CN106434684B - Scatophagus argus (Linnaeus) lutropin LH gene, Scatophagus argus (Linnaeus) LH recombinant protein and application - Google Patents
Scatophagus argus (Linnaeus) lutropin LH gene, Scatophagus argus (Linnaeus) LH recombinant protein and application Download PDFInfo
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- CN106434684B CN106434684B CN201611051640.0A CN201611051640A CN106434684B CN 106434684 B CN106434684 B CN 106434684B CN 201611051640 A CN201611051640 A CN 201611051640A CN 106434684 B CN106434684 B CN 106434684B
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses a kind of Scatophagus argus (Linnaeus) lutropin LH gene, Scatophagus argus (Linnaeus) LH recombinant protein and applications；Specifically, it is the Scatophagus argus (Linnaeus) LH recombinant protein obtained by Scatophagus argus (Linnaeus) lutropin LH gene by prokaryotic expression system inducing expression and its application, not only obtain the cDNA sequence overall length of Scatophagus argus (Linnaeus) lutropin LH gene, Scatophagus argus (Linnaeus) LH recombinant protein is also obtained by prokaryotic expression, pass through affine column purification, pure LH recombinant protein is obtained, promotes fish gonad development to be applied to fish hormone, and then fish gonad development is made to show synchronism.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of cDNA sequence of Scatophagus argus (Linnaeus) lutropin LH gene
Column, there are also the pure of the Scatophagus argus (Linnaeus) LH recombinant protein obtained by it by the building of recombinant plasmid, prokaryotic expression system inducing expression
Change and applies.
Background technique
Scatophagus argus (Linnaeus) (Scatophagusargus) also known as gold drum fish are subordinate to Perciformes (Perciformes), money Gyrinocheilidae
(Scatophagidae), it is distributed in China East Sea and South China Sea, is a kind of Quality and economy fish for having both ornamental value.Money
Fish is eurysaline fishes, is distributed in the high sea area of salinity, also can grow and survive well in pure fresh water, Ke Yizhi
The seawater connect from 35 ‰ is transferred to fresh water life, raises and train without carrying out desalination, rare adaptability superpower to salinity makes it
It is highly suitable as the material of research bony fish reproduction and growth.The Scatophagus argus (Linnaeus) spermatogenesis mechanism of Scatophagus argus (Linnaeus) is studied for sea
The desalination cultivation of water economic fish is very significant.The body variation understood in seawater fish reproductive process is adapted to environment, right
In improving seawater fish breeding and growth performance, it is of great significance for aquaculture development.
Lutropin is also known as luteotropin (luteinizinghormone, LH).Anterior pituitary basophil cell divides
The hormone secreted.For glycoprotein.Molecular weight about 30000.Chemical structure: for glycoprotein, by two subunit peptide chains of α and β with covalent bond
It is combined into.In the presence of follicular stimulating hormone, conjunction with which effect stimulates ovarian estrogen secretion, makes follicle maturity and row
Ovum makes ruptured follicle form corpus luteum and secretes estrogen and progestational hormone.Stimulation interstitial glands develop and it are promoted to secrete testis
Ketone.Therefore also known as interstitial cell augmentor.LH, FSH and human chorionic gonadotrophin (HCG) belong to glycoprotein family, LH's
Structure and function is similar to HCG, and two kinds of hormones are played a role by identical receptor, but its half-life period is very short, and about 30 minutes,
Fluctuation range is larger in blood；And HCG has longer half-life period, and more firm in conjunction with receptor, is easy by radioimmunoassay institute
Measurement, so being frequently used for clinical and laboratory research.Although interstitialcellstimulating hormone (ICSH) (LH) has the infertile treatment of the mankind
It is widely applied, but it is also fewer to the research of fish and application.
The function of lutropin: (1) promote the synthesis of estrogen and progesterone.(2) triggering ovulation: in excision adenohypophysis
Patient manually gives LH i.e. and can lead to ovulation after making follicular development to certain phase with various methods.LH causes the machine of ovulation
System may be to cause the connective tissue of ovarian follicle wall to decompose and make since molten protease, amylase, the activity of hyaluronidase increase
At.Animal experiments show that: the progesterone generated under LH effect can trigger the formation and release of ovulation enzyme.Under LH effect,
Mature ovarian follicle secretes prostaglandin.(3) promote the generation of corpus luteum: the mucomembranous cell in mature ovarian follicle exists in no LH
When, also can leuteinization naturally, but immature granulosa cell, only addition LH could leuteinizations.(4) LH to ovary its
He acts on: increasing the blood flow of ovary, increases the activity that ornithine takes off antelope enzyme, reduces ovary tissue inner cholesterol concentration.LH energy
Leydig cell Proliferation and differentiation is promoted to promote to climb the generation of ketone, meanwhile, seminal vesicle and prostate also hyperplasia.
