CN102690343A

CN102690343A - Analgesic active peptide VRD, its preparation method and application

Info

Publication number: CN102690343A
Application number: CN2011100690037A
Authority: CN
Inventors: 张景海; 刘岩峰; 王月秋; 吴春福
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2011-03-22
Filing date: 2011-03-22
Publication date: 2012-09-26
Also published as: WO2012126150A1

Abstract

The invention relates to the technical field of biomedicine, specifically to the structure, the function, and the preparation method of analgesic active peptide VRD and application thereof. More specifically, the invention relates to the structure and preparation method of analgesic active peptide VRD and its derivatives, analogues, active fragments, and conservation study of the analgesic activity by means of No. 54 amino acid residue of the analgesic active peptide VRD. The analgesic active peptide VRD and its derivatives, analogues, active fragments, as well as mutants can be used as analgesic drugs, which can be mixed with pharmaceutically acceptable carriers to prepare clinically acceptable injections, oral preparations, transdermal absorption preparations, as well as transmucosal absorption preparations, and applied in the field of medicine. The active peptide of the invention has good analgesic activity, and has the advantages of conventional and simple obtaining method and high-yield.

Description

Antalgic active peptide VRD and preparation method thereof and application

Technical field:

The present invention relates to the biological medicine technology field; Relate to structure, function and preparation method and the application of antalgic active peptide VRD; Specifically; The present invention relates to structure of antalgic active peptide VRD and verivate thereof, analogue, active fragments, the 54th conservative amino acid residues and preparation method thereof, and as the application of analgesic in field of medicaments.

Background technology:

Scorpion is claimed full worm, scorpio again as traditional Chinese medical science valuable ingredient of traditional Chinese medicine, and the beginning is stated from " Kaibao Bencao ", and Compendium of Material Medica is listed in worm portion 40 volumes, apart from modern existing more than 2000 year medicinal history, has the effect of the antispastic of relieving dizziness, high fever, infantile convulsions, epilepsy, etc., dispersing pathogen accumulation, the pain relieving of pain network.Clinically be used to treat diseases such as migraine, facial paralysis, rheumatic arthralgia and pain caused by cancer, determined curative effect.The scorpion main active ingredient is the toxin of tail glandular secretion, wherein contains to have the active ingredient of pharmacy value in a large number, and be a natural radioactivity peptide storehouse of being rich in multiple functional peptides.

At present, the composition and the structure that have analgesic activities from the scorpion acquisition clearly have, as, scorpion arialgesic antitumoral peptide, scorpion analgesic antibacterial active peptide, antalgic active peptide VGG, antalgic active peptide DKK, antalgic active peptide GRR etc.

Summary of the invention:

The objective of the invention is to prove conclusively a peptide species that comes from scorpion and have analgesic activities (this bioactive peptide called after antalgic active peptide VRD) through biological activity test in the body.Simultaneously; Antalgic active peptide VRD preparation method and application category also are provided; Specifically be to utilize genetic engineering technique; From scorpion cDNA clone obtain antalgic active peptide VRD gene and express this bioactive peptide of obtaining to have analgesic activities with and verivate, analogue, active fragments, two mutants, the preparation method of antalgic active peptide VRD and verivate thereof, analogue, active fragments, two mutants is simple, can be mixed with medically acceptable formulation with any carrier.

Two of the object of the invention is the analgesic activities to antalgic active peptide VRD; Utilize genetic engineering technique to obtain verivate, analogue, active fragments, the two mutants of serial antalgic active peptide VRD; Through analgesic activities in the experimental animals of the serial expression product that obtains, with the antalgic active peptide VRD that obtains further to develop the analgesic drug and verivate thereof, analogue, active fragments, two mutants.

Three of the object of the invention is to antalgic active peptide VRD the 54th amino acids residue its analgesic activities conservative property to be studied; Utilizing genetic engineering technique to be directed against this site amino-acid residue suddenlys change to obtain the series mutation body; Reach analgesic activities in the experimental animals of product through the series mutation body surface that obtains, confirm that the analgesic activities of antalgic active peptide VRD is had conservative property.

