CN1432577A - Sheep growth hormone releasing hormone gene and its expression product and application - Google Patents
Sheep growth hormone releasing hormone gene and its expression product and application Download PDFInfo
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- CN1432577A CN1432577A CN 02100065 CN02100065A CN1432577A CN 1432577 A CN1432577 A CN 1432577A CN 02100065 CN02100065 CN 02100065 CN 02100065 A CN02100065 A CN 02100065A CN 1432577 A CN1432577 A CN 1432577A
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Abstract
The present invention aims at providing sheep growth hormone releasing hormone gene and its encoded sheep growth hormone releasing hormone. The gene can be well expressed in animal body and behaves as the obvious improvement of animal growth characteristics. The nucleotide sequence of the sheep growth hormone releasing hormone gene consists of 135 nucleotides and encodes protein comprising 44 amino acid residues. The present invention also aims at providing one guide peptide to replace GHRH to ensure the guide protein to be secrete to outside cell and enter into body fluid circulation. Medicine with the present invention as active component may be used in promoting growth hormone secretion of sheep, improving growth characteristic of sheep and treating muscular dystrophy diseases.
Description
Technical field
The present invention relates to sheep growth hormone releasing hormone gene and the expression product and the application of a kind of synthetic gene and expression product thereof and application, particularly a kind of synthetic.
Background technology
(Growth Hormone Releasing Hormone GHRH) is a kind of peptide hormone of excretory in the animal hypothalamus to growth hormone releasing hormone.Its mechanism of action is, growth hormone releasing hormone is as tethelin (the Growth Hormone of hypophysis, GH) positivity regulatory factor, can promote the synthetic and secretion tethelin of hypophysis, improve body inner growth hormone level, and (Insulin-like GrowthFactor I, IGF-I) level improves, and then improves the production performance of animal to make the general insulin-like growth factor by tethelin.Therefore, GHRH has important use value in livestock industry.
One, the rise of GHRH research
Buger in 1973 extracts from acromegaly patient's pancreatic neoplasm GH is secreted the extract that hormesis is arranged.Frohman in 1980 tumour outside acromegaly patient's hypophysis is separated to partially purified GHRH.Thorner carries out hypophysectomy to the acromegaly patient with Turner ' s disease, do not take effect, after find pancreas tumour arranged, and extract it, then the GH level is reduced to 2ng/ml by 70ng/ml in the blood.Guillemin and Vale be respectively to the tumor tissues vitro culture with analyze to obtain, and having what promote the GH release action is two different peptides, and two kinds all have activity, and they are made up of 44 and 40 amino-acid residues respectively.The structure of 44 peptides is: junket-third-asparagus fern-third-different is bright-phenylpropyl alcohol-Su-l-asparagine-Si-junket-essence-Lai-figured silk fabrics-bright-Gan-paddy ammonia-bright-Si-third-essence-Lai-bright-bright-paddy ammonia-asparagus fern-different bright-first sulphur-Si-essence-paddy ammonia-paddy ammonia-Gan-paddy-Si-l-asparagine-paddy ammonia-paddy-essence-Gan-third-essence-third-essence-bright-NH2.This material is referred to as human pancreas's somatotropin releasing factor (hpGHRH).
Vale has also reported two kinds respectively by 40 and 37 peptides that amino-acid residue is formed.The in vitro study of these two kinds of peptides has all proved biological activity, and the highest with the GHRH40 activity.Guillemin thinks that GHRH37 and GHRH40 are the degradation product of GHRH44.Confirmed that at present people's hypothalamus GHRH is 44 peptides.
Someone separates the GHRH active substance from several thousand people's hypothalamus.Guillemin in 1984 proof is from hypothalamic GHRH identical with from Vipoma.The GHRH of rat has that 15 amino acid and people's is inequality.The GHRH of rat is 43 peptides, and a free C-terminal is arranged.The hypothalamic GHRH of ox and pig is 44 peptides, carboxylic end amideization.Just data GHRH has species variation at present.
Two, GHRH distribution in vivo
GHRH is in the distribution of central nervous system
Immunohistochemistry confirms that the cell space of GHRH is positioned at arcuate nucleus, and to studies show that of people, monkey, pig and rat, the GHRH cell space is very intensive in arcuate nucleus.Electrophysiologic study confirms that the electricity irritation ventromedial nucleus can cause the GH secretion.In addition, hippocampus, almond, shell nuclear cat also have the GHRH cell space to distribute near the medial forebrain bundle of rat, dorsomedial nucleus and periventricular nucleus and the arched roof.
GHRH has distribution widely in central nervous system, and is the highest with hypothalamus concentration especially.People and cat GHRH send at neural end and are symmetrically distributed in the median eminence outside, are parallel to capillary vessel, end at the hypophyseal portal vessel blood vessel.The GHRH fiber of rat mainly is distributed in median eminence central authorities.
The distribution of GHRH beyond central nervous system
Find that monkey GHRH is distributed in the prepituitary gland cell, also have the active sample material of GHRH at people's placenta, pancreas, recording with RIA in people's the extract of jejunum, duodenum and stomach has GHRH.
Three, the physiological action of GHRH and mechanism
GHRH and Somat claim that also (Somatostatin, SS) secretion to GH plays the dual regulation effect to Somatostatin.Under the normal circumstances, GH discharges with certain hour spacing pulse formula every day.The town is about 1～3h at a cycle.Give clear-headed rat injection GHRH, minimum in the paddy phase hormesis that GH discharges, then very obvious in the peak phase.But after the processing of rat usefulness SS antiserum(antisera), GHRH discharges the release that the paddy phase also can stimulate GH at GH.Therefore illustrate that SS plays key effect to the release of GH.V.Padmanabhan etc. (1987) also obtain identical result, adenohypophysis with ox carries out vitro culture, when the GHRH that adds 10: 2～10: 7 in the medium, the secretion of GH is linear to be increased, especially reached the climax with 10: 7 o'clock, but when adding SS again in the medium then the release effects of the adenohypophysis GH negative that is subjected to SS suppress.GHRH is necessary to the pulse that disengages of GH.In vitro study shows that GHRH has hormesis to the release of GH.
People GHRH 2～20ng that male mouse tricorn injects trace causes the GH excretory to reduce and suppresses spontaneous pulse release, but when tricorn injects higher amount GHRH 0.2～2ug in 15～20 minutes blood plasma the GH secretion peak appears.Someone thinks that the interior GHRH of hypothalamus has direct effect to endogenous GHRH and SS secretion.The Katakani report, quiet notes SS antiserum(antisera) tricorn capable of blocking injects GHRH to GH excretory restraining effect, and prompting maincenter GHRH suppresses the secretion of GH by the release that stimulates SS.Think that at present SS participates in the ultrashort feedback of GHRH self.
GHRH arrives adenohypophysis through hypophyseal portal system, and combines with eosinophil, causes biological effect by cAMP in the film.May be GHRH and become hormone-receptor complex after film combines, activate earlier a kind of pungency G albumen (Stimulatory G protein, Gs).The proteic activation of G needs GTP to participate in, G albumen inactivation then when GTP becomes GDP.The catalytic subunit of AC on the Gs activated membrane makes ATP be converted into cAMP.Promote the synthetic of GH and discharge by this process.
People attempt improving in several ways GHRH level in the animal body in recent years, and then promote the synthetic and secretion of hypophysis GH, thereby realize improving the speed of growth and the carcass lean meat percentage of animal.Studies show that injection GHRH can significantly improve the speed of growth and the lean ratio of animal.But the transformation period of GHRH is very short, has only tens minutes in vivo, and therefore, in the application of reality, injection GHRH has brought inconvenience to production.Therefore, improving animal self GHRH by some short synthesis capabilities is trend of the times.The interpolation growth additive that has increases the synthetic of GHRH in the animal hypothalamus; What have then is the level that increases GHRH in the body by the application of carrier for expression of eukaryon.
Because GHRH can promote the growth of muscle, this is just implying another very important potential---the treatment of muscular dystrophy of GHRH.
Amyotrophy is meant the voluntary muscle malnutrition, and muscle volume is little than normal volume, and myofiber attenuates even disappears.Neuromuscular disease sex change hypertrophy.The muscle nutrition situation more has substantial connection with neural system except that the pathological change of muscle tissue itself.Diseases of spinal cord often causes muscular dystrophy and amyotrophy takes place, and they are a kind of common symptons neural, muscle disease.
