CN101875691A - 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 the biological medicine technology field, relating to scorpion arialgesic antitumoral peptide mutant and derivative thereof, analogue and preparation method thereof, exactly is exactly to make up that a series of scorpion arialgesic antitumoral peptide analgesic activities are improved and the expression vector of the mutant protein that the intramolecularly halfcystine is transformed.Specifically, the present invention relates to the preparation method of the mutant protein that scorpion arialgesic antitumoral peptide analgesic activities conservative property halfcystine and analgesic activities be improved and as analgesic in the treatment Application for Field.
Background technology
Scorpion claims full worm again, scorpio as the medicinal history in existing more than 2000 year of traditional Chinese medicine, in medical science works such as " Book of Songs ", " Compendium of Materia Medica ", its medical has promptly been carried out system description, be traditional rare Chinese medicine of China, have dispel the wind relieving convulsion, function such as remove obstruction in channels to relieve pain.Be used for inclined to one side, aching all over the head, neurodynia, body body sharp pains etc. have bigger exploitation and are worth.It has been found that multiple scorpion venom single component polypeptide in recent years 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 that has the active Buthotoxin polypeptide of pharmaceutical use with simple protokaryon or the production of eukaryote expressive host seems particularly important.
Buthus martensii Karscs arialgesic antitumoral peptide (BmKAGAP) is that separation and purification first in buthus martensii Karscs such as Liu Yanfeng obtains and obtains its coding gene sequence with engineered method, has realized that the highly-soluble in intestinal bacteria is expressed.In vivo in the model experiment of mouse acetic acid twisting, this polypeptide embodies and contrasts the strong analgesic effect of medicine morphine (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 the human a kind of offending subjective sensation that has and big individual difference is arranged, and it provides warning signal when body is on the hazard, be the indispensable a kind of protection mechanism of life.And on existing research basis, the BmKAGAP analgesic activities is improved and the further research that awaits of the structure of the recombinant expression vector of mutant protein that halfcystine is transformed and the preparation method of seeking the highly-soluble expression product.
Summary of the invention
An object of the present invention is to make up BmKAGAP mutant expression vector, bioactivity research in the expression and purification of mutant protein and the preliminary body.
The present invention is devoted to promptly that the BmKAGAP analgesic activities is improved and the structure of the recombinant expression vector of mutant protein that halfcystine is transformed and the preparation method of highly-soluble expression product, the preparation method routine of BmKAGAP series mutation body of the present invention or respective analogs, simple, output is high, research and development scorpion analgesia peptide medicament had 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 through preliminary intracorporeal active experiment checking back provides the basis, will improve patient's life quality greatly, so its application prospect will be very wide.
Another object of the present invention provides the method for producing the mutein that is defined as above, and this method comprises:
(1) provides 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) recombinant vectors with step (1) transforms appropriate 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, culturing step (2) by transformed host cells 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 and the resulting protein of purifying.
A further object of the present invention provides 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 provides the application of protein in producing analgesic that is defined as above.
The invention provides a kind of above-mentioned carrier transformed host cells.
Mutant protein provided by the invention all from e. coli host cell separation and purification obtain.Can use methods such as salt precipitation, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography and gel-filtration from the lysate of cell and nutrient solution, to separate and the required protein expression product of purifying.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 the 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 the analgesia biological activity.
The present invention makes up the procaryotic cell expression carrier of corresponding mutein first, realizes proteic highly-soluble expression behind the transformed into escherichia coli host cell.
The present invention carries out the interior analgesia of the mouse 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 a pharmaceutically acceptable inert support or vehicle.Can be suitable for the pharmaceutical composition (as referring to Remington ' s Pharmaceutical Science, 15th., Mack Publishing Company, 1980) of the outer administration of gi tract according to known fundamental principle in pharmaceutical industry field and method preparation.Can by in various route of administration, particularly intravenously, intramuscular, intraarticular, intraperitoneal, the nose, intracutaneous, the outer approach of gi tract such as the subcutaneous pharmaceutical composition of the present invention that comes into operation.