LH is promoting domestic animal sexal maturity to shift to an earlier date, the dam heat of induced lactation anestrus, is improving male animal semen quality, treatment
Deep analysis and total has been obtained in the application in the fields such as the donor superfecundation in dam disease of ovary and embryo transfer
Knot, the in recent years maturation in vitro (IVM) to embryo transfer and superfecundation (MOET), egg mother cell and (IVF) in vitro fertilization again
It has made intensive studies and explores.So that us has been further appreciated that application of the LH in animal reproduction breeding: (1) promoting domestic animal
Sexal maturity shifts to an earlier date: the breeding of certain domestic animals has seasonality, if birth is later, when sexal maturity is possible to miss mating period,
Close to the processing of sexually matured domestic animal progesterone, it is used cooperatively LH, them can be made to do sth. in advance rutting；(2) induced lactation anestrus
Dam heat: LH processing can induce the dam heat and breeding of lactation anestrus, shorten tire spacing, improve the breeding effect of dam
Rate；(3) improve male animal semen quality: male animal sperm concentration is insufficient or sperm motility difference can cause dam infertile, controls using LH and FSH
It treats, sperm concentration can be improved, improve semen quality；(4) treat dam disease of ovary: LH is not complete to Monitoring Ovarian Function, arrest of development
Or alternately development has good therapeutic effect；(5) Superovluation: in embryo transfer work, in order to obtain a large amount of ovum and embryo,
Common LH handles donor animal, its ovarian follicle is made largely to reach maturity and ovulate；(6) maturation in vitro and body of egg mother cell
Outer fertilization.Livestock Oocytes maturation in vitro and promoting sexual gland hormone have close connection.In short, lutropin is extensive because of it
And peculiar biological function, have in the fields such as aquaculture industry and animal husbandry even human disease treatment extensive and tempting
Prospect.
Problem of the existing technology: as aquaculture scale constantly expands, for the life of stable guarantee quantity
Production mode, it is ensured that breeding, the efficiency grown, many drugs are put into production industry, this can undoubtedly make environment and human body
At certain injury.
Recently as the rapid progress of biology techniques, so that the understanding that related gene occurs to fish sperm achieves
Significant progress.RT-PCR technology, hybridization in situ technique, subduction hybridization technique, differential display technique, EST microarray technology
Using many genes relevant to spermatogenesis and maturation are obtained, the understanding to spermatogenesis molecule mechanism is promoted.But
Spermatogenesis has a lots of genes expression, and these gene expressions are in phase specificity, with it is stringent when, air-conditioning control closes
System detects one piece of clone's EST microarray with testis and sperm mRNA and has been described that spermatogenetic complexity, in spermatogenesis
Many tera incognitas are still had in the research of related gene.The discovery of gene and its illustrating for function and relationship are to study essence in the future
The core missions of molecule mechanism occur for son, and fish specific gene research spermatogenetic for regulation at present still in starting and is visited
The rope stage.Further screening, identification and clone's spermatogenesis specific expression gene, for understanding spermatogenesis, subtrahend point
Molecule and its regulatory mechanism for splitting and breaking up metamorphosis are particularly significant.Since the development of androgone needs stringent particular surroundings,
Lack in-vitro culture model, restricts the research of spermatogenesis molecule mechanism for a long time.But recently as the fast of biology techniques
Speed progress, so that the understanding to spermatogenesis-related gene achieves significant progress.Subduction hybridization, differential display technique are answered
With obtaining many genes relevant with spermatogenesis and maturation.Identification adjusts spermatogenetic key gene and illustrates its function
It can be the core missions for studying spermatogenesis molecule mechanism in the future, improve the research to Scatophagus argus (Linnaeus) spermatogenesis mechanism, for research
Molecular mechanism that fish sperm occurs etc. provides basic data, thus preferably Instructing manufacture practice.
Summary of the invention
The purpose of the present invention is to provide a kind of Scatophagus argus (Linnaeus) lutropin LH gene, Scatophagus argus (Linnaeus) LH recombinant protein and answer
With；Specifically, being the Scatophagus argus (Linnaeus) LH obtained by Scatophagus argus (Linnaeus) lutropin LH gene by prokaryotic expression system inducing expression
Recombinant protein and its application not only obtain the cDNA sequence overall length of Scatophagus argus (Linnaeus) lutropin LH gene, also pass through protokaryon table
Up to Scatophagus argus (Linnaeus) LH recombinant protein is obtained, by affine column purification, pure LH recombinant protein is obtained, to be applied to infuse outside Fish
It penetrates to promote fish gonad development, and then fish gonad development is made to show synchronism.
The purpose of the present invention is achieved through the following technical solutions:
In a first aspect, cDNA sequence includes such as SEQ ID the present invention relates to a kind of Scatophagus argus (Linnaeus) lutropin LH gene
Base sequence shown in NO:1.
Second aspect, the present invention relates to a kind of Scatophagus argus (Linnaeus) LH recombinant protein, the recombinant protein includes such as SEQ ID NO:2
Shown in amino acid sequence.The gene order of the recombinant protein is encoded as shown in SEQ ID NO:1.
The third aspect, the invention further relates to a kind of preparation methods of Scatophagus argus (Linnaeus) LH recombinant protein above-mentioned, obtain above-mentioned
The cDNA sequence overall length of Scatophagus argus (Linnaeus) lutropin LH gene obtains Scatophagus argus (Linnaeus) LH recombinant protein by prokaryotic expression.
Preferably, the acquisition includes the following steps:
S1, using Trizol extraction method, carry out RNA extraction using Scatophagus argus (Linnaeus) sexual gland, then carry out by reversal agents box
The synthesis of cDNA；
S2, the cDNA conserved regions design primer synthesized according to step S1, using RT-PCR and RACE technology, clone is obtained
Obtain Scatophagus argus (Linnaeus) LH gene order overall length.
