CN108264549A - Recombinate the preparation method and application of chorionic gonadotrophin rhCG - Google Patents

Recombinate the preparation method and application of chorionic gonadotrophin rhCG Download PDF

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CN108264549A
CN108264549A CN201810271691.7A CN201810271691A CN108264549A CN 108264549 A CN108264549 A CN 108264549A CN 201810271691 A CN201810271691 A CN 201810271691A CN 108264549 A CN108264549 A CN 108264549A
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fish
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amino acid
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罗昊澍
师磊
韩国
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Beijing Wei Jie Xin Bio Technology Co Ltd
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Abstract

The present invention relates to the preparation methods of recombination chorionic gonadotrophin rhCG, and recombination chorionic gonadotrophin rhCG is expressed using mammal eukaryotic expression system；The recombination chorionic gonadotrophin rhCG includes α and β subunits, and the two is connected by Van der Waals force.The recombination chorionic gonadotrophin rhCG of the present invention hastens parturition for fish, and, purity higher more more stable than the urine source hCG mass of production at present, impurity are less, and spawning effect is more preferable, applications of the alternative urine source hCG in fish reproduction.The invention further relates to applications of the recombination hCG in fresh-water fishes artificial propagation field.

Description

Recombinate the preparation method and application of chorionic gonadotrophin rhCG
Technical field
The present invention relates to biological medicine and technical field of animal reproduction, specifically, being related to recombinating chorionic gonadotrophin rhCG's Preparation method and application.
Background technology
Fish reproduction and residing ecological environment are closely related, under normal circumstances, ecological factor, such as nutrition, temperature, light It is the determinant of fish gonad development according to, flow etc..However under the conditions of artificial propagation, shortage rivers have comprehensive in pond Ecological 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 one As 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 parturition Agent can ovulate fertilization.The purpose of manual injection's ocyodinic is：On the one hand prevent certain sexual glands under artificial condition from voluntarily into Ripe 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 ovulation Production 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 main There 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 applications Several 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 β With reference to subunit.HCG promotes the formation reached maturity with corpus luteum of female egg mother cell, promotes the life of male creature sperm Into.HCG is clinically usually used in assisted reproductive therapy in people doctor, and treatment is infertile.HCG is usually used in animal reproduction production Pig, 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, ovulates Production 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, into This 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 resource Waste.Therefore, people have utilized technique for gene engineering exploitation novel human recombination hCG products, such as Merck Xue Lannuo Ovidrel (recombination physex parenteral solution), the injection chorionic gonadotrophin (beautiful pearl is currently urine source hCG) of beautiful pearl, recombinate hCG Product 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 thin Born 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 select Expression 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 protein Processing mechanism after translating, such as glycosylation, correct folding etc. are not suitable for expression complexity and most animals cell protein.Yeast table The advantages of having protokaryon and Higher eukaryotic expression system concurrently up to system, the albumen of harvest have certain post translational processing ability, but It is that there are still deficiencies, such as easily forms protein multimers and excessive glycosylation, therefore be more suitable for expressing simple eucaryote egg In vain.Insect expression system can express higher eucaryote albumen, and albumen can be carried out correctly to fold and modify, but protein content It 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, should According to different expression requirements, suitable expression system and strategy are selected.HCG is a kind of by Human plactnta syncytiotrophoblast point The glycoprotein hormones secreted, the subunit combined including two non-covalent bonds of α and β.Theoretically with mammalian cell come Expression product Reproductive 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 products Road.
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 technology To it is uniform, purity is high, impurity is few, safety coefficient is big.Mammalian cell expression system, can compared with other expression systems It 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 mammal Eukaryotic expression system expression recombination chorionic gonadotrophin rhCG；The recombination chorionic gonadotrophin rhCG includes α and β subunits, and the two passes through model De 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 same Etc. 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 function The 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 same Etc. 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 function The 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 codons Property carry out gene order optimization, the gene order of the α and β subunits after optimization is implemented in different expression cassettes, then connects respectively Onto 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 place Chief 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, preferably Chinese 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 greatly Mould 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 systems Albumen 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 provided Can, 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 modification Albumen) 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 formed Fusion 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, sheep Animal, 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, grass Fish, silver carp and bighead) and the economic fish such as Cypriniformes, Perciformes, Silurformes, salmon shape mesh, Gadiformes, Clupeiformes, Gadiformes, more It 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 breeding Deng.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 include LRH-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 rhCG It 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/kg Weight；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 first Second 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 rhCG 500IU/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 rhCG 400IU/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 time A3, 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 mammal High 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 coefficient Greatly；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 improve Spawning 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 by Smart 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 not Sensitive perciform fishes have good spawning effect, with reference to ocyodinic LRH-A3It is used with DOM, improves the oviposition of snakehead parent population Rate 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 present The 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 made The 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 respectively 65% and 71.2%).
