CN1634985A - Pig Ghrelin derivative, its encoding gene and application - Google Patents
Pig Ghrelin derivative, its encoding gene and application Download PDFInfo
- Publication number
- CN1634985A CN1634985A CN 200310112932 CN200310112932A CN1634985A CN 1634985 A CN1634985 A CN 1634985A CN 200310112932 CN200310112932 CN 200310112932 CN 200310112932 A CN200310112932 A CN 200310112932A CN 1634985 A CN1634985 A CN 1634985A
- Authority
- CN
- China
- Prior art keywords
- ghrelin
- pig
- pgem
- derivative
- pig ghrelin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a pig Ghrelin derivative and coded genes and uses thereof. The pig Ghrelin derivative is a polypeptide at least comprising GSSF or GSWF amino acid residue sequence derived from pig Ghrelin. The invention also provides a polypeptide medicine and a gene medicine. The active constituent of said polypeptide medicine is the pig Ghrelin derivative or its soluble salt. The active constituent of said gene medicine is the coded gene of the pig Ghrelin derivative. The pig Ghrelin derivative, coded genes thereof and medicines using the genes as active constituents can be used in livestock and fowl production and clinical gene supplement and substitution treatment.
Description
Technical field
The present invention relates to pig Ghrelin derivative and encoding gene and application in the biological technical field, particularly pig Ghrelin derivative and encoding gene thereof and be the medicine of activeconstituents with it.
Background technology
(Growth hormone is the anabolic hormone of a kind of promotion GH) to tethelin, can promote protein synthesis, fat acid decomposition and bone growth, and relevant with immune adjusting.It is very crucial that the adjusting of GH path is expressed optimum linear growth (Optimal linear growth), simultaneously to Stable Carbon hydrate, protein and metabolism of fat also very important (AL.Carrel et al., Endocrine, 12,163-172,2000).Growth hormone secretion cell (Somatotroph cells) secretion GH depends on the mode effect each other with paracrine (Paracrine) or autocrine (Autocrine) of HRP, target gland hormone and multiple somatomedin, and wherein many somatomedins are still unclear so far.GH is subjected to two kinds of hypothalamic neuropeptides at least from pulsed (pulsatile) release of somatotropin secretory cell (Somatotrophs): growth hormone releasing hormone (Growth hormone releasing-hormone, GHRH) with somatostatin (Somatostatin, SS) adjusting (MF.Scanlon et al., Hormone Research, 46,149-154,1996).And, may be subjected to a kind of endogenous growth hormone to urge release peptide Ghrelin (M.Kojima et al., Nature, 402,656-660,1999) and regulate.The separation of Ghrelin deserves to be called the landmark discovery in GH field, and it is secreted for the more deep GH of understanding and body growth (Somatic growth) regulation mechanism provides possibility.
Just find successively since the 1970's people, the small-molecular peptides of many synthetic and non-peptide matters are secreted (C.Bowers et al. with the external GH that all can promote in vivo, Endocrinology, 106,663-667,1980), and with the material of this class synthetic be referred to as tethelin short secrete thing (Growth hormonesecretagogues, GHSs).Different according to GHSs promotion GH excretory signal transduction mechanism in vivo with known GHRH, infer the corresponding acceptor that exists GHS in the body (Growth hormone secretagoguesreceptor, GHS-R).1996 (Science, 273,974-977,1996) such as AD.Howards successfully have been separated to the cDNA of single coding GHS-R, and confirm that GHS-R belongs to the G protein coupling receptor that the α spiral is striden film for 7 times.Genetic comparison is analyzed demonstration GHS-R gene and is guarded at people, ape, pig, ox, rat, mouse camber, and people and pig GHS-R homologous sequence are up to 93%.1999 (Nature, 402,656-660,1999) such as M.Kojima have been reported with CHO (the Chinese hamster ovary) cell strain of the stably express GHS-R that sets up and have been sought endogenous GHS as analytical system.By detection, find that the activity of GHS in the rat stomach tissue extract is the highest to extracts such as the rat heart, brain, lung, kidney, stomach and intestine.Purify through serial chromatography, therefrom be purified into the bioactive peptide of forming by 28 amino acid, and proved that it is the endogenous aglucon (Ligand) of GHS-R, called after Ghrelin (ghre is exactly the meaning of growth at the former Europe family of languages).
The Ghrelin mature peptide comprises 28 amino-acid residues, and N holds the hydroxyl of the 3rd serine residue by the decoyl esterification, and this decoyl esterification is active essential by it.Its aminoacid sequence is: GSSFLSPEHQKAQQRKESKKPPAKLQPR.Its induced strong CHO-GHSR62 cell [Ca
2+] increase, 50% excited concentration (EC50) is 2.5 * 10
-9Mol/L, threshold concentration are 10
-11Mol/L.Rat Ghrelin precursor contains 117 amino-acid residues, and N-holds 23 peptides to present the feature of secreting signal peptide, since 24 glycine residues, directly is connected after the signal peptide.The most last two the residue dried meat-arginine of Ghrelin should be processing signals.Only there are two residues different between people's stomach Ghrelin and the rat Ghrelin.The Ghrelin of purifying from people's stomach extract confirms that also its N holds the 3rd serine residue also to exist the esterification of n-decoyl to modify.Ghrelin induces former generation pituicyte release GH, and the EC50 that discharges GH is 2.1 * 10
-9Mol/L, GHRH are 0.6 * 10
-9Mol/L, and exist dosage to rely on pattern significantly.In addition, find that Ghrelin stimulates the release of hypophysis GH specifically, and do not influence the secretion of other hormones.The Northern Blot experiment is to rat tissue's analysis revealed, and Ghrelin precursor mRNA is present in the stomach, and in situ hybridization shows that the mRNA of Ghrelin is present in the neck and the base portion of secretion hydrochloric acid in gastric juice body of gland.And proof Ghrelin cell is the incretory gland cells.Ghrelin concentration is 117 ± 37.2pmol/L in the human normal plasma.Infer that Ghrelin acts on hypophysis from stomach generation or secretion back with blood circulation probably.The group fractional analysis finds that also the immunoreactive neurone of Ghrelin is arranged in the arc nuclear of hypothalamus.In addition, RT-PCR analyzes and point out that GHS-R expresses in the heart, lung, pancreas, intestines and fatty tissue, thus think that Ghrelin may have many-sided effect, as in cardiovascular system is unified metabolism, working.
As a kind of endogenous ligands (Ligand) of GHSs acceptor (GHS-R), Ghrelin finds in the rat stomach endocrine cell at first.The Ghrelin mature peptide is made up of 28 amino acid, holds the 3rd Serine (Ser at its N-
3) a special decoyl esterification posttranslational modification being arranged on the hydroxyl, the posttranslational modification of this position is that its biological function is necessary.Decoylization is not to be its unique posttranslational modification that function is arranged yet, and the acyl esterification of other type also has function, removes to carry out the Ghrelin that the decoyl esterification modifies on decoyl esterification or other Serine beyond the 3rd and do not have biological function.Albumen or the esterification of polypeptide acyl are comparatively rare, and the decoyl esterification is unique.The peptide sequence comparative studies shows that also in the Mammals of announcing sequence, preceding 7 amino acids of Ghrelin N-end are high conservatives, and all exists N-to hold the 3rd Serine translation back acyl esterification to modify phenomenon.