Can use albumen of the present invention or contain this proteinic pharmaceutical composition, be used for the treatment of the various pain relative diseases of particular type human body as therapeutical agent.The treatment effective dose of pharmaceutical composition of the present invention generally should be according to the character of disease, severity and to the responsive adaptability of medicine, and route of administration etc. is all is multifactorly determined according to principle of individuation by the clinician.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell, and the example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell etc.
Use the resulting scorpion venom antalgic active peptide of the present invention expression amount height, be easy to purifying and obtain target protein, analgesic activities is improved preferably in the fractional mutant body.
Description of drawings
With scorpion arialgesic antitumoral peptide D8K mutant (being abbreviated as D8K) is that example describes:
Fig. 1: the synoptic diagram that shows the prokaryotic cell prokaryocyte recombinant expression plasmid pSYPU/BmKAGAP (D8K) be used to express D8K.
Fig. 2: show that the colony polymerase chain reaction (PCR) method evaluation contains the bacterial colony of recombinant plasmid.
1.5% agarose gel electrophoresis detects pcr amplification product, identifies the bacterial colony that contains recombinant plasmid.Wherein swimming lane 1 positive contrast, swimming lane 2 negative contrasts, swimming lane 3 is for containing the bacterial colony that inserts the segmental recombinant plasmid of purpose, and swimming lane M is nucleic acid molecular weight mark DL2000.
Fig. 3: the metal ion-chelant chromatography color atlas that shows the D8K mutant.Arrow indication position is a target protein place elution peak among the figure.
Fig. 4: the 15%SDS-PAGE collection of illustrative plates that shows the D8K mutant.Wherein swimming lane 1 is a D8K protein band behind the purifying, and swimming lane M is the low molecular weight protein mark.
Embodiment
The following examples can make those skilled in the art more fully understand the present invention, rather than limit the scope that the present invention gives special approval to claim by any way.
Table 1. is used to make up BmKAGAP fractional mutant primer
a
aItalic GGATCC represents 3 ' terminal BamHI restriction enzyme site.
Table 2. is used for making up transformation BmKAGAP disulfide linkage fractional mutant primer
a
aItalic GGATCC represents 3 ' terminal 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
Present embodiment is enumerated to describe and is used to express construction strategy and the basic skills of the proteic recombinant plasmid pSYPU/BmKAGAP-D8K of D8K of the present invention.
According to Asp in the BmKAGAP aminoacid sequence
8Design corresponding Oligonucleolide primers P1 and primer P2, with the pSYPU/BmKAGAP plasmid that builds 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, reclaiming product with gained again is template, is that primer carries out the third round pcr amplification with P6 and P7, reclaims corresponding product.Through behind restriction endonuclease NcoI and the BamHI double digestion with carry out the plasmid pSYPU of double digestion at T equally
4Connect under the effect of DNA ligase, thermal transition competent escherichia coli cell DH5 α, process bacterium colony PCR and restriction endonuclease enzyme cut the checking screening and obtain to submit to biotechnology service company to carry out determined dna sequence behind the positive transformant.The 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 and separating Escherichia coli host cell according to a conventional method after order-checking is confirmed.At first with positive recombinant plasmid thermal transition e. coli bl21 (λ DE3), be seeded to 3ml LB (containing that microbiotic of card, 50 μ g/ml) behind the picking list bacterium colony from this LB solid plate then, in 37 ℃, the 200r/min shaken overnight is cultivated.According to 1% inoculum size overnight culture is seeded in the triangular flask that 400ml contains corresponding antibiotic fresh LB substratum, in 37 ℃, the 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, the 50mM imidazoles) resuspended thalline, carry out ultrasonication, ultrasonic end back is in 12,000g, 4 ℃ of centrifugal 20min, get supernatant liquor, the 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, behind 5 column volumes of pH damping fluid difference thorough washing through two different stepss, 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 the explanation of SDS-PAGE collection of illustrative plates, recombinant protein obtains to efficiently express, and 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
The strategy and the basic skills of 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
