CN103102403B - Two mutants of analgesic bioactive peptide GRR, and preparation and application thereof - Google Patents
Two mutants of analgesic bioactive peptide GRR, and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicine, and relates to two mutants of analgesic bioactive peptide GRR obtained by gene engineering technology, structures and a preparation method of derivatives, analogues and active fragments of the two mutants of the analgesic bioactive peptide GRR, and application of the derivatives, analogues and active fragments of the two mutants of the analgesic bioactive peptide GRR to the field of medicine as analgesic drugs. The two mutants of the analgesic bioactive peptide GRR have amino acid sequences as follows: GRDAFIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGHACWCINLPDDKPIRIPGKCHRR, and GRDAYIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKFGHACWCINLPDDKPIRIPGKCHRR. The two mutants of the analgesic bioactive peptide GRR and the derivatives, analogues or active fragments thereof can be mixed with a pharmaceutically acceptable carrier to prepare a clinically acceptable injection, an oral preparation, a transdermal preparation and a mucosal absorption preparation for all kinds of pains. The two mutants of the analgesic bioactive peptide GRR have good analgesic activity, and can be obtained through a conventional, simple and high-yield method.
Description
technical field:
The present invention relates to biological medicine technology field, relate to two mutant of antalgic active peptide GRR and preparation method and application, specifically, the present invention relates to structure of two mutant of antalgic active peptide GRR and derivative thereof, analogue, active fragments and preparation method thereof, and the application in field of medicaments as analgesic.
background technology:
Patent-antalgic active peptide GRR and preparation and application (applicant: Shenyang Pharmaceutical University thereof; Contriver: Zhang Jinghai etc.; Number of patent application: 201010107462.5; Application publication number: CN101880319 A) in and research not mentioned its mutant and derivative, and utilize biotechnology to find and obtained two mutant of antalgic active peptide GRR and derivative thereof, analogue, active fragments is a newer technology, there is no at present people's research.
summary of the invention:
The object of this invention is to provide two mutant of antalgic active peptide GRR and derivative thereof, analogue, active fragments with and preparation and application.Specifically utilize biotechnology, screening acquisition two mutant of antalgic active peptide GRR and derivative thereof, analogue, active fragments on the basis of antalgic active peptide GRR; The analgesia biological activity of two mutant of antalgic active peptide GRR has the invention attribute significantly improving compared with antalgic active peptide GRR.Two mutant of antalgic active peptide GRR and derivative thereof, analogue, active fragments preparation method are simple, can be mixed with medically acceptable formulation with any carrier.The present invention utilizes biotechnology or chemical synthesising technology to solve preparation method, obtain still there is two mutant of antalgic active peptide GRR of analgesic activities and derivative thereof, analogue, active fragments.
The present invention is achieved through the following technical solutions, it comprises: for the analgesic activities of antalgic active peptide GRR, utilize genetic engineering technique to obtain a series of antalgic active peptide GRR mutant and Partial Fragment or derivative or analogue, by analgesic activities in the experimental animals of acquisition expression product, to obtain antalgic active peptide GRR mutant and Partial Fragment or derivative or the analogue that can further develop analgesic drug.Preparation method routine of the present invention, simple, output is high, not only has important directive significance and practical value, also for suitability for industrialized production is laid a good foundation.
The present invention be directed to the analgesic activities of antalgic active peptide GRR mutant, utilize genetic engineering technique to obtain a series of antalgic active peptide GRR mutant Partial Fragments or derivative or analogue, by analgesic activities in the experimental animals of acquisition expression product, to obtain antalgic active peptide GRR mutant and Partial Fragment or derivative or the analogue that can further develop analgesic drug.
The preparation method who the invention provides multiple antalgic active peptide GRR mutant and Partial Fragment thereof or derivative or analogue, it comprises: utilize genetic engineering technique express or obtain by chemosynthesis.Genetic engineering technique method comprises:
(1) the coding DNA of antalgic active peptide GRR mutant and Partial Fragment or derivative or analogue is recombinated to expression vector;
(2) with step recombinant expression vector (1), transform suitable host cell (protokaryon or eukaryotic cell);
(3) under applicable abduction delivering condition, the culturing step host cell being converted (2);
(4) the expression product that results purifying obtain.
The invention provides the expression product separation purification method of above-mentioned antalgic active peptide GRR mutant and Partial Fragment thereof or derivative or analogue.Can use the methods such as salt precipitation, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography and gel-filtration, from the lysate of cell and nutrient solution, separate and the required expression product of purifying.In the separation and purge process of expression product, can use sodium dodecyl sulfate-polyacrylamide gel electrophoresis method (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) or detected by Western blot (WESTERN) to detect existence and the corresponding molecular size of expression product.
