CN101875691B - Scorpion arialgesic antitumoral peptide mutant and preparation method thereof - Google Patents
Scorpion arialgesic antitumoral peptide mutant and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, relating to a scorpion arialgesic antitumoral peptide mutant, a derivative thereof, an analogue and a preparation method thereof, in particular to directional reformation to the scorpion arialgesic antitumoral peptide with a genetic engineering method. The invention utilizes the DNA recombinant technology to build a series of carriers of the scorpion arialgesic antitumoral peptide mutant protein, realizes high-efficiency soluble expression and verifies the in vivo bioactivity after obtaining the mutant protein by purification; and the experiment result shows that the in vivo arialgesic activity of the mutant protein is improved.
Description
Technical field
The present invention relates to biological medicine technology field, relate to scorpion arialgesic antitumoral peptide mutant and derivative thereof, analogue and preparation method thereof, be exactly exactly build that a series of scorpion arialgesic antitumoral peptide analgesic activities are improved and molecule in the expression vector of the mutant protein transformed of halfcystine.Specifically, the present invention relates to preparation method and the application in treatment field as analgesic thereof of the mutant protein that scorpion arialgesic antitumoral peptide analgesic activities conservative property halfcystine and analgesic activities be improved.
background technology
Scorpion, as the traditional Chinese medicine medicinal history of existing more than 2000 year, claims again full worm, scorpio, in the medical science works such as " Book of Songs ", " Compendium of Materia Medica ", its medical has been carried out to system description, traditional rare Chinese medicine of China, have dispel the wind relieving convulsion, the function such as remove obstruction in channels to relieve pain.For inclined to one side, aching all over the head, neurodynia, body body sharp pains etc., have larger exploitation and are worth.It has been found that in recent years the multiple scorpion venom single component polypeptide with pharmacological actions such as antitumor, anti-inflammatory, analgesia, anti-epileptics.Natural scorpion venom output is extremely low, therefore, adopts gene engineering method, and the method for producing the active Buthotoxin polypeptide with pharmaceutical use with simple protokaryon or eukaryote expressive host seems particularly important.
Buthus martensii Karscs arialgesic antitumoral peptide (BmKAGAP) is that the separation and purification first in buthus martensii Karscs such as Liu Yanfeng obtains and uses engineered method to obtain its coding gene sequence, has realized the solution expression with high efficiency in intestinal bacteria.In vivo in the model experiment of mouse acetic acid twisting, this polypeptide embodies and contrasts the analgesic effect that medicine morphine is strong (Liu YF, Ma RL, Wang SL, Duan ZY, Zhang JH, Wu LJ, Wu CF.Expression of an antitumor-analgesic peptide from the venom of Chinese scorpionButhus martensii Karsch in Escherichia coli.Protein Expression and Purification.2003; 27:253-8).
Pain is a kind of offending subjective sensation that the mankind had and had larger individual difference, and it provides warning signal when body is on the hazard, and is the indispensable a kind of protection mechanism of life.And on existing Research foundation, BmKAGAP analgesic activities is improved and the further research that needs of the structure of the recombinant expression vector of mutant protein that halfcystine is transformed and the preparation method of finding solution expression with high efficiency product.
summary of the invention
An object of the present invention is to build BmKAGAP mutant expression vector, the expression and purification of mutant protein and preliminary in vivo bioactivity research.
The present invention is devoted to that BmKAGAP analgesic activities is improved and the structure of recombinant expression vector and the preparation method of solution expression with high efficiency product of mutant protein that halfcystine is transformed, the preparation method of BmKAGAP series mutation body of the present invention or corresponding analogue is conventional, simple, output is high, research and development scorpion analgesia peptide medicament is had to important directive significance and practical value, for suitability for industrialized production is laid a good foundation.The treatment that is widely used in Clinical Pain relative disease for this series albumen after preliminary intracorporeal active experiment checking provides basis, will greatly improve patient's life quality, so its application prospect will be boundless.