Present embodiment is enumerated to describe and is used to express construction strategy and the basic skills of the proteic recombinant plasmid pSYPU/BmKAGAP-K-K of rBmKAGAP-K-K of the present invention.Wherein the aminoacid sequence according to AGAP mature peptide C-terminal designs corresponding Oligonucleolide primers P3, is that template is carried out pcr amplification with the pSYPU/BmKAGAP plasmid that builds, 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
The strategy and the basic skills of 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
The strategy and the basic skills of 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
The strategy and the basic skills of 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 (G) acquisition of mutant [BmKAGAP-(G)] and derivative thereof, analogue of scorpion arialgesic antitumoral peptide C end
Present embodiment is enumerated to describe and is used to express rBmKAGAP of the present invention (G) proteic recombinant plasmid pSYPU/BmKAGAP (construction strategy G) and basic skills.According to the aminoacid sequence design oligonucleotides primer P4 of AGAP mature peptide C-terminal, be template with the pSYPU/BmKAGAP plasmid that builds wherein, 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 (2G) acquisition of mutant [BmKAGAP-(2G)] and derivative thereof, analogue of scorpion arialgesic antitumoral peptide C end
Present embodiment is enumerated to describe and is used to express rBmKAGAP of the present invention (2G) proteic recombinant plasmid pSYPU/BmKAGAP (construction strategy 2G) and basic skills.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
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.Just on the basis of scorpion arialgesic antitumoral peptide-D8 (K or R) mutant, add an alkaline amino acid residue (K or R) at its C-terminal.
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
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.Just add on the basis of an alkaline amino acid residue (K or R) mutant, add an alkaline amino acid residue (K or R) again at its C-terminal at scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal.
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
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.Just add on the basis of two alkaline amino acid residues (K or R)-(K or R) mutant, add an alkaline amino acid residue (K or R) again at its C-terminal at scorpion arialgesic antitumoral peptide-D8 (K or R)-C-terminal.
The mutant of this pattern and derivative thereof, analogue comprise: 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: scorpion arialgesic antitumoral peptide-D8 (K or R)-C holds the (G) acquisition of mutant and derivative thereof, analogue
Present embodiment obtains scorpion arialgesic antitumoral peptide-D8 (K or R)-C end, and (G) mutant and derivative thereof, analogue are 1,2 and 7 strategy and basic skills in conjunction with the embodiments.Just on the basis of scorpion arialgesic antitumoral peptide-D8 (K or R) mutant and derivative thereof, analogue, remove a G residue at its C-terminal.
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: scorpion arialgesic antitumoral peptide-D8 (K or R)-C holds the (2G) acquisition of mutant and derivative thereof, analogue
Present embodiment obtains scorpion arialgesic antitumoral peptide-D8 (K or R)-C end, and (2G) mutant and derivative thereof, analogue are 1,2 and 8 strategy and basic skills in conjunction with the embodiments.Just (G) on the basis of mutant and derivative thereof, analogue, remove a G residue again at its C-terminal at scorpion arialgesic antitumoral peptide-D8 (K or R)-C end.
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: add the acquisition of an alkaline amino acid residue mutant and derivative thereof, analogue after two G of scorpion arialgesic antitumoral peptide C-terminal are removed
After being removed, two G that present embodiment obtains the 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: and BmKAGAP-(2G)-R, M-BmKAGAP-(2G)-R, BmKAGAP-(2G)-K, M-BmKAGAP-(2G)-K.
Embodiment 15: add the acquisition of two alkaline amino acid residue mutant and derivative thereof, analogue after two G of scorpion arialgesic antitumoral peptide C-terminal are removed
After being removed, two G that present embodiment obtains the 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: and 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: add the acquisition of three alkaline amino acid residue mutant and derivative thereof, analogue after two G of scorpion arialgesic antitumoral peptide C-terminal are removed
After being removed, two G that present embodiment obtains the 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: and 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 present embodiment obtains the 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)]
Present embodiment is enumerated construction strategy and the basic skills of describing the recombinant plasmid pSYPU/BmKAGAP-(C12S) be used to express BmKAGAP-of the present invention (C12S) mutant protein.