The research of muscle disease has become one of most active fields in the neuroscience at present, and this is because feasible molecular defect, the pathogenesis to muscle disease of molecular biology, immunology and biochemical progress had further research.
From the dissection aspect, muscle tissue does not belong to neural system, but muscle is body under central nervous system is regulated, and adapts to and one of the indispensable effector of transforming the objective world.And, the contraction of each muscle, directly undertaken again by motorial domination, therefore, muscle disease is that muscle itself and neuromuscular junction (cynapse) are located, because one group of disease due to inherited genetic factors, endocrine metabolism, the inflammation, reasons such as poisoning, virus infection, immunity and trace element.
The disease of muscle itself is myofibrosis, the atrophy that is caused by the change of myofiber chemistry composition, metabolic disturbance or unknown cause etc.In recent years, someone thinks that the pathology of muscle itself also may originate from neurogenic, cause the change of muscle biochemical metabolism, or because genetic sudden change causes the dyssynthesis of some corresponding enzyme system, be secondary to sex change, the atrophy, unable of the disorder generation muscle of these specific enzymes systems, a few patients is with obesity.This class patient often has the enzyme system of muscle metabolism unusual in the progressive stage of disease, particularly abnormal carbohydrate metabolism.Bibliographical information was also arranged in recent years, and the pathogenesis of myopathy is relevant with immune factor, common muscle disease, and as myasthenia gravis, the muscular strength syndromes, the morbidity of sudden muscle, dermatitis etc. is all relevant with immunological competence.
The myopathy no matter which kind of reason causes, basic clinical manifestation be nothing more than myasthenia or myotony, amyotrophy, false hypertrophy, myalgia etc.But in some neurogenic disease, the similar symptom of above-mentioned common also can appear.An important means for the treatment of such illness is the growth that recovers muscle, so GHRH is regarded as a kind of candidate's gene therapy means.
Summary of the invention
Main purpose of the present invention provides the sheep growth hormone releasing hormone of a kind of sheep growth hormone releasing hormone gene and coding thereof, and this gene is better expressed at animal capable, shows as significantly improving of the animal production traits.
The nucleotides sequence of sheep growth hormone releasing hormone gene of the present invention is classified No. 1 sequence in the sequence table as, is made up of 135 Nucleotide, and what its was encoded is the protein of sequence 2, is made up of 44 amino-acid residues.
Two of purpose of the present invention provides the guiding peptide of a kind of GHRH of replacement self.Thereby can guarantee to guide this protein excretion to the extracellular, enter the body fluid circulation.
The membranin guiding peptide nucleotide sequence of muscle is No. 3 sequences in the sequence table, is made up of 63 Nucleotide.What its was encoded is the protein of sequence 4, is made up of 21 amino-acid residues.
In the present invention, sequence 1 is chain with the DNA of sequence 3, and sequence 2 also is chain with the protein of sequence 4.
For the nucleotides sequence that promotes sheep growth hormone releasing hormone gene of the present invention is listed in intramuscular expression, be added with controlling element 5 ' and 3 ' the flanking nucleotide sequence that drives adjusting in the both sides of sheep growth hormone releasing hormone gene of the present invention.
5 ' flanking nucleotide sequence is No. 5 sequences in the sequence table, is made up of 2445 Nucleotide, is called 5 '-flanking.
3 ' flanking nucleotide sequence is No. 6 sequences in the sequence table, is made up of 805 Nucleotide, is called 3 '-flanking.
The plasmid and the biomass cells that contain sequence 1, sequence 3, sequence 5, sequence 6 all should belong to protection scope of the present invention.
Three of purpose of the present invention provides a kind of medicine that promotes growth hormone secretion, and the activeconstituents of this medicine is the sheep growth hormone releasing hormone Nucleotide of sequence 1 or the sheep growth hormone releasing hormone of sequence 2.
Four of purpose of the present invention provides a kind of medicine for the treatment of muscle disease, and the activeconstituents of this medicine is the sheep growth hormone releasing hormone Nucleotide of sequence 1 or the sheep growth hormone releasing hormone of sequence 2.
In order to improve the effectiveness of medicine, in said medicine, also comprise pharmaceutically acceptable carrier, wherein liposome preferably.
Liposome technology be called " biological missile " the 4th generation the target administration technology, liposome belongs to the nano-grade medicine carrier, this technology is utilized the monopolizing characteristic of liposome, toxic side effect is big, poor stability, the fast pharmaceutical pack of degraded are rolled in the liposome in blood, because lesions position vascular endothelial cell gap is bigger, liposome medicament can see through this gap and arrive lesions position, piles up in focus portion to discharge, and reaches targeted delivery of drugs.
Liposome is to be formed for film material inclusion by phosphatide, cholesterol etc.These two kinds of compositions still do not form the basic substance of liposome bilayer, and itself have very important physical function yet.By structure and particle diameter, liposome can be divided into unilamelar liposome, multilamelar liposome, contain the liposome of tensio-active agent.By performance, liposome can be divided into general liposome (comprising above-mentioned unilamelar liposome, multilamelar liposome and multiphasic liposomes etc.), property liposome, thermal sensitive liposome, pH sensitive liposome body, ultrasonic wave sensitive liposome body, photosensitive liposome and magnetic liposome etc.Press charge, liposome can be divided into neutral fat plastid, electronegativity liposome, positive polarity liposome.
Felgner in 1987 etc. use artificial synthetic cationic-liposome N-first, and (1-(2,3-two oleoyl oxygen) propyl group)-N1N1N-oxidation TMA (TriMethylAmine) (DOTMA) structure small unilamellar vesicle, form liposome-DNA mixture with the spontaneous effect of DNA, promoted the stably express that cell is taken in and born of the same parents are interior of DNA.Research afterwards finds further that again the cationic-liposome activity that has neutral phosphatide to participate in formation is stronger.Lip promptly is by cation lipid (DOTMA) and neutral phosphatide DOPE (DOPE) synthetic liposome.Be applicable to various nucleic acid are imported in the cultured cells.Its mechanism be cationic-liposome by electrostatic attraction effect and the electronegative spontaneous formation mixture of nucleic acid, net charge is that positive mixture is easy to be attached to electronegative cell surface again, with cytogamy, enters in the born of the same parents by endocytosis.Therefore it is different with traditional liposomal, is not that nucleic acid is wrapped in the lipid somatocyst, but is attached to the surface, only need to mix and can capture almost 100% nucleic acid, and the nucleic acid encapsulation ratio of traditional liposomal is less than 10%.Compare with calcium sulfate coprecipitation method or diethylaminoethyl-(DEAE)-dextran method, the nucleic acid transfection efficient of using Lip will exceed 5～100 times.Therefore cationic-liposome is used conveniently, and is with the obvious advantage, is widely used in recent years DNA, and RNA and protein import viable cell.But it is rare about the report of using this type of liposome-mediated micromolecular relatively antisense oligodeoxynucleotide (ASODN) transfection viable cell.Stoolman discovered in 1989, and the amount that DOTMA can make cell take in ASODN increases more than 10 times, and makes the biological activity of ASODN strengthen 100 times because significantly changed it in intracellular situation.Chiang in 1991 etc. confirm in mammalian cell that first cationic-liposome can make the effect of ASODN obviously strengthen.Nestle in 1994 etc. act on horn cells after the ASODN of bonding molecule 1 (ICAM-1) mRNA between Lip and targeted cells is mixed, and make the expression decreased 50% of ICAM-1, and only do not reduce 30% when having the Lip mediation.Inventor's experimental result shows that also the effect of the special ASODN of people DNA-pol α mRNA inhibition human liver cancer cell propagation under the Lip mediation has strengthened 10 times.Therefore particularly Lip can be as simple and effective launch vehicle for each lipoid plastid, and mediation GHRH enters vitro culture or cells in vivo, solves the problem that cell is taken in difficulty, thereby improves the action effect of GHRH greatly, reduces injection volume, reduces cost.
In addition, take in the efficient of GHRH, when injection, give the service efficiency that the suitable electric pulse stimulation in injection site also will improve GHRH for promoting muscle cell.