In vitro study shows that the N-end may be Ghrelin biological function activity " core " (Active core), and a preceding 4-5 amino acid promptly has the biological effect similar to total length Ghrelin (MA.Bednarek et al., J.Med.Chem, 43,4370-4376,2000; M.Matsumoto et al., Biochem.Biophys.Res.Commun, 287,142-146,2001).In the body and experiment in vitro confirms that all Ghrelin has tangible promotion GH release action, vein, the ventricles of the brain and abdominal cavity give Ghrelin the GH level in the blood that all can raise.Ghrelin also can stimulate human body GH to discharge (K.Takaya et al., The Journal of Clinical Endocrinology﹠amp; Metabolism, 85,4908-491,2000; V.Tolle et al., euroendocrinology, 73,54-61,2001).And stem-winding is that by identical molecular ratio calculating, Ghrelin promotes the effect of GH release obviously greater than GHRH (M.Pombo et al., Hormone Research 55 (Suppl 1), 11-16,2001).The Ghrelin also GHSs with synthetic is the same, and Ghrelin and GHRH also have summation action, and the collaborative GH of stimulation secretes.Ghrelin may discharge by more effective adjusting GH by the mechanism different with GHRH, is to regulate another important factor that GH discharges in the body, and detailed mechanism remains to be illustrated.But existing evidence has shown the short GH secretion of Ghrelin performance and may depend on a complete GHRH system (CY.Bowers et al., TheJournal of Clinical Endocrinology ﹠amp; Metabolism, 86,1464-1469,2001; J.Kamegai et al., Endocrinology, 142,4154-4157,2001; GS.Tannebaum et al., Endocrinology, 144,967-974,2003).In addition, Ghrelin also participates in energy metabolism (M.Tscho ¨ pet al., Nature, 407,908-913,2000; TL.Horvath et al., Endocrinology, 142,4163-4169,2001; DE.Cummings et al., The New England Journal of Medicine, 346,1623-1630,2002).Because the transformation period of Ghrelin in blood is shorter, studying Ghrelin in the past in vivo promotes the GH secretion activity to need subcutaneous repeatedly, vein or intracerebral ventricle injection synthetic Ghrelin or its analogue (M.Tscho ¨ p et al., Nature, 407,908-913,2000; M.Nakazato et al, Nature, 409,194-198,2001).
Genomic medicine (Gene medicines) is also referred to as the DNA medicine, it is that carrier for expression of eukaryon is advanced in the gene recombination with treatment meaning, directly shift animal or human's cell, give expression to polypeptide or protein with therapeutic action, thereby reach the treatment disease or regulate growth of animal, a kind of novel method of performances such as lactation (horse big dragon biotech drug Beijing science and technology press 2001).Genomic medicine is different from general gene therapy, and the cell of transgenosis not only will go out the protein of treatment at cell inner expression, and expressed protein also will discharge cell, enters the effect of systemic circulation competence exertion.It and traditional extraction, synthetic different with protein engineering class medicine, it need be at external synthetic and marking protein, need be, but the gene of " factory " of people's physical efficiency synthetic protein and polypeptide-reorganization-implant at in-vitro separation and purifying yet, allow cell oneself goes to synthesize, separation and protein purification, and be released into blood circulation voluntarily, in the part or remote part bring into play its function, and reach disease preventing and treating and regulate growth, purposes such as lactation.It does not need " raw material ", genetic transcription can be provided in the body voluntarily, express conditions needed, and cell self can synthesize, separation and purifying, and justacrine comes out; Gene in cell, can receptor in the adjusting of various transcription factors, regulation and control factor and internal and external environment; It is constantly produced, constantly discharges, constantly utilizes.Therefore, once implanting gene can be permanently effective, need not medication every day, has efficiently, long-acting, physiology, economy, practical advantage.It is a challenge to the past conventional medicament, is called as the 4th revolution of medicinal industry, becomes a 21 century international biological high-technology and important development of medicinal industry.The difference of genomic medicine and protein drug is as shown in table 1.Genomic medicine mainly contains the gene of cytokine, active polypeptide and acceptor thereof, in addition, also comprises the medicine of antisense nucleic acid, dna vaccination and gene transcription regulation etc.
The comparison of table 1. polypeptide drugs and genomic medicine
The polypeptide drugs genomic medicine
Product polypeptide-protein gene
Plasmid prokaryotic expression carrier no signal peptide carrier for expression of eukaryon signal peptide
Host cell intestinal bacteria, yeast (external) human body cell (in the body)
Transfection is easily difficult
Complex manufacturing is simpler
Purifying complex is simpler
The more expensive less expensive of cost
Stability is unstable stable
Preserve difficulty or ease
Transformation period is short long
The disposable injection of usage long term injections
Curative effect is short lasting
The side effect height
Utilize the amino acid replacement derivative of direct plasmid DNA injecting method ectopic expression GHRH or its proteolytic enzyme opposing to promote mouse or pig growth (R.Draghia-Akli et al (Nature Biotechnology, 15,1285-1289,1997; Nature Biotechnology, 17,1179-1183,1999; FASEB Journal, 17,526-528,2003) early report is arranged, explore applying gene pharmaceutical means ectopic expression Ghrelin or derivatives thereof and Ghrelin associating GHRH so, perhaps can find a kind of than using GHRH more effective, more physiology, more economical the growth of animals or poultry that promotes, improve food conversion ratio, increase degree meat rate improves milk production, promotes the method that high-quality, high yield animal products are produced.For genomic medicine, Ghrelin and derivative cDNA thereof can grappling (Target) to the peripheral organ, polypeptide is synthesized processing, secretion there, at last, is transported to systemic circulation, and in blood circulation be have bioactive.Skeletal muscle is the target tissue of the direct plasmid DNA transfection of ideal in the body, and plasmid DNA can be expressed (HL.Davis et al., Human Gene Therapy, 4,151-159,1993 in the long-time conspicuous level of intramuscular; SK.Tripathy et al., Proceedings of the National AcademySciences USA, 93,10876-10880,1996).Genomic medicine is a kind of replacement therapy, is different from classical gene therapy, as tumour, it does not resemble gene therapy the cell of needs 100% implant all into that gene could suppress growth of tumor.Someone points out, gene is advanced in the muscle cell transfection of main 2%-5%, and its marking protein can reach the purpose of treatment by autocrine and paracrine and circulation secretion.Genomic medicine also is different from genetically engineered and polypeptide drug, and it is a kind of factor that continues to work, and a shot can be permanently effective, but and repeated application.Therefore, it does not also require and can be integrated into genome, the lifelong expression.So not only can increase the security of genomic medicine, also improve the genomic medicine application flexibility.
The innovation and creation content
The purpose of this invention is to provide a boar Ghrelin derivative and an encoding gene thereof.
Pig Ghrelin derivative provided by the present invention, it for comprise at least GSSF or GSWF amino acid residue sequence by pig Ghrelin polypeptides derived.
Wherein, described pig Ghrelin derivative be preferably comprise at least GSSFLSP or GSWFLSP amino acid residue sequence by pig Ghrelin polypeptides derived.
Above-mentioned pig Ghrelin derivative can be synthetic by ordinary method.
The 2nd Serine among described GSSF and the GSSFLSP is the Serine of decoyl esterification; For making described pig Ghrelin derivative be secreted into blood circulation, the N end of described pig Ghrelin derivative also is connected with signal peptide/leader peptide sequence, described signal peptide/guiding peptide can be pig Ghrelin or human growth hormone releasing hormone (hGHRH) signal peptide/guiding peptide, is preferably pig Ghrelin signal peptide/guiding peptide.
