CN102580091B - Method for enhancing analgesic action of opioid analgesic and reagent - Google Patents

Abstract

The invention relates to a method for enhancing the analgesic action of an opioid analgesic and a reagent, and discloses an application of a substance for restraining the mutual action of a delta opioid receptor (DOR) and a mu opioid receptor (MOR), and a preparation for enhancing the analgesic action of the opioid analgesic. The invention further discloses a protein for enhancing the analgesic action of the opioid analgesic, and a medicinal composition.

Description

Strengthen method and the reagent of the analgesic activity of opioid analgesic

Technical field

The invention belongs to biotechnology and pharmaceutical field; More specifically, the present invention relates to method and the reagent of the analgesic activity that strengthens opioid analgesic.

Affiliated technical field personnel should understand, for convenience of the detection of groupization, in embodiments of the invention, fusion rotein TM1-TAT has added labelled protein flag, be not essential for the function of carrying out TM1, described labelled protein flag can omit or change into other labelling, such as Myc, HA etc.

The inhibitor of DOR

Inventor's discovery, the inhibitor of DOR can pass through to suppress the activity of DOR, thereby suppresses the interaction of DOR and MOR.Therefore, the inhibitor of DOR also can be for promoting the analgesic activity of opioid analgesic.

As used herein, the inhibitor of described DOR has comprised antagonist, lower adjustment, blocker, blocker etc.The material of transcribing and translating of the activity of any DOR of reduction, the stability that reduces DOR, the expression that suppresses DOR, minimizing DOR effective acting time or inhibition DOR all can be used for the present invention, as the active substance that can be used for the analgesic activity that promotes opioid analgesic.

As optimal way of the present invention, the inhibitor of described DOR is (but being not limited to): naltrindole, ICI 174,864, TIPP, Naltriben.

Pharmaceutical composition

The present invention also provides a kind of pharmaceutical composition, and it contains effective dose (as 0.000001-50wt%; Preferably 0.00001-20wt%; Better, 0.0001-10wt%) inhibition delta opiate receptor and the interactional material of mu opioid receptor, and pharmaceutically acceptable carrier.Described inhibition delta opiate receptor and the interactional material of mu opioid receptor are: MOR TM1 or its variant; Or MOR TM1 and the fusion rotein of wearing film peptide; Or the inhibitor of DOR.

As optimal way of the present invention, described pharmaceutical composition also contains: effective dose is (as 0.000001-50wt%; Preferably 0.00001-20wt%; Better, 0.0001-10wt%) opioid analgesic.Preferably refer to act on the analgesic on Mu opiate receptor, as opium alkaloid class (morphine, codeine) and synthetic product (Pethidine, buprenorphine, etorphine, fentanyl, methadone, dihydroetorphine etc.).Some other Mu opiate receptor can be referring to Clin Drug Investig.2009; 29 Suppl 1:3-16.Pain treatment with opioids:achieving the minimal effective and the minimalinteracting dose.Geppetti P, the documents such as Benemei S, these documents are introduced the present invention, so that alternative Mu opiate receptor to be provided.More preferably, described opioid analgesic is morphine.

Pharmaceutical composition of the present invention can be directly used in the analgesia of mammalian organism.In addition, also can combine use with other therapeutic agent or adjuvant simultaneously.

Conventionally, these materials can be formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably, pH is about 6-8.

As used herein, term " contains " and represents that various compositions can be applied in mixture of the present invention or compositions together.Term " mainly by ... composition " and " by ... form " be included in during term " contains ".As used herein, term " effective dose " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.

As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.

Described pharmaceutically acceptable carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Conventionally pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, for example, with normal saline or contain glucose and the aqueous solution of other adjuvant is prepared by conventional method.Described pharmaceutical composition should be manufactured under aseptic condition.The dosage of active component is treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.

The effective dose of inhibition delta opiate receptor of the present invention and the interactional material of mu opioid receptor can change with order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (for example, by clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization rate of described inhibition delta opiate receptor and the interactional material of mu opioid receptor, metabolism, half-life etc.; The order of severity of the disease that patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Conventionally, when inhibition delta opiate receptor of the present invention and the interactional material of mu opioid receptor give with the dosage of about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-10mg/kg the weight of animals) every day, can obtain gratifying effect.For example, by an urgent demand for the treatment of situation, can give the dosage that several times separate every day, or dosage is reduced pari passu.

Screening technique

The DOR described in cicada and MOR interact directly related for the sensitivity of opioid analgesic with MOR after, can screen inhibition DOR and the interactional material of MOR based on this mechanism.

Therefore, the invention provides a kind of method that screening can be used for the potential material of adjusting the analgesic activity that strengthens opioid analgesic, described method comprises: candidate substances is contacted with the interactional system of DOR and MOR; With detect candidate substances on DOR and the interactional impact of MOR; If described candidate substances can suppress (comprising: weaken, block or dissociate) DOR and MOR interacts, show that this candidate substances is the potential material that can be used for the analgesic activity that strengthens opioid analgesic.

In optimal way of the present invention, in the time screening, in order to be easier to observe DOR and the interactional change of MOR, matched group also can be set, described matched group can be DOR and the interactional system of MOR of not adding described candidate substances.

Described system includes, but is not limited to: solution system, subcellular fraction system, cell system, organizational framework, organ systems or animal system.

As optimal way of the present invention, described method also comprises: the potential material obtaining is carried out to further cell experiment and/or animal experiment, further to select and to determine for the real useful material of the analgesic activity that strengthens opioid analgesic.

On the other hand, the present invention also provides the potential material that can be used for the analgesic activity that strengthens opioid analgesic that adopts described screening technique to obtain.The material that these Preliminary screening go out can form a screening storehouse so that people finally can therefrom filter out can be for the real useful material of the analgesic activity that strengthens opioid analgesic.

Major advantage of the present invention is:

(1) clearly to have established the DOR of DOR agonist induction and the common endocytosis of MOR and co-degradation be one of reason causing MOR desensitization in the present invention, thereby a kind of New Policy that strengthens morphine analgesia effect by suppressing MOR-DOR interaction is physically provided.

(2) the present invention has found and can be used for suppressing the interactional material of MOR-DOR, can be used as analgesic for clinical.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: the condition described in lab guide (New York:Cold Spring Harbor Laboratory Press), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.

Unless otherwise defined, the same meaning that all specialties that use in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

I. materials and methods

Express plasmid with the MOR (HA-MOR) of HA label referring to Guan JS, Xu ZZ, Gao H, He SQ, Ma GQ, Sun T, Wang LH, Zhang ZN, Lena I, Kitchen I, et al:Interactionwith Vesicle Luminal Protachykinin Regulates Surface Expression of delta-OpioidReceptors and Opioid Analgesia.Cell 2005,122:619-631.

Express plasmid with the DOR (Myc-DOR) of Myc label referring to Bao L, Jin SX, ZhangC, Wang LH, Xu ZZ, Zhang FX, Wang LC, Ning FS, Cai HJ, Guan JS, et al:Activation of delta opioid receptors induces receptor insertion and neuropeptidesecretion.Neuron 2003,37:121-133.

Express plasmid with the DOR mutant [Myc-DOR (M)] of Myc label referring to Whistler, J.L., Tsao, P., and von Zastrow, M. (2001) .A phosphorylation-regulated brakemechanism controls the initial endocytosis of opioid receptors but is not requiredfor post-endocytic sorting to lysosomes.J Biol Chem 276,34331-34338.

Express the structure with the plasmid of the MOR (MOR-Flag) of Flag label: taking HA-MOR plasmid as template, carry out pcr amplification with MOR2 primer in table 1, the amplified production obtaining is inserted in the corresponding site in pEGFP-N3 (Clontech) carrier after HindIII and KpnI enzyme action, obtains the plasmid of expressing with the MOR of Flag label.

Express the structure with the plasmid of the MOR mutant [MOR (M)] of Flag label: taking HA-MOR plasmid as template, first use TM1 (circulation for the first time) and TM3 (circulation for the first time) primer (in table 1) to carry out pcr amplification, TM1 and TM3 (circulation for the second time) primer that amplified production is used of purifying again carries out respectively PCR reaction, again product is reclaimed and purified through HindIII and KpnI enzyme action, connect in pEGFP-N3 (Clontech) carrier.

Express the structure with the plasmid of GFP mark, α-CGRP (1-25)-MOR mutant [MOR (63-93)]: by α-CGRP ^1-25oligonucleotide renaturation, then connects into through EcoRI, in the pEGFP-N3 carrier that BamHI enzyme action is crossed.

Express the structure of the plasmid of MOR TM1-TAT (with GST, FLAG label): first by TM1 oligonucleotide renaturation, connect in the pGEX-KG (GE Healthcare LifeSciences) that EcoRI and SalI enzyme action are crossed, then the coding C end of renaturation is connected in the aforementioned plasmid of crossing through SalI and HindIII enzyme action with the oligonucleotide of the tat peptide of 10 Gly.After expression, can obtain following fusion rotein: DYKDDDDKPSMVTAITIMALYSIVCVVGLFGNFLVMYVIGGGGGGGGGGYGRKKRR QRRR (wherein, tat peptide section is positioned at the carboxyl terminal of TM1, before TM1, with FLAG labelling, between the first cross-film of MOR and tat peptide section, 10 glycine are added).

