.5kg or so, it is disease-free, physical strong, fish body is smooth, the intact snakehead parent population of scale, it is randomly divided into 4Group：Negative control group, uhCG (uhCG) group, rhCG-1 groups and rhCG-2 groups, every group of 10 tail raun, 5 tail milter.It is fed before hastening parturitionThe fresh food of snakehead eating, such as shrimp, tadpole, wild fishes are often washed by water, and during 25 DEG C of water temperature, are hastened parturition using two needle injections,According to following table time and dosage injection relative medicine, wherein milter injection halves, and after second is injected, records Effect time,The artificial dry method of the male and female parent population of heat is inseminated, method is as follows：Fish body is dried, the ovum for waiting to produce female parent population is squeezed into basin,Milter sperm is squeezed at once, is gently mixed with hand, smart ovum is made fully to combine and improves rate of fertilization.What is do not laid eggs in raun 6h is denoted as notIt produces, oviposition is denoted as half production on a small quantity in 6h.Parent population after hastening parturition cleans injury with 20ppm potassium permanganate, injects 30,000/tail mouldPlain anti-inflammatory.Fertilized eggs, which are placed in circular hatching channal, carries out artificial incubation, and rate of fertilization is calculated after 12h.
The results are shown in Table 2, and the Effect time for individually injecting the female fish parent fish of rhCG snakeheads is 26h, spawning rate and rate of fertilizationRespectively 50% and 63.5%, snakehead is insensitive to hCG.RhCG and LRH-A and DOM, which is shared, shows the spawning effect of snakehead parent populationWrite, the female fish parent fish Effect time (21h) of rhCG-1 group snakeheads far below negative control group (25h), uhCG groups (22.5h) andRhCG-2 groups.Female 9 tail of fish parent fish of 10 tail of rhCG-1 groups produces entirely, and 1 tail half produces, and spawning rate reaches 95%, rate of fertilization 96.3%,Higher than negative control group (70% and 78.4%), uhCG groups (75% and 87.3%) and rhCG-2 groups.
Embodiment 6 recombinates application of the hCG albumen in Pelteobagrus fulvidraco is promoted to hasten parturition
Pelteobagrus fulvidraco, is commonly called as Huang Lading, Silurformes, Pelteobagrus, is a kind of omnivorous biased physical property economic fish, fine and tender tasteDelicious, full of nutrition, the market price is high, is a kind of emerging freshwater aquiculture kind.
Selection raun 100g, milter 200g or so, the Pelteobagrus fulvidraco parent population physical strong, disease-free and injury-free, constitution is plentiful, at randomIt is divided into 4 groups：Negative control group, uhCG (uhCG) group, rhCG-1 groups and rhCG-2 groups, every group of 10 tail raun, 5 tail milter.It cultivatesPeriod, which adds, feeds the bait such as spiral shell freshwater mussel, during 25 DEG C of water temperature, is hastened parturition using two needle injections, corresponding medicine is injected according to following table time and dosageThe injection of object, wherein milter halves, and after second is injected, records Effect time, the artificial dry method of female fish parent fish of heat is awardedEssence, method are as follows：First gametocide fish takes out spermary, is placed in mortar and shreds, and it is spare to add in spermatozoa preservative fluid.It checks raun, will treatThe ovum of the female parent population of production is squeezed into basin, and sperm and ovum are mixed fertilization with feather.Being denoted as not laying eggs in raun 6h is not produced,Oviposition is denoted as half production on a small quantity in 6h.Parent population after hastening parturition cleans injury with 20ppm potassium permanganate, and injection 30,000/tail penicillin disappearsIt is scorching.Fertilized eggs are placed in hatching road and are hatched, rate of fertilization is calculated after 12h.
As a result as shown in table 3 below, Pelteobagrus fulvidraco is insensitive to various ocyodinics, individually injects the effect of rhCG Pelteobagrus fulvidraco parent populationsIt is 23.5h between seasonable, spawning rate and rate of fertilization are respectively 40% and 61.8%.RhCG and LRH-A and DOM is shared to Pelteobagrus fulvidraco parentThe spawning effect of fish is notable.The female fish parent fish Effect time (21.5h) of rhCG-1 group Pelteobagrus fulvidracos less than negative control group (for 24 hours),UhCG groups (22.5h) and rhCG-2 groups, female 8 tail of fish parent fish of 10 tails produce entirely, and 2 tails half produce, and spawning rate and rate of fertilization are 90% He85.7%, it is above negative control group (35% and 63.4%), uhCG groups (65% and 71.2%) and rhCG-2 groups.
Although above the present invention is described in detail with a general description of the specific embodiments,On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.CauseThis, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
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