(5) fish provided by the invention recombination hCG and its derived protein or the albumen by modification, to freshwater fish Spawning 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 electrophoresis Figure, 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：Albumen Marker；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, embodiment According 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 ground It carries out.
Embodiment 1 recombinates the screening of hCG protein expression systems
HCG is investigated in Escherichia coli (Escherichia coli)-BL21 (DE3), P. pastoris cell The expression of (Pichia pastoris)-GS115 and mammalian cell-Chinese hamster ovary cell (CHO).In gene The nucleotide sequence of hCG α (GenBank NM_000735.3) and hCG β (GenBank NM_000737.3) subunit is retrieved in library It is and artificial synthesized.The nucleotide sequence of hCG α subunits and β subunits is transferred to respectively expression vector pET-28a, pPIC9K and In 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；CHO Active recombination hCG is obtained in cell, but protein content is low, need to optimize.Final choice Chinese hamster ovary celI is as recombination hCG Expression 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 into Row codon optimization, α subunits nucleotide sequence such as SEQ ID NO after optimization:Shown in 2, β subunits nucleotide sequence such as SEQ ID NO: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 will Expression 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 carefully Born 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 first Analyse (such as CM FF, GE Healthcare) purifying：Loading is balanced with equilibrium liquid (20mM ammonium acetates, pH5.0), then uses eluent Eluent is collected in (50Mm PB, 3M sodium chloride, pH8.5) elution.Secondly weak cation exchange is chromatographed into collection liquid hydrophobic layer Analyse (such as Phenyl, GE Healthcare) purifying：Loading is balanced with equilibrium liquid (50mM PB, 3M sodium chloride, pH8.5), then It eluted with eluent (10mM PB, pH8.5), collect eluent.Finally hydrophobic chromatography collection liquid is exchanged (such as with strong anion QFF, 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 drug Pharmacopeia " chorionic gonadotrophin biologic assay ", method is as follows：By hCG standard items and recombination hCG samples, (estimating activity is respectively 15000IU/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 close Lattice, birth 17-23 day, weight 9-13g, same source female immature mouse, be randomly divided into 6 groups, every group 15 by weight.Daily Give the standard items or recombination hCG 0.2ml per sub-cutaneous injections respective concentration respectively in the roughly the same time, once a day, continuously Animal 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 hours Except 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 assay It 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 quotation 3.2 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 cultivated One 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 negative Control group, uhCG (uhCG) group, rhCG-1 groups and rhCG-2 groups, every group of 10 tail raun, 5 tail milter.Nurturing period adds throwing appropriate Fine 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 dosage Drug is answered, wherein milter injection halves, and after second is injected, records Effect time.The male and female parent population of heat is manually done Method 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 hand Light 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 6h Production.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 hatching Artificial 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 sensitivities It 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 groups Full 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, cultivation Density 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 4 Group：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 parturition The 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 not It 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 mould Plain 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 fertilization Respectively 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 population Write, the female fish parent fish Effect time (21h) of rhCG-1 group snakeheads far below negative control group (25h), uhCG groups (22.5h) and RhCG-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 taste Delicious, 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 random It 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 cultivates Period, 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 dosage The 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 awarded Essence, 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 treat The 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 disappears It 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 populations It 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 parent The 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% He 85.7%, 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.Cause This, 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
<110>Beijing Wei Jiexin bio tech ltd
<120>Recombinate the preparation method and application of chorionic gonadotrophin rhCG
<130> KHP181111214.0
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 116
<212> PRT
<213>People (Homo sapiens)
<400> 1
Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser
1 5 10 15
Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro
20 25 30
Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro
35 40 45
Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro
50 55 60
Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu
65 70 75 80
Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly
85 90 95
Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr
100 105 110
Tyr His Lys Ser
115
<210> 2
<211> 348
<212> DNA
<213>People (Homo sapiens)
<400> 2
atggactact accggaagta cgccgccatc ttcctggtga ccctgtccgt gttcctgcac 60
gtgctgcact ccgcccctga cgtgcaggac tgccctgagt gcaccctgca ggagaacccc 120
ttcttctccc agcccggagc ccctatcctg cagtgcatgg gctgctgctt ctccagggcc 180
taccctaccc ccctgaggag caagaagacc atgctggtgc agaagaacgt gacctccgag 240
agcacctgct gcgtggccaa gtcctacaac agggtgaccg tgatgggcgg cttcaaggtg 300
gagaaccaca ccgcctgcca ctgctccacc tgctactacc acaagtcc 348
<210> 3
<211> 165
<212> PRT
<213>People (Homo sapiens)
<400> 3
Met Glu Met Phe Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly
1 5 10 15
Gly Thr Trp Ala Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile
20 25 30
Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr
35 40 45
Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val
50 55 60
Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg
65 70 75 80
Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val
85 90 95
Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu
100 105 110
Cys Arg Arg Ser Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu
115 120 125
Thr Cys Asp Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro
130 135 140
Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr
145 150 155 160
Pro Ile Leu Pro Gln
165
<210> 4
<211> 495
<212> DNA
<213>People (Homo sapiens)
<400> 4
atggagatgt tccagggcct gctgctgctc ctgctgctca gcatgggcgg cacatgggcc 60
tccaaggagc ctctcaggcc caggtgcagg cctatcaacg ccaccctggc cgtggagaag 120
gaaggatgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccccaca 180
atgaccaggg tgctgcaagg agtgctgcct gccctgcccc aagtggtgtg caactaccgg 240
gacgtgaggt tcgagtccat caggctgcct ggctgcccta ggggagtcaa ccctgtggtc 300
tcctacgccg tggccctgtc ctgtcagtgt gccctgtgca ggcggtccac aacagactgc 360
ggcggaccca aggaccaccc tctgacctgc gacgacccca ggttccaaga ctcctcctcc 420
tccaaggctc cccctccttc cctcccttcc cctagcaggc tgcctggacc ttccgacacc 480
cccattctgc cccag 495