Another object of the present invention is to provide the method for producing the mutein being defined as above, and the method comprises:
(1) provide the D8K of BmKAGAP, D8R, C end-(K or R), C end-(K or R)-(K or R), C end-(K or R)-(K or R)-(K or R), C-terminal reduces by one or two Gly, and D8 (K or R) and C-terminal increase by one or two or three basic aminoacidss, the recombinant expression vector of the coded DNA of mutant of D8 (K or R) and one of C-terminal minimizing or two Gly and four pairs of disulfide linkage thereof;
(2) with the recombinant vectors of step (1), transform suitable host cell;
(3) at the D8K that is suitable for expressing BmKAGAP, D8R, C end-(K or R), C end-(K or R)-(K or R), C end-(K or R)-(K or R)-(K or R), C-terminal reduces by one or two Gly, and D8 (K or R) and C-terminal increase by one or two or three basic aminoacidss, the host cell being converted of culturing step (2) under the condition of the mutant of D8 (K or R) and one of C-terminal minimizing or two Gly and four pairs of disulfide linkage thereof;
(4) results the resulting protein of purifying.
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.
A further object of the present invention is to provide the application in producing analgesic of the protein that is defined as above.
The invention provides the host cell that a kind of above-mentioned carrier transforms.
Mutant protein provided by the invention all from e. coli host cell separation and purification obtain.Can use the methods such as salt precipitation, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography and gel-filtration separated and required protein expression product of purifying from the lysate of cell and nutrient solution.In the separation and purge process of 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 product.
The present invention also provides the application of said mutation body protein in field of biological pharmacy, specifically comprises analgesia biological activity.
The present invention builds the procaryotic cell expression carrier of corresponding mutein first, realizes the solution expression with high efficiency of albumen after conversion e. coli host cell.
The present invention carries out the interior analgesia of the Mice Body biologic activity experiment of this series mutation body protein first.
The invention further relates to the pharmaceutical composition that contains above-mentioned albumen and at least one pharmaceutically acceptable inert support or vehicle.Can be suitable for according to the known fundamental principle in pharmaceutical industry field and method preparation the pharmaceutical composition (as referring to Remington ' s Pharmaceutical Science, 15th., Mack Publishing Company, 1980) of the outer administration of gi tract.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.
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.
The resulting Scorpion analgesic peptide expression amount of application the present invention is high, is easy to purifying and obtains target protein, and in fractional mutant body, analgesic activities is improved preferably.
Accompanying drawing explanation
The scorpion arialgesic antitumoral peptide D8K mutant (being abbreviated as D8K) of take describes as example:
Fig. 1: show for expressing the schematic diagram of the prokaryotic cell prokaryocyte recombinant expression plasmid pSYPU/BmKAGAP (D8K) of D8K.
Fig. 2: show that colony polymerase chain reaction (PCR) method evaluation is containing the bacterial colony of recombinant plasmid.
1.5% agarose gel electrophoresis detects pcr amplification product, identifies the bacterial colony containing recombinant plasmid.The wherein positive contrast of swimming lane 1, the negative contrast of swimming lane 2, swimming lane 3 is for containing the bacterial colony of the recombinant plasmid that inserts object fragment, and swimming lane M is nucleic acid molecular weight mark DL2000.
Fig. 3: the metal ion-chelant thin layer chromatography figure that shows D8K mutant.In figure, arrow indication position is target protein place elution peak.
Fig. 4: the 15%SDS-PAGE collection of illustrative plates that shows D8K mutant.Wherein swimming lane 1 is D8K protein band after purifying, and swimming lane M is low molecular weight protein mark.
Embodiment
The following examples can make the present invention of those skilled in the art comprehend, rather than limit by any way the scope that the present invention gives special approval to claim.
Table 1. is for building BmKAGAP fractional mutant primer
a
aitalic GGATCC represents 3 ' end BamHI restriction enzyme site.
Table 2. is for building transformation BmKAGAP disulfide linkage fractional mutant primer
a
aitalic GGATCC represents 3 ' end BamHI restriction enzyme site.