For strengthening the biological activity of described pig Ghrelin derivative, can add a basic aminoacids at the C-terminal of described pig Ghrelin derivative, as Methionin.
Described pig Ghrelin derivative is preferably the SEQ ID № in the sequence table: 1, SEQ ID №: 2, SEQ ID №: 3, SEQ ID №: 4, SEQ ID №: 5, SEQ ID №: 6 or SEQ ID №: 7.
SEQ ID №: 1 is made up of 52 amino-acid residues, and holding the 1st to the 24th amino acids residue sequence from N is pig Ghrelin signal peptide sequence, and holding the 25th to the 52nd amino acids residue sequence from N is pig Ghrelin sequence; SEQ ID №: 2 are made up of 52 amino-acid residues, holding the 1st to the 24th amino acids residue sequence from N is pig Ghrelin signal peptide sequence, holds the 25th to the 52nd amino acids residue sequence for being that all the other are pig Ghrelin sequence tryptophan residue except that the 27th amino acids residue from N; SEQ ID №: 3 are made up of 32 amino-acid residues, holding the 1st to the 24th amino acids residue sequence from N is pig Ghrelin signal peptide sequence, from N hold the 25th to the 31st amino acids residue sequence be pig Ghrelin hold the 1st to the 7th amino acids residue sequence sequence from N, holding the 32nd amino acids residue from N is lysine residue; SEQ ID №: 4 are made up of 32 amino-acid residues, holding the 1st to the 24th amino acids residue sequence from N is pig Ghrelin signal peptide sequence, hold the 25th to the 31st amino acids residue sequence to hold the 1st to the 7th amino acids residue sequence for what except that the 27th amino acids residue be that all the other are pig Ghrelin tryptophan residue from N from N, holding the 32nd amino acids residue from N is lysine residue; SEQ ID №: 5 are made up of 59 amino-acid residues, hold the 1st to the 31st amino acids residue sequence behaviour GHRH signal peptide sequence from N, and holding the 32nd to the 59th amino acids residue sequence from N is pig Ghrelin sequence; SEQ ID №: 6 are made up of 59 amino-acid residues, hold the 1st to the 31st amino acids residue sequence behaviour GHRH signal peptide sequence from N, hold the 32nd to the 59th amino acids residue sequence for being that all the other are pig Ghrelin sequence tryptophan residue except that the 34th amino acids residue from N; SEQ ID №: 7 are made up of 39 amino-acid residues, hold the 1st to the 31st amino acids residue sequence behaviour GHRH signal peptide sequence from N, hold the 32nd to the 38th amino acids residue sequence to hold the 1st to the 7th amino acids residue sequence for what except that the 34th amino acids residue be that all the other are pig Ghrelin tryptophan residue from N from N, holding the 39th amino acids residue from N is lysine residue.
The gene of above-mentioned pig Ghrelin derivative of encoding also belongs to protection scope of the present invention; the expression carrier and the clone that contain the above-mentioned pig Ghrelin derivative of encoding also belong to protection scope of the present invention, (contain the SEQ ID № in the ordered list: 1 encoding sequence) as expression vector pGEM-wt-sGhln; pGEM-mt-sGhln (contains the SEQ ID № in the ordered list: 2 encoding sequences); pGEM-tmt-sGhln (contains the SEQ ID № in the ordered list: 4 encoding sequences); pGEM-twt-sGhln (contains the SEQ ID № in the ordered list: 3 encoding sequences); pGEM-wt-hGhln (contains the SEQ ID № in the ordered list: 5 encoding sequences); pGEM-mt-hGhln (contains the SEQ ID № in the ordered list: 6 encoding sequences); pGEM-tmt-hGhln (contains the SEQ ID № in the ordered list: 7 encoding sequences).
Another object of the present invention provides a kind of promotion Mammals weightening finish, improve food conversion ratio, the rising serum growth hormone level, improve organization, raising degree meat rate, the human various tethelin defectives of correction and promotion growth hormone secretion and the polypeptide drugs that increase apocleisis crowd and emaciation patient appetite, the activeconstituents of this medicine is above-mentioned pig Ghrelin derivative or its soluble salt.
A further object of the present invention provides a kind of promotion Mammals weightening finish, improve food conversion ratio, the rising serum growth hormone level, improve organization, raising degree meat rate, the human various tethelin defectives of correction and promotion growth hormone secretion and the genomic medicine that increases apocleisis crowd and emaciation patient appetite, the activeconstituents of this medicine is the encoding gene of above-mentioned pig Ghrelin derivative.
The aforementioned polypeptides medicine can carry out administration by injection; The said gene medicine can carry out administration by the expression vector that injection contains said gene.
Pig Ghrelin derivative of the present invention and encoding gene thereof and be that the medicine of activeconstituents will promote the Mammals weightening finish with it, improve food conversion ratio, the rising serum growth hormone level, improve organization, raising degree meat rate, correct human various tethelin defectives and promote growth hormone secretion and increase aspects such as apocleisis crowd and emaciation patient appetite and play a significant role, can be used for Production of Livestock and Poultry, human clinical gene replenishes and replacement therapy.
Description of drawings
Fig. 1 is pig Ghrelin and derivative cDNA pT-wt-sGhln or pT-wt-hGhln vector construction synoptic diagram
Fig. 2 is the structure synoptic diagram of myogen specific expression carrier framework
Fig. 3 is pig Ghrelin and derivative cDNA myogen expression vector establishment synoptic diagram thereof
Fig. 4 is for producing the schema of genomic medicine
Fig. 5 is a rats with left quadriceps muscle of thigh plasmid DNA single dose injection back gaining effect
Fig. 6 is a rats with left quadriceps muscle of thigh plasmid DNA single dose injection back gaining effect
Fig. 7 is a Q sepharoae XL plasmid DNA purge process
Fig. 8 is the electrophorogram of plasmid pGEM-tmt-sGhln DNA behind Q Sepharose QL purifying
Fig. 9 is a young rats with left quadriceps muscle of thigh plasmid DNA single dose injection back gaining effect
Figure 10 is a radioimmunoassay serum after the direct intramuscularly plasmid DNA of young rat
Figure 11 injected back 30 days for plasmid DNA, and RT-PCR detects pGEM-wt-sGhln, pGEM-tmt-sGhln encoding gene expression of results
Embodiment
The structure of embodiment 1, pig Ghrelin mature peptide and derivative myogen expression vector thereof
1, pig Ghrelin and derivative cDNA thereof clone
Pig Ghrelin and derivative cDNA pT-wt-sGhln thereof or pT-wt-hGhln vector construction process as shown in Figure 1, concrete steps are as follows:
The pig Ghrelin that has announced according to Gene Bank (the GeneBank number of landing: No.AB035704) and (the GeneBank number of landing: no.NM_021081) of people GHRH (hGHRH) precursor cDNA sequence, synthetic 3 oligonucleotide strands and 6 primers (worker is given birth in Shanghai), oligonucleotide chain and primer sequence are as shown in table 2.Oligonucleotide single stranded sequence 1 comprises pig Ghrelin signal peptide and the preceding 28bp cDNA sequence (justice) of mature peptide, comprising a base mutation (Ghrelin mature peptide the 5th amino acids, leucine (Leu) codon is by TTG → TTA) to be construed as limiting enzyme Afl II recognition sequence (CTTAAG); Oligonucleotide single stranded sequence 2 comprises people GHRH signal peptide and the preceding 20bp cDNA sequence (justice) of pig Ghrelin mature peptide, wherein also comprise a base mutation (Ghrelin mature peptide the 5th amino acids, leucine (Leu) codon is by TTG → TTA) to be construed as limiting enzyme Afl II recognition sequence (CTTAAG); Oligonucleotide single stranded sequence 3 comprises the preceding 84bp cDNA sequence (antisense) of pig Ghrelin mature peptide, and the front is contained 12 bases and comprised TAG Transcription Termination codon, and restriction enzyme Spe I recognition sequence (ACTAGT) and 3 enzymes are cut protection base CGT.(Ghrelin mature peptide the 5th amino acids, leucine (Leu) antisense codon is by CAA → TAA) to be construed as limiting enzyme Afl II recognition sequence (CTTAAG) comprising a base mutation.At first, sequence 1 and sequence 3 annealing, polishing is made pcr amplification with primer 1 and 3 then, and extension amplification outcome forms pT-wt-sGhln to sequencing vector pMD-18-T (Takara); Secondly, as masterplate, make pcr amplification with primer 4 and 5 with pT-wt-sGhln, the Sac I/Afl II site that the PCR product is inserted into pT-wt-sGhln produces pT-mt-sGhln; At last, with Sac I/Afl II double digestion pT-mt-sGhln, reclaim small segment, with primer 4 and 6 amplification small segments, the Sac I/SpeI site that the PCR product is inserted into pT-mt-sGhln forms pT-tmt-sGhln.With Sac I/Afl II double digestion pT-wt-sGhln, reclaim small segment, the Sac I/Afl II site that is inserted into pT-tmt-sGhln forms pT-twt-sGhln.Between sequence 2 and sequence 3, do above-mentioned same operation with primer to 2 and 3,4 and 5 and 4 and 6 successively and produce corresponding pT-wt-hGhln, pT-mt-hGhln and pT-tmt-hGhln.