The present invention carries out the interior analgesia of the body biologic activity experiment of above-mentioned antalgic active peptide GRR mutant and Partial Fragment or derivative or analogue first.
The present invention also provides the application in field of biological pharmacy of above-mentioned antalgic active peptide GRR mutant and Partial Fragment thereof or derivative or analogue, the biological activity of specifically easing pain.
A further object of the present invention is to provide and contains the protein that is defined as above and the pharmaceutical composition of one or more pharmaceutically acceptable carriers or vehicle.The pharmaceutical composition that can be suitable for the outer administration of gi tract according to the preparation of the known fundamental principle in pharmaceutical industry field and method is (as referring to Remington ' s Pharmaceutical Science, 15th., Mack Publishing Company, 1980).Can by various route of administration, particularly intravenously, intramuscular, intraarticular, intraperitoneal, nose, intracutaneous, the outer approach of gi tract such as the subcutaneous pharmaceutical composition of the present invention that comes into operation.
Another object of the present invention is to provide the protein being defined as above in the application of producing in analgesic.The pharmaceutical composition that can use albumen of the present invention or contain this protein, as therapeutical agent, is used for the treatment of the various pain relative diseases of particular type human body.The treatment effective dose of pharmaceutical composition of the present invention generally should be according to the character of disease, severity and the responsive adaptability to medicine, and the factors such as route of administration is determined according to principle of individuation by clinician.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell, and the example of conventional prokaryotic host cell comprises intestinal bacteria, Bacillus subtilus etc.Conventional eukaryotic host cell comprises yeast cell, insect cell and mammalian cell etc.
accompanying drawing explanation:
Fig. 1 is antalgic active peptide GRR mutant 1 aminoacid sequence
Fig. 2 is antalgic active peptide GRR mutant 2 aminoacid sequences
embodiment:
The following examples can make the present invention of those skilled in the art comprehend, rather than limit by any way the present invention and give special approval to the scope of claim.
Embodiment 1:
The acquisition of antalgic active peptide GRR mutant 1 gene
The present embodiment is described the construction strategy and the basic skills that for expressing, obtain one of antalgic active peptide GRR mutant of the present invention gene.
According to the aminoacid sequence of antalgic active peptide GRR and gene order thereof, for the 5th amino acids residue-Y(tyrosine), utilize conventional genetic engineering technique, with antalgic active peptide GRR gene for the gene that sets out, be converted into F(phenylalanine), thereby obtain antalgic active peptide GRR mutant 1 gene, the structure of the antalgic active peptide GRR mutant 1 of this genes encoding is as follows: GRDAFIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGHACWCINLPDDKP IRIPGKCHRR.
Embodiment 2:
The acquisition of antalgic active peptide GRR mutant 2 genes
Strategy, basic skills and the primary process of the present embodiment are with embodiment 1.
According to the aminoacid sequence of antalgic active peptide GRR and gene order thereof, for the 42 amino acids residue-Y(tyrosine), utilize conventional genetic engineering technique, with antalgic active peptide GRR gene for the gene that sets out, be converted into F(phenylalanine), thereby obtain antalgic active peptide GRR mutant 2 genes, the structure of the antalgic active peptide GRR mutant 2 of this genes encoding is as follows: GRDAYIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKFGHACWCINLPDDKP IRIPGKCHRR.
Embodiment 3:
The acquisition of antalgic active peptide GRR mutant 1 and derivative thereof, analogue, active fragments
1. the structure of antalgic active peptide GRR mutant 1 and derivative thereof, analogue, active fragments gene
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of antalgic active peptide GRR mutant 1 gene of the present invention.
According to antalgic active peptide GRR mutant 1(structure referring to Fig. 1 .) N-terminal and C-terminal aminoacid sequence, design respectively corresponding Oligonucleolide primers, simultaneously at 5 of above-mentioned two Oligonucleolide primers ' end, add respectively restriction endonuclease hydrolysis site sequence separately, so that the enforcement of gene recombination; Antalgic active peptide GRR mutant 1 gene obtaining take embodiment 1 carries out pcr amplification as template, and agarose gel electrophoresis detects product and carries out the gel recovery of nucleic acid fragment; After the above-mentioned restriction endonuclease double digestion of mentioning with the connection of recombinating under the effect of T4 DNA ligase of the same plasmid that carries out double digestion, thermal transition competent escherichia coli cell DH 5 α, cut after checking screening obtains positive transformant and submit to biotechnology service company to carry out determined dna sequence through bacterium colony PCR and restriction endonuclease enzyme.Result shows, successfully builds antalgic active peptide GRR mutant 1 express recombinant vectors by the method for said gene engineering.