Embodiment 1: the acquisition of scorpion arialgesic antitumoral peptide D8K mutant and derivative thereof, analogue
1. the structure of plasmid pSYPU/BmKAGAP-D8K
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of the recombinant plasmid pSYPU/BmKAGAP-D8K of D8K albumen of the present invention.
According to Asp in BmKAGAP aminoacid sequence
8design corresponding Oligonucleolide primers P1 and primer P2, the pSYPU/BmKAGAP plasmid building of take is template, respectively with P1 and P7, P2 and P6 are that primer carries out pcr amplification, agarose gel electrophoresis detects product and carries out the gel recovery of nucleic acid fragment, it is template that the gained of take again reclaims product, take P6 and P7 as primer carries out third round pcr amplification, reclaims corresponding product.After restriction endonuclease NcoI and BamHI double digestion with the plasmid pSYPU that carries out double digestion equally at T
4under the effect of DNA ligase, connect, thermal transition competent escherichia coli cell DH5 α, cuts after checking screening obtains positive transformant and submits to biotechnology service company to carry out determined dna sequence through bacterium colony PCR and restriction endonuclease enzyme.Result shows, by engineered method success construction recombination plasmid pSYPU/BmKAGAP-D8K.
2. the separation and purification of mutant protein
The expression product of fermentation expression separating Escherichia coli host cell according to a conventional method after order-checking is confirmed.First by positive recombinant plasmid thermal transition e. coli bl21 (λ DE3), then from this LB solid plate, after picking list bacterium colony, be seeded to 3ml LB (containing that microbiotic of card, 50 μ g/ml), in 37 ℃, 200r/min shaken overnight is cultivated.According to 1% inoculum size, overnight culture is seeded to 400ml containing in the triangular flask of corresponding antibiotic fresh LB substratum, in 37 ℃, 200r/min shaking culture to OD600 be 0.6-0.8, adding final concentration is the inductor isopropyl ss D-thiogalactoside (IPTG) of 0.166mmol/L, cultivates 4h.Finish fermentation, 3000g, 4 ℃ of centrifugal 20min, collect thalline.With 40ml lysis buffer (0.1M PBS, 0.15M NaCl, 50mM imidazoles) resuspended thalline, carry out ultrasonication, after ultrasonic end in 12,000g, 4 ℃ of centrifugal 20min, 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 0.1M PBS (pH 8.0) pre-balance, through the pH damping fluid difference of two different stepss, fully wash after 5 column volumes, use 0.5M 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.
Scorpion arialgesic antitumoral peptide D8K mutant and and the pattern of derivative, analogue comprise: BmKAGAP-D8K, M-BmKAGAP-D8K, MHHHHHHM-BmKAGAP-D8K, MHHHHHH-BmKAGAP-D8K.
Embodiment 2: the acquisition of scorpion arialgesic antitumoral peptide D8R mutant and derivative thereof, analogue
Strategy and the basic skills of the present embodiment acquisition scorpion arialgesic antitumoral peptide D8R mutant and derivative thereof, analogue, the roughly the same strategy of embodiment 1 and basic skills.
The pattern of scorpion arialgesic antitumoral peptide D8R mutant and derivative thereof, analogue comprises: BmKAGAP-D8R, M-BmKAGAP-D8R, MHHHHHHM-BmKAGAP-D8R, MHHHHHH-BmKAGAP-D8R.
Embodiment 3: the acquisition of scorpion arialgesic antitumoral peptide C end-K-K mutant [BmKAGAP-K-K] and derivative thereof, analogue
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of the recombinant plasmid pSYPU/BmKAGAP-K-K of rBmKAGAP-K-K albumen of the present invention.Wherein according to the aminoacid sequence of AGAP mature peptide C-terminal, design corresponding Oligonucleolide primers P3, the pSYPU/BmKAGAP plasmid building of take carries out pcr amplification as template, and corresponding primer is P3 and P6.Primary process is with embodiment 1.