Preferably, the cloning primer is to for 3 ' LH1,3 ' LH2 and 5 ' LH1,5 ' LH2.Its base sequence is distinguished successively such as
Shown in SEQ ID NO:3-6.
Preferably, the prokaryotic expression includes the following steps:
A1, Scatophagus argus (Linnaeus) lutropin LH gene cDNA sequence connect with pGEM-TEasy carrier after, conversion is to DH5 α
Competent cell, amplification cultivation；It is collected by centrifugation, extracting contains the plasmid of target gene (LH gene) and pGEM-TEasy carrier；
A2, according to pET-28a+Carrier selects restriction enzyme site XhoI (158), and NdeI (238) respectively extracts step A1
The Plasmid DNA arrived carries out double digestion；Recycle effective segment；
A3, purifying；Target gene is connected with the recovery product of plasmid, it is thin that connection product imports BL-21 expression competence
Born of the same parents；Centrifuge separation；Bacterium is chosen, chooses effective bacterium solution after bacterium colony PCR verifying；
A4, effective bacterium solution is transferred in the LB liquid medium containing kanamycins, 37 DEG C with volume ratio 1: 10, is shaken
Culture is swung, recombinant protein inducing expression is carried out, obtains recombination thick leach protein.
Preferably, in step A2, digestion primer pair is pET (LH)-F, pET (LH)-R；Its corresponding sequence is respectively such as
SEQ ID NO:7, shown in 8.
It preferably, further include the step of obtaining pure Scatophagus argus (Linnaeus) LH recombinant protein by affine column purification after prokaryotic expression.
Preferably, the affine column purification includes the following steps:
B1,1ml nickel Ago-Gel FF or nickel NTA Ago-Gel FF prepacked column are taken, with 10ml equilibration buffer,
Then 10ml recombinates thick leach protein sample with 0.5ml/min loading, and then 2ml/ pipe is in charge of collection；
B2, unadsorbed recombination thick leach protein sample, flow velocity 1-2ml/min, 2ml/ pipe are washed away with 15ml equilibration buffer
It collects；
B3, unadsorbed recombination thick leach protein sample, flow velocity 1-2ml/min, 2ml/ pipe are washed away with 5ml elution buffer
It collects.
Fourth aspect, the invention further relates to a kind of Scatophagus argus (Linnaeus) LH recombinant proteins above-mentioned to be used as promotion fish gonad development
Purposes in medicament.
Preferably, the promotion fish gonad development medicament is hormone liquid.
5th aspect, the invention further relates to Scatophagus argus (Linnaeus) LH recombinant proteins made from a kind of preparation method above-mentioned to be used as rush
Purposes into fish gonad development medicament.
Preferably, the promotion fish gonad development medicament is hormone liquid.
6th aspect, the invention further relates to a kind of fish external injection of recombinant protein, the hormone liquid includes obtaining
The cDNA sequence overall length for taking Scatophagus argus (Linnaeus) lutropin LH gene above-mentioned is recombinated by the Scatophagus argus (Linnaeus) LH that prokaryotic expression obtains
Albumen.
7th aspect, the invention further relates to a kind of fish preparation method of the external injection of recombinant protein, the method packets
Include following steps:
C1, using Trizol extraction method, carry out RNA extraction using Scatophagus argus (Linnaeus) sexual gland, then carry out by reversal agents box
The synthesis of cDNA；
C2, the cDNA conserved regions design primer synthesized according to step S1, using RT-PCR and RACE technology, clone is obtained
Obtain Scatophagus argus (Linnaeus) LH gene order overall length；
C3, Scatophagus argus (Linnaeus) lutropin LH gene cDNA sequence connect with pGEM-TEasy carrier after, conversion is to DH5 α
Competent cell, amplification cultivation；It is collected by centrifugation, extracting contains the plasmid of target gene (LH gene) and pGEM-TEasy carrier；
C4, according to pET-28a+Carrier selects restriction enzyme site XhoI (158), and NdeI (238) respectively extracts step A1
The Plasmid DNA arrived carries out double digestion；Recycle effective segment；
C5, purifying；Target gene is connected with the recovery product of plasmid, it is thin that connection product imports BL-21 expression competence
Born of the same parents；Centrifuge separation；Bacterium is chosen, chooses effective bacterium solution after bacterium colony PCR verifying；
C6, effective bacterium solution is transferred in the LB liquid medium containing kanamycins, 37 DEG C with volume ratio 1: 10, is shaken
Culture is swung, recombinant protein inducing expression is carried out, obtains recombination thick leach protein；
C7, recombination thick leach protein obtain pure Scatophagus argus (Linnaeus) LH recombinant protein by affine column purification, i.e., the described fish recombination
The outer injection of proteosome.
Preferably, the affine column purification includes the following steps:
B1,1ml nickel Ago-Gel FF or nickel NTA Ago-Gel FF prepacked column are taken, with 10ml equilibration buffer,
Then 10ml recombinates thick leach protein sample with 0.5ml/min loading, and then 2ml/ pipe is in charge of collection；
B2, unadsorbed recombination thick leach protein sample, flow velocity 1-2ml/min, 2ml/ pipe are washed away with 15ml equilibration buffer
It collects；
B3, unadsorbed recombination thick leach protein sample, flow velocity 1-2ml/min, 2ml/ pipe are washed away with 5ml elution buffer
It collects.