Four of the object of the invention is the preparing methods to antalgic active peptide VRD and verivate thereof, analogue, active fragments, two mutants, utilizes genetic engineering technique or chemical synthesising technology to solve antalgic active peptide VRD and verivate thereof, analogue, active fragments, two mutants.The genetic engineering technique method of antalgic active peptide VRD and verivate thereof, analogue, active fragments, two mutants comprises:

⑴ will the encode DNA of antalgic active peptide VRD or derivatives thereof or its analogue or its active fragments or its two mutants recombinates to expression vector;

⑵ transform appropriate host cell (protokaryon or eukaryotic cell) with the recombinant expression vector that step ⑴ obtains;

Under the abduction delivering condition that is fit to, culturing step ⑵ by transformed host cells;

⑷ results and the resulting protein of purifying.

Another object of the present invention provides the expression product separation purification method of above-mentioned antalgic active peptide VRD and verivate thereof, analogue, active fragments, two mutants.This method can be used methods such as salt precipitation, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography and gel-filtration, from the lysate of cell and nutrient solution, separates 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 the existence and the corresponding molecular size of expression product.

The present invention carries out analgesia BA experiment in the mouse body of above-mentioned antalgic active peptide VRD and verivate thereof, analogue, active fragments, two mutants first.

The present invention also provides the application in field of biological pharmacy of above-mentioned antalgic active peptide VRD and verivate thereof, analogue, active fragments, two mutants, specifically its analgesia biological activity.

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.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 through 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.

A further object of the present invention provides the application of protein in producing analgesic that is defined as above.Can use albumen of the present invention or contain this proteinic pharmaceutical composition, be used to treat 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 flexibility of medicine, and many factors such as route of administration are confirmed 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.

Antalgic active peptide VRD of the present invention and verivate thereof, analogue, active fragments, two mutants have analgesic activities preferably; Its preparation method is conventional, simple, output is high; Not only have important directive significance and practical value, also lay a good foundation for suitability for industrialized production.

Embodiment:

Following embodiment can make those skilled in the art more fully understand the present invention, rather than limits the scope that the present invention gives special approval to claim by any way.

Embodiment 1:

The acquisition of antalgic active peptide VRD

According to antalgic active peptide VRD ( VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNAC

WCIKLPDSVPIRVPGKCHR) N-terminal and C-terminal aminoacid sequence with and gene order, design corresponding Oligonucleolide primers respectively, simultaneously at 5 of above-mentioned two Oligonucleolide primers ' end, add restriction endonuclease respectively NdeI with BamHI hydrolysis site sequence; With scorpion cDNA is that template is carried out pcr amplification, and agarose gel electrophoresis detects product and carries out the gel recovery of nucleic acid fragment; Through restriction endonuclease NdeI with BamHBehind the I double digestion; Recombinate under the effect of T4 DNA ligase with the plasmid that carries out double digestion equally and to be connected; Thermal transition competent escherichia coli cell DH 5 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, through the method structure acquisition antalgic active peptide VRD gene of said gene engineering.

With positive recombinant plasmid thermal transition e. coli bl21 (λ DE3), be seeded to 3 ml LB (containing that microbiotic of card, 50 μ g/ml) behind the picking list bacterium colony from this LB solid plate then, in 37 ℃, 200 r/min shaken overnight are cultivated.According to 1% inoculum size overnight culture is seeded in the triangular flask that 400 ml contain 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 cf-, 4 ℃ of centrifugal 20 min collect thalline.With the resuspended thalline of 40 ml lysis buffers (0.1 M PBS, 0.15 M NaCl, 50 mM imidazoles); Carry out ultrasonication, after the ultrasonic end in 12,000g cf-, 4 ℃ of centrifugal 20 min; Get supernatant; The gained deposition repeats fragmentation once according to above-mentioned steps, merges supernatant twice, directly is splined on the good metal ion-chelant chromatography column of 0.1 M PBS (pH 8.0) pre-balance; Behind 5 column volumes of pH damping fluid difference thorough washing through two different stepss, use 0.5 M imidazoles (pH 9.0) to carry out wash-out and gather in the crops this elutriant.Expression product constitutional features in the elutriant is: MHHHHHHM VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVP IRVPGKCHRUtilize chemical method at M place fracture peptide chain, thereby obtain antalgic active peptide VRD, constitutional features is: VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVP IRVPGKCHR.

Embodiment 2:

The separation and purification of antalgic active peptide VRD

Do not reach the expection purity requirement at the expression product of embodiment 1 acquisition or the purity of antalgic active peptide VRD; Can utilize hydrophobic chromatography, ion exchange chromatography, gel permeation chromatography, reversed phase chromatography method; Carry out alone or in combination respectively, so that further separation and purification is to expecting purity requirement.

