CN101012273A - Rana grahami secretory peptide, gene thereof and application in pharmacy - Google Patents
Rana grahami secretory peptide, gene thereof and application in pharmacy Download PDFInfo
- Publication number
- CN101012273A CN101012273A CNA2007100656720A CN200710065672A CN101012273A CN 101012273 A CN101012273 A CN 101012273A CN A2007100656720 A CNA2007100656720 A CN A2007100656720A CN 200710065672 A CN200710065672 A CN 200710065672A CN 101012273 A CN101012273 A CN 101012273A
- Authority
- CN
- China
- Prior art keywords
- secretory peptide
- rana grahami
- gene
- peptide
- rana
- 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.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a non-finger frog secretory peptide and gene and application in the drug in the biological medical domain, which codes a cyclic peptide with molecular weight at 1587.98 and isoelectric point at 9.7, wherein the primary structure of peptide is Phe Met Pro Ile Leu Ser Cys Ser Arg Phe Lys Arg Cys with disulfide bond in the seventh cysteine and thirteenth cysteine; the gene of secretory peptide is composed of 300 nucleic acids, which codes 138-177th nucleic acid; the peptide possesses obvious bacteria and fungi inhibiting action, which can be applied to make microbe infection disease drug.
Description
Technical field:
The present invention relates to a kind of odorranagrahami (Rana Grahami) secretion peptide and gene and the application in pharmacy, belong to biomedical sector.
Background technology:
The discovery of penicillin makes people can tackle all diseases that all kinds of cause pathogeny imcrobe infection causes, and all kinds of microbiotic that derive have thus been made huge contribution to the protection human health especially.But, because people cause many bacteriums that they have been produced resistance to the life-time service and the abuse of these " traditional microbiotic ".So people begin to seek new antimicrobial agents.Discovering in recent years, polypeptide antibiotics not only has the broad spectrum antimicrobial activity, have " traditional microbiotic " incomparable superiority simultaneously: as when the very little concentration, just can wide spectrum and kill microorganisms (comprise at present clinically endurance strain) apace; Fungi also there is restraining effect; Be difficult to inducible strain and produce resistance; All effective for local infection and systemic infection, promise to be antimicrobial agents of new generation.At present, the development at the small peptide antimicrobial agents has been subjected to increasingly extensive attention.
Belong in traditional Chinese medicine and the national medicine in China, a lot of amphibian animals are used widely, as Chinese toad (Bufo gargarizans), Bombina maxima (Bombina maxima), Rana nigromaculata (pelophylaxnigromaculata), Rana guentheri (Hylarana guentheri) and rana limnocharis (Euphlyctislimnocharis) etc.The skin of these amphibian animals and internal organ have pharmacologically active and clinical efficacy widely.Reported that pharmacologically active has: antimicrobial, antitumor, analgesia, toponarcosis, immunomodulatory, to cardiovascular systems effect etc.Abroad, the searching of the specific pharmacology reactive monomer of batrachians skin compound has been the focus of new drug invention.According to domestic and foreign literature, separated obtaining different active polypeptide from various biogenetic derivations, and some has entered clinical stage.Active polypeptide Magainin tool wide spectrum anti-microbial effect as obtaining from Xenopus laevis (Xenopus laevis) skin juice has anti-tumor activity simultaneously, has got permission as extensive pedigree antibiotic in the U.S., and it is clinical that its gene engineering product entered for three phases; The active polypeptide IB-367 that Intrabiotics company produces has got permission to be used for the treatment of cancer patient and has infected stomatocace one clinical trial phase that causes because of various bacteria.
There is long history in China to the application of batrachians medicine, but the research of its activeconstituents and pharmacological properties is mainly concentrated on organic molecules such as alkaloid, and is few to its skin activity peptide matters research.Odorranagrahami (Rana Grahami) mainly is distributed in Sichuan, Yunnan Province, is one of China characteristic resources animal.
The contriver searches comparison with Rana grahami secretory peptide complete sequence amino acid structure of the present invention through Protein Data Bank, finds no any phase homopolypeptide.The contriver searches comparison with Rana grahami secretory peptide encoding gene of the present invention through gene database, finds no any homologous genes.