According to Cys in the BmKAGAP aminoacid sequence
12Designing corresponding Oligonucleolide primers P8 and primer P9, is template with the pSYPU/BmKAGAP plasmid that builds, 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
Present embodiment is enumerated to describe and is used to express the construction of recombinant plasmid strategy and the basic skills of BmKAGAP-of the present invention (C16S), BmKAGAP-(C22S), BmKAGAP-(C26S), BmKAGAP-(C36S), BmKAGAP-(C46S) and BmKAGAP-(C48S) mutant protein.
According to Cys in the 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
Present embodiment is enumerated construction strategy and the basic skills of describing the recombinant plasmid pSYPU/BmKAGAP (C63S) be used to express BmKAGAP-of the present invention (C63S) mutant protein.
According to Cys in the BmKAGAP aminoacid sequence
63Designing corresponding Oligonucleolide primers P22, is template with the pSYPU/BmKAGAP plasmid that builds, and is that primer carries out pcr amplification with P6 and P22.Primary process is with embodiment 18.
Embodiment 21:BmKAGAP-(C12S, C63S), BmKAGAP-(C16S, C36S), BmKAGAP-(C22S, C46S), BmKAGAP-(C26S, C48S) acquisition of mutant
Present embodiment enumerate describe be used to express BmKAGAP-of the present invention (C12S, C63S), BmKAGAP-(C16S, C36S), BmKAGAP-(C22S, C46S), BmKAGAP-(C26S, C48S) the construction of recombinant plasmid strategy and the basic skills of mutant protein.
Be template with pSYPU/BmKAGAP-(C12S), pSYPU/BmKAGAP-(C16S), pSYPU/BmKAGAP-(C22S), pSYPU/BmKAGAP-(C26S) plasmid that builds respectively, introduce catastrophe point series primer with corresponding another part desire and carry out pcr amplification.Primary process is with embodiment 1.
Embodiment 22: analgesic activities in the mutant protein body-mouse acetic acid twisting method
Present embodiment is intended to verify the analgesic activities of scorpion arialgesic antitumoral peptide mutant after the transformation of specific site amino acid by analgesic model in the mouse body.Protein concentration adopts the Lowry method to measure.
Mouse acetic acid twisting method analgesic model: Glacial acetic acid is injected the Kunming mouse intraperitoneal as chemical irritant, then cause the deep, big area and more persistent pain stimulation, cause mouse to produce " turning round body " reaction (belly indent, trunk and back leg extension, hips up).
The 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 behind the 20min, turns round the body number of times behind the 5min in the record mouse 10min.With the positive contrast of morphine, physiological saline is blank, calculates the writhing response inhibiting rate of each administration group according to the following equation.
Analgesia is the biological activity test result show: the blank group of physiological saline laboratory animal is turned round the body number of times, and (Mean ± SEM) is 43.70 ± 2.99; Positive controls (morphine, 3.51 μ mol/kg) laboratory animal is turned round the body number of times, and (Mean ± SEM) is 29.37 ± 1.85, and turning round the body inhibiting rate is 32.79%; It is 54.81% that the laboratory animal of scorpion arialgesic antitumoral peptide group (0.14 μ mol/kg) is turned round the body inhibiting rate; 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 are turned round the body inhibiting rate 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 that one or two or three basic aminoacidss, D8 (K or R) and two G of C-terminal removal add the mutant of one or two or three basic aminoacids and derivative thereof, analogue (0.14 μ mol/kg) laboratory animal is turned round body inhibiting rate 92.3%-100%; The mutant laboratory animal 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 turned round the body inhibiting rate and sees Table 3.
Mouse acetic acid twisting analgesic experiment result after the table 3. scorpion arialgesic antitumoral peptide cysteine mutation
Claims (15)
1. scorpion arialgesic antitumoral peptide mutant and derivative thereof, analogue, it is characterized in that: at least one is changed the 8th D of scorpion arialgesic antitumoral peptide and scorpion arialgesic antitumoral peptide C-terminal.
2. scorpion arialgesic antitumoral peptide mutant according to claim 1 and derivative thereof, analogue, it is characterized in that: the 8th D change of described scorpion arialgesic antitumoral peptide refers to that the 8th D replaced by K or R, obtains scorpion arialgesic antitumoral peptide D8K mutant and derivative, analogue or scorpion arialgesic antitumoral D8R mutant and derivative thereof, analogue:
VRDGYIAKDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG
VRDGYIARDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG
MHHHHHHMVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG
MHHHHHHVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG.
3. scorpion arialgesic antitumoral peptide mutant according to claim 1 and derivative thereof, analogue is characterized in that: described scorpion arialgesic antitumoral peptide C-terminal change adds alkaline amino acid residue after comprising scorpion arialgesic antitumoral peptide C-terminal increase alkaline amino acid residue or minimizing G or reducing by two G.
4. scorpion arialgesic antitumoral peptide mutant according to claim 3 and derivative thereof, analogue, it is characterized in that: the alkaline amino acid residue of described increase is one or two or three, the G of described minimizing is one or two, and the basic aminoacids that adds behind two G of described minimizing is residual to be one or two or three.
5. scorpion arialgesic antitumoral peptide mutant according to claim 3 and derivative thereof, analogue, it is characterized in that: described scorpion arialgesic antitumoral peptide C-terminal increases alkaline amino acid residue, obtains scorpion arialgesic antitumoral peptide C end-(K or R) mutant and derivative, analogue or scorpion arialgesic antitumoral peptide C end-(K or R)-(K or R) mutant and derivative, analogue or scorpion arialgesic antitumoral peptide C end-(K or R)-(K or R)-(K or R) mutant and derivative thereof, analogue:
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVP IRVPGKCNGG (K or R)
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVP IRVPGKCNGG (K or R) (K or R)
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVP IRVPGKCNGG (K or R) (K or R) (K or R)
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNA
CWCYKLPDKVPIRVPGKCNGG (K or R)
MHHHHHHMVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQW
AGVYGNACWCYKLPDKVPIRVPGKCNGG (K or R)
MHHHHHHVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCY KLPDKVPIRVPGKCNGG (K or R)
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKV PIRVPGKCNGG (K or R) (K or R)
MHHHHHHMVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWC YKLPDKVPIRVPGKCNGG (K or R) (K or R)
MHHHHHHVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCY KLPDKVPIRVPGKCNGG (K or R) (K or R)
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKV PIRVPGKCNGG (K or R) (K or R) (K or R)
MHHHHHHMVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWC YKLPDKVPIRVPGKCNGG (K or R) (K or R) (K or R)
MHHHHHHVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCY KLPDKVPIRVPGKCNGG (K or R) (K or R) (K or R).
6. scorpion arialgesic antitumoral peptide mutant according to claim 3 and derivative thereof, analogue, it is characterized in that: described scorpion arialgesic antitumoral peptide C-terminal reduces G, obtain scorpion arialgesic antitumoral peptide C end-(G) mutant and derivative thereof, analogue or scorpion arialgesic antitumoral peptide C end-(2G) mutant and derivative thereof, analogue:
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNG
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNG
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN
MHHHHHHMVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNG
MHHHHHHMVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN
MHHHHHHVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNG
MHHHHHHVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN。
7. scorpion arialgesic antitumoral peptide mutant according to claim 1 and derivative thereof, analogue is characterized in that: add one or two or three alkaline amino acid residue mutant and derivative thereof, analogue after two G of scorpion arialgesic antitumoral peptide C-terminal are removed:
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVP IRVPGKCN (K or R)
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKV PIRVPGKCN (K or R)
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVP IRVPGKCN (K or R) (K or R)
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKV PIRVPGKCN (K or R) (K or R)
VRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVP IRVPGKCN (K or R) (K or R) (K or R)
MVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKV PIRVPGKCN (K or R) (K or R) (K or R).
8. scorpion arialgesic antitumoral peptide mutant according to claim 1 and derivative thereof, analogue is characterized in that: scorpion arialgesic antitumoral peptide D8 (K or R) and C-terminal increase by one or two or three alkaline amino acid residue mutant and derivative thereof, analogue:
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG (K or R)
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG (K or R)
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG (K or R) (K or R)
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG (K or R) (K or R)
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG (K or R) (K or R) (K or R)
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN GG (K or R) (K or R) (K or R).