Hydrophobic chromatography separates:

Expression product that metal ion-chelant chromatography column among the embodiment 1 obtains or the chemical method antalgic active peptide VRD that the fracture peptide chain obtains at the M place; Transfer pH to the requirement condition scope with aqueous hydrochloric acid or phosphate aqueous solution or acidic solution or basic soln; Add neutral salt to 2M concentration; Be splined on and use 2M neutral salt-buffer soln equilibrated hydrophobic chromatography post in advance; Use 2.0M, 1.5M, 1.0M, 0.5M neutral salt-buffer solution elution respectively, use buffer solution elution at last, the elutriant that will contain target compound merges.The isolating characteristic of hydrophobic chromatography: 1. hydrophobic chromatoghaphy medium select phenyl-hydrophobic chromatography add material or octane-hydrophobic chromatography add material-or hexane-hydrophobic chromatography add material-or butane-hydrophobic chromatography add material; 2. neutral salt is selected (HN ₃) ₂SO ₄Or Na ₂SO ₄Or NaCl; 3. the pH of damping fluid is chosen in pH2-pH12, and this condition neither influences the biological activity of analgesic bioactive substance, does not influence the separation of activeconstituents again; 4. the concentration of damping fluid is chosen in 1mM-100mM, and this condition neither influences the biological activity of analgesic bioactive substance, does not influence the separation of activeconstituents again.

Uf processing analgesic bioactive substance solution

The purpose of utilizing uf processing is salt or foreign protein or the exchange buffering liquid of removing in the analgesic bioactive substance solution, in addition, analgesic bioactive substance solution is concentrated.Selection can make antalgic active peptide VRD or and the ultra-filtration membrane that sees through of verivate, analogue, active fragments carry out uf processing, collect ultrafiltration and see through liquid this moment, and foreign protein is trapped and realizes isolating purpose.Selection can hold back antalgic active peptide VRD or and the ultra-filtration membrane of verivate, analogue, active fragments carry out uf processing; Collect ultrafiltration and concentration liquid (antalgic active peptide VRD or and verivate, analogue, active fragments be trapped) this moment; And the buffering of salt, damping fluid is seen through, foreign protein, the water that can see through ultra-filtration membrane, realizes removing salt, foreign protein or exchange buffering liquid or spissated purpose.The characteristic of uf processing: 1. seeing through that ultra-filtration membrane selects is that molecular weight is the ultra-filtration membrane of 10kDa or 20kDa or 30kDa or 40kDa or 50kDa, and the ultra-filtration membrane of preferred 30kDa is greater than or less than the ultra-filtration membrane of 30kDa, its yield or ultrafiltration efficient all effected; 2. holding back that ultra-filtration membrane selects is that molecular weight is the ultra-filtration membrane of 1kDa or 2kDa or 3kDa or 5kDa, and the ultra-filtration membrane of preferred 1kDa is greater than or less than the ultra-filtration membrane of 1kDa, its yield or ultrafiltration efficient all effected; 3. the temperature of uf processing is chosen in 0 ℃-45 ℃, and this condition is neither destroyed the physico-chemical property of ultra-filtration membrane, does not influence the activity of target compound again.

Ion exchange chromatography separates

Utilize the analgesic bioactive substance solution of uf processing, further the damping fluid with the ion exchange chromatography experiment carries out desalination, buffer exchange and concentrated.Pending good sample liquid is splined on the ion exchange column of using the damping fluid balance good in advance; Earlier remove non-adsorbable foreign protein with the damping fluid thorough washing; Carry out stepwise elution with 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.4M, 0.5M salts solution respectively, the elutriant that will contain analgesic activities merges.The isolating characteristic of ion exchange chromatography: 1. chromatography media selects cation-exchange chromatography to add material; Add material or SP-ion exchange chromatography like the CM-ion exchange chromatography and add material or S-ion exchange chromatography and add cation-exchange chromatography such as material and add material, the damping fluid potential of hydrogen is chosen in pH2-pH7 at this moment; 2. chromatography media selects anion-exchange chromatography to add material, adds material or DEAE-ion exchange chromatography like the Q-ion exchange chromatography and adds material or QAE-ion exchange chromatography and add material plasma displacement chromatography and add material, and this moment, the damping fluid potential of hydrogen was chosen in pH7-pH12; 3. the concentration of damping fluid is chosen in 1mM-50mM, and this condition neither influences the biological activity of analgesic bioactive substance, does not influence the separation of activeconstituents again; 4. the selection of the salts solution of stepwise elution is the damping fluid of requirement concentration or in the 10mM damping fluid, adds the concentration of neutral salt to needs; 5. neutral salt is selected (HN ₃) ₂SO ₄Or Na ₂SO ₄Or NaCl or KCl, preferred NaCl.