Summary of the invention:
The object of the present invention is to provide a kind of new Rana grahami secretory peptide with wide spectrum antimicrobial (comprising gram-negative, positive bacteria, fungi) and gene and the application in pharmacy.
In order to realize purpose of the present invention, the invention provides following technical scheme:
Rana grahami secretory peptide:
Rana grahami secretory peptide is a kind of ring type polypeptide of Chinese batrachians Rana grahami secretory peptide genes encoding, molecular weight 1587.98 dalton, iso-electric point 9.7, polypeptide complete sequence primary structure is: Phe Met ProIle Leu Set Cys Ser Arg Phe Lys Arg Cys (FMPILSCSRFKRC), its 7th halfcystine and the 13 the halfcystine intramolecular disulfide bond that coordinates.
The clone of Rana grahami secretory peptide gene comprises:
The total RNA of Rana grahami skin extracts, the mRNA purifying, and mRNA reverse transcription and cDNA library construction, the design primer utilizes PCR method screening Rana grahami secretory peptide gene.Amplimer length is 25 Nucleotide, and its sequence is 5 ' ATGTTCACCATGAAGAAATCCCTGT3 ', and another amplimer of PCR is the SMART of CLONTECH company
TM5 ' PCR Primer primer among the cDNA Library Construction Kit, its sequence is 5 ' ATTCTAGAGGCCGAGGCGGCCGACATG3 '.The positive monoclonal that obtains carries out gene nucleotide series and measures.Gene sequencing result shows that the gene of the Rana grahami secretory peptide of encoding is made up of 300 Nucleotide, from 5 ' end to 3 ' terminal sequence is:
atgttcacca tgaagaaatc cctgttactc cttttcttcc ttgcgaccat caacttatct 60
ctctgtgagg aagagagaaa tgcagaagaa gaaagaagag atgatccaga tgaaatgaat 120
gctgaagtgg aaaaacgatt tatgccaata cttagttgtt caaggtttaa aagatgttga 180
aacttgaatt ggaaatcatc tgatgtggaa tatcatttag ctaaatgtac atcagatgtc 240
ttataaaaaa ataaacatgc tgcatacact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300
The ripe active polypeptide of coding odorranagrahami is a 138-177 position Nucleotide, and its aminoacid sequence is:
Phe Met Pro Ile Leu Ser Cys Ser Arg Phe Lys Arg Cys
5 10
The Rana grahami secretory peptide gene prepares the application of Rana grahami secretory peptide as genetically engineered.
The preparation method of Rana grahami secretory peptide:
Infer according to the gene of coding Rana grahami secretory peptide and the aminoacid sequence of Rana grahami secretory peptide to synthesize its complete sequence with automatic Peptide synthesizer.By the anti-phase C of HPLC
18Post desalination, purifying.Identify its purity with the HPLC method then, molecular weight determination adopts fast atom bombardment mass spectroscopy(FABMS) method (Fast atom bombardmentmass spectrometry, FAB-MS), isoelectric focusing electrophoresis is measured iso-electric point, measures the aminoacid sequence structure with automatic Protein Sequencer.
Rana grahami secretory peptide has the effect of significant inhibition bacterium and fungal growth, can be used as the application of preparation cause pathogeny imcrobe infection treatment of diseases medicine.
Beneficial effect of the present invention is:
By Rana grahami secretory peptide encoding gene its amino acid structure of deriving, the synthetic Rana grahami secretory peptide has the effect of significant inhibition bacterium and fungal growth.The beneficial features that this Rana grahami secretory peptide has is simple in structure, synthetic convenient, antibiotic pedigree is wide.
Embodiment:
Embodiment one:
The Rana grahami secretory peptide gene clone:
I, the total RNA of Rana grahami skin extract:
A. live body odorranagrahami water cleans up, and puts into the liquid nitrogen quick-frozen 4 hours, gets skin histology, weigh, get the 300mg skin histology, add the total RNA of 10ml and extract damping fluid (Trizol solution, U.S. GIBCOBRL company product), homogenate is 30 minutes in the 20ml glass homogenizer.
B. add equal-volume phenol/chloroformic solution, violent mixing, room temperature was placed 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm, reject precipitation.
C. supernatant adds isopyknic Virahol, and room temperature was placed 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm, precipitation is washed once with 75% ethanol, dries, and pipe end throw out is the total RNA of Rana grahami skin.