9. scorpion arialgesic antitumoral peptide mutant according to claim 1 and derivative thereof, analogue is characterized in that: scorpion arialgesic antitumoral peptide D8 (K or R) and one or two G mutant of C-terminal minimizing and derivative thereof, analogue:
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN G
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN G
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN.
10. scorpion arialgesic antitumoral peptide mutant according to claim 1 and derivative thereof, analogue is characterized in that: add one or two or three alkaline amino acid residue mutant and derivative thereof, analogue after two G of scorpion arialgesic antitumoral peptide D8 (K or R) and C-terminal are removed:
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN (K or R)
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN (K or R)
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN (K or R) (K or R)
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN (K or R) (K or R)
VRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN (K or R) (K or R) (K or R)
MVRDGYIA (K or R) DKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCN (K or R) (K or R) (K or R).
11. it is characterized in that in scorpion arialgesic antitumoral peptide according to claim 1 and derivative thereof, the analogue: the 12nd, the 16th, the 22nd, the 26th, the 36th, the 46th, the 48th of described scorpion arialgesic antitumoral peptide and the 63rd Cys form disulfide linkage, or 8 Cys of scorpion arialgesic antitumoral peptide derivative, analogue form disulfide linkage.
12. scorpion arialgesic antitumoral peptide mutant according to claim 1 and derivative thereof, analogue is characterized in that: 8 Cys of described scorpion arialgesic antitumoral peptide mutant and derivative thereof, analogue form disulfide linkage.
13. according to any one described scorpion arialgesic antitumoral peptide mutant and derivative thereof, analogue among the claim 1-12, it is characterized in that: can utilize genetic engineering technique and chemical synthesising technology to prepare.
14. a pharmaceutical composition is characterized in that: contain protein and one or more pharmaceutically acceptable carrier or vehicle just like any one qualification among the claim 1-12.
15. described scorpion arialgesic antitumoral peptide mutant of any one among the claim 1-12 and derivative thereof, analogue and the application of mutein in the preparation analgesic that is limited thereof.
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| CN102690342A (en) * | 2011-03-22 | 2012-09-26 | 沈阳药科大学 | Anti-cancer analgesic peptide VKVR, its preparation method and application |
| WO2012126150A1 (en) * | 2011-03-22 | 2012-09-27 | 沈阳药科大学 | Peptide with analgesic activity and preparation method thereof |
| CN111303264A (en) * | 2020-03-01 | 2020-06-19 | 沈阳药科大学 | Anti-inflammatory analgesic active peptide GSN and RRD, and preparation and application thereof |
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| CN1341662A (en) * | 2001-09-30 | 2002-03-27 | 沈阳药科大学 | Scorpion pain-stopping anti-tumor Val-Arg-Gly peptide and its preparation method |
| CN101041692A (en) * | 2006-10-20 | 2007-09-26 | 沈阳药科大学 | Scorpion analgesic antibacterial active peptide and preparation method thereof |
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| CN1341662A (en) * | 2001-09-30 | 2002-03-27 | 沈阳药科大学 | Scorpion pain-stopping anti-tumor Val-Arg-Gly peptide and its preparation method |
| CN101041692A (en) * | 2006-10-20 | 2007-09-26 | 沈阳药科大学 | Scorpion analgesic antibacterial active peptide and preparation method thereof |
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| CN102690342A (en) * | 2011-03-22 | 2012-09-26 | 沈阳药科大学 | Anti-cancer analgesic peptide VKVR, its preparation method and application |
| WO2012126150A1 (en) * | 2011-03-22 | 2012-09-27 | 沈阳药科大学 | Peptide with analgesic activity and preparation method thereof |
| CN102690342B (en) * | 2011-03-22 | 2014-10-29 | 沈阳药科大学 | Anti-cancer analgesic peptide VKVR, its preparation method and application |
| CN111303264A (en) * | 2020-03-01 | 2020-06-19 | 沈阳药科大学 | Anti-inflammatory analgesic active peptide GSN and RRD, and preparation and application thereof |
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