In like manner, utilize genetic engineering technique, can obtain respectively antalgic active peptide GRR mutant 1 derivative, analogue, active fragments gene.
2. the acquisition of antalgic active peptide GRR mutant 1 and derivative thereof, analogue, active fragments
Utilize gene process technology, 1 gene recombination of antalgic active peptide GRR mutant, to colibacillus expression plasmid, is extracted to plasmid and checked order.
The constitutional features of this recombinant expression plasmid coding expression product is: MHHHHHHM GRDAFIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGHACWCINLPDDKP IRIPGKCHRR.
By positive recombinant plasmid thermal transition e. coli bl21 (λ DE3), then from this LB solid plate (containing corresponding screening microbiotic), after picking list bacterium colony, be seeded to 3 ml LB (containing corresponding microbiotic), in 37 ℃, 200 r/min shaken overnight are cultivated.According to 1% inoculum size, overnight culture is seeded to 400 ml containing in the triangular flask of corresponding antibiotic fresh LB substratum, in 37 ℃, 200 r/min shaking culture to OD600 be 0.6-0.8, adding final concentration is inductor isopropyl ss-D-thiogalactoside (IPTG) of 0.166 mmol/L, cultivates 4 h.Finish fermentation, 3000 g, 4 ℃ of centrifugal 20 min, collect thalline.With 40 ml lysis buffers (0.1 M PBS, 0.15 M NaCl, 50 mM imidazoles) resuspended thalline, carry out ultrasonication, after ultrasonic end in 12,000g, 4 ℃ of centrifugal 20 min, obtain supernatant liquor, gained precipitation repeats fragmentation once according to above-mentioned steps, merge supernatant liquor twice, directly be splined on the good metal ion-chelant chromatography column of 0.1 M PBS (pH 8.0) pre-balance, through the pH damping fluid of two different stepss, fully wash after 5 column volumes respectively, use 0.5 M imidazoles (pH 9.0) to carry out wash-out and gather in the crops this elutriant.Products therefrom is verified its purity through 15% SDS-PAGE.By column chromatography chromatogram figure and SDS-PAGE collection of illustrative plates, illustrated, recombinant protein obtains high efficient expression, reaches electrophoresis purity.The expression product of above-mentioned acquisition, utilizes chemical method at M place fracture peptide chain, thereby obtains antalgic active peptide GRR mutant 1, and constitutional features is: GRDAFIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGHACWCINLPDDKP IRIPGKCHRR
.
The same principle obtains expression product, and its constitutional features is: M GRDAFIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGHACWCINLPDDKP IRIPGKCHRR
.
3. expression and the purifying thereof of antalgic active peptide GRR mutant 1 in yeast
Coding antalgic active peptide GRR mutant 1 gene is increased with Oligonucleolide primers, obtain and insert segment.PCR reacts the encoding sequence of the N-terminal region aminoacid sequence of restriction enzyme site that 5 ' Oligonucleolide primers sequence of using contains EcoR V restriction enzyme and antalgic active peptide GRR mutant 1, the encoding sequence of the C-terminal region aminoacid sequence of restriction enzyme site that 3 ' end primer sequence contains EcoR I restriction enzyme, translation stop codon and antalgic active peptide GRR mutant 1.
The restriction enzyme site of the restriction enzyme on primer is corresponding to the restriction enzyme digestion sites on Yeast expression carrier pPIC9K (wherein the restriction endonuclease otch of EcoR V and SnaB I is flat end), this plasmid vector coding antibiotics resistance (Amp
rand Kan
r), a replicon from 2 μ plasmids, an AOX1 promotor, can be by the high efficient expression of methanol induction in pichia spp, the signal peptide sequence of a α-factor, a transcription termination signal, HIS4 selected marker and integration sequence.
With SnaB I and EcoR I digestion pPIC9K carrier, with EcoR V and EcoR I digestion Insert Fragment, subsequently Insert Fragment is connected into pPIC9K carrier, connect product thermal transition E.coli DH 5 α bacterial strains, on the LB culture dish that contains Amp, screen transformant, containing Amp(100 μ g/ml) LB liquid nutrient medium in incubated overnight containing the clone of required construction.Extracting plasmid, sequence verification Insert Fragment correctly inserts.
Get after plasmid linearization, electricity is transformed into yeast cell, and linearizing recombinant vectors is by producing stable yeast conversion body with the homologous sequence generation homologous recombination of host genome.Utilize G418 to filter out integration transformation of high copy.The inoculation that screening is obtained is in being equipped with the 250ml shaking flask of 25ml MGY, BMG or BMGY substratum, and (16～18 h) in 28～30 ℃/250～300 revs/min, to be cultured to OD600=2～6; The lower 3000 revs/min of centrifugal 5min of room temperature, collect thalline, with MM, BMM or the resuspended thalline of BMMY (approximately 10～20ml) of 1/5 to 1/10 former volume of culture; The bacterium liquid of step 2 gained is placed in to the shaking flask of 100ml, with the sealing of double gauze or cheese cloth, is positioned over continued growth on the shaking table of 28～30 ℃/250～300 revs/min; Every 24h is 0.5～1.0% to adding 100% methyl alcohol to final concentration in substratum; Pigment is removed in the ultra-filtration membrane ultrafiltration that the supernatant liquor of sample separation is 3000Da with molecular weight cut-off, detects and identify the expression of recombinant protein with SDS-PAGE, Western-Blot and activity experiment.This system is expressed product structure and is characterized as: GRDAFIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGHACWCINLPDDKP IRIPGKCHRR
.
Embodiment 4:
The acquisition of antalgic active peptide GRR mutant 2 and derivative thereof, analogue, active fragments
Strategy, basic skills and the primary process of the present embodiment are with embodiment 3.
Restructuring antalgic active peptide GRR mutant 2 constitutional featuress that obtain at escherichia coli expression are: MHHHHHHMGRDAYIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKFGHACWC INLPDDKPIRIPGKCHRR.This expression product, utilizes chemical method at M place fracture peptide chain, thereby obtains antalgic active peptide GRR mutant 2, and constitutional features is: GRDAYIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKFGHACWCINLPDDKP IRIPGKCHRR.
The same principle obtains expression product, and its constitutional features is: MGRDAYIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKFGHACWCINLPDDK PIRIPGKCHRR.
The expression product constitutional features of utilizing yeast expression to obtain is: GRDAYIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKFGHACWCINLPDDKP IRIPGKCHRR.
Embodiment 5:
In two mutant molecules of antalgic active peptide GRR, cysteine residues is replaced the acquisition of expression product
In two mutant molecules of the present embodiment acquisition antalgic active peptide GRR, cysteine residues is replaced strategy and the basic skills of expression product, the roughly the same strategy of embodiment 1-4 and basic skills.
The antalgic active peptide GRR mutant 1(C12S obtaining) mutant expression product, its constitutional features is: the 12nd C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1(C16S) mutant expression product, its constitutional features is: the 16th C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1(C22S) mutant expression product, its constitutional features is: the 22nd C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1(C26S) mutant expression product, its constitutional features is: the 26th C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1(C36S) mutant expression product, its constitutional features is: the 36th C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1(C46S) mutant expression product, its constitutional features is: the 46th C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1(C48S) mutant expression product, its constitutional features is: the 48th C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1(C63S) mutant expression product, its constitutional features is: the 63rd C of antalgic active peptide GRR mutant 1 sports S
.
Obtain antalgic active peptide GRR mutant 1 (C12S, C63S) mutant expression product, its constitutional features is: the 12nd and 63 C of pain active peptide GRR mutant 1 sport S
.
Obtain antalgic active peptide GRR mutant 1 (C16S, C36S) mutant expression product, its constitutional features is: the 16th and 36 C of pain active peptide GRR mutant 1 sport S
.
Obtain antalgic active peptide GRR mutant 1 (C22S, C46S) mutant expression product, its constitutional features is: the 22nd and 46 C of pain active peptide GRR mutant 1 sport S
.
Obtain antalgic active peptide GRR mutant 1 (C26S, C48S) mutant expression product, its constitutional features is: the 26th and 48 C of pain active peptide GRR mutant 1 sport S
.