The purifying of II, Rana grahami skin mRNA:
The PolyATtract of U.S. PROMEGA company is adopted in Rana grahami skin mRNA separation and purification
MRNA Isolation Systems test kit.
A. get the total RNA 500 μ g of Rana grahami skin and be dissolved in the 500 μ l DEPC water, put into 65 ℃ of water-baths 10 minutes, add Oligo (dT) probe and 13 μ l, the 20 * SSC solution of people 3 μ l, mixing is placed the room temperature cooling, is called A liquid.
B. the washing of magnetic bead (SA-PMP): magnetic bead is flicked mixing,, abandon supernatant, add 0.5 * SSC 0.3ml,, add 0.1ml 0.5 * SSC at last and suspend, be referred to as B liquid to magnetic force frame absorption 30 seconds to magnetic force frame absorption 30 seconds.
C. A liquid is added in the B liquid, room temperature was placed 10 minutes, to magnetic force frame absorption 30 seconds, abandon supernatant, with 0.1 * SSC washing 4 times, abandon supernatant at last, add 0.1ml DEPC aqueous suspension, to the magnetic force frame, adsorbed 30 seconds, supernatant is moved to new test tube, add 0.15ml DEPC water again and suspend again, to magnetic force frame absorption 30 seconds, moving supernatant to above-mentioned test tube, then is the Rana grahami skin mRNA of purifying in the supernatant.
D. add 1/10 volume 3M sodium acetate, pH5.2, the equal-volume Virahol, in-70 ℃ of placements 30 minutes, 4 ℃, centrifugal 10 minutes of 12000rpm abandoned supernatant, and resolution of precipitate is in 10 μ l DEPC water.
III, Rana grahami skin cDNA library construction: adopt the CLONTECH Creator of company
TMSMART
TMCDNA Library Construction Kit Construction of Plasmid cDNA Library test kit.
A.cDNA first chain synthesizes (mRNA reverse transcription):
1. add 1 μ l Rana grahami skin mRNA, 1 μ lSMART IV oligonucleotide, 1 μ l CDS III/3 ' PCR primer at the aseptic centrifuge tube of 0.5ml, add 2 μ l deionized waters and make cumulative volume reach 5 μ l.
2. the reagent in the mixing centrifuge tube and centrifugal 1 minute with 12000rpm, 72 ℃ of insulations 2 minutes.
3. centrifuge tube was hatched on ice 2 minutes.
4. in centrifuge tube, add following reagent 2.0 μ l 5 * the first chains buffering, 1.0 μ l 20mM dithiothreitol (DTT), 1.0 μ l10mM dNTP mixtures, 1.0 μ l PowerScript ThermoScript II.
5. reagent and with 12000rpm centrifugal 1 minute in the mixing centrifuge tube were 42 ℃ of insulations 1 hour.
6. centrifuge tube is placed and end the synthetic of first chain on ice.
7. it is standby to get the 2 synthetic cDNA of μ l institute, first chain from centrifuge tube.
B. adopt long terminal polymerase chain reaction (LD-PCR) method second chain that increases
1.95 ℃ preheating PCR instrument.
2. 2 μ l cDNA, first chains (mRNA reverse transcription), 80 μ l deionized waters, 10 μ l, 10 * Advantage, 2 PCR buffering, 2 μ l, 50 * dNTP mixture, 2 μ l, 5 ' PCR primer, 2 μ l CDS III/3 ' PCR primers and 2 μ l Escherichia coli polymerase centrifuge tubes are reacted.
3. in the PCR instrument, increase by following program:
1. 95 ℃ of 20 second
2. 22 circulations:
95 ℃ of 5 second
68 ℃ 6 minutes
4. after the loop ends, synthetic cDNA two strands in the centrifuge tube is carried out extracting.
The C.PCR product Wizard of PROMEGA company
SV Gel and PCR Clean-UpSystem test kit carries out extracting and reclaims, and step is as follows:
1. will put upside down mixing by the isopyknic film binding buffer of the double-stranded adding of the cDNA that PCR obtains, will mix liquid then and change the centrifugal purification post over to, room temperature left standstill 5 minutes, and DNA is fully combined with pellosil.16,000g centrifugal 1 minute with 12000rpm outwells the waste liquid in the collection tube.