The pattern of scorpion arialgesic antitumoral peptide C end-K-K mutant [BmKAGAP-K-K] and derivative thereof, analogue comprises: BmKAGAP-K-K, M-BmKAGAP-K-K.
Embodiment 4: the acquisition of scorpion arialgesic antitumoral peptide C end-(K or R) mutant and derivative thereof, analogue
Strategy and the basic skills of the present embodiment acquisition scorpion arialgesic antitumoral peptide C end-(K or R) mutant and derivative thereof, analogue, the roughly the same strategy of embodiment 3 and basic skills.This pattern comprises: BmKAGAP-K, M-BmKAGAP-K, BmKAGAP-R, M-BmKAGAP-R.
Embodiment 5: the acquisition of scorpion arialgesic antitumoral peptide C end-(K or R)-(K or R) mutant and derivative thereof, analogue
Strategy and the basic skills of the present embodiment acquisition scorpion arialgesic antitumoral peptide C end-(K or R)-(K or R) mutant and derivative thereof, analogue, the roughly the same strategy of embodiment 3 and basic skills.The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-K-K, BmKAGAP-K-R, BmKAGAP-R-K, BmKAGAP-R-R, M-BmKAGAP-K-K, M-BmKAGAP-K-R, M-BmKAGAP-R-K and M-BmKAGAP-R-R.
Embodiment 6: the acquisition of scorpion arialgesic antitumoral peptide C end-(K or R)-(K or R)-(K or R) mutant and derivative thereof, analogue
Strategy and the basic skills of the present embodiment acquisition scorpion arialgesic antitumoral peptide C end-(K or R)-(K or R)-(K or R) mutant and derivative thereof, analogue, the roughly the same strategy of embodiment 3 and basic skills.The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-K-K-K, BmKAGAP-K-K-R, BmKAGAP-K-R-K, BmKAGAP-K-R-R, BmKAGAP-R-K-K, BmKAGAP-R-K-R, BmKAGAP-R-R-R, BmKAGAP-R-R-K, M-BmKAGAP-K-K-K, M-BmKAGAP-K-K-R, M-BmKAGAP-K-R-K, M-BmKAGAP-K-R-R, M-BmKAGAP-R-K-K, M-BmKAGAP-R-K-R, M-BmKAGAP-R-R-R and M-BmKAGAP-R-R-K.
Embodiment 7: the acquisition of scorpion arialgesic antitumoral peptide C end (G) mutant [BmKAGAP-(G)] and derivative thereof, analogue
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of the recombinant plasmid pSYPU/BmKAGAP (G) of rBmKAGAP of the present invention (G) albumen.Wherein, according to the aminoacid sequence design oligonucleotides primer P4 of AGAP mature peptide C-terminal, the pSYPU/BmKAGAP plasmid building of take is template, and corresponding primer is P4 and P6.Primary process is with embodiment 1.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-(G) and M-BmKAGAP-(G).
Embodiment 8: the acquisition of scorpion arialgesic antitumoral peptide C end (2G) mutant [BmKAGAP-(2G)] and derivative thereof, analogue
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of the recombinant plasmid pSYPU/BmKAGAP (2G) of rBmKAGAP of the present invention (2G) albumen.Wherein, according to the aminoacid sequence design oligonucleotides primer P5 of AGAP mature peptide C-terminal, corresponding primer is P5 and P6.Primary process is with embodiment 1.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-(2G) and M-BmKAGAP-(2G).
Embodiment 9: scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal adds the acquisition of an alkaline amino acid residue (K or R) mutant and derivative thereof, analogue
The present embodiment obtains scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal, and to add an alkaline amino acid residue (K or R) mutant and derivative thereof, analogue be in conjunction with the embodiments 1,2 and strategy and the basic skills of embodiment 4.Namely, on the basis of scorpion arialgesic antitumoral peptide-D8 (K or R) mutant, at its C-terminal, add an alkaline amino acid residue (K or R).