The hydrophobic chromatography purifying

Analgesic bioactive substance is further purified according to the basic framework of hydrophobic chromatography separation method.Analgesic bioactive substance solution adds neutral salt to 2M concentration; Be splined on and use 2M neutral salt-buffer soln equilibrated hydrophobic chromatography post in advance; (wash-out of 2.0M～0.0M) merges and collects the elution peak that contains analgesic activities, carries out that ultrafiltration is desalted and concentration to carry out the neutral salt concentration gradient.The characteristic of hydrophobic chromatography purifying: 1. hydrophobic chromatoghaphy medium select phenyl-hydrophobic chromatography add material or octane-hydrophobic chromatography add material-or hexane-hydrophobic chromatography add material-or butane-hydrophobic chromatography add material; 2. neutral salt is selected (HN ₃) ₂SO ₄Or Na ₂SO ₄Or NaCl; 3. the pH of damping fluid is chosen in pH2-pH12; 4. the concentration of damping fluid is chosen in 1mM-100mM.

The ion exchange chromatography purifying

Analgesic bioactive substance further separates according to the basic framework of ion exchange chromatography separation method.Pending good sample liquid is splined on the ion exchange column of using the damping fluid balance good in advance, earlier removes non-adsorbable foreign protein with the damping fluid thorough washing, and (wash-out of 0.0M～1.0M) merges the elution peak that collection contains analgesic activities to carry out the salt concn gradient.The characteristic of ion exchange chromatography purifying: 1. chromatography media selects cation-exchange chromatography to add material; Add material or SP-ion exchange chromatography like the CM-ion exchange chromatography and add material or S-ion exchange chromatography and add cation-exchange chromatography such as material and add material, the damping fluid potential of hydrogen is chosen in pH2-pH7 at this moment; 2. chromatography media selects anion-exchange chromatography to add material, adds material or DEAE-ion exchange chromatography like the Q-ion exchange chromatography and adds material or QAE-ion exchange chromatography and add material plasma displacement chromatography and add material, and this moment, the damping fluid potential of hydrogen was chosen in pH7-pH12; 3. the concentration of damping fluid is chosen in 1mM-50mM; 4. the selection of the salts solution of salt gradient wash-out is the damping fluid of requirement concentration or in the 10mM damping fluid, adds the concentration of neutral salt to needs; 5. neutral salt is selected (HN ₃) ₂SO ₄Or Na ₂SO ₄Or NaCl or KCl, preferred NaCl.

Gel chromatography

The solution that will contain analgesic activities carries out ultrafiltration and concentration, and sample liquid is splined in advance with the good gel permeation chromatography post of elutriant balance and carries out purifying, collects the elution peak that contains analgesic activities.Gel chromatography features: 1. Chromatographic medium can choose Sephacryl S-100 HR or Sephacryl S-200 HR or Sephadex G-50 or Sephadex G-75 or Sephadex G-100 or Sephadex G-150 or Superose 12 prep grade or Superose 6 prep grade or Superdex 30 prep grade or Superdex 75 prep grade or Superose 12 HR or Superose 6 HR or Superdex Peptide HR or Superdex75 HR or Superdex Peptide PE Tim materials such as gel chromatography; 2. eluent pH choose pH2-pH12; 3. eluent concentration in plasma concentrations greater than 0.15M selection and above .

Reversed phase chromatography

Active ingredient; Be splined on and use the good reversed phase chromatography post of 0.1% trifluoracetic acid-20% acetonitrile solution balance in advance; Add material like SOURCE 15RPC, 30RPC chromatography; With 0.1% trifluoracetic acid-20% acetonitrile solution thorough washing, then carry out acetonitrile concentration gradient (20% to 95%) wash-out earlier, merge and collect the maximum elution peak component-antalgic active peptide target compound of elution peak area.

Embodiment 3:

Expression and the purifying thereof of antalgic active peptide VRD in yeast

Utilize Oligonucleolide primers to increase the sequence of coding antalgic active peptide gene; Employed 5 ' Oligonucleolide primers the sequence of reacting PCR contains the encoding sequence of N-terminal zone aminoacid sequence of restriction enzyme site and the antalgic active peptide VRD of EcoR V restriction enzyme, and 3 ' end primer sequence contains the encoding sequence of the C-terminal zone aminoacid sequence of the restriction enzyme site of EcoR I restriction enzyme, translation stop codon and antalgic active peptide VRD.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the Yeast expression carrier pPIC9K (wherein the restriction endonuclease otch of EcoR V and SnaB I for flat terminal), this plasmid vector coding antibiotics resistance (Amp ^rAnd Kan ^r), a replicon from 2 μ plasmids, an AOX1 promotor can be efficiently expressed by 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; Antalgic active peptide VRD gene with EcoR V and EcoR I digestion pcr amplification; Subsequently it is connected into the pPIC9K carrier; Connect product thermal transition E.coli DH 5 α bacterial strains, screen transformant containing on the LB petridish of Amp, incubated overnight contains the clone of required construction in the LB liquid nutrient medium that contains Amp (100 μ g/ml).The extracting plasmid, sequence verification is inserted fragment and is correctly inserted.After getting plasmid linearization, electricity is transformed into yeast cell, and linearizing recombinant vectors produces stable yeast conversion body through the homologous sequence generation homologous recombination with host genome.Utilize G418 to filter out the integration transformant of high copy.The inoculation that screening obtains is shaken in the bottle in the 250ml that 25ml MGY, BMG or BMGY substratum are housed, be cultured to OD600=2～6 (16～18 h) in 28～30 ℃/250～300 rev/mins; The following 3000 rev/mins of centrifugal 5min of room temperature collect thalline, with the resuspended thalline of MM, BMM or BMMY (about 10～20ml) of 1/5 to 1/10 former volume of culture; The bacterium liquid of step 2 gained is placed the bottle that shakes of 100ml, seal, be positioned over continued growth on 28～30 ℃/250～300 rev/mins the shaking table with double gauze or cheese cloth; Every 24h adds 100% methyl alcohol to final concentration in substratum be 0.5～1.0%.Induce in the nutrient solution of engineering bacterium expression and contain expression product, this expression product constitutional features is: VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVP IRVPGKCHR.

To expression product in the nutrient solution of inducing engineering bacterium expression, the separation purification method that is provided according to embodiment 2, with the expression product separation and purification to expecting purity.

Embodiment 4:

The verivate of antalgic active peptide VRD, analogue, active fragments obtain

The strategy and the basic skills of the verivate of present embodiment acquisition antalgic active peptide VRD, analogue, active fragments, roughly the same strategy and the basic skills of embodiment 1, embodiment 2 and embodiment 3.

Specifically be to add one or more amino-acid residues in that the N-terminal of antalgic active peptide VRD or C-terminal are extra, the antalgic active peptide VRD verivate of acquisition, analogue, active fragments still have the analgesia biological activity.Its constitutional features is:

MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHR。

are n (n can be 1 or 2 or 3 or 4 or 5) (SGGGG).

（SGGGG）n VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVPIR

VPGKCHR (n can be 1 or 2 or 3 or 4 or 5).

Following explanation can make those skilled in the art more fully understand the constitutional features of above-mentioned antalgic active peptide VRD verivate, analogue, active fragments, rather than limits the scope that the present invention gives special approval to claim by any way.N is that the constitutional features of 3 antalgic active peptide VRD verivate, analogue, active fragments is: MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSV PIRVPGKCHRSGGGGSGGGGSGGGG;

or MSGGGGSGGGGSGGGGVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGG RYGNACWCIKLPDSVPIRVPGKCHR.

Embodiment 5:

The analgesic activities conservative property of antalgic active peptide VRD the 54th amino acids residue

Present embodiment obtains the strategy and the basic skills of the 54th series mutation body of antalgic active peptide VRD, roughly the same strategy and the basic skills of embodiment 1, embodiment 2, embodiment 3 and embodiment 4.