2. the elutriant (containing ethanol) that adds 700 μ l in the centrifugal purification post, 16,000g centrifugal 1 minute with 12000rpm outwells the waste liquid in the collection tube.
3. repeating step 2.
4.16,000g centrifugal 5 minutes with 12000rpm.
5. the centrifugal purification post is placed new centrifuge tube.
6. add 30 μ l ultrapure waters, at room temperature left standstill 5 minutes.
7.16,000g centrifugal 1 minute with 12000rpm, pipe end solution is the cDNA two strands of purified mistake.
D. the preparation of bacillus coli DH 5 alpha competent cell:
1. the single DH5 α of picking bacterium colony is inoculated in 3ml and does not contain in the LB substratum of penbritin, and 37 ℃ of overnight incubation are got above-mentioned bacterium liquid next day and are inoculated at 1: 100 in proportion in the 50ml LB nutrient solution, and 37 ℃ vibrated 2 hours.Work as OD
600Value reaches at 0.35 o'clock, the results bacterial cultures.
2. bacterium is transferred in aseptic, disposable, the ice-cold 50ml polypropylene tube, 10min puts in the side on ice, makes culture be cooled to 0 ℃.
In 4 ℃ with the centrifugal 10min of 4100r/min, to reclaim cell.
4. pour out nutrient solution, pipe is inverted 1min so that last trace nutrient solution flows to end.
5. the 0.1mol/L CaCl of every 50ml initial incubation liquid and 30ml precooling
2-MgCl
2Solution (80mmol/L MgCl
2, 20mmol/L CaCl
2) resuspended every part of cell precipitation.
In 4 ℃ with the centrifugal 10min of 4100r/min, to reclaim cell.
7. pour out nutrient solution, pipe is inverted 1min so that last trace nutrient solution flows to end.
8. every 50ml initial incubation thing ice-cold 0.1mol/L CaCl of 2ml
2Resuspended every part of cell precipitation, standby after the packing.
E. the enzyme conversion cutting, connect and connect product:
1. add 1 μ l Takara pMD18-T carrier, the double-stranded solution of 4 μ l odorranagrahami cDNA in Eppendorf tube, full dose is 5 μ l.
2. the ligase enzyme buffer mixture that adds 5 μ l (equivalent).
3.16 ℃ reaction 2 hours.
4. full dose (10 μ l) is added in the 100 μ l DH5 α competent cells, places 30 minutes in the ice.
5.42 after 90 seconds of ℃ heating, in ice, placed 1 minute again.
6. add 37 ℃ of LB substratum 890 μ l of bathing of temperature, 37 ℃ of slow shaking culture 60 minutes.
7. get 200 μ l and coat on the LB substratum that contains X-Gal, IPTG, Amp 37 ℃ and cultivated 16 hours, form single bacterium colony.
8. each LB plate washs bacterium colony with 5ml LB liquid nutrient medium, and it is frozen to add 30% glycerine.The cDNA that makes up approximately contains 1 * 10
6Individual independent clone.
IV, Rana grahami secretory peptide gene clone screening:
Amplimer length is 25 Nucleotide, and its sequence is 5 ' ATGTTCACCATGAAGAAATCCCTGT3 ', and another amplimer of PCR is the SMART of CLONTECH company
TM3 ' PCR Primer primer among the cDNA Library ConstructionKit, its sequence is 5 ' ATTCTAGAGGCCGAGGCGGCCGACATG3 '.
The PCR reaction is carried out under the following conditions: 94 ℃ of 30 second, 52 ℃ of 45 second and 72 ℃ of 2 fens 30 seconds, 35 circulations.
The bacterium cDNA library that makes up of titration at first, be diluted to suitable bacterial concentration (about 5000 bacteria/milliliters with the LB substratum that contains 100 μ g/ml penbritins then, be respectively applied for first run screening second with 30 bacteria/milliliters and take turns screening), on 96 well culture plates by 8 * 8 matrix bed boards (totally 64 holes, every hole 100 μ l), 37 ℃ of incubated overnight.Merge inoculum respectively by row, column, have 16 samples to carry out PCR and identify that the positive hole bacteria samples of intersecting enters second and takes turns screening.