Table 2. oligonucleotide sequence and primer
Oligonucleotide sequence and primer sequence (5 ' to 3 ') justice/antisense
Oligonucleotide sequence 1 ATGCCCTCCACGGGGACCATTTGC justice
AGCCTGCTGCTCCTCAGCGTGCTC
CTCATGGCAGACTTGGCCATGGCG
GGCTCCAGCTT
CCCCGAA
CACC
Oligonucleotide sequence 2 ATGCCACTCTGGGTGTTCTTCTTTG justice
TGATCCTCACCCTCAGCAACAGCT
CCCACTGCTCCCCACCTCCCCCTTT
GACCCTCAGGATGCGGCGGGGCTC
CAGCTT
CCC
TTGGCTGCTGGCTTCTTGGACTCCT
TTCTCTGCTGCACTTTCTGGTGTTCG
GGG
AAGCTGGAGCC
Primer 1 ATGCCCTCCACGGGGACCATTT justice
Primer 2 ATGCCACTCTGGGTGTTCTTCTTT justice
Primer 4 TTC
GGTACCCGG justice
Primer 6 GCA
The CTACTTGGGGCTTAA antisense
Annotate: underscore is represented restriction enzyme Afl II, Spe I and Sac I recognition sequence.Primer 5 comprises and Serine antisense codon is replaced by antisense tryptophane codon (GCT → CCA), primer 6 will be at the terminal Methionin antisense codon (CTT) that adds of Ghrelin small peptide C-.
Annealing reaction cumulative volume 20 μ l, positive-sense strand, each 2.5 μ M of antisense strand, 1mMdNTP, TagDNA polysaccharase 0.25U/ μ l, reaction conditions: 94 ℃, 4min; 58 ℃, 40s; 72 ℃, 1min; A circulation.Above-mentioned all PCR reaction conditionss all are identical: at first 94 ℃, and sex change 5min, then, and 60 ℃ of annealing 30s, 72 ℃ are extended 30s, 94 ℃ of sex change 30s, totally 30 circulations.
2, muscle specific expression vector establishment
Skeletal muscle α-actin5 ' is verified can to drive the expression of a plurality of reporter genes in vivo and in vitro, and is that muscle tissue is special.
Come from pGEM-5zf (for the pGEM-T Vector of Promega) plasmid DNA skeleton pGEM-A5f3f building process as shown in Figure 2: pGEM-A5f3f contains a 2857bp Nco I/Mlu I fragment, comprising 1910bp pig skeletal muscle α-actin (SKA) 5 ' flank, first exon and first intron.PGEM also has a 613bp Sac I/Nsi segmental people GH of I (hGH) cDNA3 ' non-translational region.5 ' the flank of plasmid DNA skeleton pGEM and 3 ' flank all are that the method by PCR increases from genome and obtains.PGEM-A5f3f expression vector skeleton is patent protection, patent publication No.: CN1432577A.In order to make up pig Ghrelin and derivative variant cDNA myogen expression vector thereof, as shown in Figure 3, at first pGEM-A5f3f is carried out Mlu I single endonuclease digestion, use Klenow Fragment (Takara) polishing then, reclaim big fragment, carry out Spe I single endonuclease digestion again.Wild-type Ghrelin and derivative cDNA thereof increase from corresponding pT-Ghlns construct by PCR, and the flush end Mlu I/Spe I that is inserted into carrier then produces corresponding pGEM-wt-sGhln, pGEM-mt-sGhln, pGEM-twt-sGhln, pGEM-tmt-sGhln and pGEM-wt-hGhln, pGEM-mt-hGhln, pGEM-tmt-hGhln.After enzyme is cut, the polishing reaction conditions: 94 ℃, 5 minutes; 68 ℃, 15 minutes; The PCR reaction conditions is the same.The carrier pGEM-wt-hGHRH that contains hGHRH cDNA has applied for Chinese patent, patent publication No.: CN1432577A.The target gene sequences of all expression vectors is all passed through sequence verification.The aminoacid sequence of Ghrelin, Ghrelin derivative and the hGHRH of pGEM-Ghlns series expression vector establishment figure and coding thereof is as shown in table 3.
Table 3. and the corresponding coding of expression vector pig Ghrelin and derivative polypeptide structure thereof
The expression vector structure
pGEM-wt-sGhln????MPSTGTICSLLLLSVLLMADLAMA
*GSSFLSPEHQKVQQRKESKK
PAAKLKPR(SEQ?ID?№：1)
pGEM-mt-sGhln????MPSTGTICSLLLLSVLLMADLAMA
*GSWFLSPEHQKVQQRKESKK
PAAKLKPR(SEQ?ID?№：2)
pGEM-twt-sGhln???MPSTGTICSLLLLSVLLMADLAMA
*GSSFLSPK(SEQ?ID?№：3)
pGEM-tmt-sGhln??MPSTGTICSLLLLSVLLMADLAMA
*GSWFLSPK(SEQ?ID?№：4)
pGEM-wt-hGhln???MPLWVFFFVILTLSNSSHCSPPPPLTLRMRR
*GSSFLSPEHQKVQQ
RKESKKPAAKLKPR(SEQ?ID?№：5)
pGEM-mt-hGhln???MPLWVFFFVILTLSNSSHCSPPPPLTLRMRR
*GSWFLSPEHQKVQQ
RKESKKPAAKLKPR(SEQ?ID?№：6)
pGEM-tmt-hGhln??MPLWVFFFVILTLSNSSHCSPPPPLTLRMRR
*GSWFLSPK(SEQ?ID?№：7)
Annotate: what pGEM-wt-sGhln, pGEM-mt-sGhln, pGEM-tmt-sGhln and pGEM-twt-sGhln carried is the signal peptide of pig Ghrelin self, is people GHRH signal peptide and pGEM-wt-hGhln, pGEM-mt-hGhln, pGEM-tmt-hGhln connect.What carrier pGEM-wt-sGhln, pGEM-wt-hGhln encoded is pig Ghrelin mature peptide; Carrier pGEM-mt-sGhln, pGEM-mt-hGhln coding be that to hold the 3rd mutant serine be the mutant of tryptophane to pig GhrelinN-; And carrier pGEM-tmt-sGhln, pGEM-tmt-hGhln coding is the Ghrelin small peptide derivative that only comprises preceding 7 amino acids of pig GhrelinN-end, comprising the 3rd mutant serine is tryptophane, and, added a basic aminoacids at the C-end, Methionin (K).* be the signal peptide aminoacid sequence before, and carrier pGEM-twt-sGhln to be the pig GhrelinN-that only encodes hold the Ghrelin small peptide of preceding 7 amino acids, only added a basic aminoacids, Methionin (K) at the C-end.
Embodiment 2, producer gene medicine
The program of producer gene medicine as shown in Figure 4, detailed process is as follows:
The preparation of sample is extracted operation according to " molecular cloning " plasmid DNA large scale: pGEM-wt-sGhln, pGEM-mt-sGhln, pGEM-tmt-sGhln, pGEM-twt-sGhln, pGEM-wt-hGhln, pGEM-mt-hGhln and the pGEM-tmt-hGhln of preparation among the embodiment 1 are imported DH5 α respectively according to a conventional method.At 37 ℃, cultivate the host bacterium to logarithmic phase for 200 rev/mins then.Then according to flow operations shown in Figure 4, wherein, CaCL
2As precipitation agent, calcium ion is used to reduce RNA in the clean lysate, chromosomal DNA and open loop plasmid; Utilize
KTA explorer system (Pharmacia Biotech Uppsala, Sweden) platform, as shown in table 4 with the sepn process that Q Sepharose XL carries out:
Table 4. plasmid DNA separating step, program and condition
Sample RNA wash-out plasmid wash-out cleans on the purifying procedure balance
Column volume (CV) 5CV Xml+5CV 3CV 5CV 5CV 5CV
Damping fluid 0.5MKAC 25mMTris-HCl, 25mMTris-HCl, 50mM phosphoric acid salt 0.5MNaOH
Ph5.5?????????????????????0.75M?NaCl，??0.5M?NaCl，????1MNaCL????????2MNaCL
pH8.0?????????pH8.0??????????pH10.00
Annotate: the volume of CV=column volume X=cleared lysate
Through after the anionresin, the target purity that super coiled DNA and impurity can reach in the plasmid sample is as shown in table 5.The plasmid DNA of purifying does not contain pollutents such as all bacterial chromosomal dnas, RNA, albumen and intracellular toxin.Expression vector behind the purifying is correct through sequence verification.
The target content of superhelix and impurity in the table 5. plasmid preparation process
The content target
Superhelix (Supercoied plasmid)＞95% nucleic acid
RNA＜2% nucleic acid
Chromosomal DNA＜1% plasmid
Intracellular toxin (Endotoxins) 100EU/mg plasmid
The structure of pig Ghrelin and derivative myogen expression vector thereof is as described in the embodiment 1.Plasmid DNA (genomic medicine) prepares in a large number according to Science Press's " molecular cloning " alkaline lysis, and through polyoxyethylene glycol (PEG) purifying.
Male SPF level Wistar rat is injected 200 μ g pGEM-wt-sGhln, pGEM-mt-sGhln and pGEM-twt-sGhln respectively, is contrast with the pGEM-A5f3f empty carrier.。After the plasmid injection, Ghrelin, Ghrelin Serine (Ser
3) substitute mutant and Ghrelin small peptide variant is secreted into blood circulation.The promotes growth effect is as shown in Figure 5: intramuscularly pGEM-wt-sGhln promotes that rat about 4 weeks of growing are many, accumulation weightening finish peak appears at back 21 days of injection respectively, accumulation weightening finish average specific control group is higher than 9.3% (139.06 ± 13.15vs.127.24 ± 13.58, p＜0.26), show that skeletal muscle is like exercising posttranslational modification mechanism to the Ghrelin in this expression, because the verified Ghrelin that removes decoylization does not have function; Injection pGEM-twt-sGhln also can significantly promote the rat weightening finish, accumulation weightening finish peak appears at back 14 days of injection respectively, the average specific control group is higher than 12.3% (107.62 ± 7.15vs.95.79 ± 9.67, p＜0.05), shows that Ghrelin small peptide derivative can simulate total length Ghrelin biological effect; And injection pGEM-mt-sGhln promotes growth effect is of short duration relatively, and accumulation weightening finish peak appears at back 7 days of injection respectively, and accumulation weightening finish average specific control group is higher than 6.3% (49.66 ± 4.54vs.46.75 ± 4.56, p＜0.37), shows Ghrelin Serine Ser
3) be replaced by tryptophane Trp
3) mutant also can promote growth, but effect is undesirable.Among Fig. 5 the result be expressed as mean number (Mean) ± standard error (Standard error, SE); Injection pGEM-wt-sGhln (n=18), pGEM-mt-sGhln (n=18), pGEM-twt-sGhln (n=18), the control animal (n=18) that the age is similar; Inject and observed pGEM-twt-sGhln in back 14 days with respect to control group remarkable * p≤0.05 of increasing weight.
Embodiment 4, experimentation on animals
The structure of pig Ghrelin or derivatives thereof myogen expression vector is as described in the EXAMPLE l.Plasmid DNA prepares in a large number according to Science Press's " molecular cloning " alkaline lysis, and through polyoxyethylene glycol (PEG) purifying.
Male SPF level Wistar rat is injected 200 μ g pGEM-wt-hGhln, pGEM-tmt-sGhln, pGEM-tmt-hGhln or joint injection pGEM-wt-sGhln ﹠amp respectively; PGEM-wt-hGHRH (containing 100 μ gpGEM-wt-sGhln and 100 μ g pGEM-wt-hGHRH) is contrast with the pGEM-A5f3f empty carrier.After the plasmid injection, Ghrelin, Ghrelin small peptide derivative and GHRH are secreted into blood circulation.The promotes growth effect is as shown in Figure 6: pGEM-tmt-sGhln and pGEM-wt-sGhln ﹠amp; PGEM-wt-hGHRH joint injection group can powerful stimulate normal rat 3 weeks of growing many, the optimal stimulus effect appears at back 14 days of injection, the average accumulated weightening finish is respectively than control group high 19% and 15% (79.84 ± 4.29g, versus 66.96 ± 4.57g, p＜0.05; 76.71 ± 4.17 versus 66.96 ± 4.57g, p＜0.05).And plasmid DNA injection group does not organize hypertrophy (Orgamegaly) and related pathologies to change.By contrast, the gaining effect of pGEM-wt-hGhln and pGEM-tmt-hGhln injection group be it would be better control group in the same time period.The result shows: in genomic medicine, Ghrelin small peptide derivative can be simulated total length Ghrelin biological effect, has strong stimulation growth result; On the genomic medicine method, Ghrelin and GHRH have the effect of collaborative stimulating growth in this system; In direct plasmid DNA injecting method, the effect of the signal peptide of Ghrelin/lead peptide itself is better than the signal peptide of hGHRH/lead peptide.Among Fig. 6 the result be expressed as mean number (Mean) ± standard error (Standard error, SE); Injection pGEM-wt-hGhln (n=18),, pGEM-tmt-hGhln (n=16), pGEM-tmt-sGhln (n=16) and pGEM-wt-sGhln﹠amp; PGEM-wt-hG HRH joint injection (n=16), the control animal (n=16) that the age is similar; Inject and observed pGEM-tmt-sGhln and pGEM-wt-hGhln ﹠amp in back 14 days; PGEM-wt-hGHRH joint injection group is with respect to control group remarkable * p≤0.05 of increasing weight.
For the promotes growth of further checking carrier pGEM-wt-sGhln and pGEM-tmt-sGhln is renderd a service and enhancing gene importing efficient, at first, the Q Sepharose XL anion-exchange column that utilizes peace agate West Asia company to provide exists
KTA purifying platform has carried out purifying (column volume 5ml, sample 20ml) to the injection plasmid DNA, and to remove impurity such as bacterial chromosomal dna, RNA, albumen and intracellular toxin, the enrichment super coiled DNA uses for genomic medicine, reduces plasmid injection consumption.The result that anion-exchange column Q Sepharose XL carries out purifying to the injection plasmid DNA as shown in Figure 7, the electrophoresis of plasmid pGEM-tmt-sGhln DNA behind the purifying as shown in Figure 8, among Fig. 8, the pipe number that digitized representation receives, the super coiled DNA that shown enrichment, reduced chromosomal DNA, the SDA required standard is satisfied in RNA, proteic interference, particularly endotoxin content substantially.Absorbance behind the purifying is as shown in table 6:
Plasmid DNA absorbance behind table 6. purifying
Plasmid DNA OD260 OD280 OD260/Od280 plasmid content (mg/ml)
pGEM-wt-sGhln????0.366??0.200????1.83?????????0.92
pGEM-tmt-sGhln????????0.440?????0.246??????1.79?????????1.10
Injected preceding 3 days in plasmid DNA, take the lead in future the plasmid DNA injection site injected 100ul 0.75% bupivacaine to promote the picked-up of muscle cell to plasmid DNA, improve gene transfering efficiency.
Male SPF level SD (Sprague Dawley) rats with left quadriceps muscle of thigh is injected pGEM-wt-sGhln or pGEM-tmt-sGhln respectively, 150 μ g plasmid DNA/only, control group injection pGEM-A5f3f empty carrier.Animal is conventional raises free choice feeding and drinking-water.Weigh: 1 time/5 days; Take a blood sample/adopt and organize sample: heart extracting blood 1ml after the Animal Anesthesia, 4 ℃ are spent the night, centrifugal preparation serum ,-70 ℃ are frozen, in order to detection GH level (RIA), 1 time/10 days, 10/group/times.Put to death rat after getting blood, gather the injection site tissue sample, liquid nitrogen is preserved.
Rat GH (rGH) serum-concentration detects and still adopts by NHPP, NIDDK, and (Radioimmunoassay RIA) measures (the Chinese Academy of Agricultural Sciences atom the subject of knowledge and the object of knowledge mark chamber) to the test kit that USDA, USA provide by radioimmunology.Data as standard (rendeing a service 2IU/mg), are expressed as ng/ml with rat GH-RP-2, and the susceptibility of reagent is 0.1ng/ml.For fear of the error between measuring, all laboratory samples that a measuring obtains.Each sample is provided with a repetition.
Utilize RT-PCR to analyze plasmid DNA and inject back 30 days injection group destination gene expression situations.The total RNA of muscle tissue extracts and the synthetic of cDNA first chain operated (Gibco) according to the test kit specification sheets.Design special Oligonucleolide primers amplification pGEM-wt-sGhln 168bp fragment 5 '-ATGCCCTCCACGGGGACCATTT-3 ' (justice), 5 '-GCAACTAGTCTAC CGGGGCTTC-3 ' (antisense) or amplification pGEM-tmt-sGhln 108bp fragment 5 '-ATGCCCTC CACGGGGACCATTT-3 ' (justice), 5 '-GCAACTAGTCTACTTGGGGCTTAA-3 ' (antisense).Reaction conditions: 94 ℃, 30s, 60 ℃ of annealing 30s, 72 ℃ of 30s, 25 μ l systems, 30 circulations.Linear shape model as one of The data SAS software (GLM) is analyzed.Numeric representation is mean number (Mean) ± standard error (Standard error, SE), use Ducan ' s multiple ratio than the statistical significant difference between variance analysis evaluation plasmid injection group and control group body weight, weightening finish, average daily gain, Serum GH level and the variation of Serum GH level.P＜0.05, significant difference.
After the plasmid injection, Ghrelin, its small peptide derivative are secreted into blood circulation.PGEM-wt-sGhln, pGEM-tmt-sGhln injection group can powerfully stimulate normal rat 3 weeks of growing many, gaining effect as shown in Figure 9, the optimal stimulus effect appears at injection back 15 and 20 days respectively, the average accumulated weightening finish is respectively than control group high 11% and 21% (145.61 ± 2.37g, versus 131.76 ± 4.56g; 207.81 ± 9.01 versus 171.75 ± 5.62g, p＜0.05).Behind the sacrifice of animal, quantity is cut apart each organ (heart, lung, liver, spleen, brain, kidney stomach, testis etc.), shows injection each organ is consistent behind the plasmid to grow, and the injection group is not organized hypertrophy (Orgamegaly) and related pathologies pathology.The carcass weight analysis shows that also plasmid DNA injection group genetic ability for carcass weight is better than control group.Among Fig. 9, the result is expressed as mean number (Mean) ± average mistake (Standard error); Injection pGEM-wt-sGhln (n=10), pGEM-tmt-sGhln (n=10), the control animal (n=10) that the age is similar; Inject and observed pGEM-tmt-sGhln in back 20 days significantly, * p≤0.05 with respect to the control group weightening finish.
Plasmid DNA injection back 10,20,30 days has been gathered the blood sample of plasmid DNA injection group and control animals respectively.Serum GH radioimmunoassay result as shown in figure 10, show with respect to control group, pGEM-tmt-sGhln injection group Serum GH level significantly rises, reached peak value (73.04 ± 17.02ng/ml Vs.39.94 ± 14.44ng/ml in back 20 days in injection, n=10, p＜0.043), injects and got back to basal level in back 30 days.In the identical time period, pGEM-wt-sGhln injection group Serum GH level also is higher than control animals.The result shows, utilizes the expression vector ectopic expression Ghrelin of muscle specific or its small peptide derivative can induce GH to discharge rising Serum GH level.Among Figure 10, the result is expressed as mean number (Mean) average mistake, and (Standard error SE), injects pGEM-wt-sGhln (n=10), pGEM-tmt-sGhln (n=10), the contrast (n=10) that the age is similar.Injected back 20 days, the change of plasmid pGEM-tmt-sGhln DNA injection group Serum GH level is significantly higher than control group, * p≤0.05.
After the plasmid DNA injection, gathered injection site muscle in 3,7,10,14,20,30 days respectively after injection, extracted the total RNA of muscle tissue then, total RNA handles to eliminate plasmid through DNase I.Destination gene expression situation that the design special primer has utilized the RT-PCR technical Analysis.The result shows basically in the injection site muscle tissue of gathering in the whole experiment after the plasmid DNA injection as shown in figure 11, detects its intended purposes expression of gene.At least the illustration purpose gene can be kept after the plasmid DNA injection and express 1 month.Among Figure 11, Gln1 represents pGEM-wt-sGhln, numeral plasmid DNA injection back fate; Gln5 represents that pGEM-tmt-sGhln injected back 30 days;-, do not add ThermoScript II; M, and molecular weight marker (Marker, PBR322).
Embodiment 6, please replenish and improve the experimentation on animals of polypeptide drugs
Male SPF level Wistar subcutaneous rat is injected the pig Ghrelin derivative in the table 7 of solid phase synthesis respectively, 100mg/kg, 2 times/day, control group injecting normal saline, 18 of every group of injections.Use the significant difference between Ducan ' s multiple comparisons variance analysis evaluation pig Ghrelin derivative injection group and the control group average daily gain.Its promotes growth effect is as shown in table 7, shows the Ghrelin mutant, and small peptide and small peptide variant can promote the rat growth, and gaining effect is obvious.In the table 7, the result is expressed as mean number (Mean) ± average mistake, and (Standard error, SE), significant difference is represented in * p≤0.05.
The effect of table 7. polypeptide drugs (
#The esterification of expression acyl)
| Pig Ghrelin derivative | Inject average weight gain after 3 days | Inject average weight gain after 5 days | Inject average weight gain after 7 days | Inject average weight gain after 10 days |
| It is the pig Ghrelin of tryptophane that N holds the 3rd amino acids residue | ?24.3±1.13g | ?41.2±2.14g | ?58.6±3.24g | ?84.6±5.19g |
| ?GSSF | ?22.1±0.98g | ?37.3±1.93g | ?51.3±2.43g | ?75.3±4.15g |
| ?GS #SF | ?22.8±1.03g | ?39.2±2.03g | ?55.1±3.10g | ?79.5±4.02g |
| ?GSWF | ?23.2±1.01g | ?38.8±1.87g | ?54.9±3.69g | ?79.3±3.95g |
| ?GSSFLSPK | ?22.7±1.20g | ?38.1±1.74g | ?52.3±2.89g | ?76.0±4.34g |
| ?GS #SFLSPK | ?24.6±1.15g | ?41.7±2.10g * | ?59.6±3.13g * | ?83.5±4.21g * |
| ?GSWFLSPK | ?23.8±1.17g | ?41.0±1.79g * | ?58.9±3.27g * | ?84.3±4.43g * |
| Contrast | ?22.3±1.12g | ?37.5±1.65g | ?52.2±2.24g | ?75.4±4.32g |
Sequence table
<160>7
<210>1
<211>52
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Met?Pro?Ser?Thr?Gly?Thr?Ile?Cys?Ser?Leu?Leu?Leu?Leu?Ser?Val?Leu
1???????????????5???????????????????10??????????????????15
Leu?Met?Ala?Asp?Leu?Ala?Met?Ala?Gly?Ser?Ser?Phe?Leu?Ser?Pro?Glu
20??????????????????25??????????????????30
His?Gln?Lys?Val?Gln?Gln?Arg?Lys?Glu?Ser?Lys?Lys?Pro?Ala?Ala?Lys
35??????????????????40??????????????????45
Leu?Lys?Pro?Arg
50
<210>2
<211>52
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>2
Met?Pro?Ser?Thr?Gly?Thr?Ile?Cys?Ser?Leu?Leu?Leu?Leu?Ser?Val?Leu
1???????????????5???????????????????10??????????????????15
Leu?Met?Ala?Asp?Leu?Ala?Met?Ala?Gly?Ser?Trp?Phe?Leu?Ser?Pro?Glu
20??????????????????25??????????????????30
His?Gln?Lys?Val?Gln?Gln?Arg?Lys?Glu?Ser?Lys?Lys?Pro?Ala?Ala?Lys
35??????????????????40??????????????????45
Leu?Lys?Pro?Arg
50
<210>3
<211>32
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>3
Met?Pro?Ser?Thr?Gly?Thr?Ile?Cys?Ser?Leu?Leu?Leu?Leu?Ser?Val?Leu
1???????????????5???????????????????10??????????????????15
Leu?Met?Ala?Asp?Leu?Ala?Met?Ala?Gly?Ser?Ser?Phe?Leu?Ser?Pro?Lys
20??????????????????25??????????????????30
<210>4
<211>32
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>4
Met?Pro?Ser?Thr?Gly?Thr?Ile?Cys?Ser?Leu?Leu?Leu?Leu?Ser?Val?Leu
1???????????????5???????????????????10??????????????????15
Leu?Met?Ala?Asp?Leu?Ala?Met?Ala?Gly?Ser?Trp?Phe?Leu?Ser?Pro?Lys
20??????????????????25??????????????????30
<210>5
<211>59
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>5
Met?Pro?Leu?Trp?Val?Phe?Phe?Phe?Val?Ile?Leu?Thr?Leu?Ser?Asn?Ser
1???????????????5???????????????????10??????????????????15
Ser?His?Cys?Ser?Pro?Pro?Pro?Pro?Leu?Thr?Leu?Arg?Met?Arg?Arg?Gly
20??????????????????25??????????????????30
Ser?Ser?Phe?Leu?Ser?Pro?Glu?His?Gln?Lys?Val?Gln?Gln?Arg?Lys?Glu
35??????????????????40??????????????????45
Ser?Lys?Lys?Pro?Ala?Ala?Lys?Leu?Lys?Pro?Arg
50??????????????????55
<210>6
<211>59
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>6
Met?Pro?Leu?Trp?Val?Phe?Phe?Phe?Val?Ile?Leu?Thr?Leu?Ser?Asn?Ser
1???????????????5???????????????????10??????????????????15
Ser?His?Cys?Ser?Pro?Pro?Pro?Pro?Leu?Thr?Leu?Arg?Met?Arg?Arg?Gly
20??????????????????25??????????????????30
Ser?Trp?Phe?Leu?Ser?Pro?Glu?His?Gln?Lys?Val?Gln?Gln?Arg?Lys?Glu
35??????????????????40??????????????????45
Ser?Lys?Lys?Pro?Ala?Ala?Lys?Leu?Lys?Pro?Arg
50??????????????????55
<210>7
<211>39
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>7
Met?Pro?Leu?Trp?Val?Phe?Phe?Phe?Val?Ile?Leu?Thr?Leu?Ser?Asn?Ser
1???????????????5???????????????????10??????????????????15
Ser?His?Cys?Ser?Pro?Pro?Pro?Pro?Leu?Thr?Leu?Arg?Met?Arg?Arg?Gly
20??????????????????25??????????????????30
Ser?Trp?Phe?Leu?Ser?Pro?Lys
35
Claims (10)
1, a boar Ghrelin derivative, it for comprise at least GSSF or GSWF amino acid residue sequence by pig Ghrelin polypeptides derived.
2, pig Ghrelin derivative according to claim 1 is characterized in that: described pig Ghrelin derivative for comprise at least GSSFLSP or GSWFLSP amino acid residue sequence by pig Ghrelin polypeptides derived.
3, pig Ghrelin derivative according to claim 1 and 2 is characterized in that: the 2nd Serine among the described GSSF is the Serine of decoyl esterification.
4, pig Ghrelin derivative according to claim 1 and 2 is characterized in that: the N end of described pig Ghrelin derivative also is connected with signal peptide sequence; Described signal peptide is pig Ghrelin or human growth hormone releasing hormone (hGHRH) signal peptide preferably.
5, pig Ghrelin derivative according to claim 1 and 2, it is characterized in that: the C-terminal of described pig Ghrelin derivative is added with a basic aminoacids.
6, pig Ghrelin derivative according to claim 1 and 2 is characterized in that: described pig Ghrelin derivative is the SEQ ID № in the sequence table: 1, SEQ ID №: 2, SEQ ID №: 3, SEQ ID №: 4, SEQ ID №: 5, SEQ ID №: 6 or SEQ ID №: 7.
7, the gene of the described pig Ghrelin of coding claim 1 derivative.
8, contain described expression carrier of claim 7 and clone.
9, a kind of genomic medicine, its activeconstituents is the gene of coding pig Ghrelin derivative; Described pig Ghrelin derivative for comprise at least GSSF or GSWF amino acid residue sequence by pig Ghrelin polypeptides derived.
10, a kind of polypeptide drugs, its activeconstituents are pig Ghrelin derivative or its soluble salt; Described pig Ghrelin derivative for comprise at least GSSF or GSWF amino acid residue sequence by pig Ghrelin polypeptides derived.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101129327A CN1321131C (en) | 2003-12-26 | 2003-12-26 | Pig Ghrelin derivative, its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101129327A CN1321131C (en) | 2003-12-26 | 2003-12-26 | Pig Ghrelin derivative, its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1634985A true CN1634985A (en) | 2005-07-06 |
| CN1321131C CN1321131C (en) | 2007-06-13 |
Family
ID=34843368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101129327A Expired - Fee Related CN1321131C (en) | 2003-12-26 | 2003-12-26 | Pig Ghrelin derivative, its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1321131C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676449A (en) * | 2012-05-14 | 2012-09-19 | 浙江大学 | Ghrelin-containing sheep embryo in-vitro culture solution and culture method thereof |
| CN107253984A (en) * | 2017-05-08 | 2017-10-17 | 河南金大众生物工程有限公司 | Growth promotion complex polypeptide and its application |
| CN111286485A (en) * | 2016-08-01 | 2020-06-16 | 北京市农林科学院 | Application of caprylated Ghrelin in inhibiting secretion of cumulus cell complex cAMP and MAPK in vitro |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK1197496T3 (en) * | 1999-07-23 | 2007-10-22 | Kenji Kangawa | New peptides |
| WO2001092292A2 (en) * | 2000-05-30 | 2001-12-06 | Merck & Co., Inc. | Ghrelin analogs |
| JP4493913B2 (en) * | 2001-01-31 | 2010-06-30 | 中外製薬株式会社 | Undernutrition Symptom Treatment Agent |
| EP1385879A4 (en) * | 2001-05-10 | 2005-02-02 | Univ Queensland | Reproductive cancer diagnosis and therapy |
| JP3454269B1 (en) * | 2002-03-26 | 2003-10-06 | セイコーエプソン株式会社 | Radio-controlled clock and method of controlling radio-controlled clock |
| BR0306685A (en) * | 2002-05-21 | 2005-04-26 | Daiichi Suntory Pharma Co Ltd | Pharmaceutical composition containing ghrelin |
-
2003
- 2003-12-26 CN CNB2003101129327A patent/CN1321131C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676449A (en) * | 2012-05-14 | 2012-09-19 | 浙江大学 | Ghrelin-containing sheep embryo in-vitro culture solution and culture method thereof |
| CN102676449B (en) * | 2012-05-14 | 2015-06-10 | 浙江大学 | Ghrelin-containing sheep embryo in-vitro culture solution and culture method thereof |
| CN111286485A (en) * | 2016-08-01 | 2020-06-16 | 北京市农林科学院 | Application of caprylated Ghrelin in inhibiting secretion of cumulus cell complex cAMP and MAPK in vitro |
| CN111304159A (en) * | 2016-08-01 | 2020-06-19 | 北京市农林科学院 | Application of octanoylated Ghrelin in inhibiting bovine oocyte meiosis in vitro |
| CN111304158A (en) * | 2016-08-01 | 2020-06-19 | 北京市农林科学院 | Application of caprylylated Ghrelin in promoting secretion of estradiol and progesterone of oocyte cumulus cell complex of cow |
| CN111304158B (en) * | 2016-08-01 | 2022-01-21 | 北京市农林科学院 | Application of caprylylated Ghrelin in promoting secretion of estradiol and progesterone of oocyte cumulus cell complex of cow |
| CN107253984A (en) * | 2017-05-08 | 2017-10-17 | 河南金大众生物工程有限公司 | Growth promotion complex polypeptide and its application |
| CN107253984B (en) * | 2017-05-08 | 2020-09-01 | 河南金大众生物工程有限公司 | Growth-promoting composite polypeptide and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1321131C (en) | 2007-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1241942C (en) | Methods for treatment of diabetes using peptide analogues of insulin | |
| CN1362968A (en) | Novel peptides | |
| CN1582295A (en) | Novel epidermal growth factor protein and gene, and methods of use therefor | |
| CN101035806A (en) | Amylin family polypeptide-6 (AFP-6) analogs and methods of making and using them | |
| CN1320117C (en) | Adiponectin-Glucagon-like peptide-1-like peptide recombinant protein expression vector and construction | |
| CN1654656A (en) | FenneropenaeusChinensis Crustin gene and coded protein and cloning method therefor | |
| CN1149258A (en) | Inhibin compositions and methods of use thereof | |
| CN1634985A (en) | Pig Ghrelin derivative, its encoding gene and application | |
| CN1254375A (en) | Motilin homologs | |
| CN1119650A (en) | Motilin-like polypeptides with gastrointestinal motor stimulating activity | |
| CN101045745A (en) | Scale preparation method of bursin and application of used as avian influema vaccine adjuvant | |
| CN1171998C (en) | Antibacterial peptide gene of Chinese prawn and its colon technique | |
| CN1191271C (en) | Thymic peptide fusion protein as one new interferon and its prepn. and use | |
| CN100335636C (en) | Thioredoxin gene of Schistosoma japonicum (Chinese Mainland Strain) and its cloning expression method and application | |
| CN1746188A (en) | Analog of GLP-1 | |
| CN1657617A (en) | Clone, expression and anti-tick immanoprotection action of falcate rhipicephalus RhcA and RhcB genes | |
| CN1854295A (en) | Production of specific micro-antibody for oarium cancer | |
| CN1274827C (en) | Cancer gene and its coded protein and special cell line | |
| CN1367180A (en) | Novel natural antibacterial peptide, its code sequence and application | |
| CN101050448A (en) | Oral recombined DNA vaccine for accelerating growth of animal, and application | |
| CN1247616C (en) | New monomeric insulin and its medicinal composition and prepn process | |
| CN1803194A (en) | Immunity regulating type DNA vaccine for preventing and treating chicken coccidiosis | |
| CN1733807A (en) | A kind of fusion rotein and encoding gene and application with growth facilitation action | |
| CN1289527C (en) | Horny cell growth factor mutant with high bioactivity and its preparation process and use thereof | |
| CN101057970A (en) | Therapeutic vaccine for Myostatin specific antibody and its preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070613 Termination date: 20141226 |
|
| EXPY | Termination of patent right or utility model |