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP (D8R)-K, M-BmKAGAP (D8R)-K, BmKAGAP (D8R)-R, M-BmKAGAP (D8R)-R, BmKAGAP (D8K)-R, M-BmKAGAP (D8K)-R, BmKAGAP (D8K)-K, M-BmKAGAP (D8K)-K.
Embodiment 10: scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal adds the acquisition of two alkaline amino acid residues (K or R)-(K or R) mutant and derivative thereof, analogue
The present embodiment obtains scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal, and to add two alkaline amino acid residues (K or R)-(K or R) mutant and derivative thereof, analogue be 9 and 4 strategy and basic skills in conjunction with the embodiments.Namely at scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal, add on the basis of an alkaline amino acid residue (K or R) mutant, at its C-terminal, add again an alkaline amino acid residue (K or R).
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP (D8R)-K-(K or R), M-BmKAGAP (D8R)-K-(K or R), BmKAGAP (D8R)-R-(K or R), M-BmKAGAP (D8R)-R-(K or R), BmKAGAP (D8K)-R-(K or R), M-BmKAGAP (D8K)-R-(K or R), BmKAGAP (D8K)-K-(K or R), M-BmKAGAP (D8K)-K-(K or R).
Embodiment 11: scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal adds the acquisition of three alkaline amino acid residues (K or R)-(K or R)-(K or R) mutant and derivative thereof, analogue
The present embodiment obtains scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal, and to add three alkaline amino acid residues (K or R)-(K or R) mutant and derivative thereof, analogue be 10 and 4 strategy and basic skills in conjunction with the embodiments.Namely at scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal, add on the basis of two alkaline amino acid residues (K or R)-(K or R) mutant, at its C-terminal, add again an alkaline amino acid residue (K or R).
The mutant of this pattern and derivative thereof, analogue comprises: BmKAGAP (D8R)-K-(K or R)-(K or R), M-BmKAGAP (D8R)-K-(K or R)-(K or R), BmKAGAP (D8R)-R-(K or R)-(K or R), M-BmKAGAP (D8R)-R-(K or R)-(K or R), BmKAGAP (D8K)-R-(K or R)-(K or R), M-BmKAGAP (D8K)-R-(K or R)-(K or R), BmKAGAP (D8K)-K-(K or R)-(K or R), M-BmKAGAP (D8K)-K-(K or R)-(K or R).
Embodiment 12: the acquisition of scorpion arialgesic antitumoral peptide-D8 (K or R)-C end (G) mutant and derivative thereof, analogue
It is 1,2 and 7 strategy and basic skills in conjunction with the embodiments that the present embodiment obtains scorpion arialgesic antitumoral peptide-D8 (K or R)-C end (G) mutant and derivative thereof, analogue.Namely, on the basis of scorpion arialgesic antitumoral peptide-D8 (K or R) mutant and derivative thereof, analogue, at its C-terminal, remove a G residue.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP (D8R)-(G), M-BmKAGAP (D8R)-(G), BmKAGAP (D8K)-(G), M-BmKAGAP (D8K)-(G).
Embodiment 13: the acquisition of scorpion arialgesic antitumoral peptide-D8 (K or R)-C end (2G) mutant and derivative thereof, analogue
It is 1,2 and 8 strategy and basic skills in conjunction with the embodiments that the present embodiment obtains scorpion arialgesic antitumoral peptide-D8 (K or R)-C end (2G) mutant and derivative thereof, analogue.Namely, on the basis of scorpion arialgesic antitumoral peptide-D8 (K or R)-C end (G) mutant and derivative thereof, analogue, at its C-terminal, remove again a G residue.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP (D8R)-(2G), M-BmKAGAP (D8R)-(2G), BmKAGAP (D8K)-(2G), M-BmKAGAP (D8K)-(2G).
Embodiment 14: after two G of scorpion arialgesic antitumoral peptide C-terminal are removed, add the acquisition of an alkaline amino acid residue mutant and derivative thereof, analogue
After being removed, two G that the present embodiment obtains scorpion arialgesic antitumoral peptide C-terminal add an alkaline amino acid residue mutant and derivative thereof, analogue and are 8 and 9 strategy and basic skills in conjunction with the embodiments.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-(2G)-R, M-BmKAGAP-(2G)-R, BmKAGAP-(2G)-K, M-BmKAGAP-(2G)-K.
Embodiment 15: after two G of scorpion arialgesic antitumoral peptide C-terminal are removed, add the acquisition of two alkaline amino acid residue mutant and derivative thereof, analogue
After being removed, two G that the present embodiment obtains scorpion arialgesic antitumoral peptide C-terminal add two alkaline amino acid residue mutant and derivative thereof, analogue and are 8,9 and 14 strategy and basic skills in conjunction with the embodiments.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-(2G)-R-(R or K), M-BmKAGAP-(2G)-R-(R or K), BmKAGAP-(2G)-K-(R or K), M-BmKAGAP-(2G)-K-(R or K).
Embodiment 16: after two G of scorpion arialgesic antitumoral peptide C-terminal are removed, add the acquisition of three alkaline amino acid residue mutant and derivative thereof, analogue
After being removed, two G that the present embodiment obtains scorpion arialgesic antitumoral peptide C-terminal add two alkaline amino acid residue mutant and derivative thereof, analogue and are 8,9,14 and 15 strategy and basic skills in conjunction with the embodiments.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-(2G)-R-(R or K)-(R or K), M-BmKAGAP-(2G)-R-(R or K)-(R or K), BmKAGAP-(2G)-K-(R or K)-(R or K), M-BmKAGAP-(2G)-K-(R or K)-(R or K).
Embodiment 17: scorpion arialgesic antitumoral peptide D8 (K or R) and C-terminal increase the acquisition of or two or three alkaline amino acid residue mutant and derivative thereof, analogue
After being removed, two G that the present embodiment obtains scorpion arialgesic antitumoral peptide C-terminal add two alkaline amino acid residue mutant and derivative thereof, analogue and are 13,14,15 and 16 strategy and basic skills in conjunction with the embodiments.
The mutant of this pattern and derivative thereof, analogue comprise: BmKAGAP-D8 (K or R)-(2G)-(R or K), M-BmKAGAP-D8 (K or R)-(2G)-(R or K), BmKAGAP-D8 (K or R)-(2G)-(R or K)-(R or K), M-BmKAGAP-D8 (K or R)-(2G)-(R or K)-(R or K), BmKAGAP-D8 (K or R)-(2G)-(R or K)-(R or K)-(R or K), M-BmKAGAP-D8 (K or R)-(2G)-(R or K)-(R or K)-(R or K).
Embodiment 18: scorpion arialgesic antitumoral peptide-(C12S) acquisition of mutant [BmKAGAP-(C12S)]
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of the recombinant plasmid pSYPU/BmKAGAP-(C12S) of BmKAGAP-of the present invention (C12S) mutant protein.
According to Cys in BmKAGAP aminoacid sequence
12design corresponding Oligonucleolide primers P8 and primer P9, the pSYPU/BmKAGAP plasmid building of take is template, and respectively with P7 and P8, P6 and P9 are that primer carries out pcr amplification.Primary process is with embodiment 1.
The acquisition of embodiment 19:BmKAGAP-(C16S), BmKAGAP-(C22S), BmKAGAP-(C26S), BmKAGAP-(C36S), BmKAGAP-(C46S) and BmKAGAP-(C48S) mutant
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of the recombinant plasmid of BmKAGAP-of the present invention (C16S), BmKAGAP-(C22S), BmKAGAP-(C26S), BmKAGAP-(C36S), BmKAGAP-(C46S) and BmKAGAP-(C48S) mutant protein.
According to Cys in BmKAGAP aminoacid sequence
16, Cys
22, Cys
26, Cys
36, Cys
46, Cys
48design corresponding Oligonucleolide primers P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20, P21.Primary process is with embodiment 18.