Compared with prior art, the invention has the following beneficial effects:
1) present invention is using molecules biological operation technologies such as RT-PCR and RACE, and clone obtains the cDNA of lutropin
Then sequence is made by the technologies construction recombination plasmid such as digestion by recombinant plasmid transformed to Escherichia coli in IPTG
With the expression of lower induction recombinant protein, then extracts, purifies.The present invention put forth effort on obtain it is a kind of it is safer, healthy, have
The hormone night of effect, in the process building, separation and the purifying of mentioned recombinant plasmid are external in aquaculture before being
What injection was not involved with.
2) present invention has filled up domestic and international and has prepared proteosome using the building of Scatophagus argus (Linnaeus) lutropin (LH) recombinant plasmid
Outer injection solves the problems, such as that Scatophagus argus (Linnaeus) gonad development is nonsynchronous.
3) due in feed addition feed recombinant protein exist generated by feedstuff feeding recombinant protein waste, albumen benefit
The problems such as not high with rate；And Gonadotropic Hormone in Fish is glycoprotein substance, is easy had the intracorporal protease of stomach fish to degrade,
To reduce its physiological function.Therefore, the present invention can more directly be such that vitro recombination LH acts on using injection induction mode
Fish body, using intuitively research LH to the regulating and controlling effect of the generation of fish gamete.
4) present invention has recognized Scatophagus argus (Linnaeus) lutropin LH recombinant protein to maturity period money using injection abductive approach
The influence of fish gamete development parameter.It can be from gene expression dose, serum associated hormone level and scanning tunneling microscope angle point
Analysis, thus it is speculated that its possible mechanism for the deep regulatory mechanism for recognizing Scatophagus argus (Linnaeus) Reproductive Axis (BPG-axis) and establishes practical gold
Money fish culture technology provides basis.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is pET-28a (+) vector plasmid map；
Fig. 2 is pET-28a vector multiple cloning site schematic diagram；
Fig. 3 is that enzyme-linked immunization (ELISA) measures hormonal readiness schematic diagram, wherein A is 11-KT (testosterone) female ♀ water
Flat schematic diagram, B are Estrogen (estradiol) female ♀ level schematic diagram, and C is 11-KT (testosterone) male ♂ level schematic diagram, D
For Estrogen (estradiol) male ♂ level schematic diagram；
Fig. 4 is scanning electron microscopic observation, shooting sexual gland structural schematic diagram；Wherein, A, B, E, F are control group；C, D, G, H are
Test group；
Fig. 5 is LH clone PCR schematic diagram；
Fig. 6 is that LH glue telegram in reply is swum, PCR send and surveys positive colony product electrophoresis verifying schematic diagram；Wherein, a is the telegram in reply swimming of LH glue
Schematic diagram, b are positive colony product electrophoresis schematic diagram；
Fig. 7 is double digestion pET-LH electrophoretogram；Wherein, in a pET-LH, b, upper left pET-28a, lower-left LH, right pET-
LH；
Fig. 8 is pET-LH recombinant protein Western test map；Wherein a is colour developing shooting figure, and the road 1-5 is coomassie
Brilliant blue dyes shooting figure, and 6,7 be Westernblot transferring film ECL colour developing shooting figure, and b is recombinant protein purification result schematic diagram；
Specific embodiment
The invention firstly uses the molecules biological operation technologies such as RT-PCR and RACE, obtain Scatophagus argus (Linnaeus) lutropin LH
The cDNA sequence overall length of gene obtains Scatophagus argus (Linnaeus) by prokaryotic expression system inducing expression and slightly mentions LH recombinant protein, by affine
Column purification obtains pure LH recombinant protein, is applied to fish hormone, promotes fish gonad development, makes fish gonad development
Show synchronism.
Specific step is as follows:
1) Trizol extraction method is utilized, carries out RNA extraction using Scatophagus argus (Linnaeus) sexual gland, then pass through reversal agents box (Roche)
The synthesis of cDNA is carried out, the cDNA of synthesis is stored in -20 DEG C, for use.
(Fig. 5 is LH clone PCR schematic diagram to the cDNA overall length 608bp of LH gene；As shown in Figure 5, LH clip size
608bp (Marker used is 2000bp, Takara)), sequence is as shown in SEQ ID NO:1, and wherein open reading frame is
438bp (the 106-543 base) encodes 145 amino acid, and the 1-22 amino acid is signal peptide, and there is an os osseum
The region fish paraspecific Cys-Ser-Gly-His (CSGH).In addition, having found a N- glycosyl in LH amino acid sequence
Change site (NHT), contains a tailing signal AAATAA in 3 ' end noncoding regions.
NCBI, ORFfinder LH cDNA sequence testing result are as follows:
ORF encodes amino acid: the 1-22 amino acid is signal peptide, and particular sequence is as shown in SEQ ID NO:2: MMAA
QVSRATFPLILFLEFTAAFQLPPCQLVNHTVSLEKEGCPKCHPVETTICSGHCLTK DPVIKIPFSNVYQHVCTYR
DLYYKTFELPDCPPGVDPTVTYPVALSCHCGRCAMDTSD CTFESLHPDFCMNDIPFYYPSEGMSLY。
2) by biosoftware BioEdit, the biosoftwares such as Primer5.0 utilize RT- according to conserved regions design primer
PCR and RACE technology, clone obtain Scatophagus argus (Linnaeus) LH (lutropin) gene order overall length.
Cloning primer:
| 3′LH1 | GTCTCTGGAGAAGGAGGGCTGT (SEQ ID NO:3) |
| 3′LH2 | TACAAGACATTTGAGCTTCCCGA (SEQ ID NO:4) |
| 5′LH1 | CAGGCTCTCGAAGGTGCAGTC (SEQ ID NO:5) |
| 5′LH2 | TCGGGAAGCTCAAATGTCTTGTA (SEQ ID NO:6) |
Again by being connect overnight, so with 16 DEG C of pGEM-TEasy carrier (Pu Luomaige (Beijing) Bioisystech Co., Ltd)
It is converted again afterwards into DH5 α competent cell (TIANGEN Biotech (Beijing) Co., Ltd.), after then shaking bacterium 3-6h, carries out bacterium
PCR verifying is fallen, effective bacterium solution is inoculated into fresh LB liquid medium, allows large intestine bar using enough LB liquid mediums
Bacterium massive amplification culture.
3) extract plasmid: 2) shaken bacterium bag includes the competent cell for being transferred to target gene and expression vector, amplification cultivation in
Later, first the Escherichia coli of amplification cultivation are collected by centrifugation, then utilize Takara plasmid extraction kit (the white good science and technology in Shanghai
Development Co., Ltd) a large amount of plasmid for extracting target gene and carrier.
4) double digestion: according to pET-28a+Carrier (Beijing Quanshijin Biotechnology Co., Ltd) selects restriction enzyme site
(carrier pET-28a (+) map is as shown in Figure 1, carrier size 5369bp by XhoI (158), NdeI (238)；Carrier pET-28a
The restriction enzyme site of selection is as shown in Figure 2 in (+)), double digestion is carried out to the Plasmid DNA of extraction respectively, by target fragment and expression
Plasmid carries out double digestion with restriction enzyme, and for digestion products after agarose electrophoresis detects, 2% agarose gel runs glue, uses
Promega glue goes back to kit (Pu Luomaige (Beijing) Bioisystech Co., Ltd) and recycles effective segment.
Digestion primer:
| pET(LH)-F | CATATGTTCCAGCTGCCGCCATGC (SEQ ID NO:7) |
| pET(LH)-R | CTCGAGTTAGTACAGAGACATCCCTTCAGACGG (SEQ ID NO:8) |
Fig. 7 is double digestion pET-LH electrophoretogram；By Fig. 7-a it is found that pET-LH size is about 6000bp, digestion is correct, returns
Receiving product is purpose segment (Marker used is 5000bp)；By Fig. 7-b it is found that because purchase pET-28a carrier segments size
For 5369bp, pET-LH size is about 6000bp in figure, meets digestion and is intended to (Marker used is 5000bp, Takara).
5) it purifies: glue being carried out by agarose and is returned, target gene recycling is lesser segment, and plasmid is then biggish
Section.Fig. 6 is LH glue telegram in reply swimming schematic diagram；By Fig. 6-a, b it is found that LH clip size 608bp (Marker used is 2000bp,
Takara)。
6) it connects: target gene is connected with the recovery product of plasmid, with 10: 1 ratio, 16 DEG C of connections are overnight.Agar
After sugared electroresis appraisal is correct, connection product is imported BL-21 expression competent cell, and (BL21 (DE3) competent cell Tiangeng is raw
Change scientific and technological (Beijing) Co., Ltd) in, it is added in the EP pipe of the 1.5ml equipped with fresh 1mlLB fluid nutrient medium, shakes bacterium.
7) digestion detects: carrying out double digestion and single endonuclease digestion (being all 2) detection respectively to connection product.It will carry purposeful
The bacterium solution of the plasmid of gene and carrier is inoculated in 50mlLB fluid nutrient medium (Amp+Working solution concentration 50ug/ml), 37 DEG C,
200rpm, shakes bacterium 6-8h, and double digestion group compares mutually with single endonuclease digestion group.
8) bacterium solution shaken is collected by centrifugation, is then applied to Kana+LB plate, every group of 10 samples carry out choosing bacterium,
It is placed in equipped with 1mlKana+LB liquid medium 1.5mlEP pipe in shake bacterium 3-6h, carry out bacterium colony PCR verifying, choose above-mentioned
Effective bacterium solution in step carries out next inoculation work.
9) save strain: by 50% 8. effective bacterium solution mix with 50% glycerol, shake up, (glycerol concentration exists for -80 DEG C of preservations
30-70%.)
10) above-mentioned 8) bacterium solution is transferred in new LB, 37 DEG C, 200rpm with 1: 100, carries out mass propgation 2-3h；When
After bacterium grows to OD=0.4-0.6, IPTG working solution is added to its final concentration of 1mM, 37 DEG C of Fiber differentiation 6-8h, collects bacterium solution
(being control group, no IPTG).