V, Rana grahami secretory peptide gene sequencing and result:
Extract plasmid DNA and measure nucleotide sequence with dideoxy method, use instrument to be the full-automatic nucleotide sequencing instrument of U.S. AppliedBiosystems373A, sequencing primer is BcaBEST
TMSequencingPrimer RV-M and BcaBEST
TMSequencing Primer M13-47, BcaBEST
TMSequencingPrimer RV-M sequence: 5 ' GAGCGGATAACAATTTCACACAGG3 ', BcaBEST
TMSequencing Primer M13-47:5 ' CGCCAGGGTTTTCCCAGTCACGAC3 '.Gene sequencing result from 5 ' end to 3 ' terminal sequence is:
atgttcacca tgaagaaatc cctgttactc cttttcttcc ttgcgaccat caacttatct 60
ctctgtgagg aagagagaaa tgcagaagaa gaaagaagag atgatccaga tgaaatgaat 120
gctgaagtgg aaaaacgatt tatgccaata cttagttgtt caaggtttaa aagatgttga 180
aacttgaatt ggaaatcatc tgatgtggaa tatcatttag ctaaatgtac atcagatgtc 240
ttataaaaaa ataaacatgc tgcatacact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300
The sequence table of Rana grahami secretory peptide gene nucleotide is: sequence length is 300 bases, sequence type: nucleic acid, chain number: strand, topology: straight chain shape, sequence kind: cDNA, source: Rana grahami skin.
The ripe active polypeptide of coding odorranagrahami is a 138-177 position Nucleotide, and its aminoacid sequence is:
Phe Met Pro Ile Leu Ser Cys Ser Arg Phe Lys Arg Cys
5 10
The Rana grahami secretory peptide gene prepares the application of Rana grahami secretory peptide as genetically engineered.
Embodiment two:
The preparation Rana grahami secretory peptide:
The preparation method of I, Rana grahami secretory peptide: infer according to the gene of coding Rana grahami secretory peptide and the aminoacid sequence of Rana grahami secretory peptide to synthesize its complete sequence with automatic Peptide synthesizer.By the anti-phase C of HPLC
18Column chromatography desalination, purifying.
II, molecular weight determination adopt the fast atom bombardment mass spectroscopy(FABMS) method, and (Fast atom bombardment massspectrometry, FAB-MS), with glycerine: m-nitrobenzyl alcohol: methyl-sulphoxide (1: 1: 1, V: V: V, volume ratio) is a substrate, Cs
+As projectile, electric current is 1 μ A, and emission voltage is 25Ky.
The Rana grahami secretory peptide of III, purifying is identified its purity with the high-efficient liquid phase chromatogram HPLC method, and molecular weight determination adopts the fast atom bombardment mass spectroscopy(FABMS) method, and isoelectric focusing electrophoresis is measured iso-electric point, measures the aminoacid sequence structure with automatic Protein Sequencer.
Rana grahami secretory peptide is a kind of ring type polypeptide of Chinese amphibian animal Rana grahami secretory peptide genes encoding, molecular weight 1587.98 dalton, iso-electric point 9.7, polypeptide complete sequence primary structure is: phenylalanine-methionine(Met)-proline(Pro)-Isoleucine-leucine-Serine-halfcystine-Serine-arginine-phenylalanine-Methionin-arginine-halfcystine (FMPILSCSRFKRC), its 7th halfcystine and the 13 the halfcystine intramolecular disulfide bond that coordinates.
Embodiment three:
The effect of Rana grahami secretory peptide bacteria growing inhibiting:
Anti-microbial activity detects, and adopts cylinder plate method, and substratum is the plain agar substratum.The substratum 20ml that injects heating and melting respectively as bottom, makes its even stand cloth at the bottom of the ware, after solidifying in plate, after other gets an amount of heating and melting of substratum, in every ware, add the 5ml bacteria suspension respectively, shake up, make it on bottom, evenly spread out cloth, as the bacterium layer.After the cooling, evenly put into 6 of disinfectant stainless steel cups in the plate moderate distance.First steel bowl adds testing compound solution 0.1 ml of 0.1-0.3 mg/ml concentration, and all the other steel bowls adopt doubling dilution to add sample liquid, and the inhibition zone size is observed in 37 ℃ of cultivations.Inhibition zone 10mm above as minimal inhibitory concentration (minimal inhibitory concentration, MIC).Bacterial isolates derives from No.1 Hospital Attached to Kunming Medical College, and this test repeats four times, averages result such as table 1.