The acquisition of embodiment 20:BmKAGAP-(C63S) mutant
The present embodiment is enumerated and is described for expressing construction strategy and the basic skills of the recombinant plasmid pSYPU/BmKAGAP (C63S) of BmKAGAP-of the present invention (C63S) mutant protein.
According to Cys in BmKAGAP aminoacid sequence
63design corresponding Oligonucleolide primers P22, the pSYPU/BmKAGAP plasmid building of take is template, take P6 and P22 to carry out pcr amplification as primer.Primary process is with embodiment 18.
The acquisition of embodiment 21:BmKAGAP-(C12S, C63S), BmKAGAP-(C16S, C36S), BmKAGAP-(C22S, C46S), BmKAGAP-(C26S, C48S) mutant
The present embodiment is enumerated and is described for expressing BmKAGAP-(C12S of the present invention, C63S), BmKAGAP-(C16S, C36S), construction strategy and the basic skills of the recombinant plasmid of BmKAGAP-(C22S, C46S), BmKAGAP-(C26S, C48S) mutant protein.
PSYPU/BmKAGAP-(C12S), pSYPU/BmKAGAP-(C16S), pSYPU/BmKAGAP-(C22S), pSYPU/BmKAGAP-(C26S) plasmid building of take is respectively template, introduces catastrophe point series primer carry out pcr amplification with corresponding another part wish.Primary process is with embodiment 1.
Embodiment 22: analgesic activities-mouse acetic acid twisting method in mutant protein body
The present embodiment is intended to verify the analgesic activities of the rear scorpion arialgesic antitumoral peptide mutant of specific site amino acid transformation by analgesic model in Mice Body.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).
18-22g Kunming mouse, male and female half and half, random packet, 8 every group, abdominal injection mutant protein sample, causes Encelialgia by 0.2ml/20g abdominal injection 0.6% (v/v) acetic acid after 20min, records the writhing number of times in mouse 10min after 5min.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: physiological saline blank group laboratory animal writhing number of times (Mean ± SEM) is 43.70 ± 2.99; Positive controls (morphine, 3.51 μ mol/kg) laboratory animal writhing number of times (Mean ± SEM) is 29.37 ± 1.85, and writhing inhibiting rate is 32.79%; The laboratory animal writhing inhibiting rate of scorpion arialgesic antitumoral peptide group (0.14 μ mol/kg) is 54.81%; The mutant of scorpion arialgesic antitumoral peptide D8K, D8R, C end-(K or R), C end-(K or R)-(K or R), C end-(K or R)-(K or R)-(K or R), one or two G of C-terminal minimizing and derivative thereof, analogue (0.14 μ mol/kg) laboratory animal writhing inhibiting rate are at 67.0%-81.7%; Scorpion arialgesic antitumoral peptide D8 (K or R) and C-terminal increase by one or two or three basic aminoacidss, D8 (K or R) and C-terminal and reduce by one or two G, C-terminal and remove two G and add one or two or three basic aminoacidss, D8 (K or R) and two G of C-terminal removal and add the mutant of one or two or three basic aminoacids and derivative thereof, analogue (0.14 μ mol/kg) laboratory animal writhing inhibiting rate 92.3%-100%; The mutant laboratory animal writhing inhibiting rate of the 12nd, the 16th, the 22nd, the 26th, the 36th, the 46th, the 48th and the 63rd Cys of scorpion arialgesic antitumoral peptide is in Table 3.
Mouse acetic acid twisting analgesic experiment result after table 3. scorpion arialgesic antitumoral peptide cysteine mutation
Claims (3)
1. the scorpion arialgesic antitumoral peptide mutant of sequence as shown in SEQ ID NO:1-120.
2. a pharmaceutical composition, is characterized in that: one or more in the mutant that contains claim 1 and pharmaceutically acceptable carrier or vehicle.
3. the application of scorpion arialgesic antitumoral peptide mutant claimed in claim 1 in preparing analgesic.
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