11) Protein Detection: 4 DEG C, 12,000g centrifugation 5min, bacterial precipitation is collected, 1 × PBS rinsing (is careful not to molten
Solution), then dissolution precipitating, adds 2 × LoadingBuffer, and 100 DEG C are boiled 5min, is cooled to room temperature, and then 10,000g is centrifuged
10min takes 5ul supernatant to carry out SDS-PAGE electrophoresis.Examine dye or WesternBlot analysis, purpose band occur, protein abundance compared with
Height carries out inducible protein amplification cultivation.Fig. 8 is pET-LH recombinant protein Western test map；By Fig. 8-a, b is it is found that albumen
Gene expression abundance is higher, and size is about 16kDa.
12) recombinant protein inducing expression: take it is above-mentioned 9) in -80 DEG C preservation pET-LH/BL and pET-28a/BL freeze bacterium
Liquid 100ul is connected to 1ml (Kana containing kanamycins+) fresh LB liquid medium in, 37 DEG C, shaken cultivation 1h.Then draw
Appropriate bacterium solution is applied to containing kanamycins (Kana+) LB solid plate culture medium on, 37 DEG C of culture 10-14h.
13) picking monoclonal is connected to lml (Kana containing kanamycins+) LB liquid medium 1.5mlEP pipe in, 37 DEG C,
Shaken cultivation 8-10h is to allow Escherichia coli massive amplification (growing to Escherichia coli logarithmic growth phase).It, will after bacterium solution PCR verifying
Bacterium solution containing target gene Insert Fragment is inoculated in the fresh (Kana of 400ml with the ratio of 1: 100 (v/v)+Final concentration 50ug/
Ml) in LB liquid medium, 37 DEG C of amplification cultivations.
14) recombination thick leach protein obtains: the bacterium solution of collection being centrifuged 10min in 4000-5500rpm, culture medium is removed, uses
Thallus is resuspended in the PBS buffer solution of pH=7.4.Ice-bath ultrasonic is crushed thallus.Parameter is set as voltage 200V, and 2s is broken, between 2s
Every limpid to bacterium solution repeatedly.4000-5500rpm is centrifuged 10min, is precipitated as cell fragment, and supernatant is the thick egg of thallus
It is white.Aspirate supernatant, -20 DEG C save backup.
15) solution reagent: (purifying agents useful for same is purchased from raw work bioengineering Shanghai limited liability company) equalizing and buffering
Liquid, the 50mM phosphate buffer NaCl containing 0.5M of pH=7.4, imidazoles containing 20mM.Elution buffer, the 50mM phosphorus of pH=7.4
Acid buffer NaCl containing 0.5M, imidazoles containing 500mM.
16) recombinant protein isolates and purifies: taking 1ml nickel Ago-Gel FF or nickel NTA Ago-Gel FF prepacked column, uses
Then 10ml equilibration buffer takes broken supernatant 10ml sample with 0.5ml/min loading, then 2ml/ pipe is in charge of collection.
Unadsorbed sample is washed away with 15ml equilibration buffer, flow velocity 1-2ml/min, 2ml/ pipe is collected.It is washed with 5ml elution buffer
Unadsorbed sample is removed, flow velocity 1-2ml/min, 2ml/ pipe is collected.5ml equilibration buffer pillar is used again, fills 20% second
Alcohol, closing, in case next time uses.The part of collection Kao Masi brilliant blue method, SDS-Page electrophoresis, Western-Blot detection.
The following describes the present invention in detail with reference to examples.Following embodiment will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that those skilled in the art
For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to guarantor of the invention
Protect range.
Embodiment
1, recombinant protein inducing expression: the pET-LH/BL and pET-28a/BL of above-mentioned -80 DEG C of preservations is taken to freeze bacterium solution
100ul is connected to 1ml (Kana containing kanamycins+) fresh LB liquid medium in, 37 DEG C, shaken cultivation 1-3h.Then draw
Appropriate bacterium solution is applied to containing kanamycins (Kana+) LB solid plate culture medium on, 37 DEG C of culture 10-14h.Picking monoclonal connects
In 1ml (Kana containing kanamycins+) LB liquid medium 1.5mlEP pipe in, 37 DEG C, shaken cultivation 8-10h is to allow large intestine bar
Bacterium massive amplification (grows to Escherichia coli logarithmic growth phase).
2, after bacterium solution PCR verifying, the bacterium solution containing target gene Insert Fragment is inoculated in the ratio of 1: 100 (v/v)
Fresh (the Kana of 400ml+Final concentration 50ug/m1) in LB liquid medium, 37 DEG C of amplification cultivations.
3, recombination thick leach protein obtains: the bacterium solution of collection being centrifuged 10min in 4000-5500rpm, culture medium is removed, uses
Thallus is resuspended in the PBS buffer solution of pH=7.4.Ice-bath ultrasonic is crushed thallus.Parameter is set as voltage 200V, and 2s is broken, between 2s
Every limpid to bacterium solution repeatedly.4000-5500rpm is centrifuged 10min, is precipitated as cell fragment, and supernatant is the thick egg of thallus
It is white.Aspirate supernatant, -20 DEG C save backup；
4, solution reagent: (purifying agents useful for same is purchased from raw work bioengineering Shanghai limited liability company) equalizing and buffering
Liquid: the 50mM phosphate buffer NaCl containing 0.5M of pH=7.4, imidazoles containing 20mM.Elution buffer: the 50mM phosphorus of pH=7.4
Acid buffer NaCl containing 0.5M, imidazoles containing 500mM.
5, recombinant protein isolates and purifies: 1ml nickel Ago-Gel FF or nickel NTA Ago-Gel FF prepacked column are taken first,
With 10ml equilibration buffer, then take broken supernatant 10ml sample with 0.5ml/min loading, then 2ml/ pipe is in charge of receipts
Collection.Unadsorbed sample is washed away with 15ml equilibration buffer, flow velocity 1-2ml/min, 2ml/ pipe is collected.With 5ml elution buffer
Unadsorbed sample is washed away, flow velocity 1-2ml/min, 2ml/ pipe is collected.5ml equilibration buffer pillar is used again, fills 20%
Ethyl alcohol, closing, in case next time uses.The part of collection Kao Masi brilliant blue method, SDS-Page electrophoresis, Western-Blot inspection
It surveys.
6, hormone: being diluted using physiological saline, and configuration concentration is the recombinant protein injection of 5-10ng/ml,
Respectively to three groups (blank group: do not inject any drug and drug, control group: injection and the physiological saline of experimental group same dose,
Experimental group: injection concentration is the recombinant protein of 5-10ng/ml, injection volume 1.5-2ml/100g) Scatophagus argus (Linnaeus) progress intramuscular injection
(dorsal fin) takes sexual gland, serum if sampling time point is set as 0d, 7d, 14d.
7, result detects: the detection of molecular level is carried out to the sample adopted, via enzyme-linked immunization (ELISA reagent
Cayman company, box Germany) to the testosterone in serum, estradiol carries out Concentration Testing.Separately by the sexual gland adopted by handling,
It is observed and is shot under scanning electron microscope.
It is as shown in Figure 3 that enzyme-linked immunization (ELISA) measures hormonal readiness, it is seen that in 0d, 7d, 14d, Scatophagus argus (Linnaeus) blank group,
Control group, the testosterone in experimental group serum, estradiol concentration difference.
Estradiol content 391pg/ml at 0 day in the female money fish serum of experimental group, content is 578pg/ at 7 days
Ml significantly rises, and content is reduced to 305pg/ml at 14 days, and decreases.And testosterone concentration is from 0 day to 14 day in serum
Content is between 11-17pg/ml, before and after the processing without significant change (* p < 0.05).
Testosterone content 50pg/ml or so at 0 day and 7 days in the male money fish serum of experimental group, content is at 14 days
132pg/ml significantly rises.And day content is between 6.5-7.5pg/ml from 0 day to 14 for estradiol content in serum, processing
Front and back is without significant change (* p < 0.05).
Scanning electron microscopic observation, shooting sexual gland structural schematic diagram are as shown in figure 4, Scanning Tunneling Microscopy discovery, processing
Front and back ovary tissue experimental group and control group are without significant change.It is asynchronous that sperm development occurs in control group spermary, experimental group sperm
It develops more consistent.Illustrate that hormone LH recombinant protein can promote Scatophagus argus (Linnaeus) gamete and generate and develop.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
SEQUENCE LISTING
<110>Shanghai Ocean University
<120>Scatophagus argus (Linnaeus) lutropin LH gene, Scatophagus argus (Linnaeus) LH recombinant protein and application
<130> 2016
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 608
<212> DNA
<213> Scatophagus argus
<400> 1
ggaaactgcc tgcaggctgc agacgggact gttcaaaaca cgcccactag ctggcacacc 60
tgacaatcaa cagaacaact aagtaaatta ttgactgact tcaggatgat ggctgcacag 120
gtcagcagag cgacgttccc cttgatactg tttctggaat tcacagcggc cttccagctg 180
ccgccatgcc agctcgtcaa ccacacggtg tctctggaga aggagggctg tcccaagtgt 240
cacccggtgg aaacaaccat ctgcagtggg cactgcctca ccaaggatcc tgtcattaag 300
ataccgttca gcaacgttta ccagcacgtc tgcacgtacc gggacttgta ctacaagaca 360
tttgagcttc ccgactgtcc acccggcgtg gacccgacgg tcacctaccc cgtggctctg 420
agctgccact gcggccgctg cgccatggac acatccgact gcaccttcga gagcctgcac 480
ccagacttct gcatgaacga catacctttc tactacccgt ctgaagggat gtctctgtac 540
taaagcacac gacacgtaca aaaactgtgc tgacttcaaa tattgtaaat aaagattgtt 600
gcatttac 608
<210> 2
<211> 145
<212> PRT
<213> Scatophagus argus
<400> 2
Met Met Ala Ala Gln Val Ser Arg Ala Thr Phe Pro Leu Ile Leu Phe
1 5 10 15
Leu Glu Phe Thr Ala Ala Phe Gln Leu Pro Pro Cys Gln Leu Val Asn
20 25 30
His Thr Val Ser Leu Glu Lys Glu Gly Cys Pro Lys Cys His Pro Val
35 40 45
Glu Thr Thr Ile Cys Ser Gly His Cys Leu Thr Lys Asp Pro Val Ile
50 55 60
Lys Ile Pro Phe Ser Asn Val Tyr Gln His Val Cys Thr Tyr Arg Asp
65 70 75 80
Leu Tyr Tyr Lys Thr Phe Glu Leu Pro Asp Cys Pro Pro Gly Val Asp
85 90 95
Pro Thr Val Thr Tyr Pro Val Ala Leu Ser Cys His Cys Gly Arg Cys
100 105 110
Ala Met Asp Thr Ser Asp Cys Thr Phe Glu Ser Leu His Pro Asp Phe
115 120 125
Cys Met Asn Asp Ile Pro Phe Tyr Tyr Pro Ser Glu Gly Met Ser Leu
130 135 140
Tyr
145
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 3
gtctctggag aaggagggct gt 22
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 4
tacaagacat ttgagcttcc cga 23
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 5
caggctctcg aaggtgcagt c 21
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 6
tcgggaagct caaatgtctt gta 23
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 7
catatgttcc agctgccgcc atgc 24
<210> 8
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 8
ctcgagttag tacagagaca tcccttcaga cgg 33
Claims (8)
1. a kind of Scatophagus argus (Linnaeus) lutropin LH gene, cDNA sequence is the base sequence as shown in SEQ ID NO:1.
2. a kind of Scatophagus argus (Linnaeus) LH recombinant protein, which is characterized in that the recombinant protein is the amino acid as shown in SEQ ID NO:2
Sequence.
3. a kind of preparation method of Scatophagus argus (Linnaeus) LH recombinant protein as claimed in claim 2, which is characterized in that obtain as right is wanted
The cDNA sequence overall length of Scatophagus argus (Linnaeus) lutropin LH gene described in asking 1 obtains Scatophagus argus (Linnaeus) LH by prokaryotic expression and recombinates egg
It is white.
4. preparation method according to claim 3, which is characterized in that the acquisition includes the following steps:
S1, using Trizol extraction method, carry out RNA extraction using Scatophagus argus (Linnaeus) sexual gland, then carry out cDNA's by reversal agents box
Synthesis；
S2, the cDNA conserved regions design primer synthesized according to step S1, using RT-PCR and RACE technology, clone obtains gold
Money fish LH gene order overall length.
5. preparation method according to claim 3, which is characterized in that the prokaryotic expression includes the following steps:
A1, Scatophagus argus (Linnaeus) lutropin LH gene cDNA sequence connect with pGEM-TEasy carrier after, conversion to DH5 α experience
State cell, amplification cultivation；It is collected by centrifugation, extracts the plasmid containing target gene and pGEM-TEasy carrier；
A2, according to pET-28a+Carrier selects restriction enzyme site XhoI, NdeI, the Plasmid DNA that step A1 is extracted respectively into
Row double digestion；Recycle effective segment；
A3, purifying；Target gene is connected with the recovery product of plasmid, connection product imports BL-21 and expresses competent cell；From
Heart separation；Bacterium is chosen, chooses effective bacterium solution after bacterium colony PCR verifying；
A4, effective bacterium solution is transferred in the LB liquid medium containing kanamycins with volume ratio 1:10,37 DEG C, oscillation training
It supports, carries out recombinant protein inducing expression, obtain recombination thick leach protein.
6. preparation method according to claim 3, which is characterized in that further include after prokaryotic expression by affine column purification,
The step of obtaining pure Scatophagus argus (Linnaeus) LH recombinant protein.
7. preparation method according to claim 6, which is characterized in that the affine column purification includes the following steps:
B1,1mL nickel Ago-Gel FF or nickel NTA Ago-Gel FF prepacked column are taken, with 10mL equilibration buffer, then
10mL recombinates thick leach protein sample with 0.5mL/min loading, and then 2mL/ pipe is in charge of collection；
B2, unadsorbed recombination thick leach protein sample is washed away with 15mL equilibration buffer, flow velocity 1-2mL/min, 2mL/ pipe is collected；
B3, the recombination thick leach protein sample adsorbed is washed away with 5 mL elution buffers, flow velocity 1-2mL/min, 2mL/ pipe is collected.
8. a kind of fish external injection of recombinant protein, which is characterized in that the hormone liquid includes Scatophagus argus (Linnaeus) LH recombination egg
It is white；The Scatophagus argus (Linnaeus) LH recombinant protein is the cDNA sequence for obtaining Scatophagus argus (Linnaeus) lutropin LH gene as described in claim 1
Column overall length is obtained by prokaryotic expression.
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| CN102080095A (en) * | 2010-12-03 | 2011-06-01 | 中山大学 | Eukaryotic expression vector containing mature peptide gene of epinephelus coioids gonadotropin hormone I, recombinant strain, recombinant protein and application thereof |
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Non-Patent Citations (5)
| Title |
|---|
| Scatophagus argus luteinizing hormone mRNA, complete cds;Wang,W.;《GenBank》;20170402;全文 |
| 草鱼促性腺激素及其受体基因的克隆及表达分析;周毅;《中国优秀硕士论文全文数据库 农业科技辑》;20091115;第2009年卷(第11期);全文 |
| 金钱鱼促性腺激素基因的克隆、原核表达及功能验证;王武;《中国优秀硕士论文全文数据库 农业科技辑》;20180215;第2018年卷(第2期);全文 |
| 鱼类促性腺激素基因研究进展;吕绍巾;《生物学杂志》;20170630;第34卷(第3期);全文 |
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