Express the structure of the plasmid of TAT-MOR TM1 (with GST, FLAG label): first by TM1 oligonucleotide renaturation, connect in the pGEX-KG (GE Healthcare LifeSciences) that EcoRI and SalI enzyme action are crossed, then the C end of renaturation coding is connected in the plasmid just now building of crossing through BamHI and HindIII enzyme action with the oligonucleotide of the tat peptide of 10 Gly.

Express the structure of the plasmid of MOR TM3-TAT (with GST, FLAG label): and the structure of the plasmid of MORTM1-TAT (with GST, FLAG label) is similar, just changes TM1 oligonucleotide into TM3 oligonucleotide.

Express the structure of the plasmid of TAT-MOR TM3 (with GST, FLAG label): and the structure of the plasmid of TAT-MORTM1 (with GST, FLAG label) is similar, just changes TM1 oligonucleotide into TM3 oligonucleotide.

Table 1, plasmid construction primer and oligonucleotide used

Agonist-L-ENK (Sigma), the Delt I of DOR be referring to Wang, H.B., Zhao, B., Zhong, Y.Q., Li, K.C., Li, Z.Y., Wang, Q., Lu, Y.J., Zhang, Z.N., He, S.Q., Zheng, H.C., et al. (2010) .Coexpression of δ-and μ-opioid receptors innociceptive sensory neurons.Proc Natl Acad Sci U S A 107,13117-13122; MOR agonist DAMGO is available from Sigma; The inhibitor naltrindole of DOR is available from Tocris.

Various antibody

HA and Myc antibody are respectively purchased from Roche and Sigma company.

DOR antibody is purchased from Santa Cruz.

MOR antibody is purchased from Dia Sorin.

DOR ^13-17antibody available from Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455, USA.

The biotin labeled method of cell surface

Before 1 micromole Delt stimulation process 30 minutes or afterwards, add biotin and cell to hatch.Use RIPA liquid to blow and beat in cell, cracking one hour in the refrigerator of 4 DEG C.Use the pearl of streptavidin by under biotin labeled albumen collection at the lysate of cell.In order to detect the phosphorylation situation of receptor, cultured cells is used 1 micromolar Delt or DAMGO stimulation within 30 minutes, to carry out cracking afterwards again.All (150mM NaCl, 30mM HEPES, 10mM NaF, the 1%Triton and 0.01%SDS) cracking in the RIPA of ice bath buffer of all cells.After drug treating, first pass through ultrasonic homogenate from the sample of mouse spinal cord, then centrifugal.The sample of handling well carries out electrophoresis at SDS-PAGE, and transferring film adds primary antibodie, then uses enhanced chemiluminescence (ECL; Thermo Scientific) colour developing.Use Flag (1: 1,000, Sigma), Myc (1: 2,000), p-DOR (1: 1000, Neuromics), p-MOR (1: 1000, Neuromics), DOR (1: 3,000, Santa Cruz), MOR (1: 1,000) and the various antibody such as actin (1: 10,000, Santa Cruz).Each experiment at least in triplicate.Result is used the quantitative analysis of Scion Image software.

In situ hybridization

Being used for detecting the probe that mice DOR1 and MOR1 express first uses specific primer separately to increase.The probe of DOR1 is with on digoxigenin labelling, and the probe of MOR1 is to use on fluorescein (fluorescein) labelling.The dorsal root ganglion (DRG) of the L4 of adult mice and L5 (referring to the 4th and the 5th pair of waist section) is processed 30 minutes in 10 μ g/ml protein kinase Ks, and in the hybridization solution of 67 DEG C prehybridization 2.5 hours.Section is afterwards used the probe of 1 μ g/ml DOR1 and 2 μ g/ml MOR1 under the condition of 67 DEG C, to hybridize 18 hours.Using after 1% confining liquid processes, section anti-fluorescein two anti-in 4 DEG C spend the night.Then cut into slices and strengthen in liquid and process 20 minutes at tyramide signal.After signal is enhanced, section is hatched 24 hours in the confining liquid that contains anti-digoxigenin alkali phosphatase and anti-dinitrophenyl (dinitrophenyl) Alexa488 under the condition of 4 DEG C again.After section is used alkali phosphatase buffer solution for cleaning, then add 1 μ l/mlnitroblue tetrazolium and the chloro-3-indole of the bromo-4-of 3.5 μ l/ml5-phosphoric acid (5-bromo-4-chloro-3-indolyl phosphate) to develop the color.

Histogenic immunity group/Immunohistochemistry method

The HEK293 cell of transfection uses the anti-HA in rabbit source (1: 500 at 37 DEG C, or the anti-Myc in mice source (1: 200 Clontech), Developmental Studies Hybridoma Bank) antibody, or LysoTracker Red DND-99 (1: 500, Molecular Probes), hatch in advance 30 minutes.Next cell uses 1 micromolar Delt I or DAMGO to stimulate 30 or 90 minutes.The cell of handling is fixed 15 minutes through 4% paraformaldehyde and 0.2% picric acid, is cleaning after twice, uses two corresponding separately anti-hatching.Neuron is cultivated in the incubator of 37 DEG C, adds the tat peptide albumen of 0.5nM to process after 12 hours, uses the antibody of the anti-GST in rabbit source (1: 1000, Proteintech Group) to hatch 30 minutes in 37 DEG C of water-baths.Be fixed through the neuron of so processing, and add two anti-with fluorescein (fluorescein), then mounting.Picture obtains through Leica SP2 Laser Scanning Confocal Microscope, and carries out quantitative analysis through Scion Image software.

Mice carries out frozen section after 4% paraformaldehyde and 0.2% picric fixative perfusion fixation, selects the tissue of spinal cord L4-5 position and cuts into slices.Use the anti-Mu opiate receptor of Cavia porcellus (1: 400, Neuromics), the anti-Delta opiate receptor in rabbit source (1: 400, Neuromics), the anti-CGRP in mice source (1: 400, Celltech) process, then add accordingly and resist with two of fluorescein or rhodamine or CY5 (1: 100, Jackson ImmunoResearch).Dying lustful section uses Laser Scanning Confocal Microscope to gather picture.Some section is used indirectly Immunoperoxidase histochemical method.

The experiment of mice whipping

All zooperies are all carried out under the specification of the Society for Neuroscience (USA).The male mice growing up was raised under the condition of daily cycle taking 12 hours.All experiments are all carried out by day.Experiment a few days ago, makes mice adapt to, stability fundamental pain threshold in experimental situation.Analgesic effect tests to detect with the mice whipping of 52 DEG C of water-baths.Holding gently mice, mouse tail 1/3 part is vertically immersed in 52 DEG C of hot baths, if mice has obvious whipping reaction, record is from entering the response time of water to whipping, as pain threshold.Screening basic threshold value (Lbase) is that normal reaction mice carries out subsequent experimental the mice of 2-3s left and right, for avoiding mice to be burned, select 10s as ultimate value (cut off), when same mice METHOD FOR CONTINUOUS DETERMINATION, be 15-20s interval time.The terminal that starts most the each use of response time point conduct timing of the quick whipping of mice.The fiducial time that the response time of carrying out recording before drug treating reacts as mice whipping.Delt or morphine are all dissolved in the normal saline of 5 microlitres, use special syringe needle to be expelled in spinal cavity through aperture between the vertebra of waist section.TAT albumen is processed first 2.5 hours at morphine, and 1.5 hours, 0.5 hour through lumbar injection 200 microgram/2 milliliter.The inventor uses 10 seconds maximums as the reaction of mice whipping by the time.Analgesic effect is used formula to calculate: maximum possible effect (MPE)=100 × (record response time-benchmark response time/(the 10-benchmark response time).Data are used the mode of mean value ± SEM to represent.After detecting, the data of each dosage t between group use one-way ANOVA to analyze.

II. embodiment

Background technology

Delta opiate receptor (DOR) and mu opioid receptor (MOR) all belong to the member of g protein coupled receptor superfamily, and they work in the regulation and control of pain.It is generally acknowledged, DOR imports the inhibition effect of the last endogenous opiatepeptide that has slightly mainly mediated dorsal horn neurons secretion in the pain sensation, and MOR is opioid analgesic---the main drug target of morphine.Being formed in g protein coupled receptor superfamily member of receptor oligomeric body is ubiquitous, and early stage pharmaceutical research finds that the ligand molecular of DOR can regulate and control the activity of MOR, implied interrelated between DOR and MOR.Utilize biochemical method, researcher finds not only all to exist in MOR and the cell of DOR cotransfection but also the membrane structure that separates from spinal cord the oligomeric body of the two formation.What is more important, pharmacological method blocking-up or genetic method knock out DOR can both strengthen the analgesia that MOR mediates.But the interaction physically importance in pain sensation regulation and control still needs further to determine between MOR and DOR.In addition, understand the MOR activity inhibition mechanism of DOR agonist induction, can provide a kind of new thinking and method for strengthening the effectiveness taking MOR as target spot analgesics and reducing side effect.

After selective agonist stimulates, g protein coupled receptor is activated and endocytosis, then multiple quick by recirculation approach or enter degradation pathway and cause receptor down-regulated.Research shows, the MOR after endocytosis and DOR experience the different courses of processing.After MOR agonist DAMGO processes, the MOR of endocytosis mainly gets back on cell membrane multiple quick by recirculation approach, and after the processing of the agonist of DOR, the DOR of endocytosis is mainly transported to lysosome degraded.The phosphorylation of the opiate receptor of agonist induction and ubiquitination have play a part important in the endocytosis of receptor and degraded.But former research is all to carry out on the cell of the independent transfection MOR of external source or DOR, so in the transfectional cell of two kinds of receptors of coexpression or neuron the transport of MOR whether to be subject to the regulation and control of DOR not clear.Former research hint is when two kinds of receptors are altogether when endocytosis, approach after their shared identical endocytosis, and this approach is wherein not adopted when a kind of receptor in single expression, therefore the oligomericization of receptor may be one mechanism wherein.

To sum up, also there is many parts of not knowing for interaction and their the endocytosis mechanism in cell of DOR and MOR at present in this area.Therefore, this area is also necessary this further to study, thereby develops for the useful product of the regulation and control pain sensation.

Summary of the invention

The object of the present invention is to provide method and the reagent of the analgesic activity that strengthens opioid analgesic.

In a first aspect of the present invention, provide and suppress delta opiate receptor (DOR) and the purposes of the interactional material of mu opioid receptor (MOR), for the preparation of the preparation of analgesic activity that strengthens opioid analgesic.

In another preference, described inhibition delta opiate receptor and the interactional material of mu opioid receptor are selected from:

The inhibitor of delta opiate receptor;

The first membrane spaning domain albumen (MOR TM1) of mu opioid receptor; Or

C-terminus is connected with the first membrane spaning domain albumen of the mu opioid receptor of wearing film peptide.

In another preference, described to suppressing delta opiate receptor (DOR) and the interactional material of mu opioid receptor (MOR) also for the preparation of the preparation for the treatment of pain.

In another preference, the first membrane spaning domain albumen that described c-terminus is connected with the mu opioid receptor of wearing film peptide is a fusion rotein, the first membrane spaning domain albumen that its aminoterminal is mu opioid receptor, and c-terminus is for wearing film peptide.

In another preference, the first membrane spaning domain albumen that the aminoterminal of described fusion rotein is mu opioid receptor, c-terminus is trans-activator (Transactivator, TAT), Polyarginine, Penetratin, MAP or Transportan.

In another preference, the inhibitor of described delta opiate receptor is selected from: naltrindole, ICI 174,864, TIPP, Naltriben.

In another preference, described opioid analgesic is the analgesic acting on Mu opiate receptor.

In another preference, described opioid analgesic includes but not limited to: opium alkaloid class (as morphine, codeine) or synthetic product (as Pethidine, buprenorphine, etorphine, fentanyl, methadone, dihydroetorphine etc.).

In another preference, described opioid analgesic is morphine.

In another preference, described analgesic activity comprises: the analgesic activity of spinal levels, burning pain, inflammatory pain and machinery pain etc.

In another preference, described pain is: chronic pain, acute pain.

In another preference, described pain is: nociceptor pain, neuropathic pain.

In another preference, described pain includes but not limited to: headache, face pain, cervicodynia, shoulder pain, backache, chest pain, stomachache, back pain, lumbago, leg pain, muscle and skeleton pain, body pain, blood vessel pain, gout, arthritis ache, the pain relevant with somatoform disorders, visceral pain, the pain that infectious disease (as AIDS and postherpetic neuralgia) causes, boniness pain, reaping hook cell anemia, autoimmune disease, the pain of multiple sclerosis or inflammation-related, active chronic inflammation pain, cancerous pain, neuropathic pain, the pain that damage or operation cause, carcinomas pain, nociceptive pain, diabetes, peripheral neurophaty, neuralgia after scar rash, trigeminal neuralgia, waist or cervix uteri radiculopathy, glossopharyngeal neuralgia, autonomic reflex pain, sympathetic reflex dystrophy, nerve root avulsion, cancer, chemical damage, toxin, malnutrition, virus or antibacterial infect, burn or the neuropathic pain of its cooperative programs about causing, the degenerative osteoarthritis that aging causes, or their cooperative programs.

, provide and suppress delta opiate receptor and the interactional albumen of mu opioid receptor on the other hand in the present invention, described albumen is:

The first membrane spaning domain albumen of mu opioid receptor; Or

C-terminus is connected with the first membrane spaning domain albumen of the mu opioid receptor of wearing film peptide.

In another preference, the first membrane spaning domain albumen that described c-terminus is connected with the mu opioid receptor of wearing film peptide is a fusion rotein, the first membrane spaning domain albumen that its aminoterminal is mu opioid receptor, and c-terminus is for wearing film peptide.

In another preference, the described film peptide of wearing is trans-activator, Penetratin, polyarginine, MAP or Transportan.

In another preference, the first membrane spaning domain albumen that described c-terminus is connected with the mu opioid receptor of wearing film peptide is:

The albumen of aminoacid sequence as shown in connection 50-60 position in 9-39 position in SEQ ID NO:4;

The albumen of aminoacid sequence as shown in 9-60 position in SEQ ID NO:4; Or

The albumen of aminoacid sequence as shown in SEQ ID NO:4.

To on the other hand, provide a kind of polynucleotide of separation in the present invention, inhibition delta opiate receptor and the interactional albumen of mu opioid receptor described in described polynucleotide encoding.

To on the other hand, provide the purposes of described polynucleotide in the present invention, for the preparation of the preparation of analgesic activity that strengthens opioid analgesic.

To on the other hand, provide a kind of plasmid vector in the present invention, it contains described polynucleotide.

To on the other hand, provide a kind of genetically engineered host cell in the present invention, it contains described carrier; Or in its genome, be integrated with described polynucleotide.

To on the other hand, provide a kind of described inhibition delta opiate receptor and the preparation method of the interactional albumen of mu opioid receptor in the present invention, the method comprises: (a) under conditions suitable for the expression, cultivate described host cell; (b) from culture, isolate described albumen.

To on the other hand, provide a kind of for analgesic pharmaceutical composition in the present invention, described pharmaceutical composition contains:

(1) material of group under being selected from of effective dose: described albumen or the inhibitor of delta opiate receptor; With

(2) pharmaceutically acceptable carrier.

In another preference, described pharmaceutical composition also contains:

(3) opioid analgesic of effective dose.

In another preference, described opioid analgesic is morphine.

, provide a kind of medicine box is also provided on the other hand in the present invention, in described medicine box, contain described for analgesic pharmaceutical composition.

To on the other hand, provide a kind of method of the potential material that screens the analgesic activity that strengthens opioid analgesic in the present invention, described method comprises:

(1) candidate substances is contacted with the interactional system of delta opiate receptor and mu opioid receptor;

(2) detect candidate substances to delta opiate receptor and the interactional impact of mu opioid receptor;

If described candidate substances can suppress (weaken, block or dissociate) delta opiate receptor and mu opioid receptor interacts, show that this candidate substances is the potential material that strengthens the analgesic activity of opioid analgesic.

In another preference, in test group, candidate substances is joined in delta opiate receptor and the interactional system of mu opioid receptor; And/or

Step (2) comprising: delta opiate receptor and mu opioid receptor interaction situation in the system of detection test group, and with matched group comparison, wherein said matched group is delta opiate receptor and the interactional system of mu opioid receptor of not adding described candidate substances;

If in test group, the interaction of delta opiate receptor and mu opioid receptor is weaker than statistically (be preferably significantly smaller than, as more than 20%, preferably weak more than 50% in weak; Better is weak more than 80%) matched group, just show this candidate substances be strengthen opioid analgesic analgesic activity potential material.

In another preference, described system is selected from: cell system (or cell culture objects system), subcellular fraction system, solution system, organizational framework, organ systems or animal system.

In another preference, described method also comprises: the potential material obtaining is carried out to further cell experiment and/or animal experiment, further to select from candidate substances and to determine for the useful material of analgesic activity that strengthens opioid analgesic.

To on the other hand, provide a kind of method of the analgesic activity that strengthens opioid analgesic in the present invention, described method comprises: the interaction of delta opiate receptor and mu opioid receptor in inhibition object.

To on the other hand, provide a kind of analgesic method in the present invention, described method comprises: the inhibition delta opiate receptor and the interactional material of mu opioid receptor that first need analgesic object effective dose; Need afterwards the opioid analgesic of analgesic object effective dose.

To on the other hand, provide the purposes of wearing film peptide in the present invention, insert with the direction identical with wild type transmembrane protein on cell membrane for the cross-film region (comprising transmembrane protein or its fragment containing this cross-film region) that guides transmembrane protein.

In another preference, the described film peptide of wearing is selected from: trans-activator, Penetratin, polyarginine, MAP or Transportan.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Brief description of the drawings

Fig. 1. the agonist induction DOR of receptor-specific and the common endocytosis of MOR.

(A) aminoterminal is entered in HEK293 cell with the plasmid corotation of the DOR (Myc-DOR) of Myc label with MOR (HA-MOR) and the aminoterminal of HA label.In order to be marked at the receptor on cell membrane, first cultured cells is hatched to half an hour in the mixture with the anti-HA antibody of rabbit and the anti-Myc antibody of mice, after agonist is processed, fixed cell carries out the two marks of immunofluorescence (MOR is for green, and DOR is red).In the Cytoplasm of matched group (control), can only see on little capsule balloon-shaped structure and have positive mark, these are the receptors that continue endocytosis.At 1 μ M Delt I, SNC-80, under the stimulation of Delt II or DAMGO, was originally arranged in the DOR and the MOR endocytosis that on cell membrane, are labeled at living cells state and entered the capsule balloon-shaped structure (shown in arrow) coexisting.The statistics that ratio between total receptor signal of putting on according to the receptor signal of endocytosis and surface markers method is made shows, the DORs being caused by Delt I and DAMGO and the common endocytosis of MORs can be respectively by the NTI of 5 μ M, and naloxone (NLX) or CTOP weaken (figure below).

(B, C) c-terminus is entered in HEK293 cell with MOR (MOR-Flag) and the Myc-DOR plasmid corotation of Flag label, after Delt I processes, the albumen of surface of cell membrane is by biotin institute labelling, and it is collected that the albumen on these labellings is fixed on streptavidin on globule.Process after 30 minutes at 1 μ M Delt I or SNC-80, significantly decline at the DOR of surface of cell membrane and the level of MOR.The experiment of immunoblotting shows, the Delt I of 30 minutes processes the phosphorylation level that can only improve DOR, on the not impact of the phosphorylation level of MOR.And DAMGO can only affect the phosphorylation of MOR.

(D) expressing MOR-Flag and DOR mutant (T352A, T353A, T358A, T361A and S363A) in the HEK293 cell of [Myc-DOR (M)], two kinds of receptors do not change under 1 μ M Delt I and SNC-80 stimulate in the expression of surface of cell membrane.The result of each immunoblotting represents three independently experiments, and the data of statistics all represent with the percent of matched group, and * * represents P < 0.01 compared with not giving cell that Delt I processes.

Fig. 2. sorting approach after the MOR of agonist induction and DOR endocytosis.

(A, B) enters HA-MOR and Myc-DOR corotation in HEK293 cell, carries out preliminary making with anti-HA antibody and anti-Myc antibody, and lysosome structure is used LysoTracker to carry out labelling (redness).Under the stimulation of the Delt I of 90 minutes or DAMGO, fixed cell carries out the two marks of immunofluorescence.In the cell of processing at Delt I, the DOR of endocytosis and MOR enter in the structure of LysoTracker labelling (shown in arrow) jointly, stimulate two receptoroids of endocytosis not by LysoTracker institute labelling and be subject to DAMGO.Scale is 8 μ m.

(C) MOR-Flag and Myc-DOR corotation are entered in HEK293 cell, the experiment of co-immunoprecipitation shows, the stimulation of Delt I can increase the ubiquitination level of DOR and MOR, and DAMGO reduces.The result of each immunoblotting represents three independently experiments.

(D) the HEK293 cell of expressing DOR and MOR before drug treating is by biotin institute labelling, under the processing through 90 minutes 1 μ M Delt I or DAMGO, cell lysis, collected by being fixed on streptavidin on globule by biotin labeled albumen.The experiment of immunoblotting shows, the DOR and the MOR quantity that under DeltI stimulates, are labeled significantly reduce.The Leupeptin+MG132 of 100 μ M can suppress this process.Statistics data all represent with the percent of matched group, * * represent with do not give Delt I process cell compared with P < 0.01.

Fig. 3. activate at mouse spinal cord the analgesic activity that DOR can lower the level of MOR and reduce morphine.

(A) two target in situ hybridizations show, the mRNA of DOR and MOR coexist in mice DRG small neuron (arrow).What small arrow was pointed to is the small neuron of only expressing MOR.Scale is 8 μ m.

(B) use DOR ^13-17the mouse spinal cord tissue sample that knocks out at Oprd1 First Exon of antibody in can't detect the signal of immunoblotting.

(C) the immunostaining signal that uses DOR antibody to detect in the nerve fiber of wild type mouse spinal cord section I and II layer has disappeared in the section of Oprd1 First Exon knock out mice, and this dyeing signal also can be sponged by corresponding antigen simultaneously.

(D) experiment of immunofluorescence three labellings shows, MOR (redness), DOR (green) and CGRP (blueness) coexist at I and the II layer of cornu dorsale medullae spinalis.The figure of magnification at high multiple shows that three coexists at the whole end of nerve fiber.Scale is 8 μ m.

(E) experiment of co-immunoprecipitation shows, the interaction after the Delt I of intrathecal injection 2 μ g is 15 minutes between DOR and MOR significantly strengthens.The result of each immunoblotting represents three independently experiments.

(F) in the mouse spinal cord tissue sample that the antibody of the DOR using knocks out at Oprd1 First Exon, also can't detect the signal of immunoblotting in co-immunoprecipitation experiment.

(G) use the Delt I of 5 μ g or L-ENK and SNC-80 to stimulate after 15 minutes, can make the MOR ubiquitination level of cornu dorsale medullae spinalis significantly improve.The result of each immunoblotting represents three independently experiments.

(H) mice is after intrathecal injection (5x1 μ g, every 15 minutes 1 time) Delt I processes, and the dyeing that immunofluorescence label is presented at mouse spinal cord I and II layer MOR obviously reduces.Carry out quantitative analysis according to 3 mices and every mice 3-5 section, * * represents P < 0.01 compared with giving the mice of saline group.

52 DEG C of water-bath whipping experiments of (I, J, K) mice show in sheath, give in advance Delt I and can significantly reduce the analgesic activity that gives morphine in sheath, and this effect to have dose dependent.The antagonist NTI of DOR, the effectiveness (J) that can stop DOR agonist Delt I to reduce the analgesic activity of morphine.SNC-80 also has the effect same with Delt I (J).* represents P < 0.01 compared with giving the mice of saline group (every group of 10 mices).

First of Fig. 4 .MOR worn membrane structure territory and mediated the interaction of MOR and DOR.

(A) illustrate the structure (wherein grey area segment table shows membrane spaning domain) of various MOR or variant, comprising: wild type MOR; Replace the MOR mutant [MOR (M)] of the first membrane spaning domain with the 3rd membrane spaning domain of MOR; Containing the MOR mutant [MOR (63-93)] of TM1.

(B, C) co-immunoprecipitation experiment shows, DOR can with MOR on first wear membrane structure territory interact, DOR only exists and interacts with the MOR of wild type, and and mutant between not do not interact, the overexpression of first membrane spaning domain of MOR can significantly reduce the interaction between DOR and MOR.

(D) at corotation in the cell of DOR and MOR mutant, 1 μ M Delt I can reduce the expression of DOR on cell membrane, and the mutant of MOR is not acted on.The result of each immunoblotting represents three independently experiments.

(E) turn three in the cell of (simultaneously proceeding to HA-MOR, Myc-DOR, MOR (63-93)), stimulate the DOR and the common endocytosis of MOR that cause to be stoped by first membrane spaning domain of the MOR of overexpression by agonist.The cell number of every group of statistics is 30, and * * represents P < 0.01 compared with the cell of first membrane spaning domain of corotation MOR not.Scale is 4 μ m.

(F) immunohistochemical experiment, first membrane spaning domain of the MOR of overexpression can disturb by Delt I or make the receptor that DAMGO causes be total to endocytosis.

Fig. 5. interrupt the interaction between DOR and MOR with TM1-TAT fusion rotein.

(A) four kinds of fusion rotein that contain TM1 and TM3 of diagram, TAT is at aminoterminal or the c-terminus of TM1 and TM3.The mice dorsal root ganglion neurons of cultivating is after the processing of TAT fusion rotein, after permeable membrane, GST antibody labeling cell shows that all TAT fusion rotein are all mainly positioned on cell membrane, with the non-antibody labeling that penetrates film method GST is the GST albumen being exposed to outside cell membrane, show to only have tat peptide to be inserted into direction on neuronal cell film at the fusion rotein of the c-terminus of cross-film section consistent with wild type MOR, the opposite direction that tat peptide inserts at the aminoterminal of cross-film section.Scale is 8 μ m.Anti-GST represents anti-GST antibody.

(B) mice immune-gold labeled-Yin before the processing of TM1-TAT albumen is carried out embedding in 2.5 hours later strengthens labelling.Under light microscopic, TM1-TAT albumen is mainly distributed in the whole end of nerve fiber of cornu dorsale medullae spinalis shallow-layer, and Electronic Speculum Image Display TM1-TAT albumen is mainly distributed on the film at the whole end of nerve fiber of spinal cord synaptic glomerulus.What arrow represented is postsynaptic region.

(C, D) experiment of co-immunoprecipitation shows, after the TM1-TAT albumen of 2.5 hours are processed, between DOR and MOR, the interaction of foundation level significantly reduces, and makes the increase of the ubiquitination level of MOR also can return to foundation level by intrathecal injection 5 μ g Delt I.In statistics, add the co-immunoprecipitation signal of α 2A-AR and NK1-R and MOR, the in the situation that of the processing of MOR TM1-TAT albumen and unaffected this result.The result of each immunoblotting represents three independently experiments.

Fig. 6. can improve the analgesic effect of morphine with TM1-TAT fusion rotein.

(A) 52 DEG C of water-bath whipping experiments of mice show, the analgesic effect of subcutaneous injection morphine can be by lumbar injection TM1-TAT albumen (in 2.5h/day, 10mg/kg/ is each or 5mg/kg/ is each, inject altogether three times) significantly strengthen, injection TAT-TM1 and TM3-TAT do not affect, and this effect also has dose dependent.In addition, the analgesic effect of intrathecal injection Delt I 2.5 μ g/kg can not be strengthened (B) by lumbar injection TM1-TAT albumen.(C) at the morphine of mouse subcutaneous injection 5mg/kg, at 5 days, the analgesic effect of morphine disappeared completely later, showed the generation of morphine effect.And if every day, mice all first passed through the processing (in 2.5h/day, 10mg/kg/ is each, injects altogether three times) of TM1-TAT before morphine in injection, the tolerance situation of morphine can be eased.And under similarity condition, use TM3-TAT that mice is processed the tolerance effect of morphine is not affected.

Detailed description of the invention

The inventor is through research widely, find that the interaction between delta opiate receptor (DOR) and mu opioid receptor (MOR) is directly related for the sensitivity of opioid analgesic with MOR, the interaction of blocking-up or reduction DOR and MOR can strengthen the analgesic activity of opioid analgesic.The inventor has also found the reagent that can strengthen the analgesic activity of opioid analgesic on this basis.

As used herein, have comprised " containing ", " having " or " comprising " " comprising ", " mainly by ... form ", " substantially by ... form " and " by ... form "; " mainly by ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".

As used herein, " separation " refers to that material separates (if crude, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.

As used herein, term " pain " or " bitterly " refer to the offending reaction of one that body produces damaged tissue or potential damage, are a kind of Physiological Psychology activities of complexity, are also one of modal symptoms clinically.Pain can be divided into body source property (somatogenic) pain and psychogenic (psychogenic) pain.Body source property pain can be divided again susceptibility pain (as nociceptive pain, nociceptive pain) and neuropathic pain (Neuropathic Pain).Pain can be divided into according to its generation and lasting time and process: acute pain and chronic pain, chronic pain also comprises chronic susceptibility pain or chronic neuropathic pain model.

As used herein, " neuropathic pain " is to cause due to the damage of maincenter or peripheral nervous system or pathological change, include but not limited to lumbago and backache (lower back pain), neuralgia (neuralgia), fibromyalgia (Fibromyalgia), neuralgia (the diabetic neuropathic pain that diabetes are relevant, DNP), the neuralgia (pain associated with multiple sclerosis) that multiple sclerosis is relevant, postherpetic neuralgia (PHN) and HIV related (neurogenic) neuralgia (HIVNP) etc.

As used herein, " nociceptor pain " is to cause because the nociceptor in human body is subject to machinery, heat, chemical stimulation or damage, body nociceptor pain and internal organs nociceptor pain be can be divided into, cancer pain (cancer-related pain), arthritis pain (arthritic pain), postoperative pain (post-operative pain) and HIV related (nociceptor) neuralgia (HIVNP) etc. included but not limited to.Wherein, neuralgia (neuralgia) includes but not limited to trigeminal neuralgia (Trigeminal neuralgia), sciatica (sciatic neuralgia) etc.

Pain can be divided into according to its generation and lasting time and process: acute pain and chronic pain.

Acute pain is a group offending impression, consciousness and affectional experience with the rapid complexity occurring of the obvious stimulation factors such as damage or various emergency cases, with autonomous, psychology and reaction behavior, it comprises pain, burn and various Medicine and Surgery emergency case after operation, after wound, for example: heart infarction, acute pancreatitis, biliary colic, renal colic, acute appendicitis, pathologisch Bruch, there is acute pain etc. in acute ileus patient.The Therapeutic Method of acute pain comprises: controlled analgesia (PCA) is carried out in ward, by local anaesthetics and/or opiates medicine in epidural space infusion etc.In mechanism or animal experiment, the normal models such as formaldehyde method, hot plate method or the method for adopting causes the acute pain of animal, and gives immediately medicine to be measured, to observe the mitigation of medicine to be measured to acute pain.

Chronic pain refers to that pain continues the long period (as exceed several weeks～1 month), exceed the progress of general acute disease, or exceed the reasonable time of wound healing, or, or the interval time recurrent pain of process several months or several years relevant with the chronic pathology process that causes constant pain.In mechanism or animal experiment, often adopt the inductions such as complete Freund's adjuvant to cause the chronic pain of animal, and give medicine to be measured after induction a few days and even several weeks, to observe the mitigation of medicine to be measured to chronic pain.

The course of disease of chronic pain is long, the cause of disease is complicated; The different structure of maincenter and peripheral nervous system and function abnormal; The unify variation of emotion of autonomic nervous system; And social adaptation, life are or/and the reduction of ability to work; The feature of spirit, the many-sided mental maladjustment of family, has determined the significance of rehabilitation means for treatment of chronic pain.

Chronic pain is not alleviated, can be psychologically, cause harmful effect to patient on physiology; The pain that can not alleviate, by postponing patient's recovery, extends the date of being in hospital, and increases burden to patient and family members, strengthens the cost of medical insurance, makes it forfeiture work, family, dignity, causes depression, anxiety, suicide, and the maimed colony of psychology is expanded.Moreover, also increased social factor leading to social instability.Comparatively speaking, it is generally acknowledged now that acute pain is a symptom of disease, and chronic pain is a class disease.

Chronic pain includes but not limited to: 1. soft tissue, joint and bone pain: pain after deformity pain, skeletal muscle pain, lumbago, myofasical pain syndrome, headache, burn after various osteoarthritis, wound.2. deep tissue and Encelialgia: cardiovascular pain, ophthalmalgia, mouthful face ache, chronic gynecological pain, sexual anhedonia, genitourinary system chronic pain.3. neural and nerve root injury pain: phantom limb pain after amputation, peripheral nerve pain, sympathetic reflex dystrophy and sympathetic nerve lasting pain, trigeminal neuralgia and atypical face pain, nerve root injury and arachnoiditis, postherpetic neuralgia.4. central pain: the blood vessel injury of brain, spinal cord, as hemorrhage, infraction, vascular malformation; Multiple sclerosis; Traumatic spinal cord injury, brain injury; Syringomyelia and syringobulbia; Tumor; Parkinson disease.5. children with pain: growing pain.6. old people's pain: various cervical spondylosiss, prolapse of lumbar intervertebral disc, lumbar spondylolisthesis etc. are pain caused.7. cancerous pain.

" bitterly " described in the present invention includes but not limited to: headache, face pain, cervicodynia, shoulder pain, backache, chest pain, stomachache, back pain, lumbago, leg pain, muscle and skeleton pain, body pain, blood vessel pain, gout, arthritis ache, the pain relevant with somatoform disorders, visceral pain, the pain that infectious disease (as AIDS and postherpetic neuralgia) causes, boniness pain, reaping hook cell anemia, autoimmune disease, the pain of multiple sclerosis or inflammation-related, active chronic inflammation pain, cancerous pain, neuropathic pain, the pain that damage or operation cause, carcinomas pain, nociceptive pain, diabetes, peripheral neurophaty, neuralgia after scar rash, trigeminal neuralgia, waist or cervix uteri radiculopathy, glossopharyngeal neuralgia, autonomic reflex pain, sympathetic reflex dystrophy, nerve root avulsion, cancer, chemical damage, toxin, malnutrition, virus or antibacterial infect, burn or the neuropathic pain of its cooperative programs about causing, the degenerative osteoarthritis that aging causes, or their cooperative programs.

DOR, MOR and fragment thereof

DOR and MOR are well known to those skilled in the art, and they all belong to the member of g protein coupled receptor superfamily, in the regulation and control of pain, work.Wherein, MOR is the main drug target of opioid analgesic (as morphine).Usually, people's DOR albumen has the sequence shown in SEQ ID NO:2 or referring to the sequence shown in GenBank accession number AAA18789.2, its coded sequence is referring to the sequence shown in GenBank accession number NM_000911.3.Usually, people's MOR albumen has the sequence shown in SEQ ID NO:1 or referring to the sequence shown in GenBank accession number AAH74927.1, its coded sequence is referring to the sequence shown in GenBank accession number AY521028.1.Replacement, disappearance or the interpolation of the one or more amino acid residues of process of DOR or MOR and the variant form that forms are also included within the present invention.Its variant form comprises the alternative sequence of a part of conserved amino acid, and the described sequence through amino acid substitution does not affect its activity or retained the activity of its part.Suitably replacing aminoacid is technology well known in the art, and described technology can be implemented easily, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, substantially can not change biological activity at the inessential area change single amino acids of a peptide species.See the Molecular Biology of The Gene such as Watson, the 4th edition, 1987, The Benjamin/Cummings Pub.Co.P224.

The present invention also provides the bioactive fragment of a kind of MOR of deriving from, and it is the first membrane spaning domain protein fragments (MOR TM1) of MOR.The inventor's research finds, activate DOR and can make MOR in DOR-MOR complex enter into degradation pathway after endocytosis together with DOR, thereby caused MOR with respect to its agonist as the desensitization of morphine.These processes depend on MOR wears membrane structure territory and DOR interaction by its first.The MOR TM1 protein fragments of expressing can weaken the interaction of DOR and MOR competitively.Therefore, described MOR TM1 protein fragments can promote opioid analgesic as the analgesic effect of morphine by weakening the interaction of DOR and MOR.

The sequence of the first membrane spaning domain protein fragments of described MOR is as shown in SEQ ID NO:3.

In addition, the invention still further relates to and derive from protein fragments MOR albumen, that there is sequence shown in SEQ ID NO:3.Also including aminoacid sequence shown in SEQ ID NO:3 and one or both ends is still available with other amino acid whose MOR protein fragments, as long as it also has the function of competitive binding DOR.

Replacement, disappearance or the interpolation of the one or more amino acid residues of process of MOR TM1 and the variant form that forms are also included within the present invention.Its variant form comprises the alternative sequence of a part of conserved amino acid, and the described sequence through amino acid substitution does not affect its activity or retained the activity of its part.Suitably replacing aminoacid is technology well known in the art, and described technology can be implemented easily, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, substantially can not change biological activity at the inessential area change single amino acids of a peptide species.See the MolecularBiology of The Gene such as Watson, the 4th edition, 1987, The Beniamin/Cummings Pub.Co.P224.

Once separate the sequence that has obtained described MOR TM1 albumen or its variant, just can obtain in large quantity this albumen with recombination method.This is normally cloned into carrier by its encoding gene, then proceeds to cell, is then separated and obtains from the host cell propagation by conventional method.In addition, for the albumen compared with short, also can adopt the method for synthetic (as synthesized by Peptide synthesizer) to synthesize relevant sequence, the method for synthetic can obtain needed albumen easy and rapidly.

The present invention has also comprised the nucleic acid of the separation of MOR TM1 albumen that coding is described or its variant, and it can complete sequence synthetic, also can obtain by the method for pcr amplification.After the nucleotide sequence described in having obtained coding, be connected into suitable expression vector, then proceeded to suitable host cell.Host cell after finally transforming by cultivation, obtains desired albumen by separation and purification.

The present invention has also comprised the carrier of the nucleic acid molecules that comprises the described MOR TM1 albumen of coding or its variant.Described carrier also can comprise the expression regulation sequence being connected with the series of operations of described nucleic acid molecules, so that the expression of albumen.Described " operability is connected " or " being operationally connected in " refer to so a kind of situation, and some part of linear DNA sequence can regulate or control the activity of same linear DNA sequence other parts.For example, if the transcribing of promoter control sequence, it is exactly to be operationally connected in coded sequence so.In addition the reconstitution cell that, contains the described MOR TM1 albumen of coding or its variant nucleotide sequence is also included within the present invention.

Fusion rotein

The present invention also provides a kind of fusion rotein, and it comprises successively from aminoterminal to c-terminus: MOR TM1 and wear film peptide.As used herein, described " wearing film peptide " refers to that a class has the polypeptide of cell-penetrating effect, and the fusion rotein of himself or itself and other albumen can enter in cell by cell membrane.

As optimal way of the present invention, the described film peptide of wearing is trans-activator (Transactivator, TAT).The inventor finds, after TAT protein fusion TM1, can make allogenic polypeptide TM1 optionally be inserted on cell membrane, body inject this fusion rotein can suppress spinal cord competitively in the interaction of MOR-DOR, thereby strengthen the analgesic effect of morphine.

As optimal way of the present invention, the aminoacid sequence of described TAT can be substantially the same with the sequence shown in 50-60 position in SEQ ID NO:4.

Described MOR TM1 and wear between film peptide and can directly be connected, or connect by polypeptide connexon (connection peptides).Connexon itself do not have connect beyond with other functions, therefore under technical field personnel understand possible aminoacid composition.Described connexon comprises 0-20 aminoacid; Be preferably 0-15 aminoacid; As 10.Described connection peptides is for example made up of 10 Gly.

Fusion rotein at body injection MOR TM1 and TAT can be transported to the end that the mouse spinal cord dorsal horn pain sensation is imported into, the interaction of blocking-up DOR-MOR, thus promote morphine analgesia effect.

The present invention also finds in the time that TAT merges the c-terminus at TM1 and TM3, immune labeled test shows that TM1 and TM3 can be similar to the such insertion of wild type on cell membrane, can't cause the cellular uptake of these transmembrane proteins, therefore, tat peptide not only can be used as a carrier of wearing film, and because this peptide section of existence of hydrophobic cross-film section can also guide the direction of insertion of transmembrane protein on cell membrane.

Embodiment 1, under the activation of agonist DOR and MOR endocytosis jointly

In order to understand whether the transhipment of MOR is subject to the adjusting of DOR, the inventor in HEK293 cell (purchased from ATCC) corotation with the MOR of HA label protein with the DOR of Myc label protein, under specific agonist stimulates separately, DOR or MOR can endocytosis.Because HA or Myc labelling are connected on the aminoterminal of receptor, outside Cell-oriented, can use HA and Myc antibody, at the state of living cells, the receptor on labeled cell film is fixed cell after agonist stimulates, add corresponding two to resist, can detect receptor and be subject to the post-stimulatory heavy distribution situation of agonist.Under normal circumstances, DOR and MOR are mainly distributed on cell membrane.1 μ M DOR agonist Delt I, under the stimulation of Delt II or SNC-80, the DOR being labeled on cell membrane and MOR be endocytosis jointly, coexists in same capsule balloon-shaped structure.In independent transfection in the cell of MOR, Delt I can not cause the endocytosis of MOR on film, shows that the endocytosis of the MOR that DeltI causes needs the co expression of DOR.When under the stimulation of 1 μ M MOR agonist DAMGO, also endocytosis jointly of two kinds of receptors of corotation, and this common endocytosis can be respectively by the NTI of 5 μ M, naloxone (NLX) or CTOP weaken.See Figure 1A.

The inventor has further confirmed by the biotin labeled method of cell surface the result obtaining by immunohistochemical experiment.After the stimulation of agonist, rest on the receptor of cell surface of transfection by biotin institute labelling, and collected by being fixed on streptavidin on globule, the stimulation of Delt I and SNC-80 can make to be positioned at the DOR of cell surface and the level of MOR reduces greatly, sees Figure 1B.These results show, the cell of two kinds of receptor co expression is under the specific agonist of opiate receptor stimulates, and DOR and MOR be endocytosis jointly.

The phosphorylation of receptor plays an important role in the endocytosis process of opiate receptor.The inventor finds that the phosphorylation of receptor participates in the receptor endocytosis process that agonist causes.The experiment of immunoblotting shows, the phosphorylation of DOR and MOR background level is very low, and the stimulation of Delt I can increase the phosphorylation level of DOR specifically, and the stimulation of DAMGO can improve the phosphorylation level of MOR specifically, sees Fig. 1 C.

The inventor is further by corotation MOR with there are five phosphorylation sites sudden change (T352A, T353A, T358A, the calculating in T361A and S363A site is based on SEQ ID NO:2) DOR mutant DOR (M), verified the importance of phosphorylation in two receptor endocytosis processes.This mutant can not be phosphorylated after being upset.At corotation in the cell line of two kinds of receptors, under the stimulation of Delt I and SNC-80, DOR (M) and MOR all can not be by endocytosis, see Fig. 1 D.The above results shows that the DOR activating is essential for the common endocytosis of MOR and DOR.

The MOR of embodiment 2, activation guiding surface of cell membrane by DOR enters degradation pathway

In order to determine these last situations of the MOR of endocytosis together with DOR, activate DOR guiding MOR and enter together with DOR in lysosome structure.Use the method for immune three labellings, the inventor finds that Delt I can induce DOR and MOR to enter into the lysosome structure with LysoTracker labelling, but DAMGO stimulate can not, see Fig. 2 A-B.

The experiment of co-immunoprecipitation shows, in 293 cell lines of co expression DOR and MOR, DeltI processes the level that improves DOR and MOR ubiquitination, and DAMGO does not affect the level of DOR and MOR ubiquitination, sees Fig. 2 C.

Whether the inventor further uses biochemical method, detect by the receptor of endocytosis and really degraded.Before making treated with medicaments cell, first use biotin labeling living cells surface.Use again Delt I or DAMGO irritation cell 90min, by lysis, and collect by biotin labeled albumen, found that Delt I can significantly reduce the acceptor quantity on biotin labeling, but stimulating, DAMGO can not, protease inhibitor Leupeptin in lysosome and MG132 can block the receptor degraded that Delt I causes completely, see Fig. 2 D.More than experiment shows, the DOR of common endocytosis and MOR enter into lysosome and degrade.

Embodiment 3, activate DOR in spinal levels and cause the downward of MOR and reduce the analgesic effect of morphine

Two target in situ hybridizations show, the mRNA of DOR and MOR coexists (arrow) in mice DRG small neuron, sees Fig. 3 A.

And use DOR ^13-17mice (purchased from JacksonLaboratory) the myeloid tissue sample that knocks out at Oprd1 First Exon of antibody in can't detect the signal of immunoblotting, this has proved the specificity of this antibody using, and sees Fig. 3 B.

The immunostaining signal that uses DOR antibody to detect in the nerve fiber of wild type mouse spinal cord section I and II layer has disappeared in the section of Oprd1 First Exon knock out mice, and this dyeing signal also can be sponged by corresponding antigen simultaneously, this has just verified the specificity problem of the antibody staining using in experiment, sees Fig. 3 C.

DOR and MOR have expression in the dorsal root ganglion small neuron of expressing neuropeptide substance p and CGRP, and these neuronic centripetal fibers end at I and the II layer of spinal cord.Three labelling experiment of immunostaining show, DOR and MOR coexist with CGRP in the centripetal fiber of mouse spinal cord and whole end, see Fig. 3 D.

The experiment of co-immunoprecipitation shows, the interaction of DOR and MOR still exists in spinal levels, and this acts under the stimulation of Delt I and can be enhanced, and sees Fig. 3 E.

Simultaneously the inventor has also verified the signal that also can't detect immunoblotting in co-immunoprecipitation experiment in the mouse spinal cord tissue sample that the antibody of the DOR using knocks out at Oprd1 First Exon, Fig. 3 F.

In addition, agonist-L-ENK (2 times, 5 μ g/ time, 15 minutes, interval) intrathecal injection of 5 μ g Delt I (2 times, 5 μ g/ time, 15 minutes, interval) or the endogenic DOR of 5 μ g can significantly improve the ubiquitination level of MOR, sees Fig. 3 G.

Consistent with biochemical test, the experiment of SABC also shows, when intrathecal injection Delt I (2 μ g/15 minute), the MOR immunostaining of spinal cord I and II layer significantly weakens, and sees Fig. 3 H.These results all show, the DOR activating in the afferent sensory fiber of cornu dorsale medullae spinalis causes the expression of MOR to be lowered.

The inventor further tests discovery, and the activation of DOR can reduce the analgesic activity of morphine in spinal levels.The whipping experiment that mice is 52 DEG C shows, 30min before intrathecal morphine, intrathecal injection Delt I in advance, the analgesic activity of morphine will decline greatly, and the effect of Delt I has dose dependent, and this similar effect also can be by injecting L-ENK in sheath and SNC-80 obtains in advance, NTI and Delt I share the inhibition that can block the effect of Delt I On Morphine Analgesia, see Fig. 3 I, J, K and table 2.The inhibitor naltrindole of intrathecal injection DOR can improve the analgesic activity of morphine in advance, in table 3.

Table 2, before morphine is processed, (1.5 μ g, i.t.) 30 minutes mices are accepted Delt I or L-ENK and SNC-80 and process (i.t.) and can weaken significantly the spinal cord analgesic activity that morphine causes

Note: compared with accepting normal saline pretreated group before giving morphine, ^*p < 0.05, ^*p < 0.01 and ^{* *}p < 0.001.

The processing (NTI, i.t.) of table 3, DOR antagonist naltrindole strengthens the spinal cord analgesic activity that morphine causes

Note: compared with accepting normal saline pretreated group before giving morphine, ^*p < 0.05 He ^{* *}p < 0.001.

Above-mentioned a series of experiment shows, causes the decline of the analgesic activity of common mediation in the downward of experiencing the MOR of DOR mediation in centripetal fiber.

First on embodiment 4, MOR worn membrane structure territory and mediated the interaction between MOR and DOR

In order to evaluate more all sidedly the interaction between DOR and MOR, the effect in the negative regulation of MOR activity, the inventor seeks the region of Physical interaction between DOR and MOR.By computer Simulation calculation, first on prediction MOR worn the interface that membrane structure territory is most possible and DOR combination.

In the plasmid that the inventor builds, first of MOR worn to membrane structure territory (TM1, be positioned at the amino acid whose 63-93 of MOR position, on MOR albumen, sequence location is counted based on sequence shown in SEQ ID NO:1) the signal peptide of N termination the preceding paragraph CGRP, so that wearing membrane structure territory albumen, first that is fused to green fluorescent protein GFP can correctly be inserted on cell membrane, this plasmid enters in HEK293 cell with myc-DOR plasmid corotation, interaction between research MOR and DOR, is shown in Fig. 4 A.The experiment of co-immunoprecipitation shows, what this segment table reached wear, and membrane structure territory can interact with the DOR of total length, further experiment confirms, if the section of the cross-film for the third time (TM3 by first membrane spaning domain on MOR with it, be positioned at the amino acid whose 144-163 of MOR position) replace this mutant, i.e. Fig. 4 A, MOR (M), and the interaction of DOR weakens greatly.If first of expressing again MOR simultaneously at co expression DOR and MOR worn the peptide section in membrane structure territory, interaction between two receptors is also significantly weakened, this shows that first overexpression of wearing membrane structure territory can disturb the interaction between DOR and MOR, sees Fig. 4 B and Fig. 4 C.Above result shows, first of MOR worn membrane structure territory and really mediated the interaction of itself and DOR.

Use the mutant of MOR and first peptide section of wearing membrane structure territory of MOR as instrument, the inventor has further verified interaction between DOR and the MOR importance for two kinds of common endocytosis of opiate receptor.See Fig. 4 D, the processing of Delt I (1 μ M) can make DOR endocytosis, and do not affect (Fig. 4 E) to expressing on the film of MOR mutant, the inefficacy of this common endocytosis, be not because the transhipment of mutant itself is out of joint, because DAMGO (1 μ M) processes the mutant endocytosis that can make MOR.

The inventor is further by the experiment discovery of SABC, and first membrane spaning domain of the MOR of overexpression can disturb by Delt I or make the receptor that DAMGO causes be total to endocytosis, sees Fig. 4 F.

This series of experiment shows, first membrane spaning domain of MOR can separate DOR with MOR, thereby affects the common endocytosis of receptor.

The correct insertion of the membrane spaning domain of embodiment 5, tat peptide section guiding MOR on cell membrane

The physiological significance of evaluating the oligomeric body of receptor just needs one to interrupt physically interactional method of g protein coupled receptor.Albumen and tat peptide section can be merged to reach the object of carrying in albuminous cell, tat peptide is one section of transduction peptide section (sequence is YGRKKRRQRRR) that has 11 acidic amino acids, and it derives from the mankind's immunodeficiency poisonous carrier.The experiment in people early stage shows, the peptide section of this section short, as a kind of carrier of penetration cell, can be brought into little material or large protein molecular in cell.

First the inventor is connected on tat peptide section the c-terminus of the TM1 of MOR, merges GST and Flag label protein at the aminoterminal of TM1-TAT polypeptide, can use the antibody of anti-GST and Flag to detect.In order to detect the location of TM1-TAT fusion rotein in cell, the inventor cultivates the dorsal root ganglion neurons of mice, then add with the fusion rotein of TAT and process, use the antibody of GST to carry out the dialytic or non-dialytic labelling of hatching.Found that, TAT albumen is distributed on the cell membrane of dorsal root ganglion small neuron (91%) mostly, also has small part to be positioned, in the structure of small neuron kytoplasm vesicle shape, to see Fig. 5 A.Meanwhile, the inventor also finds, the ratio that dorsal root ganglion large neuron is labeled is much smaller, only has 43%.Therefore, these TAT fusion rotein can be inserted on the cell membrane of small neuron very efficiently, and this phenomenon may be the common formation of the stagnation on film due to the film ability of wearing of tat peptide and the hydrophobicity of cross-film section.

Meanwhile, the inventor also finds that the insertion of TM1 albumen on cell membrane that TAT merges has directivity.The direction of insertion on cell membrane is identical with the direction of insertion of cross-film section in MOR at the fusion rotein of TM1 c-terminus for TAT, can see by the method for the non-permeable membrane viable cell labelling of GST antibody, TAT is obvious at the GST labelling of the TM1 of c-terminus albumen, illustrates that GST is mainly positioned at outside cell membrane; And form with it sharp contrast, and the GST labelling that TAT is positioned at N-terminal TM1 albumen is not strong, illustrates that GST is mainly in cell, and in the direction that this albumen inserts in cell membrane and MOR, the direction of insertion of TM1 is contrary.On the tat peptide fusion rotein of the 3rd membrane spaning domain (TM3) of MOR, also find same distribution situation, seen Fig. 5 A.

Therefore, tat peptide not only can be used as a carrier of wearing film, and because this peptide section of existence of hydrophobic cross-film section can also guide the direction of insertion of transmembrane protein on cell membrane.

Embodiment 6, injection TM1-TAT albumen can strengthen the analgesic effect of morphine

The inventor has further checked TM1-TAT albumen (TAT is at TM1 c-terminus) whether can interrupt the interaction between DOR and MOR at cornu dorsale medullae spinalis.Experiment confirms in lumbar injection TM1-TAT albumen (3 times, 10mg/kg/ time) (this albumen is dissolved in conventional GST albumen purification eluent, 0.1 milligram every milliliter of concentration) expression of albumen after 2.5 hours, can be detected in spinal levels, the experiment that immune-gold labeled before embedding-Yin strengthens labelling is presented at cornu dorsale medullae spinalis I and II layer elementaryly feels to import into TM1-TAT in tip and is positioned on cell membrane, 68.8 ± 7.9% marking particle is positioned on the film of experiencing the whole end of centripetal fiber of mouse spinal cord I and II layer, sees Fig. 5 B.This results suggest, the TAT fusion rotein giving in mice can be transported to cornu dorsale medullae spinalis, and is inserted on the film at the whole end of afferent sensory fiber.Process and within first 2.5 hours, give the TM1-TAT albumen enhancing spinal cord analgesic activity that morphine causes at morphine, in table 4.

Table 4, within first 2.5 hours, give TM1-TAT (i.p.3 time) in morphine processing (2mg/kg, s.c.) and strengthen the spinal cord analgesic activity that causes of morphine

Note: compared with accepting normal saline pretreated group before giving morphine, ^*p < 0.05 He ^*p < 0.01.

Meanwhile, the inventor finds to give TM1-TAT albumen and can reduce the inhibition of the On Morphine Analgesia effect of DOR mediation.The experiment of co-immunoprecipitation confirms, at lumbar injection TM1-TAT albumen after 2.5 hours, interaction between DOR and MOR has significantly reduced, and same processing also can make the increase of the spinal cord MOR ubiquitination due to intrathecal injection DeltI stimulation return to foundation level, sees Fig. 5 C-D.These results all show, TM1-TAT can interrupt the interaction between DOR and MOR after on the film that is inserted into sensation Afferent Terminals.

The experiment of the further behavioristics of the inventor is found, about 2.5 hours lumbar injection TM1-TAT albumen before giving injected in mice morphine, can significantly strengthen the analgesic effect of subcutaneous injection morphine, its reinforced effects reaches more than three times, the analgesic activity of DOR mediation is at all unaffected, sees Fig. 6 B and table 5.And the effect that TAT-TM1 albumen causes has specificity, because TAT merges and is connected on TM3 c-terminus or N-terminal fusion rotein at aminoterminal, the TAT of TM1 and all can not affects the inhibitory action of the On Morphine Analgesia of DOR mediation, see Fig. 6 A.These results suggest, the function of spinal levels MOR is just subject to the inhibition of DOR under normal circumstances, and the analgesic activity of morphine can interact and be enhanced by interrupting between DOR and MOR.The inventor also finds the processing (in 2.5h/ days, 10mg/kg/ is each, injects altogether three times) of TM1-TAT simultaneously, can alleviate 1 of morphine effect and produce.And use TM3-TAT to process mice under similarity condition, the tolerance effect of morphine is not affected Fig. 6 C and table 6.

Table 5, process (2.5 μ g, i.t.) front 2.5h give TM1-TAT albumen (10mg/kg, i.p.) at Delt I, the spinal cord analgesic activity of DOR mediation is unaffected

Table 6, morphine process before 2.5h (5mg/kg, i.p.) give TM1-TAT albumen (10mg/kg, i.p.), can alleviate the generation of morphine effect

Note: compared with accepting normal saline (control) pretreated group before giving morphine, ^*p < 0.05, ^*p < 0.01and ^{* *}p < 0.001.

Embodiment 8. screening of medicaments

The corotation building using previous embodiment 1 with the MOR of HA label protein and with the HEK293 cell of the DOR of Myc label protein as the cell model for screening.

Test group: with the culture of the above-mentioned cell of candidate substances processing;

Matched group: without the culture of the above-mentioned cell of candidate substances processing.

By the interaction situation of co-immunoprecipitation method identification of M OR and DOR, if compared with matched group, in test group, the interaction of MOR and DOR weakens (weak 50% or more than), show that candidate substances is to suppress the interactional material of MOR and DOR, it is useful for the analgesic activity that strengthens opioid analgesic.

Particularly, the TM1-TAT albumen that the inventor utilizes previous embodiment 5 to prepare, and TM3-TAT albumen is as candidate substances, found that, TM1-TAT albumen can suppress the interaction of MOR and DOR significantly; And TM3-TAT effect is not obvious.Therefore visible TM1-TAT albumen is useful for the analgesic activity that strengthens opioid analgesic.

Discuss

The inventor's research shows, in DOR-MOR complex, the activation of DOR can cause the endocytosis of two kinds of opiate receptors on cell membrane, and the opiate receptor of common endocytosis enters into degradation pathway.First of MOR worn membrane structure territory and mediated the interaction between DOR and MOR, tat peptide section is merged at first of MOR and wears the c-terminus in membrane structure territory, the direction of insertion of fusion rotein in cell is identical with first direction of wearing membrane structure territory in MOR overall molecule, give mice TM1-TAT fusion rotein and can interrupt the interaction between spinal levels DOR and MOR, thus the analgesic effect of enhancing morphine.These results show, the interaction that interrupts physical property between DOR and MOR can be used as a kind of strategy of analgesic activity of the MOR of raising mediation.

Known in the time of single expression DOR and MOR, two kinds be sorted in different transporting pathway by cognition.The DOR of endocytosis often enters in lysosome and degrades, and gets back to sensitization again on cell membrane and the MOR of endocytosis enters into Regeneration Ways.The inventor's experiment shows, in the time of two kinds of receptor coexpressions, optionally MOR or DOR agonist can make the common endocytosis of two receptoroids, and are sorted to identical approach.This effect is subject to the impact of different biochemical processes, opiate receptor optionally agonist causes the phosphorylation of corresponding receptor, thatly once appeared between MOR and somatostatin receptor or Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 receptor across the phosphorylation modification between receptor, but do not occur between DOR and MOR.Further experiment shows, the stimulation of DOR selective agonist can cause the ubiquitination of two receptoroids, and DAMGO has reduced the foundation level of two kinds of receptor ubiquitinations, this discovery also with the existing cognitive consonance of people, the endocytosis of receptor can mode dependent by ubiquitin or independent form carry out.

Constantly the experimental data of accumulation shows that DOR has multiple action in pain sensation regulation and control.The agonist of DOR only has very weak or relatively mild analgesic activity, but DOR shows the negative regulation effect to MOR.On the cell membrane causing based on acceptor interaction, the common minimizing of DOR and MOR number may be a kind of important mechanisms of neuron to opioid peptide sensitivity that regulate.In dorsal root ganglion small neuron, MOR is distributed on cell membrane, and DOR is mainly arranged in the large compactness vesicle that kytoplasm contains neuropeptide, the processing of DOR agonist can make intracellular DOR be inserted on cell membrane, causes the ubiquitination level of MOR in cornu dorsale medullae spinalis nerve fiber to increase and the reduction of receptor number.Therefore, due to the upper film of DOR in large compactness vesicle, the interaction between DOR and MOR significantly increases, and this process may be a kind of inherent mechanism to MOR activity inhibition in spinal levels DOR mediation.

Suppress the active of DOR or knock out DOR gene all can strengthen the effect of morphine with medicine.In this article, the inventor interrupts the interaction between DOR and MOR by a kind of method, detects the impact of On Morphine Analgesia effect.The TM1-TAT of lumbar injection can be transported in spinal cord, and is inserted on the after birth at the whole end of afferent sensory fiber, weakens the interaction in DOR and MOR foundation level, and the analgesic effect of morphine in spinal levels significantly strengthened.These find prompting, the DOR that is arranged in DOR-MOR complex is just activated by endogenic opioid peptide in the time of foundation level, as the endorphins (enkephalin) that can be discharged by dorsal horn neurons under in pain stimulus, the release of these peptide classes can make just to make under normal circumstances DOR to produce inhibitory action to the function of MOR.Before morphine is processed, give TM1-TAT albumen, can be competitively in conjunction with the DOR on cell membrane that is inserted into due to being processed by pain stimulus or morphine, thereby reduce DOR and the MOR combination at surface of cell membrane.

Have in the present invention an important discovery, tat peptide can be served as a guiding composition in TM1-TAT albumen, and the TM1 polypeptide of guiding external source is inserted on cell membrane by correct direction, and this is extremely important to TM1 functionating.This method can provide a kind of new means to the inventor, in body situation, analyzing the impact on neuron activity by interrupting between two class g protein coupled receptors the interaction of physical property on cell membrane, and the normal function major part of each autoreceptor is not being exerted an influence simultaneously.In order to design the molecular probe that can effectively interrupt acceptor interaction, first the inventor must confirm to interact and form the molecule interface of heteromultimers between G protein coupling receptor.The inventor's experiment shows, what mediate acceptor interaction by confirmation wears membrane structure territory, and tat peptide is connected to and wears the aminoterminal in membrane structure territory or carboxyl and bring in to control and wear the direction of insertion of membrane structure territory on cell membrane.The physical property interactional method of this interference g protein coupled receptor with high selectivity on cell membrane, for the function that the inventor studies acceptor interaction provides a kind of strong instrument, is not only also the New Policy that a kind of medicine is interfered simultaneously.

All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (11)

1. suppress the purposes of delta opiate receptor and the interactional material of mu opioid receptor, for the preparation of the preparation of the analgesic activity of enhancing opioid analgesic; Described inhibition delta opiate receptor and the interactional material of mu opioid receptor are selected from:

The inhibitor of delta opiate receptor, is selected from: naltrindole, ICI 174,864, TIPP or Naltriben;

The first membrane spaning domain albumen of mu opioid receptor; Or

C-terminus is connected with the first membrane spaning domain albumen of the mu opioid receptor of wearing film peptide; The described film peptide of wearing is trans-activator;

Wherein, described, the aminoacid sequence of the first membrane spaning domain albumen of mu opioid receptor is as shown in SEQ ID NO:3.

2. purposes as claimed in claim 1, is characterized in that, described opioid analgesic is the analgesic acting on Mu opiate receptor.

3. purposes as claimed in claim 1, is characterized in that, described opioid analgesic is morphine.

4. suppress delta opiate receptor and the interactional albumen of mu opioid receptor, described albumen is:

The first membrane spaning domain albumen of mu opioid receptor; Or

C-terminus is connected with the first membrane spaning domain albumen of the mu opioid receptor of wearing film peptide; The described film peptide of wearing is trans-activator;

Wherein, described, the aminoacid sequence of the first membrane spaning domain albumen of mu opioid receptor is as shown in SEQ ID NO:3.

5. albumen as claimed in claim 4, is characterized in that, described albumen is:

The albumen of aminoacid sequence as shown in connection 50-60 position in 9-39 position in SEQ ID NO:4;

The albumen of aminoacid sequence as shown in 9-60 position in SEQ ID NO:4; Or

The albumen of aminoacid sequence as shown in SEQ ID NO:4.

6. polynucleotide for separation, inhibition delta opiate receptor and the interactional albumen of mu opioid receptor described in described polynucleotide encoding claim 4 or 5.

7. the purposes of polynucleotide claimed in claim 6, for the preparation of the preparation of analgesic activity that strengthens opioid analgesic.

8. a plasmid vector, it contains the polynucleotide described in claim 6 or 7.

9. a genetically engineered host cell, it contains carrier claimed in claim 8; Or in its genome, be integrated with the polynucleotide described in claim 6 or 7.

10. for an analgesic pharmaceutical composition, it is characterized in that, described pharmaceutical composition contains:

(1) be selected from the material of lower group: the albumen described in claim 4 or 5 or the inhibitor of delta opiate receptor; The inhibitor of described delta opiate receptor is selected from: naltrindole, ICI 174,864, TIPP or Naltriben and

(2) pharmaceutically acceptable carrier.

11. pharmaceutical compositions as claimed in claim 10, is characterized in that, described pharmaceutical composition also contains:

(3) opioid analgesic.