Table 1, the effect of Rana grahami secretory peptide bacteria growing inhibiting:
| Bacterial strain | Minimal inhibitory concentration (ug/ml) |
| Rana grahami secretory peptide | |
| Escherichia coli ATCC25922 (Escherichia coli ATCC25922) staphylococcus aureus ATCC2592 (Staphylococcus auneus ATCC2592) hay bacillus (Bacillus sub tilis) | 4.68 9.37 37.5 |
By table 1 as seen, the synthetic Rana grahami secretory peptide has the effect of significant bacteria growing inhibiting.
Rana grahami secretory peptide suppresses the effect of fungal growth:
Anti-mycotic activity detects and adopts cylinder plate method, and substratum is improvement husky Bao Shi (Sabousand) substratum.Inject respectively substratum 20ml that heating dissolves in plate as bottom, make its even stand cloth at the bottom of ware, get an amount of heating of substratum after solidifying in addition and dissolve, in every ware, add the 5ml bacteria suspension respectively, shake up, make its even stand cloth on bottom, as the bacterium layer.After the cooling, put into 5 of disinfectant stainless steel cups in the plate moderate distance.First steel bowl adds the testing compound solution 0.1ml of 0.3mg/ml concentration, and all the other steel bowls adopt doubling dilution to add sample liquid, and the inhibition zone size is measured in 37 ℃ of cultivations behind the 24h-48h.Inhibition zone 10mm with as minimal inhibitory concentration (minimal inhibitory concentration, MIC).Bacterial isolates derives from institute of microbiology of Yunnan University, this experiment do three parallel, get geometrical mean, result such as table 2.
Table 2. Rana grahami secretory peptide suppresses the effect of fungal growth:
| Bacterial strain | Minimal inhibitory concentration (ug/ml) |
| Rana grahami secretory peptide | |
| Candida albicans ATCC2002 (Candia albicans ATCC2002) flavus IFFI4015 (Aspergillus flavus sp ATCC2592) | 1.1 3.5 |
By table 2 as seen, the synthetic Rana grahami secretory peptide has the effect of significant inhibition fungal growth.
Rana grahami secretory peptide and gene thereof and the application _ ST25 in pharmacy
SEQUENCE LISTING
＜110〉Kunming Institute of Zoology, Chinese Academy of Sciences
＜120〉Rana grahami secretory peptide and gene thereof and the application in pharmacy
<130>1
<160>2
<170>PatentIn version 3.4
<210>1
<211>300
<212>DNA
<213>Rana grahami
<400>l
atgttcacca tgaagaaatc cctgttactc cttttcttcc ttgcgaccat caacttatct 60
ctctgtgagg aagagagaaa tgcagaagaa gaaagaagag atgatccaga tgaaatgaat 120
gctgaagtgg aaaaacgatt tatgccaata cttagttgtt caaggtttaa aagatgttga 180
aacttgaatt ggaaatcatc tgatgtggaa tatcatttag ctaaatgtac atcagatgtc 240
ttataaaaaa ataaacatgc tgcatacact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300
<210>2
<211>13
<212>PRT
<213>Rana grahami
<400>2
Phe Met Pro Ile Leu Ser Cys Ser Arg Phe Lys Arg Cys
l 5 10
Claims (4)
1. Rana grahami secretory peptide, it is characterized in that this polypeptide is a kind of ring type polypeptide of Chinese batrachians Rana grahami secretory peptide genes encoding, molecular weight 1587.98 dalton, iso-electric point 9.7, polypeptide complete sequence primary structure is: Phe Met Pro Ile Leu Ser Cys Ser Arg Phe Lys Arg Cys, its 7th halfcystine and the 13 the halfcystine intramolecular disulfide bond that coordinates.