The antalgic active peptide GRR mutant 2(C12S obtaining) mutant expression product, its constitutional features is: the 12nd C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2(C16S) mutant expression product, its constitutional features is: the 16th C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2(C22S) mutant expression product, its constitutional features is: the 22nd C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2(C26S) mutant expression product, its constitutional features is: the 26th C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2(C36S) mutant expression product, its constitutional features is: the 36th C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2(C46S) mutant expression product, its constitutional features is: the 46th C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2(C48S) mutant expression product, its constitutional features is: the 48th C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2(C63S) mutant expression product, its constitutional features is: the 63rd C of antalgic active peptide GRR mutant 2 sports S
.
Obtain antalgic active peptide GRR mutant 2 (C12S, C63S) mutant expression product, its constitutional features is: the 12nd and 63 C of pain active peptide GRR mutant 2 sport S
.
Obtain antalgic active peptide GRR mutant 2 (C16S, C36S) mutant expression product, its constitutional features is: the 16th and 36 C of pain active peptide GRR mutant 2 sport S
.
Obtain antalgic active peptide GRR mutant 2 (C22S, C46S) mutant expression product, its constitutional features is: the 22nd and 46 C of pain active peptide GRR mutant 2 sport S
.
Obtain antalgic active peptide GRR mutant 2 (C26S, C48S) mutant expression product, its constitutional features is: the 26th and 48 C of pain active peptide GRR mutant 2 sport S
.
Embodiment 6:
Analgesic activities-mouse acetic acid twisting method in body
The present embodiment is determined the in vivo bioactivity-analgesic activities of antalgic active peptide GRR mutant by analgesic model in Mice Body.In addition be also intended to verify the analgesic activities of antalgic active peptide GRR mutant derivative, analogue, active fragments.Protein concentration adopts Lowry method to measure.
Mouse acetic acid twisting method analgesic model: inject Kunming mouse intraperitoneal using Glacial acetic acid as chemical irritant, then cause deep, big area and more lasting pain stimulation, cause mouse to produce " writhing " reaction (belly indent, trunk and back leg extension, hips up).
18-22 g Kunming mouse, male and female half and half, random packet, 8 every group, tail vein injection sample, causes Encelialgia by 0.2 ml/20 g abdominal injection 0.6% (v/v) acetic acid after 20 min, records the writhing number of times in mouse 10 min after 5 min.With the positive contrast of morphine, physiological saline is blank, calculates according to the following equation the writhing response inhibiting rate of each administration group.
Analgesia biological activity test result shows:
1. physiological saline blank group laboratory animal writhing number of times (Mean ± SEM) is 38.1 ± 2.8.
2. the laboratory animal writhing response inhibiting rate of antalgic active peptide GRR (dosage, 0.102 μ mol/kg) group is 52.7%.
3. antalgic active peptide GRR mutant 1 and derivative thereof, analogue, active fragments (dosage, 0.102 μ mol/kg) laboratory animal writhing response inhibiting rate all more than 80.3%, the analgesia biological activity of relative analgesia active peptide GRR increases tool significance.
4. antalgic active peptide GRR mutant 2 and derivative thereof, analogue, active fragments (dosage, 0.102 μ mol/kg) laboratory animal writhing response inhibiting rate all more than 72.6%, the analgesia biological activity of relative analgesia active peptide GRR increases tool significance.
5. in two mutant molecules of antalgic active peptide GRR that embodiment 5 obtains, cysteine residues is replaced expression product (dosage, 0.102 μ mol/kg) laboratory animal writhing response inhibiting rate all below 45%, the analgesia biological activity with respect to corresponding antalgic active peptide GRR mutant having declines, tool significance.