The present invention by design artificial gene order, realizes producing GHRH by the muscle cell of animal body self, and is discharged in the body fluid circulation, the adjustment of the participation animal production traits according to GHRH can promote growth of animal on physiology regulating effect.Considered following factor during design: guarantee that 1, the GHRH gene can express in muscle cell, select for use the protein expression element that abundance is high in the muscle to drive the GHRH expression of gene.2, for guarantee that the GHRH that expresses can pass cytolemma and enter the body fluid circulation in muscle cell, selected the suitable guide peptide.What guiding GHRH secreted membrane is muscle M-Cadherin (muscle calcium-dependent intercellularadhesion) guiding peptide, and M-Cadherin is that calcium dependent iuntercellular is sticking plain.The collaborative guiding of sheep growth hormone releasing hormone gene peptide gene synthetic together is comparatively ideal scheme.The guiding peptide of muscle cell internal secretion albumen and membrane-spanning protein all can be used to use.
Below in conjunction with accompanying drawing specific embodiments of the invention are described further.
Description of drawings
Fig. 1 is the structure synoptic diagram of expression vector.
Fig. 2 is the structure synoptic diagram of cloning by expression of the present invention.
Fig. 3 is the evaluation respectively of the plasmid pGEM-A3F that contains the plasmid pGEM-A5F of 5 ' flanking nucleotide sequence (2442bp) and 3 ' flanking nucleotide sequence (805bp).
Fig. 4 is the plasmid pGEM-A3F of 3 ' flanking nucleotide sequence and the result after two the cutting of 5 ' flanking nucleotide sequence plasmid pGEM-A5F process Nde I+Nsi I.
Fig. 5 makes up for expression vector pGEM-A5F3F.
Fig. 6 is the evaluation of expression vector plasmid pGEM-A5F3F.
Fig. 7 identifies for the splicing that artificial synthetic contains the GHRH sequence that guides peptide.
Fig. 8-A is structure and the evaluation of expression plasmid pGEM-A5F3F-M-GHRH.
Fig. 8-B is structure and the evaluation of expression plasmid pGEM-A5F3F-M-GHRH.
Embodiment
Material and reagent:
1, bacterial strain and plasmid
Bacillus coli DH 5 alpha and carrier pGEM-T vector.PGEM-T vector carrier is the carrier of the clone PCR products of Promega company production, and it has Amp screening resistance and lac Z selection markers, is being used down convenient screening positive plasmid with host bacterium DH5 α.
Expression vector is connected with goal gene:
A, the expression vector pGEM-A5f3f that makes up is carried out enzyme with Sna B1 cut, then mend and put down.And then utilize SalI that it is carried out enzyme and cut.
B, utilize Sal I that synthetic GHRH fragment enzyme is cut.
The result that C, connection A, B enzyme are cut forms cloning by expression.
2, reagent and other material
Cloning vector pGEM-T vector, ligase enzyme, agarose and molecular weight of albumen standard are available from Promega company; Restriction enzyme is available from Biolab company and Promega company, and it is that worker SK 131 products are given birth in Shanghai that DNA reclaims test kit.
Various damping fluids such as TE, the STE of solution I, II, III and pH8.0 that plasmid extracts, Tris, edta buffer liquid are all according to the formulated in Science Press's publication " molecular cloning experiment guide " (second edition).
3, approaches and methods:
The preparation of the alkaline lysis of plasmid be utilize plasmid DNA by behind the alkali environment than the easier renaturation of genomic dna, and can remove simultaneously that a large amount of proteic principles carry out.After sex change and renaturation, plasmid can get off by ethanol or isopropanol precipitating.
(1) a large amount of preparations of plasmid DNA and purifying (alkaline lysis):
One single bacterium colony access is contained in the 200mlLB liquid nutrient medium of Amp 37 ℃ distant bed overnight incubation.
↓
Inoculum is moved into the centrifuge tube of 50ml, reclaimed bacterium in centrifugal 5 minutes in 4 ℃ of 8000rpm.
↓
Outwell supernatant, bacterial precipitation is resuspended among the STE of 10ml ice precooling fully vibration washing.
↓
4 ℃, 8000rpm reclaimed bacterium in centrifugal 5 minutes again.
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The solution I that adds the precooling of 1ml ice, thermal agitation.
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The solution II that adds the new preparation of 2ml is gently put upside down centrifuge tube for several times, with abundant mixing content, in the chamber
Temperature was placed 6 minutes.
↓
Add the solution III of 1.5ml ice precooling, slowly soak into the whole tube walls of centrifuge tube from bottom to top, as far as possible with mixed solution
Avoid thalline to be scattered on the tube wall, placed on ice 10 minutes, occur white flocks in the pipe.
↓
Spend centrifugal 15 minutes of 12000rpm for 4 ℃.
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With 4 metafiltration paper supernatant is filled in the centrifuge tube of another 50ml, adds the Virahol of 0.6 times of volume, fully mix
Even, placed 10 minutes in room temperature.
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Room temperature, centrifugal 15 minutes of 12000rpm, precipitate nucleic acids.
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Supernatant is abandoned in suction, in room temperature with 70% washing with alcohol precipitation and tube wall number minute.Pour out ethanol, inhale with vacuum unit
Get tube wall and pipe end liquid.
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With 350ul sterilization distilled water dissolving nucleic acid precipitation.
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Change dna solution over to the 1.5ml centrifuge tube, add isopyknic 5Mol/L lithium chloride solution again, abundant mixing, in
Room temperature centrifugal 10 minutes with 12000rpm.
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Supernatant is transferred in another 1.5ml centrifuge tube, is added the Virahol of equivalent, abundant mixing, in room temperature with 12000rpm
Centrifugal 10 minutes, to reclaim the nucleic acid precipitation.
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Supernatant is abandoned in suction, with 70% washing with alcohol precipitation and tube wall number minute, pours out ethanol in room temperature, inhales with vacuum unit
Remove tube wall and bottom liquid.
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With 100ul sterilization distilled water dissolution precipitation, add the Rnase that 10ul 10mg/ml does not have DNase, put in 37 ℃
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Add 400ul sterilization distilled water, use isopyknic phenol, phenol: chloroform, each extracting of chloroform one time.
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Water is transferred to another centrifuge tube, add 1/4 volume NaAc (3M, pH5.2), fully mixing adds 2 times of volume of ethanol, placed 10 minutes in room temperature, in 4 ℃ with 12000rpm centrifugal 15 minutes, the recovery plasmid DNA.
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Supernatant is abandoned in suction, adds the ethanol of 1ml 70%, washing precipitation 5 minutes.
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Blot precipitation with vacuum unit, and be dissolved in 50ul sterilization distilled water.
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Get 1ul and go up the sample electrophoresis, check extraction effect.
(2) purifying of plasmid
Large-scale purifying mainly relies on column chromatography, and dependence post bed to the affinity degree difference of plasmid, makes plasmid separate with other impurity under different condition.
(3) detection of plasmid DNA concentration and purity well.The value of this moment can reflect the concentration of sample, and the relation between the concentration is 1 OD260=50ug/ml.In the practical application, the ratio of OD260/OD280 need not be 1.8, but approaching more expression purity is good more.
The structure of embodiment 1, cloning by expression
One, the structure element of cloning by expression
1,5 ' flanking nucleotide sequence
5 ' flanking nucleotide sequence is to be transformed by ox skeletal muscle actin promoter
Design of primers is as follows:
up：??????CTGGGGAGGAGGGTTTCGTATGT
LENGTH：??23nt??ΔG＝-3.6kcal/mol???(G+C)％＝56.5％???Tm＝72
Low：?????
Sna?BI
LENGTH：??28nt??
ΔG＝-6.9kcal/mol?
(G+C)％＝50.0％??
Tm＝72
Below for contrasting by 5 ' the flanking nucleotide sequence of above primer PCR acquisition and the ox skeletal muscle actin promoter sequence (the GENEBANK sequence number is U02285) of report, homology is 99%.
Upper line: by 5 ' flanking nucleotide sequence of above primer PCR acquisition
from?1?to?2445
Lower line: the ox skeletal muscle actin promoter of report
from?1?to?2441
1????CTGGGGAGGAGGGTTTCGTATGTGCTTTTATATCCCTCTTCGAGGACCCGCACCTGTCCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1????CTGGGGAGGAGGGTTTCGTATGTGCTTTTATATCCCTCTTCGAGGACCC
TCCC
61???AGTTGCTGAGTTCCACCACCGAGTTCCTATTCTGGGAACACTTGAGCACATCAGAAAAAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
61???AGTTGCTGAGTTCCACCACCGAGTTCCTATTCTGGGAACACTTGAGCACATCAGAAAAAT
121??GAGTGGTTCCATTCTGGCTCACATCACATCACTGATGCACCCCTTAAAGCATGTCCCTGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||121????GAGTGGTTCCATTCTGGCTCACATCACATCACTGATGCACCCCTTAAAGCATGTCCCTGA181????GTTCATTGCAGAAAATTGTTCCTCCTTGTACCTTCCACAGCAAGGTTAGAACTGTTCCCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||181????GTTCATTGCAGAAAATTGTTCCTCCTTGTACCTTCCACAGCAAGGTTAGAACTGTTCCCC241????TCAGGGGAAAAAACAGTGAGAAGCACCAACTTAATAACCTCCTCTGACCCCTACTCCACC
||||||||||||||||||||||||||||||||||||||||||?|||||||||||||||||241????TCAGGGGAAAAAACAGTGAGAAGCACCAACTTAATAACCTCCTTTGACCCCTACTCCACC301????TTTACCATAAGTAGATCCAAATCCTTCTAGAAAATCAGAAAGACATATCCCCGTGTATCA
|||||||||||||||||||||||||||||||||||||||||||||?||||||?|||||||301????TTTACCATAAGTAGATCCAAATCCTTCTAGAAAATCAGAAAGACAGATCCCCATGTATCA361????GCGGTATAAATAGAACCGCTCTGCAGACTCTGGTGGACGGTGACTCTCCAAGGTGGATTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||361????GCGGTATAAATAGAACCGCTCTGCAGACTCTGGTGGACGGTGACTCTCCAAGGTGGATTG421????
GCCGGCCTTGGCTGGGCATCACCCTCTAAATATAACGATGAGTTTGTTCAGCC
|||?|||||?||||||||||||||||||||||||||||||||||||||||||||||||||421????GGAAT
GCCTTGGCTGGGCATCACCCTCTAAATATAACGATGAGTTTGTTCAGCC481????TTTGCAGAAGGGAAAGGTTTTGCCCATCCTAGAGCGCGATGCCCTTGTCCTCCTTACAGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||481????TTTGCAGAAGGGAAAGGTTTTGCCCATCCTAGAGCGCGATGCCCTTGTCCTCCTTACAGG541????GAGGAGAGACGGTTGAGGCTTCATCTAGTAAGAGCACTTCTCAGTTCCCATCCTAGGGAT
|||||||||||||||||||||||||||||||||||||?||||||||||||?|||||||?|541????GAGGAGAGACGGTTGAGGCTTCATCTAGTAAGAGCACCTCTCAGTTCCCACCCTAGGGCT601????ATGACACTTGCCTTTCCTCCCCAGGACTGGGAAGTCGGTGAGCCCCGCCAAGGATGCGGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||601?????ATGACACTTGCCTTTCCTCCCCAGGACTGGGAAGTCGGTGAGCCCCGCCAAGGATGCGGG661?????AGTAGGGTGCTCAGCTCGGCCTGCCATACTCCAGAGCCGGCCAGTTAGTCACTCAACTTC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||661?????AGTAGGGTGCTCAGCTCGGCCTGCCATACTCCAGAGCCGGCCAGTTAGTCACTCAACTTC721?????ACTCCCTTCATGAGCTCCCGAGCCCACAACACGTCCCCGAGACGGGCAGCTCTGGGTGTC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||721?????ACTCCCTTCATGAGCTCCCGAGCCCACAACACGTCCCCGAGACGGGCAGCTCTGGGTGTC781?????CTGGCTCAGTGCCAGAGGCTGCGAGAGCCGCTGCGGGGCCTGTGCCGGGAGCCCAGCGCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||781?????CTGGCTCAGTGCCAGAGGCTGCGAGAGCCGCTGCGGGGCCTGTGCCGGGAGCCCAGCGCC841?????TCCTCCCCGGACTCTCCACAGTTGTCCTCGCGACAGGTGCGCGCCCGCCGGCCTCCGGTG
||||||||||||||||||||||||||||||||||||?||||||||||||||||?||||||841?????TCCTCCCCGGACTCTCCACAGTTGTCCTCGCGA
CGCGCCCGCCGGCCGCCGGTG901?????ACCCTCTCCGGTGGGGGTGGGGGCCGACGGTGTCAGCCCTCTGGATTGGGGAGCTCGGGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||901?????ACCCTCTCCGGTGGGGGTGGGGGCCGACGGTGTCAGCCCTCTGGATTGGGGAGCTCGGGG961?????TTGGGGGAGAGACCGAGTTCGCTGGGAGTCTGGGGGGAGGGATCGCCTGCCTCCCTGCCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||961?????TTGGGGGAGAGACCGAGTTCGCTGGGAGTCTGGGGGGAGGGATCGCCTGCCTCCCTGCCC1021????GGGACTCCGGGAGTGACTCCATCTCCGGTCCTCTTGGCCCCACCACTAGGATATAGAGTC
||||||||||||||||||||||||||||||||||||?|||||||||||||||||||||||1021????GGGACTCCGGGAGTGACTCCATCTCCGGTCCTCTTGTCCCCACCACTAGGATATAGAGTC1081?????CTCCTCGGTGGAGTCACACTTAAGGAGTTGGAGGTGGGGGGTAAGGG
ACCCGAA
|||||||||||||||||||||||||||||||||||||||||||?|||||||?||||||||1081?????CTCCTCGGTGGAGTCACACTTAAGGAGTTGGAGGTGGGGGGTAGGGGTGA
CGAA1141?????GAAGAGGTGAAATGTGGGCGCACCTTCCCGAGGCTCGGAGAACACCGAATGGCCGGGGGC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1141?????GAAGAGGTGAAATGTGGGCGCACCTTCCCGAGGCTCGGAGAACACCGAATGGCCGGGGGC1201?????GGGGCGGCTGCGGACAGGTGCAGCCCGGGCGCAGGCTCACTGGCGCACCCCGAACACTCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1201?????GGGGCGGCTGCGGACAGGTGCAGCCCGGGCGCAGGCTCACTGGCGCACCCCGAACACTCG1261?????GTGACCCTCGCCGCACCCCAGCCCCTCCGCCAGGCAACCGGGCGGGGTCGGGAGGGGCCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1261?????GTGACCCTCGCCGCACCCCAGCCCCTCCGCCAGGCAACCGGGCGGGGTCGGGAGGGGCCG1321?????ACCAGCGGGCAGACACTCCATATACGGCCCGGCCCGTGTTACCTGGGCTCAGGCCAGGCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1321?????ACCAGCGGGCAGACACTCCATATACGGCCCGGCCCGTGTTACCTGGGCTCAGGCCAGGCC1381?????TCTCCTTCTTTGGTCAGCGCAGGGGACCCGGGCGGGGACCCAGGCCTTGAACTGGTCGGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1381?????TCTCCTTCTTTGGTCAGCGCAGGGGACCCGGGCGGGGACCCAGGCCTTGAACTGGTCGGG1441?????GGAGGGGGCTCTAGTGCCCAACACCCAAATATGGCTCGAGAAGGGGAGCG
TGC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1441?????GGAGGGGGCTCTAGTGCCCAACACCCAAATATGGCTCGAGAAGGGGAGCGACA.TCCTGC1501?????GGGGCGGCGCGAGGGGAGCGCCCGAGGGCTATATAAAACCTGAGCAGGGGGACGAGCGGC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1500?????GGGGCGGCGCGAGGGGAGCGCCCGAGGGCTATATAAAACCTGAGCAGGGGGACGAGCGGC1561??????CACCGCAGCGGATAGCGCCGAGAGAAGTCTCGCTTCCTTCCCACGGTGACCGGGACCGGA
||||||||||||?|||||||||||||||||||||||||||||||||||||||||||||||1560??????CACCGCAGCGGACAGCGCCGAGAGAAGTCTCGCTTCCTTCCCACGGTGACCGGGACCGGA1621??????GCCGGAGAGCCGCAGGTGTAGCCACCCGCCCAGGTAGGCAGGGGCGGGGTGGGGACAGTA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||?|1620??????GCCGGAGAGCCGCAGGTGTAGCCACCCGCCCAGGTAGGCAGGGGCGGGGTGGGGACAGAA1681??????GGACGTGTCCCACCGCGGCAGCCTGGAGAACTCCAGGTAGAGCGCGGCCCCTCACTGCAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1680??????GGACGTGTCCCACCGCGGCAGCCTGGAGAACTCCAGGTAGAGCGCGGCCCCTCACTGCAT1741??????CAGCCCCAGCCCCGCGTCCTGTGAGCCGCGCCTCTGGCCCCAGAGTCACGTGTGCTTTAA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1740??????CAGCCCCAGCCCCGCGTCCTGTGAGCCGCGCCTCTGGCCCCAGAGTCACGTGTGCTTTAA1801??????AGAAGAGACAGGAAGCCGGGTCCTTTGGAAAGATCTCCAAATTTATTCCCAGCCCTTTGG
||||||||||||||||||||||||||||||||||||||||||||||||||||?|||||||1800??????AGAAGAGACAGGAAGCCGGGTCCTTTGGAAAGATCTCCAAATTTATTCCCAG.CCTTTGG1861??????GGCAGCTCTCCTTGACACTTCGTGCGTGCCGAGGTAGATGAAAGGTTTTGCTTTAGTCCT
||||||||||||||||||||||||||||||||||||||||||||||||||||||\\||||1859??????GGCAGCTCTCCTTGACACTTCGTGCGTGCCGAGGTAGATGAAAGGTTTTGCTTTAGTCCT1921??????CCCCAGGGCAGTTCCTTCCCAGCAGAACCAAAGGACAGTAGAAGGGACAATTTCCCCTGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1919??????CCCCAGGGCAGTTCCTTCCCAGCAGAACCAAAGGACAGTAGAAGGGACAATTTCCCCTGG1981??????CTCGCCATCAGTTACAAGGGCGCTTCGGCTGAGTCTGAAGTGCCATGGAGGTCCAAAATA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||1979??????CTCGCCATCAGTTACAAGGGCGCTTCGGCTGAGTCTGAAGTGCCATGGAGGTCCAAAATA2041?????AGCCATTCGTGTCCTGGTGCAGTGCCCAAGCCCTTTCCACGGCGTCTCCTTCCACCCTCA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||2039?????AGCCATTCGTGTCCTGGTGCAGTGCCCAAGCCCTTTCCACGGCGTCTCCTTCCACCCTCA2101?????CAGTGGCCCAGAAATCAGGGTTCGCGGACCTTGACCCCCATGCGATGCCAGGGAGGGGCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||2099?????CAGTGGCCCAGAAATCAGGGTTCGCGGACCTTGACCCCCATGCGATGCCAGGGAGGGGCG2161?????GAGAGGGCGGTGCCTGCAGCGAA.GCAGACAACTCTCGGGTCTGTGTGTCCTGAGTTCCC
|||||||||||||||||||||||?||||||||||||||||||||||||||||||||||||2159?????GAGAGGGCGGTGCCTGCAGCGAAGGCAGACAACTCTCGGGTCTGTGTGTCCTGAGTTCCC2220?????AGATGGAGAAACGCTCTCTGTGGTCTCCTCGCAGTAGAGCTCGCAGCAGCGAAGCTCCGC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||2219?????AGATGGAGAAACGCTCTCTGTGGTCTCCTCGCAGTAGAGCTCGCAGCAGCGAACGTCCGC2280?????CCGCGCGGCTTTGATTGCTTCCCCCGCAGGATGGCGCCGGGCTGCGGGCTGGAGGCTGGA
||||||?|||||||||||||||||||||?|||||||||||||||||||||||||||||||2279?????CCGCGC.GCTTTGATTGCTTCCCCCGCA.GATGGCGCCGGGCTGCGGGCTGGAGGCTGGA2340?????GGTGTCCATAGGTCCCCGGGGAGGGAGGGCGCTCCAGCCAGCGGGGCCTCTTGCGTGAAA
||||||||||||||||||||||?|||||||||||||||||||||||||||||||?|||||2337?????GGTGTCCATAGGTCCCCGGGGA.GGAGGGCGCTCCAGCCAGCGGGGCCTCTTGCCTGAAA2400?????CACTGAGCGTCTCCATGGCATTCCTGTCCTTGCAGAAACCAGATAC
||||||||||||||||||||||||||||||||||||||||||||||2396?????CACTGAGCGTCTCCATGGCATTCCTGTCCTTGCAGAAACCAGATAC
Table 1: protein binding site comparing result
| 5 ' flanking nucleotide sequence | The ox skeletal muscle actin promoter of report | ||||
| ??Name | ?Position | ??Site | ?Name | Position | ????Site |
| ??ICSBP | ?12 | ??ggtttc | ?ICSBP | 12 | ????ggtttc |
| ????AR | ????21 | ????tgtgct | ??AR | ????21 | ????tgtgct |
| ????AR | ????95 | ????ggaaca | ??NF-uE5 | ????50 | ????acacctg |
| ????AR | ????105 | ????agcaca | ??AR | ????95 | ????ggaaca |
| ????AR | ????113 | ????agaaaa | ??AR | ????105 | ????agcaca |
| ????NFIII | ????117 | ????aaatga | ??AR | ????113 | ????agaaaa |
| ????AP-1 | ????135 | ????tggctca | ??NFIII | ????117 | ????aaatga |
| ????COUP | ????180 | ????agttca | ??AP-1 | ????135 | ????tggctca |
| ????AR | ????190 | ????agaaaa | ??COUP | ????180 | ????agttca |
| ????AR | ????197 | ????tgttcc | ??AR | ????190 | ????agaaaa |
| ????PU.1 | ????199 | ????ttcctc | ??AR | ????197 | ????tgttcc |
| ????AR | ????228 | ????agaact | ??PU.1 | ????199 | ????ttcctc |
| ????AR | ????233 | ????tgttcc | ??AR | ????228 | ????agaact |
| ????AR | ????250 | ????aaaaca | ??AR | ????233 | ????tgttcc |
| ????COUP | ????285 | ????tgaccc | ??AR | ????250 | ????aaaaca |
| ????AR | ????329 | ????agaaaa | ??COUP | ????285 | ????tgaccc |
| ????TFIID | ????365 | ????tataaa | ??AR | ????329 | ????agaaaa |
| ????AR | ????372 | ????agaacc | ??TFIID | ????365 | ????tataaa |
| ????AP-1 | ????421 | ????ggagtca | ??AR | ????372 | ????agaacc |
| ????AR | ????472 | ????tgttca | ??GT-2B | ????426 | ????cagctg |
| ????AR | ????526 | ????tgtcct | ??AR | ????472 | ????tgttca |
| ????AR | ????571 | ????agagca | ??AR | ????526 | ????tgtcct |
| ????PU.1 | ????614 | ????ttcctc | ??AR | ????571 | ????agagca |
| ????AP-1 | ????705 | ????ttagtca | ??PU.1 | ????614 | ????ttcctc |
| ????PEA1 | ????705 | ????ttagtca | ??PEA1 | ????705 | ????ttagtca |
| ????MLTF | ????708 | ????gtcact | ??AP-1 | ????705 | ????ttagtca |
| ????AP-1 | ????709 | ????tcactca | ??MLTF | ????708 | ????gtcact |
| ????AR | ????746 | ????acaaca | ??AP-1 | ????709 | ????tcactca |
| ????SIF | ????761 | ????gacggg | ??AR | ????746 | ????acaaca |
| ????AR | ????777 | ????tgtcct | ??SIF | ????761 | ????gacggg |
| ????AP-1 | ????782 | ????tggctca | ??AR | ????777 | ????tgtcct |
| ????AR | ????863 | ????tgtcct | ??AP-1 | ????782 | ????tggctca |
| ????COUP | ????899 | ????tgaccc | ????AR | ??863 | ????tgtcct |
| ????BPV-E2 | ????901 | ?accctctccggt | ????GT-2B | ??874 | ????cagctg |
| ????MLTF | ????1032 | ????agtgac | ????COUP | ??899 | ????tgaccc |
| ????AP-1 | ????1034 | ????tgactcc | ????BPV-E2 | ??901 | ????accctctccggt |
| ????AP-1 | ????1090 | ????ggagtca | ????MLTF | ??1032 | ????agtgac |
| ????AR | ????1128 | ????tgaaca | ????AP-1 | ??1034 | ????tgactcc |
| ????AR | ????1140 | ????agaaga | ????AP-1 | ??1090 | ????ggagtca |
| ????AP-2 | ????1167 | ????cccgaggc | ????AR | ??1131 | ????agaacc |
| ????AR | ????1179 | ????agaaca | ????AR | ??1140 | ????agaaga |
| ????Sp1 | ????1197 | ????gggcgg | ????AP-2 | ??1167 | ????cccgaggc |
| ????Sp1 | ????1202 | ????gggcgg | ????AR | ??1179 | ????agaaca |
| ????COUP | ????1262 | ????tgaccc | ????Sp1 | ??1197 | ????gggcgg |
| ????AP-2 | ????1276 | ????ccccagcc | ????Sp1 | ??1202 | ????gggcgg |
| ????BPV-E2 | ????1297 | ?accgggcggggt | ????COUP | ??1262 | ????tgaccc |
| ????Sp1 | ????1300 | ????gggcgg | ????AP-2 | ??1276 | ????ccccagcc |
| ????SRF | ????1338 | ??ccatatacgg | ????BPV-E2 | ??1297 | ????accgggcggggt |
| ????SRF | ????1384 | ??ccttctttgg | ????Sp1 | ??1300 | ????gggcgg |
| ????Sp1 | ????1410 | ????gggcgg | ????SRF | ??1338 | ????ccatatacgg |
| ????COUP | ????1428 | ????tgaact | ????SRF | ??1384 | ????ccttctttgg |
| ????SRF | ????1465 | ??ccaaatatgg | ????Sp1 | ??1410 | ????gggcgg |
| ????GT-IIC | ????1491 | ????acattcc | ????COUP | ??1428 | ????tgaact |
| ????Sp1 | ????1502 | ????gggcgg | ????SRF | ??1465 | ????ccaaatatgg |
| ????TFIID | ????1532 | ????tataaa | ????Sp1 | ??1501 | ????gggcgg |
| ????COUP | ????1607 | ????tgaccg | ????TFIID | ??1531 | ????tataaa |
| ????NF-uE5 | ????1633 | ????caggtgt | ????COUP | ??1606 | ????tgaccg |
| ????BPV-E2 | ????1644 | ?acccgcccaggt | ????NF-uE5 | ??1632 | ????caggtgt |
| ????Sp1 | ????1646 | ????ccgccc | ????BPV-E2 | ??1643 | ????acccgcccaggt |
| ????Sp1 | ????1662 | ????gggcgg | ????Sp1 | ??1645 | ????ccgccc |
| ????AP-2 | ????1701 | ????gcctggag | ????Sp1 | ??1661 | ????gggcgg |
| ????AR | ????1707 | ????agaact | ????AP-2 | ??1700 | ????gcctggag |
| ????AP-2 | ????1744 | ????ccccagcc | ????AR | ??1706 | ????agaact |
| ????MLTF | ????1785 | ????gtcacg | ????AP-2 | ????1743 | ????ccccagcc |
| ????AR | ????1791 | ????tgtgct | ????MLTF | ????1784 | ????gtcacg |
| ????AR | ????1801 | ????agaaga | ????AR | ????1790 | ????tgtgct |
| ????AR | ????1944 | ????agaacc | ????AR | ????1800 | ????agaaga |
| ????AR | ????1952 | ????aggaca | ????AR | ????1942 | ????agaacc |
| ????AP-2 | ????1974 | ????cccctggc | ????AR | ????1950 | ????aggaca |
| ????AR | ????2050 | ????tgtcct | ????AP-2 | ????1972 | ????cccctggc |
| ????COUP | ????2132 | ????tgaccc | ????AR | ????2048 | ????tgtcct |
| ????Sp1 | ????2156 | ????gggcgg | ????COUP | ????2130 | ????tgaccc |
| ????Sp1 | ????2165 | ????gggcgg | ????Sp1 | ????2154 | ????gggcgg |
| ????AR | ????2206 | ????tgtcct | ????Sp1 | ????2163 | ????gggcgg |
| ????Sp1 | ????2276 | ????ccgccc | ????AR | ????2205 | ????tgtcct |
| ????AR | ????2424 | ????tgtcct | ????Sp1 | ????2275 | ????ccgccc |
| ????ICSBP | ????2434 | ????gaaacc | ????AR | ????2420 | ????tgtcct |
| ????ICSBP | ????2430 | ????gaaacc |
By above-mentioned sequence and form more as can be seen, some base mutations by 5 ' the flanking nucleotide sequence that PCR method obtained used in the present invention have caused the change of transcription factor binding site point, have wherein increased a GT-IIC binding site in strong promoter region 1491.
2,3 ' flanking nucleotide sequence
3 ' flanking nucleotide sequence is formed by the transformation of ox skeletal muscle Actin muscle poly (A) tailing signal:
up：???
Nde?I
LENGTH：??28nt?
ΔG＝-10.3kcal/mol??????(G+C)％＝60.7％????Tm＝70
Low：??
Nsi?I
LENGTH：??28nt?
ΔG＝-9.9kcal/mol???????(G+C)％＝60.7％????Tm＝66
3, comprise the GHRH mature peptide encoding gene (goat, sheep) that guides peptide
The chain nucleotide sequence of artificial synthesized sequence 1 and sequence 3, sequence 3 are positioned at 5 ' end of sequence 1.
Two, the structure of cloning by expression
1, the structure of carrier (pGEM-A5f3f)
As shown in Figure 1:
5 ' flanking nucleotide sequence of pcr amplification is inserted between the multiple clone site of T-vector, is designated as pGEM-A5F.PGEM-A5F and 3 ' flanking nucleotide sequence PCR product are all cut with Nde I and Nsi I enzyme, connect and compose carrier for expression of eukaryon pGEM-A5F3F.
As shown in Figure 3, evaluation respectively for the plasmid pGEM-A3F of the plasmid pGEM-A5F that contains 5 ' flanking nucleotide sequence (2442bp) and 3 ' flanking nucleotide sequence (805bp), wherein, 1, being that the Apa I+Sac I of plasmid pGEM-A3F of 3 ' flanking nucleotide sequence is two cuts the result, and the little band of cutting-out is the carrier band of a 800bp and a 3kb; 2, being that the Nde I+Nsi I of plasmid pGEM-A3F of 3 ' flanking nucleotide sequence is two cuts the result, and the little band of cutting-out is the carrier band of a 800bp and a 3kb; 3, be that the NsiI of the plasmid pGEM-A3F of 3 ' flanking nucleotide sequence singly cuts the result, the little band of cutting-out is the carrier band of a 800bp and a 3kb; 4, be that the Nde I of the plasmid pGEM-A3F of 3 ' flanking nucleotide sequence singly cuts the result, plasmid be cut to linear that size is 3.8kb, does not have the little band that downcuts; 5, be that the two results that cut of the Nde I+Nsi I of plasmid pGEM-A5F of 5 ' flanking nucleotide sequence are cut to plasmid linear, size is 5.5kb; 6, be that the Nsi I of the plasmid pGEM-A5F of 5 ' flanking nucleotide sequence singly cuts the result and plasmid is cut to linear, size is 5.5kb; 7, be that the Nde I of the plasmid pGEM-A5F of 5 ' flanking nucleotide sequence singly cuts the result and plasmid is cut to linear, size is 5.5kb; 8, being that the Sph I+SnaB I of plasmid pGEM-A5F of 5 ' flanking nucleotide sequence is two cuts the result plasmid is cut to two bands, and size is 2.5kb and 3.0kb; 9 is 1kb Marker; 10, be the plasmid pGEM-A3F of 3 ' flanking nucleotide sequence; 11, be the plasmid pGEM-A5F of 5 ' flanking nucleotide sequence; 12, be empty plasmid, size is 3.0kb; 13, cut for the empty plasmid list, size is 3.0kb (in contrast).
As shown in Figure 4, being the plasmid pGEM-A3F of 3 ' flanking nucleotide sequence and 5 ' flanking nucleotide sequence plasmid pGEM-A5F cuts the results that reclaim the back through Nde I+Nsi I is two, wherein 1, for the plasmid pGEM-A3F that contains 3 '-flanking through the two recovery electrophoresis of cutting the back size for the band of 800bp of Nde I+Nsi I; 2, be the result after two the cutting of plasmid pGEM-A5F process Nde I+Nsi I of containing 5 '-flanking, size is 5.5bp; M, be 1kbMarker.
As shown in Figure 5, be that expression vector pGEM-A5F3F makes up, wherein, 1, for inserting the expression vector plasmid pGEM-A5F3F (6.3kb) of 5 '-flanking (2.5kb) and 3 '-flanking (800bp); 2, for only inserting the plasmid pGEM-A5F (5.5kb) (in contrast) of 5 '-flanking (2.5kb).
As shown in Figure 6, be the evaluation of expression vector plasmid pGEM-A5F3F, wherein, 1,1kb Marker; 2, singly cut plasmid pGEM-A5F3F for Nde I, size is 6.3kb; 3, singly cut plasmid pGEM-A5F3F for SnaB I, size is 6.3kb; 4, be the two results that cut plasmid pGEM-A5F3F of Nde I+SnaB I, the size of the band of cutting-out is 2.5kb+3.8kb; 5, be the two results that cut plasmid pGEM-A5F3F of Nde I+Nsi I, the size of the band of cutting-out is 800bp+5.5kb; 6, plasmid pGEM-A5F3F, size is 6.3kb; 7,1kb Marker; 8, plasmid pGEM-A5F, size is 5.5kb.
Utilize three restriction enzyme site Spe I, Not I that this carrier provides, Sal I as the segmental insertion of external source site.
Complete for guaranteeing segmental expression of external source and 5-UTR, before ATG, need to add a base " C " (the 5-UTR ending of actine).
2, the GHRH gene fragment is synthetic
Fragment is a synthetic, total length 198bp.For ease of connecting, 5 ' end is designed to flush end, and 3 ' end is designed to have the cohesive end of Sal I.
In view of synthetic segmental limitation, adopt stepwise synthesis in its concrete operations.
Complete sequence:
CATGGGTTCTGCTCTGCTCCTCGCCCTCGGGCTGCTTGCCCAGAGCCTTGGCCTGTCCTGGGCATACGCTGATGCTATCTTCACCAACTCCTA
AAAATCCTGGGCCAGCTGTCCGCTCGGAAGCTGCTGCAGGATATCATGAACCGGCAGCAGGGCGAGCGGAACCAGGAGCAGGGCGCCAAGGTGCGGCTGTAG
Utilize a distinguished sequence-ccgg-at middle part in the fragment to make up restriction enzyme site, and then connect synthetic fragment.5 ' the end of synthetic article one chain: GHRH is an end, mends with 3 ' the end Sna BI point of contact of the 5-flanking of carrier to be connected after flat.CATGGGTTCTGCTCTGCTCCTCGCCCTCGGGCTGCTTGCCCAGAGCCTTGGCCTGTCCTGGGCATACGCTGATGCTATCTTCACCAACTCCT
ccg
Mend and be the primer of doubly-linked
5’??CATGGGTTCTGCTCTGCTCC?3’
5’??GGACCGGTAGGAGTTGGTGA?3’
Synthetic second chain:
tct
AAATCCTGGGCCAGCTGTCCGCTCGGAAGCTGCTGCAGGATATCATGAACCGGCAGCAGGGCGAGCGGAACCAGGAGCAGGGCGCCAAGGTGCGGCTGTA
ttt
Be Acc III site
For Sal I site, be used to insert carrier.
Mend and be the primer of doubly-linked
5’TCTTCCGGAAAATCCTGGG?3’
5’AAAGTCGACTAGAGCCGCA?3’
3 ' the end of GHRH for Sal I Not I the cohesive end of Spe I, with 5 ' the end SalI of the 3-flanking of carrier Not I Spe I point of contact be connected.
As shown in Figure 7, for artificial synthetic contains the splicing evaluation of the GHRH sequence that guide peptide, wherein, 1, Hae IIIMarker; 2, Kuo Zeng fragment II (FII): splicing M-cadherin guiding peptide and GHRH sequence, size is 118bp; 3, Kuo Zeng fragment I (FI): splicing M-cadherin guiding peptide and GHRH sequence, size is 105bp.
Shown in Fig. 8-A and 8-B, be structure and the evaluation of expression plasmid pGEM-A5F3F-M-GHRH, wherein, and among Fig. 8-A, 1,1kb Marker; 2, be the two results that cut vector plasmid pGEM-A5F3F of SnaB I+Sal I, the size of the band of cutting-out is 6.3kb; 3, clone (FI-II) via Nco I and the two results that cut of Sal I for the fragment I and the II splicing of guiding peptide and GHRH sequence, size is 198bp; 4, be Hae III Marker; 5, the result who clones (F I-II) PCR for the fragment I and the II splicing of guiding peptide and GHRH sequence, size is 213bp.Among Fig. 8-B, 1, splicing clone (F I-II) PCR result, size is 213bp; 2, blank; 3, positive plasmid PCR result is 213bp; 4, negative plasmid contrast; 5, the PCR result of expression plasmid pGEM-A5F3F-M-GHRH, 213bp; 6, vector plasmid plasmid pGEM-A5F3FPCR contrast; 7, Hae III Marker; 8,1kb Marker; 9, cut contrast for the Nco I+Nde I of negative plasmid pGEM-A5F (5.5kb) pair; 10, the Nco I+Nde I of expression plasmid pGEM-A5F3F-M-GHRH two cut the result for 2.4kb 3.8kb 256bp 387bp; 11, the Nco I+Nde I of vector plasmid pGEM-A5F3F two cut the result for 2.4kb 3.8kb 387bp.
3, GHRH genetic expression clone explanation:
As shown in Figure 2,
A, the expression vector pGEM-A5f3f that makes up is carried out enzyme with Sna BI cut, then mend and put down.And then utilize SalI that it is carried out enzyme and cut.
B, utilize Sal I that synthetic GHRH gene fragment enzyme is cut.
The result that C, connection A, B enzyme are cut forms cloning by expression.
Injection system: disposable buttocks both sides muscle injects expression plasmid 1.2-1.5mg/ head of the present invention.Injection back promotes growth effect is as shown in table 2.
Concrete operation method is referring to the corresponding section in " molecular cloning experiment guide " (second edition) of Science Press's publication in the present embodiment.In the experiment, ram, ewe respectively are half, adopt random packet.Feeding manner is essentially herds, and seldom replenishes fine fodder.
Table 2: expression plasmid live body intramuscular injection promotes growth effect table
| Varietal character | Kazak, Xinjiang sheep | The black pearl Mu Qin sheep of Mongolia | The little tail in the Shandong sheep that trembles with fear | |||
| Experimental group | Control group | Experimental group | Control group | Experimental group | Control group | |
| Number | ??15 | ??20 | ??12 | ??22 | ??20 | ??25 |
| Body weight kg before the experiment | ??12.62 | ??12.75 | ??13.25 | ??13.38 | ??15.80 | ??15.92 |
| 5 monthly age body weight kg | ??23.50 | ??20.34 | ??40.86 | ??36.68 | ??45.76 | ??40.82 |
| Body age in October heavy kg | ??38.28 | ??32.86 | ??54.52 | ??46.89 | ??70.05 | ??60.40 |
| 10 monthly age carcass weight kg | ??19.95 | ??14.80 | ??29.16 | ??22.50 | ??40.65 | ??31.58 |
| Dressing percentage | ??52％ | ??45％ | ??53.6％ | ??48％ | ??55.8％ | ??52.6％ |
| Total raising rate | ??25.49％ | ??21.27％ | ??22.34％ | |||
Wherein, heavy body weight when mainly being wean before the experiment, injection liquid of the present invention uses and is the lamb injection in preceding 5 days of weaning; Total raising rate refers to the raising of gross production efficiency, combines the factor of day weight gain and meat productivity.
From the table the result as can be seen, injection liquid of the present invention all has significant promoter action for sheep not of the same race at the weight increase of different times.
Sequence table＜160〉6＜210〉1＜211〉135＜212〉DNA＜213〉artificial sequence＜220〉＜223＜400〉1tacgctgatg ctatcttcac caactcctac cggaaaatcc tgggccagct gtccgctcgg 60aagctgctgc aggatatcat gaaccggcag cagggcgagc ggaaccagga gcagggcgcc 120aaggtgcggc tgtag 135＜210〉2＜211〉44＜212〉PRT＜213〉artificial sequence＜220〉＜223＜400〉2Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Ile Leu Gly
5??????????????????10??????????????????15Gln?Leu?Ser?Ala?Arg?Lys?Leu?Leu?Gln?Asp?Ile?Met?Asn?Arg?Gln
20??????????????????25??????????????????30Gln?Gly?Glu?Arg?Asn?Gln?Glu?Gln?Gly?Ala?Lys?Val?Arg?Leu
35 40 44＜210〉3＜211〉63＜212〉DNA＜213〉artificial sequence＜220〉＜223〉＜400〉3atgggttctg ctctgctcct cgccctcggg ctgcttgccc agagccttgg cctgtcctgg 60gca 63＜210〉4＜211〉21＜212〉PRT＜213〉artificial sequence＜220〉＜223＜400〉4Met Gly Ser Ala Leu Leu Leu Ala Leu Gly Leu Leu Ala Gln Ser
5???????????????????10?????????????????15Leu?Gly?Leu?Ser?Trp?Ala
20 21＜210〉5＜211〉2445＜212〉DNA＜213〉＜220〉＜223〉＜400〉5ctggggagga gggtttcgta tgtgctttta tatccctctt cgaggacccg cacctgtccc 60agttgctgag ttccaccacc gagttcctat tctgggaaca cttgagcaca tcagaaaaat 120gagtggttcc attctggctc acatcacatc actgatgcac cccttaaagc atgtccctga 180gttcattgca gaaaattgtt cctccttgta ccttccacag caaggttaga actgttcccc 240tcaggggaaa aaacagtgag aagcaccaac ttaataacct cctctgaccc ctactccacc 300tttaccataa gtagatccaa atccttctag aaaatcagaa agacatatcc ccgtgtatca 360gcggtataaa tagaaccgct ctgcagactc tggtggacgg tgactctcca aggtggattg 420ggagtcagcc ggccttggct gggcatcacc ctctaaatat aacgatgagt ttgttcagcc 480tttgcagaag ggaaaggttt tgcccatcct agagcgcgat gcccttgtcc tccttacagg 540gaggagagac ggttgaggct tcatctagta agagcacttc tcagttccca tcctagggat 600atgacacttg cctttcctcc ccaggactgg gaagtcggtg agccccgcca aggatgcggg 660agtagggtgc tcagctcggc ctgccatact ccagagccgg ccagttagtc actcaacttc 720actcccttca tgagctcccg agcccacaac acgtccccga gacgggcagc tctgggtgtc 780ctggctcagt gccagaggct gcgagagccg ctgcggggcc tgtgccggga gcccagcgcc 840tcctccccgg actctccaca gttgtcctcg cgacaggtgc gcgcccgccg gcctccggtg 900accctctccg gtgggggtgg gggccgacgg tgtcagccct ctggattggg gagctcgggg 960ttgggggaga gaccgagttc gctgggagtc tggggggagg gatcgcctgc ctccctgccc 1020gggactccgg gagtgactcc atctccggtc ctcttggccc caccactagg atatagagtc 1080ctcctcggtg gagtcacact taaggagttg gaggtggggg gtaagggtga acaacccgaa 1140gaagaggtga aatgtgggcg caccttcccg aggctcggag aacaccgaat ggccgggggc 1200ggggcggctg cggacaggtg cagcccgggc gcaggctcac tggcgcaccc cgaacactcg 1260gtgaccctcg ccgcacccca gcccctccgc caggcaaccg ggcggggtcg ggaggggccg 1320accagcgggc agacactcca tatacggccc ggcccgtgtt acctgggctc aggccaggcc 1380tctccttctt tggtcagcgc aggggacccg ggcggggacc caggccttga actggtcggg 1440ggagggggct ctagtgccca acacccaaat atggctcgag aaggggagcg acattcctgc 1500ggggcggcgc gaggggagcg cccgagggct atataaaacc tgagcagggg gacgagcggc 1560caccgcagcg gatagcgccg agagaagtct cgcttccttc ccacggtgac cgggaccgga 1620gccggagagc cgcaggtgta gccacccgcc caggtaggca ggggcggggt ggggacagta 1680ggacgtgtcc caccgcggca gcctggagaa ctccaggtag agcgcggccc ctcactgcat 1740cagccccagc cccgcgtcct gtgagccgcg cctctggccc cagagtcacg tgtgctttaa 1800agaagagaca ggaagccggg tcctttggaa agatctccaa atttattccc agccctttgg 1860ggcagctctc cttgacactt cgtgcgtgcc gaggtagatg aaaggttttg ctttagtcct 1920ccccagggca gttccttccc agcagaacca aaggacagta gaagggacaa tttcccctgg 1980ctcgccatca gttacaaggg cgcttcggct gagtctgaag tgccatggag gtccaaaata 2040agccattcgt gtcctggtgc agtgcccaag ccctttccac ggcgtctcct tccaccctca 2100cagtggccca gaaatcaggg ttcgcggacc ttgaccccca tgcgatgcca gggaggggcg 2160gagagggcgg tgcctgcagc gaagcagaca actctcgggt ctgtgtgtcc tgagttccca 2220gatggagaaa cgctctctgt ggtctcctcg cagtagagct cgcagcagcg aagctccgcc 2281cgcgcggctt tgattgcttc ccccgcagga tggcgccggg ctgcgggctg gaggctggag 2341gtgtccatag gtccccgggg agggagggcg ctccagccag cggggcctct tgcgtgaaac 2401actgagcgtc tccatggcat tcctgtcctt gcagaaacca gatac 2445＜210〉6＜211〉805＜212〉DNA＜213〉＜220〉＜223〉＜400〉6gcctctccga cagccgcgac ttctccccag gacgacgaat ctgctcgcgg cgcaggcggc 60tgacctcgag ccaccgcgca gcagctgctc tccaaccatc agcgttgccg ctgccgcaaa 120ctgacacact gtttataatg tgtacataca ttgtttacct cattttgtta tttttaaaac 180gaagccctgt ggaaggaaat ggaaaacttg aagcattaaa ctcagccgtt ctgttttgct 240gcgtaaagtt gtctgctgtg tttctttgcg ggggcgggga gctgtgcgga taggggagta 300aaagggactc cgtcttcctc cgtcactttt cacaatactc taaatgagtg caggccttac 360aaggccgtga gttaggtctt ccgcagctca cggccctcgc ctctggctca gatctattcc 420tcagactggc acctgcgccg tggcccgact tgacctctgg cgtgtcccct gtgcagggct 480atctgccgga ggcgccgggg cggcgctcgg gtggccaggg cgcgcctcgg gcagggcgct 540gaccgtggtg ctggcctggg gtcccgagag caaagcgcgc tcccgctcca cccgcggggg 600cgtggcctta ctgaagctcg gttccctcct caatctgaga tctaaactga aaagtgtgcg 660ttctgcattt gaggaaccag ttctcctttg ttaagtgaag ataacccctt ctcgggaagg 720cctttgcttc gtctgggtga cgaagcgccc tttccagagt ggggagggga aggagccgcg 780cccagaggtg ggaggaggcg ggaaa 805
Claims (9)
1, the sheep growth hormone releasing hormone gene of sequence 1.
2, gene according to claim 1 is characterized in that: its 5 ' end also is connected with the guiding peptide gene of sequence 3.
3, gene according to claim 1 and 2 is characterized in that: its 5 ' end also is connected with 5 ' flanking nucleotide sequence of sequence 5.
4, gene according to claim 1 and 2 is characterized in that: its 3 ' end also is connected with 3 ' flanking nucleotide sequence of sequence 6.
5, the sheep growth hormone releasing hormone of sequence 2.
6, hormone according to claim 5 is characterized in that: its nitrogen end also is connected with the guiding peptide of sequence 4.
7, the plasmid that contains sequence 1.
8, a kind of medicine that promotes growth hormone secretion, its activeconstituents are the sheep growth hormone releasing hormone Nucleotide of sequence 1 or the sheep growth hormone releasing hormone of sequence 2.
9, a kind of medicine for the treatment of disease of muscular dystrophy, its activeconstituents are the sheep growth hormone releasing hormone Nucleotide of sequence 1 or the sheep growth hormone releasing hormone of sequence 2.
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| CNB021000654A CN1319982C (en) | 2002-01-14 | 2002-01-14 | Sheep growth hormone releasing hormone gene and its expression product and application |
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| CNB021000654A CN1319982C (en) | 2002-01-14 | 2002-01-14 | Sheep growth hormone releasing hormone gene and its expression product and application |
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| CN1432577A true CN1432577A (en) | 2003-07-30 |
| CN1319982C CN1319982C (en) | 2007-06-06 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651365A (en) * | 2015-01-29 | 2015-05-27 | 东北农业大学 | Promoter of bovine alpha-actin gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AU8589998A (en) * | 1997-07-24 | 1999-02-16 | Valentis, Inc. | Ghrh expression system and methods of use |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651365A (en) * | 2015-01-29 | 2015-05-27 | 东北农业大学 | Promoter of bovine alpha-actin gene and application thereof |
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| CN1319982C (en) | 2007-06-06 |
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