2. Rana grahami secretory peptide gene nucleotide series, it is characterized in that: cDNA is made up of 300 Nucleotide, and it from 5 ' end to 3 ' terminal sequence is:
atgttcacca tgaagaaatc cctgttactc cttttcttcc ttgcgaccat caacttatct 60
ctctgtgagg aagagagaaa tgcagaagaa gaaagaagag atgatccaga tgaaatgaat 120
gctgaagtgg aaaaacgatt tatgccaata cttagttgtt caaggtttaa aagatgttga 180
aacttgaatt ggaaatcatc tgatgtggaa tatcatttag ctaaatgtac atcagatgtc 240
ttataaaaaa ataaacatgc tgcatacact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300
3. the described Rana grahami secretory peptide gene nucleotide series of claim 2 is characterized in that, the ripe active polypeptide of coding odorranagrahami is a 138-177 position Nucleotide, and its aminoacid sequence is:
Phe Met Pro Ile Leu Ser Cys Ser Arg Phe Lys Arg Cys
5 10
4. the described Rana grahami secretory peptide of claim 1 is as the application of preparation cause pathogeny imcrobe infection treatment of diseases medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100656720A CN101012273A (en) | 2007-02-12 | 2007-02-12 | Rana grahami secretory peptide, gene thereof and application in pharmacy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100656720A CN101012273A (en) | 2007-02-12 | 2007-02-12 | Rana grahami secretory peptide, gene thereof and application in pharmacy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101012273A true CN101012273A (en) | 2007-08-08 |
Family
ID=38700013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100656720A Pending CN101012273A (en) | 2007-02-12 | 2007-02-12 | Rana grahami secretory peptide, gene thereof and application in pharmacy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101012273A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749535A (en) * | 2016-12-20 | 2017-05-31 | 中国科学院昆明动物研究所 | The smelly frog blood sugar reducing peptide in Yunnan and preparation method and applications |
-
2007
- 2007-02-12 CN CNA2007100656720A patent/CN101012273A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749535A (en) * | 2016-12-20 | 2017-05-31 | 中国科学院昆明动物研究所 | The smelly frog blood sugar reducing peptide in Yunnan and preparation method and applications |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101638432B (en) | Rana pleuraden antioxidant peptide and genes | |
| CN106749595B (en) | Tree frog defense peptides cathelicidin-PP and its gene and application | |
| CN104497119A (en) | Natural antibacterial peptide and application thereof | |
| CN105566452A (en) | Antibacterial peptide with annular structure and preparation method and application thereof | |
| CN100351270C (en) | Antibacterial peptide of brown-spotted torrential frog, its gene and application in drug preparation | |
| CN104031135B (en) | Rhacophorus defense peptides PopuDef and gene thereof and application | |
| CN102250217A (en) | Hyla simplex skin injury repair promotion polypeptides and genes as well as application thereof | |
| CN101824077A (en) | Large green frog antioxidant peptide (antioxidin-RL) as well as gene and application thereof | |
| CN102229659A (en) | Rana nigromaculata antibacterial peptide and gene and application thereof | |
| CN101475630B (en) | Bloody noun antibacterial peptide temporin-La, genes thereof and use in pharmacy | |
| CN100581584C (en) | Serine protease inhibitor of Rana grahami, and its application | |
| CN101012275A (en) | Rana grahami skin peptide, gene thereof and application in pharmacy | |
| CN101012273A (en) | Rana grahami secretory peptide, gene thereof and application in pharmacy | |
| CN101724632B (en) | Bullfrog skin active peptide, gene and application thereof in pharmacy | |
| CN101012274A (en) | Rana grahami peptide, gene thereof and application in pharmacy | |
| CN100351271C (en) | Active polypeptide of brown-spotted torrential frog, its gene and application in drug preparation | |
| CN102796176B (en) | Kunyuenin, preparation method and application | |
| CN100389125C (en) | Rana grahami propyl sperm peptide, gene and variant and its pharmaceutical use | |
| CN104356216B (en) | Version receives rope buffalo gnat defense peptides SibaDef and its gene and application | |
| CN110294808B (en) | Polypeptide SR4 with anti-inflammatory effect and application thereof | |
| CN101012277A (en) | Rana grahami skin active peptide, gene thereof and application in pharmacy | |
| CN101012278A (en) | Rana grahami anti-microorganism polypeptide, gene thereof and application in pharmacy | |
| CN102115496A (en) | Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof | |
| CN106496317B (en) | Rana chensinensis secretory peptide, gene thereof and application thereof in pharmacy | |
| CN101012276A (en) | Rana grahami antiblastic peptide, gene and variant thereof, and application in pharmacy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |