CN101805402B - Anti-opioid peptide active fragment - Google Patents
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- CN101805402B CN101805402B CN201010122575A CN201010122575A CN101805402B CN 101805402 B CN101805402 B CN 101805402B CN 201010122575 A CN201010122575 A CN 201010122575A CN 201010122575 A CN201010122575 A CN 201010122575A CN 101805402 B CN101805402 B CN 101805402B
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Abstract
The invention discloses an anti-opioid peptide, an antagonist peptide thereof and use thereof. The anti-opioid peptide has an amino acid residue sequence represented by: 1) an amino acid residue sequence SEQ ID No.1 in a sequence table; 2) an amino acid residue sequence SEQ ID No.3-5; and 3) an amino acid residue sequence obtained by substituting, losing or adding 1 to 10 amino acid residues in the amino acid residue sequence SEQ ID No.1 and the amino acid residue sequence SEQ ID No.3-5 in the sequence table. The anti-opioid peptide is a polypeptide having the antinociptive function of antagonist morphine. The antagonist peptide is has amino acid residue sequence represented by: 1) an amino acid residue sequence SEQ ID No.9-11 in a sequence table; and 2) an amino acid residue sequence obtained by substituting, losing or adding 1 to 10 amino acid residues in the amino acid residue sequence SEQ ID No.9-11 in the sequence table. The antagonist peptide is a polypeptide having the functions of strengthening the pain-relieving effect of morphine, reversing morphine tolerance and/or weakening or eliminating abstinence syndrome. The anti-opioid peptide, the active fragment thereof and the antagonist peptide are expected to play significant roles in the field of medicine and the field of the preparation of medicaments for treating drug addition and have wide application range.
Description
The application is that application number is 200710118104.2, the applying date is to divide an application for " application of a kind of anti-opioid and antagonism peptide thereof and this antagonism peptide " on 06 28th, 2007, invention and created name.
Technical field
The present invention relates to peptide section and encoding sox thereof and application; Particularly relate to a kind of anti-opioid and antagonism peptide thereof, have in preparation with this antagonism peptide and strengthen morphine analgesia, reverse the morphine tolerance and/or weaken or eliminate the application in the medicine of Withrawal symptom effect with antagonism morphine antinociceptic effect.
Background technology
As everyone knows, worldwide rampant drug trafficking and addiction activity has become a malignant tumor of harm society, has seriously disturbed orderly economic construction and social stability, and in addition, IDU has also caused the soaring of AIDS crowd's infection rate.At present; Though all strengthening, national governments unite the dynamics of combatting drug trafficking; But take drugs, narco-trafficking still remains incessant after repeated prohibition, one of them important reasons is exactly that morphine tolerance and the principle that relies on really or are not fully illustrated as yet, does not still have effective drug rehabilitation means in the world.External employing at first " alternative medicine " be that the medicine with one type of methadone replaces opium in withdrawal, this therapy will cause tolerance and the dependence of patient to methadone class medicine.At home, Mr. Yang Guodong of Ningbo narcotic house adopts hyoscine to cooperate the other medicines drug rehabilitation, does not also separate resolution addiction problem fully.Professor Han Jisheng adopts acupuncture and moxibustion therapeutic apparatus treatment Withrawal symptom; That attempts that electric pulse stimulation through CF improves endogenous opiate in the body synthesizes to come mitigation symptoms, but its greatest drawback be the efficient limited of acupuncture analgesia among the crowd and also constantly acupuncture also have the tolerance problem.Recently, Shijiazhuang tcm-expert Wang Yan debates disease executing to the opium Withrawal symptom and controls, and adopts " tortoise beetle ball " treatment withdrawal syndrome, through clinical further check and perfect.Therefore, press for a kind of can and the dependence from thorough elimination morphine tolerance on the source, and the anti-additive medicament that weakens or eliminate Withrawal symptom.
The albumen of known PEBP family extensively is present in yeast, nematode, fruit bat and each kind of plant and mammiferous Different Organs in nature, they have conservative property, do not have homology with the proteic sequence of other known structure or function.The proteic biological function of PEBP is very various, and the signal that it is grown to bloom signal and meristematic tissue plant plays regulating effect.Mammal; PEBP albumen can make it not to be activated through the phosphorylation that Raf-1 suppresses MEK, so the title of Raf kinase inhibition albumen (RKIP) is arranged again, it regulates one group of signal of Raf/MEK/ERK; Relevant with the mitotic division of cell with differentiation, relate to the transfer of tumour cell.In cns, PEBP albumen not only stimulates predecessor's albumen of cholinergic nerve peptide (HCNP) as hippocampus, and as ATP, opium and phosphatidyl ethanol ammonia conjugated protein.People (Ojika K. such as Ojika K.; Mitake S.; Tohdoh N., Appel S.H., Otsuka Y.; Katada E.And Matsukawa N. (2000) Hippocampal cholinergic neurostimulating peptides (HCNP) .Prog.Neurobiol.60; 37-83.Iwase T, Ojika K, Matsukewa N; Et al.Muscarinic cholinergic and glutamatergic reciprocal regulation of expressionof hippocampal cholinergic neurostimulating peptide precursor protein gene inrat hippocampus.Neuroscience; 2001,102 (2): point out at stripped medial septal nuclear that 341-352) the hippocampus choline peptide (HCNP) that can excite nerve improves the product of choline acetylase (ChAT) rather than the product of choline lipase (AchE).HCNP is at the stronger position of the 11 day rat brain immunoreation positives; Mainly contain pallium, hippocampal dentate, amygdala, spinal cord nuclei quintus and posterior horn of spinal cord etc.; And strong at the relative mark of Adult Rat Brain HCNP precursor messenger RNA(mRNA), mainly be distributed in pallium, hippocampus, amygdala, at a distance from positions such as the purkinje's cell of nuclear, HM, black substance, cerebellum and granular layer cells.
Summary of the invention
The purpose of this invention is to provide a kind of anti-opioid with antagonism morphine antinociceptic effect.
Anti-opioid provided by the present invention, called after 21Kd is one of following amino acid residue sequences:
1) the SEQ ID NO:1 in the sequence table;
2), has the polypeptide of antagonism morphine antinociceptic effect with replacement, disappearance or the interpolation of the amino acid residue sequence of SEQ ID NO:1 in the sequence table through one to ten amino-acid residue.
One to ten amino-acid residue of said replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function, for example, and increase histidine-tagged etc.
SEQ ID NO:1 in the sequence table is made up of 186 amino-acid residues.
The gene of code book invention anti-opioid 21Kd is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID NO:2 in the sequence table;
2) dna sequence dna of SEQ ID NO:1 in the code sequence tabulation;
3) with sequence table in the nucleotide sequence of the nucleotide sequence that limits of SEQ ID NO:2 with 90% above homology;
The nucleotide sequence of the dna sequence dna hybridization that 4) under the rigorous condition of height, can limit with the SEQ ID NO:2 in the sequence table.
The rigorous condition of said height is: (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.
SEQ ID NO:2 in the sequence table is by 558 based compositions, and coding has the protein of the amino acid residue sequence of SEQ ID NO:1 in the sequence table.
Test shows that above-mentioned anti-opioid 21Kd has a series of active fragments, and they can be one of following amino acid residue sequences:
1) the SEQ ID NO:3 in the sequence table;
2) the SEQ ID NO:4 in the sequence table;
3) the SEQ ID NO:5 in the sequence table;
4), and has the polypeptide of antagonism morphine antinociceptic effect with replacement, disappearance or the interpolation of the amino acid residue sequence of SEQ ID NO:3-5 in the sequence table through one to ten amino-acid residue.
One to ten amino-acid residue of said replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.
SEQ ID NO:3 in the sequence table is made up of 13 amino-acid residues, with its called after 21Kd-13; SEQ ID NO:4 in the sequence table is made up of 14 amino-acid residues, with its called after 21Kd-14; SEQID NO:5 in the sequence table is made up of 21 amino-acid residues, with its called after 21Kd-21.
The nucleotide sequence that encode the gene of above-mentioned anti-opioid active fragments, has the nucleotide sequence of 90% above homology with this gene or under the rigorous condition of height, can hybridize with the dna sequence dna of its qualification also receives protection of the present invention.
The rigorous condition of said height is: (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.
SEQ ID NO:6 in the sequence table is by 39 based compositions, the anti-opioid 21Kd active fragments shown in the coding SEQ ID NO:3; SEQ ID NO:7 in the sequence table is by 42 based compositions, the anti-opioid 21Kd active fragments shown in the coding SEQ ID NO:4; SEQ ID NO:8 in the sequence table is by 63 based compositions, the anti-opioid 21Kd active fragments shown in the coding SEQ ID NO:5.
The antagonism peptide of anti-opioid 21Kd of the present invention is one of following amino acid residue sequences:
1) the SEQ ID NO:9 in the sequence table;
2) the SEQ ID NO:10 in the sequence table;
3) the SEQ ID NO:11 in the sequence table;
4) amino acid residue sequence of SEQ ID NO:9-11 in the sequence table is had through replacement, disappearance or the interpolation of one to ten amino-acid residue strengthen morphine analgesia, reverse the morphine tolerance and/or weaken or eliminate the polypeptide of Withrawal symptom effect.
One to ten amino-acid residue of said replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.
SEQ ID NO:9 in the sequence table is made up of 13 amino-acid residues, with its called after CP-21Kd-13; SEQ ID NO:10 in the sequence table is made up of 14 amino-acid residues, with its called after CP-21Kd-14; SEQ ID NO:11 in the sequence table is made up of 21 amino-acid residues, with its called after CP-21Kd-21.
The nucleotide sequence that encode the gene of above-mentioned anti-opioid 21Kd antagonism peptide, has the nucleotide sequence of 90% above homology with this gene or under the rigorous condition of height, can hybridize with the dna sequence dna of its qualification also receives protection of the present invention.
The rigorous condition of said height is: (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and is washed film with 0.1 * SSPE.
SEQ ID NO:12 in the sequence table is by 39 based compositions, the antagonism peptide of the anti-opioid 21Kd shown in the coding SEQ ID NO:9; SEQ ID NO:13 in the sequence table is by 42 based compositions, the antagonism peptide of the anti-opioid 21Kd shown in the coding SEQ ID NO:10; SEQ ID NO:14 in the sequence table is by 63 based compositions, the antagonism peptide of the anti-opioid 21Kd shown in the coding SEQ ID NO:11.
Contain that arbitrary segmental primer also belongs to protection scope of the present invention in antagonism peptide expression carrier, transgenic cell line and the host bacterium of above-mentioned anti-opioid 21Kd and this gene that increases.
The antagonism peptide of above-mentioned anti-opioid 21Kd can adopt conventional artificial synthesis directly to obtain; Method synthetic such as solid-phase synthesis like available fmoc protection; Maybe can entrust the biotech firm of specialty synthetic (dodging the synthetic ltd of brilliant biological polypeptide etc.) like U.S.'s couplet (Xi'an) bio tech ltd or Shanghai; In addition, the method for microbial fermentation expression also capable of using obtains.The method of the above-mentioned antagonism peptide of expression provided by the present invention is with the recombinant expression vector importing host cell of the antagonism peptide gene that contains above-mentioned anti-opioid 21Kd, expresses the antagonism peptide that obtains anti-opioid 21Kd.
Said host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Said intestinal bacteria can be E.coli BL21 (DE3), E.coli DH5 α or E.coli Top10 etc.
Be used to make up the said carrier that sets out that contains the antagonism peptide expression carrier of anti-opioid 21Kd can be any one can be at the prokaryotic expression carrier of expression in escherichia coli foreign gene, like pET-22b, pET-11c, pET-30a, pET-28a, pET-28b or pET-28c etc.
Wherein, be the carrier that sets out with pET-22b, the antagonism peptide expression carrier that contains anti-opioid 21Kd of structure is pET-CP-21Kd-8+5, pET-CP-21Kd-13, pET-CP-21Kd-14 or pET-CP-21Kd-21.
Above-mentioned recombinant expression vector all can make up according to ordinary method.
The method that above-mentioned recombinant expression vector is transformed the host bacterium can be method for transformation commonly used in the bioengineering field, like the protoplast transformation method of heat shock method, electrotransformation, joint conversion method or PEG mediation etc.
Cultivation contains the substratum and the culture condition of host cell of the antagonism peptide gene of anti-opioid 21Kd, all can be substratum and the culture condition of cultivating the host that sets out.Wherein, need add inductor when cultivating said recombination bacillus coli host, like IPTG etc.; Add IPTG concentration can be 0.1-1.0mmol/L, be preferably 0.2mmol/L, inducing temperature can be 16-37 ℃; Be preferably 30 ℃, induction time can be 2-4 hour, is preferably 3 hours.
The antagonism peptide of said anti-opioid 21Kd strengthens morphine analgesia in preparation, reverse the morphine tolerance and/or weaken or the application eliminated in the medicine of Withrawal symptom effect also is that the present invention will protect.Certainly, be the medicine of activeconstituents with the antagonism peptide of said anti-opioid 21Kd, the content that needs protection of the present invention especially, this medicine has and strengthens morphine analgesia, reverses the morphine tolerance and/or weaken or eliminate the effect of Withrawal symptom.
Antagonism peptide with anti-opioid 21Kd is in the medicine of activeconstituents, and its activeconstituents can be selected from one or several in the following amino acid residue sequence:
1) the SEQ ID NO:9 in the sequence table;
2) the SEQ ID NO:10 in the sequence table;
3) the SEQ ID NO:11 in the sequence table;
4) amino acid residue sequence of SEQ ID NO:9-11 in the sequence table is had through replacement, disappearance or the interpolation of one to ten amino-acid residue strengthen morphine analgesia, reverse the morphine tolerance and/or weaken or eliminate the polypeptide of Withrawal symptom effect.
When needing; In said medicine, can also add one or more acceptable accessories, said auxiliary material comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, absorption enhancer, tensio-active agent, lubricant and the stablizer etc. that pharmaceutical field is conventional.
Medicine of the present invention can be processed various ways such as injection liquid, dry powder injection, tablet or granula.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
Adult's consumption of said medicine is generally 0.01-0.5mg/kg body weight/inferior, can use by one or many, is generally the course of treatment 10 to 20 days.
The invention provides a kind of separation of pure dissolves from the pig brain anti-opioid 21Kd and active fragments thereof, it has antagonism morphine antinociceptic effect.The increase that the drug abuse process is accompanied by drugs dosage can impel this proteic synthetic increase, the formation that causes the morphine tolerance and rely on.The present invention utilizes and has interactional principle between the protein molecule, according to the sequence of its three active fragmentss, and the antagonism peptide of this anti-opioid 21Kd that has designed.The experiment proof; The antagonism peptide of synthetic can be used as the suppressor factor of endogenous anti-opioid, through the antagonism peptide of three active fragmentss of intravenous injection, can improve the effect of morphine antinociceptic effect; Reduce the morphine consumption; In addition, the molecular weight of these antagonism peptides is easier to through hemato encephalic barrier less than 2000 dalton; Region of interest in cns plays the effect of offsetting endogenous anti-opioid effect, thereby can it be prepared into the medicine that strengthens morphine analgesia, reverses the morphine tolerance and/or weaken or eliminate the Withrawal symptom effect as activeconstituents.This medicine has the following advantages: 1) evident in efficacy: activeconstituents antagonism peptide is to reach the purpose that strengthens morphine analgesia, the tolerance of reverse morphine and eliminate or weaken junkies's Withrawal symptom through eliminating endogenous anti-opioid unnecessary in junkies's body and effect thereof, can eliminate Withrawal symptom from the source; 2) the antagonism peptide that obtains all is that some amino-acid residue quantity are no more than 25 small peptide, and is synthetic easily, is convenient to development research; 3) these small peptides get in the body and are degraded easily, do not possess the spinoff that produces antibody, and are safe; 4) medication is convenient, can be through multiple mode administrations such as intravenous injection mode or sublingual lozenges, to eliminate or to weaken the symptom of morphine abstinence syndrome; 5) this small peptide is stable, the preparation method is simple and easy to do, can be applicable to large-scale industrial production, and low production cost, can alleviate patient's economical load.Anti-opioid 21Kd of the present invention and active fragments thereof and its antagonism peptide will play a significant role in the preparation field of medical science and anti-additive medicament, have a extensive future.
Below in conjunction with specific embodiment the present invention is explained further details.
Description of drawings
Fig. 1 causes the detected result of mouse vas deferens effect of contraction to electricimpulse for Pedis Canitis extract
Fig. 2 injects the detected result of dog brain crude extract to rat acupuncture analgesia effects in the tricorn
Fig. 3 A and Fig. 3 B are the detected result of anti-opioid 21Kd to the antagonistic effect of morphine antinociceptic effect
Fig. 4 A-Fig. 4 C is the active peptide segment 21Kd-21 of anti-opioid 21Kd, and 21Kd-14 and 21Kd-13 are to the detected result of morphine antinociceptic effect influence
Fig. 5 is the detected result of ScFv to the influence of morphine antinociceptic effect
Fig. 6 injects the detected result of the ScFv of various dose to the influence of challenge dose morphine (50mg/kgi.p.) antinociceptic effect in morphine tolerance mouse tricorn
Fig. 7 is the detected result of nmda receptor and the influence of NOS in anti-opioid 21Kd and active fragments antagonism morphine antinociceptic effect thereof
Fig. 8 A-Fig. 8 C is CP-21Kd-21, and CP-21Kd-14 and CP-21Kd-13 are to the detected result of morphine antinociceptic effect influence
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual>(Sambrook, J., Russell, DavidW., Molecular Cloning:A Laboratory Manual, 3
RdEdition, 2001, NY, Cold SpringHarbor).
The primer and dna sequence dna are given birth to worker's biotechnology ltd by Shanghai and are synthesized.
Used experiment mice is the Kunming male mice (available from Institute of Zoology, Academia Sinica) with age in 8-10 week, body weight 20-22g among the following embodiment
The acquisition and the functional verification of embodiment 1, anti-opioid 21Kd and active fragments thereof
One, the acquisition of pig brain anti-opioid 21Kd
1, the discovery of anti-opioid
The mouse vas deferens sample that is exsomatizing; (the 0.1MS ripple is wide to give square-wave pulse; 1 time/second) stimulate and can cause contraction (concrete grammar is referring to document: J.Hughes et al (1975) Effect of morphine on adrenergictransmission in the Mouse Vas deferens.Assessment of agonist and antagonistpotencies of Narcotic analgesics 53; 371-381); Then, successively in perfusate, add Srm-Rhotaard (3.2nM) or Pedis Canitis extract (AOS, 200 μ g/mL) (for the dog brain extracts liquid concentrator earlier after Sephadex G-50 gel (available from pharmacia company) filtration; Ethyloic-Mierocrystalline cellulose (CM-C; Available from H.Reeve-Angel&.Co.Ltd company) behind the cation-exchange chromatography, obtain sample), observe the influence that the electricity irritation mouse vas deferens is caused shrinking effect.It is as shown in Figure 1 that (M represents to morphine; MAP represents to Pedis Canitis extract), experiment is found in dog brain crude extract, to exist the composition that can strengthen the contraction of electricity irritation mouse vas deferens; And the effect that the enhancing shrinking effect of this sample and morphine inhibition electricity irritation mouse vas deferens shrink is on the contrary; Therefore, after in this dog brain crude extract injection mouse tricorn, the antinociceptic effect that can be observed morphine is suppressed significantly.
In addition; Above-mentioned dog brain crude extract is injected the tricorn (400 μ g/20 μ l/ only) of the effective rat of acupuncture analgesia (6), is contrast (6) with saline water (Saline), and the beginning acusector is induced stimulation after 10 minutes; The shout reaction of rat to noxious stimulus observed at every interval ten minutes.The result is as shown in Figure 2, and (I.C.V. represents through burying the conduit micro-injection sample in tricorn in advance, and on behalf of acusector, EA induce acupuncture analgesia; X-coordinate is the dog brain crude extract injection back time, and ordinate zou is the variation of vocalization threshold), (Saline) is contrast with saline water, after acupuncture induction, the acupuncture analgesia effect appears in rat; After the injection of dog brain crude extract, carry out acupuncture induction again, the acupuncture analgesia effect of rat then disappears, and the AOS that exists in the above-mentioned experimental result prompting brain possibly be the infull internal factor of needle anaesthesia analgesia.
2, the acquisition of pig brain anti-opioid 21Kd
The method of separation and purification anti-opioid-21Kd from the pig brain; Be also to be improvement (the long people (1997) of grade of willow slightly equally with reference to the long people's of grade of willow method; The separation and purification of the anti-morphine analgesia peptide of pig brain and antiserum(antisera) thereof are to the preliminary study of morphine tolerance influence; Chinese biological The Chemicals 13 volume 3 phase 322-326), concrete grammar is: with the liquid concentrator of Medulla sus domestica extract earlier after the Sephadex-G50 gel-filtration, after S-sepharose Fast Flow ion exchange chromatography (S-sepharose Fast Flow chromatography column is available from Pharmacia company) separates; Get the sample of the 4th elution peak; After FPLC monos positively charged ion (FPLC monos chromatography column is available from Pharmacia company) exchange,, obtain purified anti-opioid material with micro-reversed-phase HPLC purifying.Because 20 amino-acid residues that this albumen N-is terminal and the pulsating amino acid residue sequence of partly hydrolysed, there is the homology of height with the ox brain.In 106 amino-acid residues having measured, only terminal the 5th, 95,103 residue of N-there are differences.Its molecular weight is 20932 dalton; (claim phosphatidyl ethanol ammonia conjugated protein PhosphatidylethanolamineBinding Protein again with the 21Kd albumen of ox brain; PEBP) molecular weight 20,847 dalton are suitable, so this albumen is called 21Kd (claiming PEBP or RKIP again).Therefore, above-mentioned pig brain 21Kd albumen possibly belong to the PEBP protein family.Anti-opioid to obtaining carries out complete sequence determination; Sequencing result shows that this anti-opioid has the amino acid residue sequence of SEQ ID NO:l in the sequence table; Form by 186 amino-acid residues; Its encoding sox has the nucleotide sequence of SEQ ID NO:2 in the sequence table, by 558 based compositions.
Two, pig brain anti-opioid 21Kd antagonism morphine antinociceptic effect detects
1, the intracerebroventricular injection approach detects pig brain anti-opioid 21Kd antagonism morphine antinociceptic effect
The 21Kd albumen of purifying was injected morphine (20mg/kg) after 10 minutes in the abdominal cavity; Do injection in the mouse tricorn; ID is respectively 0.24nM, 0.48nM and 0.96nM, is contrast with the saline water (Saline) of equal volume, detects the antinociceptic effect of morphine then.The result is (X-coordinate is represented the time behind the injection of morphia, and ordinate zou is represented the variation (%) of vocalization threshold, and n is every group of mouse quantity, and i.p. representes abdominal injection, and i.c.v. representes injection in the tricorn) shown in Fig. 3 A.21Kd albumen is to the no effect of basis pain reaction of mouse; But the antinociceptic effect to morphine (20mg/kg i.p.) has significant antagonistic effect; And receive different concns 21Kd albumen (0.24nM; 0.48nM influence and 0.96nM), the anti-injury of morphine area under a curve is respectively 98.5 ± 9.5,67.7 ± 8.0 and 55.6 ± 6.6 (cm under the different injection concentrations of 21Kd albumen
2), and the area of control group is 134.3 ± 6.7 (cm
2), The results of analysis of variance show exist between experimental group and the control group significant significant difference (P＜0.05, P＜0.001 and P＜0.001, df=28).
2, the tail vein injection approach detects pig brain anti-opioid 21Kd antagonism morphine antinociceptic effect
At mouse peritoneal injection 20mg/kg morphine (M
20) after 10 minutes, in the mouse tricorn, injecting the 21Kd albumen of purifying by the tail vein, ID is respectively 1mg/kg, 2mg/kg and 4mg/kg, is contrast with the saline water (Saline) of equal volume, detects the antinociceptic effect of morphine then.(X-coordinate is represented the time behind the injection of morphia to the result shown in Fig. 3 B; Ordinate zou is represented the variation (%) of vocalization threshold; N is every group of mouse quantity, and i.p. representes abdominal injection, and i.v. representes the tail intravenous injection); The 21Kd albumen of tail intravenous injection different concns also has significant antagonistic effect to the antinociceptic effect of morphine (20mg/kg i.p.), and relevant with the proteic concentration of 21Kd.The anti-injury of morphine area under a curve is respectively 111.2 ± 8.5,61.7 ± 5.4 and 36.5 ± 6.1 (cm under the different injection concentrations of 21Kd albumen
2), with control group 128.0 ± 5.0 (cm
2) compare, there is significant significant difference (P＜0.1, P＜0.001 and P＜0.001).
Above-mentioned test-results proves that anti-opioid 21Kd of the present invention has significant antagonism morphine antinociceptic effect.
Three, the acquisition of anti-opioid 21Kd active fragments and active detection the thereof
1, the acquisition of anti-opioid 21Kd active fragments
Supposition confirms that by step 2 it is because of 21Kd albumen is degraded to less segment in blood to the antagonistic effect of morphine antinociceptic effect that the tail vein injection approach awards anti-opioid 21Kd, through hemato encephalic barrier in the acting result of maincenter.For verifying above-mentioned supposition; Reference (Zh.W.Chen etal (1988) Isolation andcharacterization of porcine diazepam-binding inhibitor; A polypeptide not onlyof cerebral occurrence but also common in intestinal tissues and with effectson regulation of insulin release Eur.J.Biochem 174,239-245), with 21Kd albumen with after the pancreatin degraded; Obtain 21 peak samples through micro-HPLC separation; Use the method identical with step 2 to detect the antagonism morphine antinociceptic effect of wherein several staple samples, the result obtains three compositions with antagonism morphine antinociceptic effect, measures its amino acid residue sequence; The peptide section called after 21Kd-13 that will have the amino acid residue sequence of SEQ ID NO:3 in the sequence table; Be made up of 13 amino-acid residues, its encoding sox has the nucleotide sequence of SEQ ID NO:6 in the sequence table, by 39 based compositions; To have the peptide section called after 21Kd-14 of the amino acid residue sequence of SEQ ID NO:4 in the sequence table, and be made up of 14 amino-acid residues, its encoding sox has the nucleotide sequence of SEQ ID NO:7 in the sequence table, by 42 based compositions; To have the peptide section called after 21Kd-21 of the amino acid residue sequence of SEQ ID NO:5 in the sequence table, and be made up of 21 amino-acid residues, its encoding sox has the nucleotide sequence of SEQ IDNO:8 in the sequence table, by 63 based compositions.Because the amino acid residue sequence of these three peptide sections and the amino acid residue sequence of the corresponding peptide section of ox brain PEBP albumen are in full accord, therefore prove that 21Kd albumen of the present invention belongs to the PEBP protein family really.
2, anti-opioid 21Kd active peptide segment 21Kd-21, the activity of 21Kd-14 and 21Kd-13 detects
Dodge the synthetic synthetic 21Kd-21 of ltd of brilliant biological polypeptide, these three peptide sections of 21Kd-14 and 21Kd-13 by U.S.'s couplet (Xi'an) bio tech ltd and Shanghai.Use active peptide segment 21Kd-21 (1.0,2.0,4.0nM), 21Kd-14 (0.64,3.2,6.4nM) and 21Kd-13 (5.7,11.4,22.7nM) that the method identical with step 2 detect various dose to morphine (M20; 20mg/kg i.P.) influence of antinociceptic effect is contrast with saline water (Saline).(X-coordinate is represented the time behind the injection of morphia to detected result shown in Fig. 4 A-Fig. 4 C; Ordinate zou is represented the variation (%) of vocalization threshold, and n representes respectively to organize experiment mice quantity, and i.p. representes intraperitoneal injection; I.c.v. intracerebral ventricle injection is surveyed in expression); 21Kd-21, the dosage range of 21Kd-14 and 21Kd-13 antagonism morphine antinociceptic effect respectively is 1.0-2.0-4.0nM, 1.6-3.2-6.4nM and 5.7-11.4-22.7nM, and their antagonism morphine antinociceptic effects are all relevant with dosage
One, the acquisition and the Function detection thereof of the soluble single-chain variable region antibody fragment of anti-opioid 21Kd
Utilize display technique of bacteriophage; [construction process is referring to Sblattero D to use people prophage antibody library that Italian International School for Advanced Studies (SISSA) Daniele Sblattero and Andrew Bradbury etc. make up; Lou J L; Marzari R; Et al.In vivo recombination as atool to generate molecular diversity in phage antibody libraries.Reviews inMolecular Biotechnology.2001,74:303～315], from phage antibody library, filter out molecular weight about 20; The soluble single-chain variable region antibody fragment of 000 daltonian anti-opioid 21Kd abbreviates ScFv as.Inject morphine (M in the abdominal cavity
10,10mg/kg) after 10 minutes, ScFv is injected separately in the tricorn of normal mouse, ID is 4.8 μ g/6 μ l, is contrast with the phosphoric acid buffer (PBS) of equal volume, detects the antinociceptic effect of morphine then.Injecting ScFv in the detected result (n is every group of mouse quantity, and i.p. representes intraperitoneal injection, and i.c.v. representes to survey intracerebral ventricle injection) as shown in Figure 5, tricorn can make the antinociceptic effect of morphine (10mg/kg i.p.) significantly strengthen.In 110 minutes, the anti-injury of experimental mice morphine area under a curve is 603.8 ± 38.4, is 2.5 times of control group (239.4 ± 16.1), has significant significant difference (P＜0.001, t=8.7407, t
0.001=4.140, df=14), this experimental result further proves and has the restraining effect of 21Kd albumen to the morphine antinociceptic effect in the normal mouse brain really.
Two, detect the relation of anti-opioid 21Kd and morphine tolerance
Choose 20 healthy mices, every day three times, the Srm-Rhotaard of subcutaneous injection ascending-dose, ID is incremented to 60mg/kg by 10mg/kg, continues 10 days formation morphine tolerance mouse.The 21Kd-ScFv antibody group (5 every group) that then 20 morphine tolerance mouse is divided into morphine tolerance control group (not injecting 21Kd-ScFv) and injects three different concns.At the 11st day; Inject the morphine (50mg/kg) of challenge dose earlier by the abdominal cavity; After 10 minutes, in tricorn, inject 4 μ g/6 μ l more respectively, the 21Kd-ScFv of 6 μ g/6 μ l and three kinds of dosage of 8 μ g/6 μ l; Observe their morphine antinociceptic effect respectively, the healthy mice that tolerates with non-morphine is the blank group.The result is as shown in Figure 6, and (n is a mouse quantity; Ordinate zou is a morphine antinociceptic effect area under a curve; X-coordinate is the different experiments group), at the control group that forms the morphine tolerance, challenge dose morphine antinociceptic effect area under a curve is 121.52 ± 21.2 (cm in 90 minutes
2), morphine (50mg/kgi.p.) the antinociceptic effect area under a curve of three dose groups of 21Kd-ScFv (4.0 μ g/6 μ l, 6 μ g/6 μ l and 8 μ g/6 μ l) is respectively 170.18 ± 25.3,224.25 ± 49.6 and 269.97 ± 31.3 (cm
2).The 21Kd-ScFv of various dose has reversed the antinociceptic effect of challenge dose morphine to some extent; Wherein, the reverse morphine antinociceptic effect of 21Kd-ScFv 6 μ g/6 μ l and 8 μ g/6 μ l dose groups has statistical significance (* representative is compared p＜0.05 with the control group of tolerance; The * representative is compared p＜0.01 with the control group of tolerance).Blank group (261.65 ± 27cm with non-tolerance
2) compare, 21Kd-ScFv (8 μ g/6 μ l) group has reached the degree that reverses the morphine tolerance fully.Above-mentioned experimental result shows that in the forming process of morphine tolerance, the resultant quantity of endogenous anti-opioid 21Kd increases, and possibly be one of important factor that forms the morphine tolerance.
Three, detect the relation that anti-opioid 21Kd and morphine rely on
Choose 12 healthy mices, every day three times, the morphine of subcutaneous injection ascending-dose, ID is incremented to 80mg/kg by 10mg/kg, continues the tolerance of 12 days formation morphines and relies on mouse.Then 12 morphine tolerances and dependence mouse are divided into control group and experimental group.Wherein, control group is the last time after the injection of morphine during 6-8 hour, and abdominal injection opioid peptides antagonist Narlan (Naloxone) (available from Sigma company, 10mg/kg i.p.) is observed the number of times of Withrawal symptom-jump that mouse possibly bring out.Experimental group and control group are to match the observation that experimentizes fully, and operational difference only is that abdominal injection Naloxone (10mg/kg) is preceding, inject 21Kd-ScFv (8 μ g/10 μ l) through tricorn earlier.The result is as shown in table 1, and the number of times that jumping appears in control group mice is followed successively by 0,1,15,36,89,90; And the inferior number average that jumping appears in experimental mice disappears; But the amount of injecting 21Kd-ScFv antibody through tricorn does not wait; Jump just disappears after having three tricorns of 1 need to inject 21Kd-ScFv; There are 2 after twice tricorn injects 21Kd-ScFv, to jump and disappear, just no longer occur jumping after having 3 only once to inject 21Kd-ScFv.Above-mentioned experimental result explanation; Every day three times; There is bigger difference between individuals in the mouse that continues tolerance of 12 days formation morphines and dependence with ascending-dose subcutaneous injection morphine, and the number of times of the jump that Naloxone brought out can reach 0-90 time, and tricorn injection 21Kd-ScFv can eliminate time Withrawal symptom-jump; But needed 21Kd-ScFv antibody amount does not wait, thus proof morphine tolerance and the formation that relies on also with brain in the increase of 21Kd albumen resultant quantity inseparable closely.
Table 1 is in morphine tolerance and rely on mouse, and 21Kd albumen soluble single-chain antibody (ScFv i.c.v.) back of injecting various dose in the tricorn is to the influence of the Withrawal symptom-number of skips of being brought out by Narlan (10mg/kg i.p.)
| Numbering | Control group | Numbering | Experimental group |
| No.5 | 0 | No.1 | 0 ScFv(i.C.v.)8μg×1 |
| No.3 | 1 | No.11 | 0 ScFv(i.C.v.)8μg×1 |
| No.9 | 15 | No.12 | 0 ScFv(i.C.v.)8μg×1 |
| No.6 | 36 | No.10 | 6→0?ScFv(i.C.v.)8μg×2 |
| No.7 | 89 | No.4 | 8→0?ScFv(i.C.v.)8μg×2 |
| No.8 | 90 | No.2 | 45→0?ScFv(i.C.v.)8μg×3 |
Embodiment 3, detection nmda receptor and the effect of NOS in anti-opioid 21Kd and active fragments antagonism morphine antinociceptic effect thereof
Research shows; Excitatory amino acid nmda receptor and nitric oxide synthetase system (NOS) play crucial effects in tolerance of adult mice morphine and dependence process; After with nmda receptor antagonist or no inhibitor mouse being carried out pre-treatment, the formation that can suppress the morphine tolerance and rely on prevents appearance [the Adams ML of Withrawal symptom; Kalicki JM; Meyer ER, Cicero TJ (1993) Inhibition of the morphine withdrawal syndrome bynitric oxide synthase inhibitor, NG-Nitro-L-arginine methyl ester.Life Sci52:PL245-PL249; Cappendijk SL; Vries R, Dzoljic MR (1993) Inhibitory effectof nitric oxide (NO) synthase inhibitors on naloxone-precipitated withdrawalsyndrome in morphine-dependent mice.Neurosci Lett 162:97-100; Herman BH; Vocci F, Bridge P (1995) The effect of NMDA receptor antagonists and nitricoxide synthase inhibitors on opioid tolerance and withdrawal.Neuropsychopharmacology 13:269-293; Vaupel DB; Kimes AS, London ED (1995b) Nitric oxide synthase inhibitors:preclinical studies of potential use fortreatment of opiate addiction.Neuropsychopharmacology 13:315-322; Trujillo K.A. (2000) A review of preclinical studies:Are NMDA receptorsinvolved in opiate-induced neural and behavioral plasticity? Psychopharmacology 151:121-141; Heinzen EL, Pollack G.M. (2004) Thedevelopment of morphine antinociceptive tolerance in nitric oxidesynthase-deficient mice.Biochem.Pharmacology vol 67 (4): 735-741].Use excitatory amino acid nmda receptor antagonist MK-801 (available from Sigma company respectively; 0.1mg/kg i.p.) in preceding 15 minutes of injection of morphine and with nitric oxide synthetase no inhibitor L-NAME (available from Sigma; 45mg/kg i.p.) in injection of morphine preceding 20 minute; Mouse is carried out pre-treatment; To be blank (Blank) without pretreated mouse, use 21Kd (0.96nM), 21Kd-13 (22.7nM), 21Kd-14 (6.4nM) and 21Kd-21 (4.0nM) to detect influence again to morphine (20mg/kg i.P.) antinociceptic effect, be contrast (Control) with saline water (Saline).The result is as shown in Figure 7, and (ordinate zou is a morphine antinociceptic effect area under a curve, the cm of unit
2, X-coordinate projects are three kinds of contrasts, 21Kd; 21Kd-13; 21Kd-14 and 21Kd-21), anti-opioid 21Kd and active fragments 21Kd-13 thereof, 21Kd-14; 21Kd-21 peptide section is disengaged the antagonistic action of morphine antinociceptic effect, explains that anti-opioid 21Kd and bioactive peptide antagonism morphine antinociceptic effect thereof need the mediation of nmda receptor and NOS.Above-mentioned experimental result shows; Handle with nmda receptor antagonist and no inhibitor; Can suppress morphine tolerance and the formation that relies on and prevent the appearance of Withrawal symptom; Possibly be that 21Kd albumen and active fragments thereof are disengaged the retarding effect of morphine antinociceptic effect in the brain, further specify in the brain 21Kd albumen and tolerate with morphine and rely on closely related.
The acquisition of embodiment 4, anti-opioid 21Kd antagonism peptide and active detection the thereof
One, the acquisition of CP-21Kd-13, CP-21Kd-14 and CP-21Kd-21 antagonism peptide
According to there being interactional principle between the protein molecule; Reference (J.R.Heal et al (2002) Specific interactions between sense and complementary peptides:The Basis forthe proteomic code ChemBIOChem 3; 136-151) design the antagonism peptide of 21Kd-13,21Kd-14, three small peptides of 21Kd-21, wherein, with the antagonism peptide called after CP-21Kd-13 of 21Kd-13; Amino acid residue sequence with SEQ ID NO:9; Be made up of 13 amino-acid residues, its encoding sequence has the nucleotide sequence of the SEQ ID NO:12 in the sequence table, by 49 based compositions; With the antagonism peptide called after CP-21Kd-14 of 21Kd-14, have the amino acid residue sequence of SEQ ID NO:10, to form by 14 amino-acid residues, its encoding sequence has the nucleotide sequence of the SEQ ID NO:13 in the sequence table, by 42 based compositions; With the antagonism peptide called after CP-21Kd-21 of 21Kd-21, have the amino acid residue sequence of SEQ ID NO:11, to form by 21 amino-acid residues, its encoding sequence has the nucleotide sequence of the SEQ ID NO:14 in the sequence table, by 63 based compositions;
Two, the activity of CP-21Kd-13, CP-21Kd-14 and CP-21Kd-21 antagonism peptide detects
1, detects CP-21Kd-13, CP-21Kd-14 and CP-21Kd-21 influence to the morphine antinociceptic effect
Use with embodiment 1 step 2 in identical method detect CP-21Kd-21, CP-21Kd-14, CP-21Kd-13 peptide section influence to the morphine antinociceptic effect, method is: choose normal kunming mice, divide three groups (5 or 6 every group; N=5 or 6), injects CP-21Kd-13 (6mg/Kg i.v.), CP-21Kd-14 (5mg/Kg i.v.) and CP-21Kd-21 (5.6mg/Kg i.v.) by the tail vein respectively, detect influence again morphine (20mg/kg i.P.) antinociceptic effect; With saline water is contrast (Saline 50 μ l i.V.); CP-21Kd-13 wherein, (X-coordinate is represented the time (min) behind the injection of morphia to the detected result of CP-21Kd-14 and CP-21Kd-21 injection groups, and ordinate zou is represented the variation (%) of vocalization threshold shown in Fig. 8 A-Fig. 8 C; N representes respectively to organize experiment mice quantity; I.p. represent intraperitoneal injection, i.v. representes the tail intravenous injection), compare with control group; Injection CP-21Kd-21, CP-21Kd-14 or and CP-21Kd-13 after; The antinociceptic effect of morphine strengthens significantly, and obviously these antagonism peptides play a part the anti-opioid suppressor factor, has offset the effect of endogenous anti-opioid.In addition; Because these antagonism peptide molecular weights are less than 2000 dalton; Inject in the body and can get into cns by the tail vein through hemato encephalic barrier; Establish antagonism peptide CP-21Kd-21, CP-21Kd-14, CP-21Kd-13 and had the medicine that preparation strengthens morphine analgesia, the tolerance of reverse morphine and/or weakens or eliminate the Withrawal symptom effect, and the prospect that can carry out clinical application to said medicine.
2, CP-21Kd-21, CP-21Kd-14 and CP-21Kd-13 are to the influence of the morphine abstinence syndrome effect of being brought out by Narlan (Naloxone)
Choose healthy mice, every day three times, the morphine of subcutaneous injection ascending-dose, ID are that 10mg/kg is incremented to 80mg/kg, continue the tolerance of 12 days formation morphines and rely on mouse.Then morphine tolerance and dependence mouse are divided into control group (injecting normal saline) and experimental group.After the 13rd day last injection of morphine 6-8 hour; The tail vein is injected antagonism peptide CP-21Kd-21 (15mg/kg), CP-21Kd-14 (15mg/kg) and CP-21Kd-13 (15.5mg/kg) respectively; (Saline 80 μ l i.V.) are contrast with saline water; 5 minutes pneumoretroperitoneum injection Naloxone (10mg/kg) observe the number of times that mouse is brought out Withrawal symptom-jump.The experimental result of CP-21Kd-21, CP-21Kd-14 and CP-21Kd-13 group is shown in table 2 and table 3; Explanation is injected antagonism peptide CP-21Kd-21 (15mg/kg), CP-21Kd-14 (15mg/kg) and CP-21Kd-13 (15.5mg/kg) respectively by the tail vein, and the effect-jump of giving up that Naloxone (10mg/kg i.P.) brings out can be eliminated or obviously weaken.In addition; Rank test is the result show: compare with control group-1; All there are significant difference (P=1-0.025=0.975) in CP-21Kd-21, CP-21Kd-14 and CP-21Kd-13 experimental group; The CP-21Kd-14 of escalated dose compares with contrast-3 with the CP-21Kd-13 group and also has extremely significant difference, and therefore, antagonism peptide CP-21Kd-21, CP-21Kd-14 and CP-21Kd-13 all can effectively suppress or eliminate and give up effect.
Table 2 tail vein injection antagonism peptide CP-21Kd-21, CP-21Kd-14 and CP-21Kd-13 bring out morphine abstinence syndrome symptom--the influence of jump to Narlan (Naloxone 10mg/kg i.P.)
| No.1 | No.2 | No.3 | No.4 | No.5 | No.6 | No.7 | |
| Control group-1 ( |
8 | 18 | 25 | 35 | 38 | 51 | 71 |
| CP-21Kd-21***(15mg/kg?i.V.) | 0 | 1 | 5 | 11 | 18 | 18 | 25 |
| CP-21Kd-14***(15mg/kg?i.V.) | 0 | 0 | 3 | 3 | 7 | 10 | 21 |
| CP-21Kd-13***(15.5mg/kg?i.V.) | 0 | 0 | 0 | 1 | 6 | 9 | 56 |
* * rank test shows: compare with control group, difference is (P=1-0.025=0.975) extremely significantly.
Table 3 tail vein injection antagonism peptide CP-21Kd-14 (64mg/kg i.v.) and CP-21Kd-13 (48mg/kg i.v.) bring out morphine abstinence syndrome symptom--the influence of jump to Narlan (Naloxone 10mg/kg i.P.)
| No.1 | No.2 | No.3 | No.4 | No.5 | No.6 | No.7 | |
| Control group-3 ( |
66 | 83 | 91 | 97 | 114 | 147 | 554 |
| CP-21Kd-14***(64mg/kg?i.V.) | 0 | 1 | 4 | 23 | 39 | 57 | 62 |
| CP-21Kd-13***(48mg/kg?i.V.) | 0 | 0 | 3 | 8 | 23 | 36 | / |
* * rank test shows: compare with control group, difference is (P=1-0.025=0.975) extremely significantly.
Sequence table
< 110>Peking University
< 120>a kind of active fragments of anti-opioid
<130>CCGNAZ102106
<160>14
<210>1
<211>186
<212>PRT
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>1
Pro?Val?Asp?Leu?Ser?Lys?Trp?Ser?Gly?Pro?Leu?Ser?Leu?Gln?Glu?Val
1 5 10 15
Asp?Glu?Arg?Pro?Gln?His?Pro?Leu?Gln?Val?Lys?Tyr?Gly?Gly?Ala?Glu
20 25 30
Val?Asp?Glu?Leu?Gly?Lys?Val?Leu?Thr?Pro?Thr?Gln?Val?Lys?Asn?Arg
35 40 45
Pro?Thr?Ser?Ile?Thr?Trp?Asp?Gly?Leu?Asp?Pro?Gly?Lys?Leu?Tyr?Thr
50 55 60
Leu?Val?Leu?Thr?Asp?Pro?Asp?Ala?Pro?Ser?Arg?Lys?Asp?Pro?Lys?Tyr
65 70 75 80
Arg?Glu?Trp?His?His?Phe?Leu?Val?Val?Asn?Met?Lys?Gly?Asn?Asn?Ile
85 90 95
Ser?Ser?Gly?Thr?Val?Leu?Ser?Asp?Tyr?Val?Gly?Ser?Gly?Pro?Pro?Lys
100 105 110
Gly?Thr?Gly?Leu?His?Arg?Tyr?Val?Trp?Leu?Val?Tyr?Glu?Gln?Glu?Gly
115 120 125
Pro?Leu?Lys?Cys?Asp?Glu?Pro?Ile?Leu?Ser?Asn?Arg?Ser?Gly?Asp?His
130 135 140
Arg?Gly?Lys?Phe?Lys?Val?Ala?Ser?Phe?Arg?Lys?Lys?Tyr?Glu?Leu?Gly
145 150 155 160
Ala?Pro?Val?Ala?Gly?Thr?Cys?Tyr?Gln?Ala?Glu?Trp?Asp?Asp?Tyr?Val
165 170 175
Pro?Lys?Leu?Tyr?Glu?Gln?Leu?Ser?Gly?Lys
180 185
<210>2
<211>558
<212>DNA
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>2
ccggtggacc?ttagcaagtg?gtccgggcct?ctgagcctgc?aggaagtgga?tgagcggccg 60
cagcacccgc?tgcaggtcaa?atacggcggg?gcggaggtcg?acgaactggg?caaagtgctg 120
acacccaccc?aggttaaaaa?ccggcccacc?agcattacat?gggatggcct?tgatccaggt 180
aaattgtaca?ccttggtctt?gacagatccg?gatgctccca?gcaggaagga?ccccaaatac 240
agggaatggc?accatttcct?ggtggtcaac?atgaagggca?acaacatcag?cagtggcacg 300
gttctctccg?attatgtggg?ctctgggcct?cccaagggca?caggcctgcg?ccgctatgtc 360
tggctggttt?acgagcagga?aggaccactg?aagtgtgatg?agcccattct?cagcaaccga 420
tctggagacc?accgtggcaa?attcaaggtg?gcctctttcc?gcaaaaagta?cgagcttggg 480
gccccagtgg?ccggcacgtg?ttaccaggcc?gaatgggatg?attatgtgcc?caagctctac 540
gagcagctgt?ctgggaag 558
<210>3
<211>13
<212>PRT
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>3
Tyr?Arg?Glu?Trp?His?His?Phe?Leu?Val?Val?Asn?Met?Lys
1 5 10
<210>4
<211>14
<212>PRT
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>4
Leu?Tyr?Thr?Leu?Val?Leu?Thr?Asp?Pro?Asp?Ala?Pro?Ser?Arg
1 5 10
<210>5
<211>21
<212>PRT
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>5
Trp?Ser?Gly?Pro?Leu?Ser?Leu?Gln?Glu?Val?Asp?Glu?Arg?Pro?Gln?His
1 5 10 15
Pro?Leu?Gln?Val?Lys
20
<210>6
<211>39
<212>DNA
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>6
tacagagagt?ggcaccactt?cctggtggtg?aacatgaag 39
<210>7
<211>42
<212>DNA
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>7
ctgtacaccc?tggtgctgac?cgaccccgac?gcccccagca?ga 42
<210>8
<211>63
<212>DNA
< 213>wild boar be born in the year of pig (Sus scrofa domestica Brisson)
<400>8
tggagcggcc?ccctgagcct?gcaggaggtg?gacgagagac?cccagcaccc?cctgcaggtg 60
aag 63
<210>9
<211>13
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>9
Val?Ser?Leu?Pro?Val?Val?Glu?Gln?His?His?Val?His?Leu
1 5 10
<210>10
<211>14
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>10
Gln?Val?Gly?Gln?His?Gln?Gly?Val?Gly?Val?Gly?Gly?Ala?Ser
1 5 10
<210>11
<211>21
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>11
Pro?Ala?Ala?Gly?Gln?Ala?Gln?Leu?Leu?His?Val?Leu?Ser?Gly?Leu?Val
1 5 10 15
Gly?Gln?Leu?His?Leu
20
<210>12
<211>39
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>12
gtctctctcc?cagtcgtcga?acagcatcat gtccatctc 39
<210>13
<211>42
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>13
caggtcgggc?agcatcaggg?ggtcggggtc?gggggggctt?ct 42
<210>14
<211>63
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>14
ccagctgctg?ggcaggctca?gctcctccat?gtcctctctg?ggctcgtcgg?gcagctccat 60
ctc 63
Claims (4)
1. the active fragments of an anti-opioid, its amino acid residue sequence is shown in the SEQ ID NO:5 in the sequence table.
2. the gene of the active fragments of coding claim 1 described anti-opioid.
3. gene according to claim 2 is characterized in that: the nucleotide sequence of said gene is shown in the dna sequence dna of SEQ ID NO:8 in the sequence table.
4. the host bacterium that contains claim 2 or 3 described genes.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141636A (en) * | 1994-02-21 | 1997-01-29 | 阿斯特拉公司 | Novel opioid peptides for treating pain, and use therefor |
| CN1582332A (en) * | 2001-12-29 | 2005-02-16 | Seoul大学校产学协力财团 | GFP-transfected clon pig, GT knoc-out clon pig and methos for production thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141636A (en) * | 1994-02-21 | 1997-01-29 | 阿斯特拉公司 | Novel opioid peptides for treating pain, and use therefor |
| CN1582332A (en) * | 2001-12-29 | 2005-02-16 | Seoul大学校产学协力财团 | GFP-transfected clon pig, GT knoc-out clon pig and methos for production thereof |
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