Claims (10)

1. recombinate the preparation method of chorionic gonadotrophin rhCG, which is characterized in that express and recombinate using mammal eukaryotic expression system Chorionic gonadotrophin rhCG；The recombination chorionic gonadotrophin rhCG includes α and β subunits, and the two is connected by Van der Waals force；
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 equal work( The albumen as derived from i) of energy；Or
Iii) with SEQ ID NO:Amino acid sequence homology shown in 1 is more than 90%, and the amino acid with equal function The albumen of Sequence composition；
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 equal work( The albumen as derived from i) of energy；Or
Iii) with SEQ ID NO:Amino acid sequence homology shown in 3 is more than 90%, and the amino acid with equal function The albumen of Sequence composition.
2. according to the method described in claim 1, it is characterized in that, according to NCBI announce hCG α and β subunits gene order, Gene order optimization is carried out according to mammalian codons preferences, the gene order of the α and β subunits after optimization is implemented in respectively Different expression cassettes is then attached on same expression vector, converts host cell, and expressed in host cell, and separation is pure Change target protein；Alternatively,
The gene order of α and β subunits after optimization is building up on expression vector respectively, gained recombinant vector cotransformation host Cell, and expressed in host cell, isolate and purify target protein；
Wherein, the gene order of the α and β subunits after optimization is respectively such as SEQ ID NO:Shown in 2 and 4.
3. according to the method described in claim 2, it is characterized in that, the expression vector is selected from pCG, pcDNA3.1, pMS2- GFP, pCMV5, preferably pcDNA3.1.
4. according to the method described in claim 2, it is characterized in that, the host cell CHO or 293 cells.
5. following any application of recombination chorionic gonadotrophin rhCG prepared by any one of claim 1-4 the methods：
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.
6. application according to claim 5, which is characterized in that the animal is mammal or fish；Wherein, the food in one's mouth Newborn animal is pig, ox, sheep, horse or dog；The fish are fresh water or marine fishes, preferably freshwater fish.
7. application according to claim 6, which is characterized in that it is described promotion animal reproduction refer to promote egg mother cell into It is ripe, promote estrus synchronization, superfecundation, induced breeding.
8. the method that fish are hastened parturition, which is characterized in that hastened parturition by injecting ocyodinic to fish；Wherein, the ocyodinic Active constituent at least containing any one of claim 1-4 the methods prepare recombination chorionic gonadotrophin rhCG；
The preferred freshwater fish of fish, including four large Chinese carps, Cypriniformes, Perciformes, Silurformes, salmon shape mesh, Gadiformes, Pacific herring shape The economic fish of mesh and Gadiformes, the economic fish of more preferable Cypriniformes, Perciformes and Silurformes.
9. according to the method described in claim 8, it is characterized in that, the injection dosage of rhCG is 200-2000IU/kg weight.
10. according to the method described in claim 8, it is characterized in that, hastened parturition using two needle injections, the ocyodinic injected for the first time For LRH-A, injection dosage is 1-2 μ g/kg weight；Second of injection is carried out after injecting 10-12h for the first time, second of injection is urged It is LRH-A 2-10 μ g/kg weight, DOM 5-6 μ g/kg weight and rhCG 400-600IU/kg weight to produce agent.
CN201810271691.7A 2018-03-29 2018-03-29 Recombinate the preparation method and application of chorionic gonadotrophin rhCG Pending CN108264549A (en)

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Application publication date: 20180710