Specifically be 54 S residues,, 54 S residues be mutated into respectively through the transgenation technology to antalgic active peptide VRD A, Y, E, K, F, I, T, D, RResidue obtains corresponding two mutants through gene engineering expression, and its constitutional features is:

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDAVPIRVPGKCHR。

。

To the two mutants of antalgic active peptide VRD verivate, analogue, active fragments, its constitutional features is:

MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPD (A or Y or E or K or F or I or T or D or R) VPIRVPGKCHR.

(A or Y or E or K or F or I or T or D or R) VPIRVPGKCHR (SGGGG) n (n can be 1 or 2 or 3 or 4 or 5).

are n VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPD (A or Y or E or K or F or I or T or D or R) VPIRVPGKCHR (n can be 1 or 2 or 3 or 4 or 5) (SGGGG).

Embodiment 6:

Analgesic activities in the body-mouse acetic acid twisting method

Present embodiment is confirmed biological activity-analgesic activities in the body of antalgic active peptide VRD and verivate thereof, analogue, active fragments through analgesic model in the mouse body.In addition, be intended to also to verify that conservative amino acid residues is to the influence of analgesic activities in antalgic active peptide VRD and verivate thereof, analogue, the active fragments.Protein concentration adopts the Lowry method to measure.

Mouse acetic acid twisting method analgesic model: Glacial acetic acid min. 99.5 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).

18-22 g Kunming mouse, male and female half and half, random packet, 10 every group, the tail vein injection sample causes Encelialgia by 0.2 ml/20 g abdominal injection 0.6% (v/v) acetic acid behind 20 min, turns round the body number of times behind 5 min in record mouse 10 min.With the positive contrast of morphine, saline water is blank, calculates the writhing response inhibiting rate of each administration group according to the following equation.

The analgesia biological activity test is the result show:

1. saline water blank control group laboratory animal is turned round the body number of times (Mean ± SEM) is 41.75 ± 2.98; Positive controls (morphine, 1.00mg/kg) turn round the body number of times (Mean ± SEM) be 23.67 ± 1.95, and turning round the body inhibiting rate is 43.30% by laboratory animal.

2. (laboratory animal of 0.5mg/kg) is turned round the body number of times, and (Mean ± SEM) is 22.00 ± 1.79 to antalgic active peptide VRD sample sets, and turning round the body inhibiting rate is 47.30%.

3. (laboratory animal of 0.5mg/kg) is turned round the body inhibiting rate all more than 45% for antalgic active peptide VRD verivate, analogue, each sample sets of active fragments.

4. antalgic active peptide VRD the 54th amino acids residue two mutants sample sets (the laboratory animal analgesia biological activity of 0.5mg/kg):

What A. the 54th S of antalgic active peptide VRD sported E or K or Y or T or D or R two mutants and corresponding derivative thereof, analogue, active fragments respectively turns round the body inhibiting rate all more than 38%; The analgesia biological activity of (antalgic active peptide VRD) before suddenling change relatively, variation does not between the two have statistical significance;

What B. the 54th S of antalgic active peptide VRD sported A or F or I two mutants and corresponding derivative thereof, analogue, active fragments respectively turns round the body inhibiting rate all below 32%; The analgesia biological activity of (antalgic active peptide VRD) shows as reduction before the sudden change relatively, and this reduction has statistical significance;

State and learn 6 method IKLPDS

The above results shows: the 54th S of antalgic active peptide VRD or E or K or Y or T or D or R are the analgesic activities conservative amino acid residues of antalgic active peptide VRD and verivate thereof, analogue, active fragments.

SEQUENCE LISTING

< 110>Shenyang Pharmaceutical University

< 120>antalgic active peptide VRD and preparation method thereof and application

< 130>antalgic active peptide VRD and preparation method thereof and application

<160> 1

<170> PatentIn Version 3.1

<210> 1

<211> 65

<212> PRT

<213> Mesobuthus martensi

<400> 1

Val Arg Asp Ala Tyr Ile Ala Lys Pro Glu Asn Cys Val Tyr His

1 5 10

Cys Ala Thr Asn Glu Gly Cys Asn Lys Leu Cys Thr Asp Asn Gly

16 20 25

Ala Glu Ser Gly Tyr Cys Gln Trp Gly Gly Arg Tyr Gly Asn Ala

31 35 40

Cys Trp Cys Ile Lys Leu Pro Asp Ser Val Pro Ile Arg Val Pro

46 50 55

Gly Lys Cys His Arg

61

Claims

1. antalgic active peptide VRD is characterized in that, its constitutional features is:

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHR。

2. antalgic active peptide VRD according to claim 1 is characterized in that, the 54th amino acids residue of described antalgic active peptide VRD is S or E or K or Y or T or R or D.

3. the verivate of antalgic active peptide VRD or analogue or active fragments or two mutants is characterized in that, it comprises any one described aminoacid sequence total order or fragment in claim 1 or 2.

4. according to verivate or analogue or active fragments or the two mutants of claim 1 or 2 or 3 described antalgic active peptide VRD, it is characterized in that it comprises verivate or analogue or active fragments or the two mutants of following several antalgic active peptide VRD:

MHHHHHHMVRDAYIAKPENCVYHCATNEGCNKLCT

DNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHR；

MVRDAYIAKPENCVYHCATNEGCNKLCT

DNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHR；

MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYG

NACWCIKLPDSVPIRVPGKCHR (SGGGG) n(n can be 1 or 2 or 3 or 4 or 5)

M（SGGGG）n VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYG

NACWCIKLPDSVPIRVPGKCHR (n can be 1 or 2 or 3 or 4 or 5)

MVRDAYIAKPENCVYHCATNEGCNKLCT

DNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHRSGGGGSGGGGSGGGG；

MSGGGGSGGGGSGGGGVRDAYIAKPENCVYHCAT

NEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDSVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDAVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDYVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDEVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDFVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDIVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDKVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDTVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDDVPIRVPGKCHR；

VRDAYIAKPENCVYHCATNEGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPDRVPIRVPGKCHR；

MVRDAYIAKPENCVYHCATNEGCNKLCTDNGAES

GYCQWGGRYGNACWCIKLPD (A or Y or E or K or F or I or T or D or R) VPIRVPGKCHR;

MVRDAYIAKPENCVYHCATNEGCNKLCTDNGA

ESGYCQWGGRYGNACWCIKLPD (A or Y or E or K or F or I

Or T or D or R) VPIRVPGKCHR (SGGGG) n (n can be 1 or 2 or 3 or 4 or 5);

M（SGGGG）n VRDAYIAKPENCVYHCATN

EGCNKLCTDNGAESGYCQWGGRYGNACWCIKLPD (A or Y or E or K

or F or I or T or D or R) VPIRVPGKCHR (n can be 1 or 2 or 3 or 4 or 5).

5. the preparation method of claim 1 or 2 or 3 or 4 described antalgic active peptide VRD and verivate or analogue or active fragments or two mutants; Can utilize genetic engineering technique to express acquisition; It is characterized in that expression host cell is prokaryotic cell prokaryocyte or eukaryotic cell.

6. like claim 1 or 2 or 3 or 4 described antalgic active peptide VRD and verivate or analogue or active fragments or two mutants, can utilize chemical synthesising technology to obtain, it is characterized in that, chemosynthesis is that synthetic or synthesizer are synthetic.

7. claim 1 or 2 or 3 described antalgic active peptide VRD and verivate thereof or analogue or active fragments or the two mutants application in the preparation analgesic.

8. the preparation method of described antalgic active peptide VRD of claim 5 and verivate thereof or analogue or active fragments or two mutants; It is characterized in that; Behind the gene engineering expression through following method separation and purification; It comprises the genetically engineered thalline of (1) expressing after fragmentation, the centrifugal supernatant of removing the insolubles acquisition; Or centrifugal supernatant (2) supernatant of removing the insolubles acquisition obtains the target compound active ingredient through the separation of hydrophobic chromatography post behind the genetic engineering bacterium secreting, expressing; (3) obtain active ingredient through ultrafiltration to remove salt and foreign protein; Concentrate simultaneously, separate obtaining the target compound active ingredient through ion exchange column, (4) obtain active ingredient through the hydrophobic chromatography post further the separation; (5) through ultrafiltration to remove salt and to concentrate; Obtain the target compound active ingredient through the ion exchange column purifying, (6) are further purified through the gel permeation chromatography post and obtain the target compound active ingredient, and (7) obtain highly purified target compound through reverse chromatography column.

9. application according to claim 7 is characterized in that: described antalgic active peptide VRD and verivate thereof or analogue or active fragments or two mutants can be mixed with acceptable injection, oral prepns, transdermal absorption formulation, mucosa absorption preparation clinically with pharmaceutically acceptable carrier.