SEQUENCE LISTING
<110> Shenyang Pharmaceutical University
Two mutant of <120> antalgic active peptide GRR and preparation and application thereof
Two mutant of <130> antalgic active peptide GRR and preparation and application thereof
<160> 2
<170> PatentIn Version 3.1
<210> 1
<211> 66
<212> PRT
<213> Mesobuthus martensi
<400> 1
Gly Arg Asp Ala Phe Ile Ala Gln Asn Tyr Asn Cys Val Tyr His
1 5 10
Cys Phe Arg Asp Asp Tyr Cys Asn Gly Leu Cys Thr Glu Asn Gly
16 20 25
Ala Asp Ser Gly Tyr Cys Tyr Leu Ala Gly Lys Tyr Gly His Ala
31 35 40
Cys Trp Cys Ile Asn Leu Pro Asp Asp Lys Pro Ile Arg Ile Pro
46 50 55
Gly Lys Cys His Arg Arg
61
<210> 2
<211> 66
<212> PRT
<213> Mesobuthus martensi
<400> 2
Gly Arg Asp Ala Tyr Ile Ala Gln Asn Tyr Asn Cys Val Tyr His
1 5 10
Cys Phe Arg Asp Asp Tyr Cys Asn Gly Leu Cys Thr Glu Asn Gly
16 20 25
Ala Asp Ser Gly Tyr Cys Tyr Leu Ala Gly Lys Phe Gly His Ala
31 35 40
Cys Trp Cys Ile Asn Leu Pro Asp Asp Lys Pro Ile Arg Ile Pro
46 50 55
Gly Lys Cys His Arg Arg
61
Claims (9)
1. two mutant of antalgic active peptide GRR, is characterized in that, aminoacid sequence is as follows:
GRDAFIAQNYNCVYHCFRDDYCNGLCTENGA
DSGYCYLAGKYGHACWCINLPDDKPIRIPGKCHRR；
GRDAYIAQNYNCVYHCFRDDYCNGLCTENGA
DSGYCYLAGKFGHACWCINLPDDKPIRIPGKCHRR。
2. two mutant of antalgic active peptide GRR according to claim 1, is characterized in that, described mutant is to suddenly change respectively and obtain for the 5th of antalgic active peptide GRR, the 42 amino acids residue.
3. two mutant of antalgic active peptide GRR according to claim 1, it is characterized in that, the analgesic activities conservative amino acid residue of described two mutant of antalgic active peptide GRR comprises the 12nd, the 16th, the 22nd, the 26th, the 36th, the 46th, the 48th and the 63rd these eight cysteine residues of N-end of described two mutant of antalgic active peptide GRR.
4. the derivative of two mutant of antalgic active peptide GRR, is characterized in that, sequence is as shown in SEQ ID NO:1-26.
5. the preparation method of the derivative of two mutant of antalgic active peptide GRR as claimed in claim 1 or two mutant of antalgic active peptide GRR claimed in claim 4, utilize genetic engineering technique to express acquisition, it is characterized in that, expression host cell is prokaryotic cell prokaryocyte or eukaryotic cell.
6. preparation method according to claim 5, is characterized in that:
(1) the coding DNA of antalgic active peptide GRR mutant or derivative is recombinated to expression vector;
(2) with step recombinant expression vector (1), transform suitable host cell;
(3) under applicable abduction delivering condition, the culturing step host cell being converted (2);
(4) the expression product that results purifying obtain.
7. the preparation method of two sudden changes of two mutant of antalgic active peptide GRR as claimed in claim 1 or antalgic active peptide GRR claimed in claim 4 derivative originally, can utilize chemical synthesising technology to obtain, it is characterized in that, chemosynthesis is that synthetic or synthesizer are synthetic.
8. two mutant of antalgic active peptide GRR claimed in claim 1 or derivative claimed in claim 4 are in the application of preparing in analgesic.
9. application according to claim 8, is characterized in that: described two mutant of antalgic active peptide GRR or derivative and pharmaceutically acceptable carrier are mixed with acceptable injection, oral preparations, transdermal absorption formulation, mucosal absorption preparation clinically.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100857389B1 (en) * | 2006-06-30 | 2008-09-11 | (주)아모레퍼시픽 | AP-GRR peptide or peptide chain containing AP-GRR peptide, and drug-delivery carrier comprising the same |
| CN101880319A (en) * | 2010-02-09 | 2010-11-10 | 沈阳药科大学 | Antalgic active peptide GRR and preparation and application thereof |
| CN101880327A (en) * | 2010-02-09 | 2010-11-10 | 沈阳药科大学 | Scorpion arialgesic anti-tumoral peptide fusion and acquisition method thereof |
| WO2011041426A1 (en) * | 2009-09-29 | 2011-04-07 | Butamax(Tm) Advanced Biofuels Llc | Improved yeast production host cells |
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2011
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100857389B1 (en) * | 2006-06-30 | 2008-09-11 | (주)아모레퍼시픽 | AP-GRR peptide or peptide chain containing AP-GRR peptide, and drug-delivery carrier comprising the same |
| WO2011041426A1 (en) * | 2009-09-29 | 2011-04-07 | Butamax(Tm) Advanced Biofuels Llc | Improved yeast production host cells |
| CN101880319A (en) * | 2010-02-09 | 2010-11-10 | 沈阳药科大学 | Antalgic active peptide GRR and preparation and application thereof |
| CN101880327A (en) * | 2010-02-09 | 2010-11-10 | 沈阳药科大学 | Scorpion arialgesic anti-tumoral peptide fusion and acquisition method thereof |
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