CN102276731B - High-motion-activity polypeptide capable of inhibiting cocaine induction and application thereof - Google Patents
High-motion-activity polypeptide capable of inhibiting cocaine induction and application thereof Download PDFInfo
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Abstract
The invention discloses a high-motion-activity polypeptide capable of inhibiting cocaine induction and application thereof. The polypeptide disclosed by the invention is a polypeptide of (a), (b), (c) or (d) shown in the specification, wherein the (a) is a polypeptide consisting of an amino acid sequence shown in a sequence 2 in a sequence table; the (b) is a polypeptide consisting of an amino acid sequence shown as 412th to 425th amino acid residues from 5' terminal of a sequence 1 in the sequence table; the (c) is a polypeptide consisting of an amino acid sequence shown as 329th to 446th amino acid residues from the 5' terminal of the sequence 1 in the sequence table; and the (d) is a polypeptide derived from (a), (b) or (c) defined polypeptide with the same function in an amino acid sequence of the (a), (b) or (c) defined polypeptide through substitution and/or deletion and/or addition of one or more amino acid residues. Experiments of the invention prove that: the polypeptide disclosed by the invention has magnificent meaning in reducing the behavioral sensitization of a human body to cocaine stimulation.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of polypeptide and application thereof that suppresses the high locomotor activity that Cocaine brings out.
Background technology
Cocaine is to suck at present very general a kind of habituation mental excitation medicine.Cocaine habituation also is that human health and social stability are had the disease of serious harm.The Cocaine acute irritation can the induced movement activity increase.This mainly is because Cocaine can suppress the DAT of presynaptic membrane, thereby the reuptake of blocking-up Dopamine HCL causes cynapse position dopamine concentration to increase.The degree of Cocaine induced movement activity then can reflect body for the study of behaviour susceptibility of Cocaine, so for research cocaine habituation molecular mechanism and treatment thereof some enlightenment is provided.
Dopamine HCL stimulates, and can activate the Dopamine Receptors of postsynaptic membrane.Dopamine Receptors belongs to typical g protein coupled receptor, has typical seven cross-film structures.Dopamine Receptors mainly contains 5 kinds of hypotypes, be D1, D2, D3, D4 and D5 acceptor, according to the G albumen type of its coupling and for the effect of adenylate cyclase, can be divided into D1 sample acceptor, comprise D1 and D5 acceptor, and the D2 sample acceptor that comprises D2, D3 and D4 acceptor.D1 sample acceptor and excitability G α
sAlbumen coupling, activated adenyl cyclase; D2 sample acceptor is then opposite, with inhibition G α
iAlbumen coupling, the activity of inhibition adenylate cyclase.
Dopamine system mainly concentrates on the Basal ganglia position for the adjusting of motion.The extrapyramidal system motion is subject to the neural loop regulation and control of basal ganglion, and black substance-striatum DA system is wherein important neural link, and striatum is the regulation and control centres.Basal ganglion includes directly and indirect two kinds of neural loops.Directly loop is by pallium → striatum → black substance webbed region (SNR) → thalamus → pallium; Indirect loop: pallium → striatum → pallidum LHA (GPe) → end thalamus (STN) → pallidum medial area (GPi) → thalamus → pallium.Under the physiological conditions, inhibited one's impulses by black substance webbed region and pallidum medial area output tonus, act on thalamus.The time phase heat, activate direct loop, thalamus is disinthibited, it is movable to strengthen thalamus-cortical neuron; And activate indirect loop, it is movable further to suppress thalamus-cortical neuron.Consequently, directly loop can strengthen motor function, and loop suppresses motor function indirectly.They are subject to black substance-striatum DA system regulation, the direct loop transfer of D1 acceptor facilitation, and the D2 acceptor weakens indirect loop transfer.Although synaptic function is not identical, DA is consistent to two loop produce an effects, weakens the inhibition of thalamus-cortical neuron, the motor function that facilitation starts from cortex.And existing research proof, the Cocaine sensitization phenomenon mainly tends to activate the D1 acceptor of direct loop.
The research evidence shows, activates the D1 acceptor and can strengthen the locomotor activity that Cocaine brings out.Cocaine stimulates, and the dopamine level at cynapse position increases, and then can activate the D1 acceptor in the nigrostriatum neurone.By the cAMP/PKA signal path, the D1 acceptor can strengthen these neurones for the excitability of L-glutamic acid, thereby the signal of nigrostriatum direct path spreads out of and the locomotor activity that brings out.Otherwise the mouse that the D1 receptor knockout removes weakens even disappears for the locomotor activity intensified response of Cocaine.
Summary of the invention
The purpose of this invention is to provide a kind of polypeptide and application thereof that Cocaine brings out high locomotor activity that suppress.
Polypeptide provided by the invention, for as follows a) or b) c) or d):
A), the polypeptide that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
B), the polypeptide that is formed from the aminoacid sequence shown in 5 ' the terminal 412-425 amino acids residue by sequence in the sequence table 1;
C), the polypeptide that is formed from the aminoacid sequence shown in 5 ' the terminal 329-446 amino acids residue by sequence in the sequence table 1;
D), with a) or b) or the aminoacid sequence of the polypeptide that c) limits through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have having a) respectively or b of identical function) or the polypeptide that c) the limits polypeptide of deriving.
The replacement of described one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
Sequence 2 in the sequence table is comprised of 23 amino-acid residues, and the sequence 1 in the sequence table is comprised of 446 amino-acid residues.
The encoding gene of described polypeptide also is the scope of protection of the invention.
Described gene is following 1)-5) in any one dna molecular:
1) dna molecular shown in the sequence 5 in the sequence table;
2) in the sequence table sequence 6 from 5 ' terminal 1234-1275 position Nucleotide;
3) in the sequence table sequence 6 from 5 ' terminal 985-1338 position Nucleotide;
4) under stringent condition with 1) or 2) or 3) dna molecular with polypeptide of identical function of the dna sequence dna hybridization that limits and coding;
5) with 1) or 2) or 3) dna sequence dna that limits has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least a dna molecular that 99% homology and coding have the polypeptide of identical function.
Described stringent condition is: at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of lower hybridization, then use 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Sequence 5 in the sequence table is comprised of 72 Nucleotide, and the sequence 6 in the sequence table is comprised of 1341 Nucleotide.
Recombinant vectors, recombinant bacterium, expression cassette or the transgenic cell line that contains described encoding gene also is the scope of protection of the invention.
The application that described polypeptide, described encoding gene, described recombinant vectors, recombinant bacterium, expression cassette or transgenic cell tie up in the high locomotor activity medicine of the preparation inhibition drug-induced human or animal of habituation also is the scope of protection of the invention.
Described dependence producing drug is Cocaine, and described animal is rat, and described high locomotor activity embodies by improving motion distance value.
The high locomotor activity of the drug-induced animal of described inhibition habituation is by following 1)-3) at least a realization:
1) phosphorylation of inhibition I type Dopamine Receptors;
2) cytoplasmic membrane that suppresses I type Dopamine Receptors is located;
3) phosphorylation of the downstream signaling molecule of inhibition I type Dopamine Receptors;
Described I type Dopamine Receptors aminoacid sequence be sequence 1 in the sequence table;
Described downstream signaling molecule is ERK (downstream signaling molecule of I type Dopamine Receptors) and/or CREB (transcription factor cyclisation AMP response element binding protein (cAMP response elements binding protein, hereinafter to be referred as CREB)), the aminoacid sequence of described ERK is the sequence 7 in the sequence table; The aminoacid sequence of described CREB is the sequence 8 in the sequence table.
The phosphorylation of described inhibition I type Dopamine Receptors is that the phosphorylation by the 421st Serine of described polypeptide competitive inhibition I type Dopamine Receptors realizes.
The 421st Serine of described I type Dopamine Receptors is sequence table sequence 1 from 5 ' terminal the 412nd amino acids or sequence table sequence 2 from 5 ' terminal the 19th amino acids.
Another object of the present invention provides a kind of medicine that suppresses the high locomotor activity of human or animal that Cocaine brings out.
Medicine provided by the invention, its active ingredient are described polypeptide, described encoding gene, described recombinant vectors, recombinant bacterium, expression cassette or transgenic cell line.
Of the present invention experimental results show that, the present invention finds I type Dopamine Receptors (D1 dopamine receptor, hereinafter to be referred as D1R, amino acid residue sequence is sequence 1) can be by protein kinase D1 (protein kinase D1, hereinafter to be referred as PKD1) phosphorylation, because 229 Serines of ring have sported L-Ala in the three categories of overseas Chinese in the human sequence, can not be phosphorylated, therefore only considered 421 Serines of C-terminal.D1R-CT (D1 acceptor C-terminal)-S421 is according near the amino acid residue sequence 421 Serines, comprised the core sequence that the phosphorylation Preference order of PKD1 is chosen, sequence accordingly makes up and has the TAT fusogenic peptide Tat-D1R-CT-S421 that wears the film ability.The experiment of vitro kinase phosphorylation shows that Tat-D1R-CT-S421 can disturb PKD1 to the phosphorylation of D1R-CT, makes its phosphorylation degree be down to 53.39 ± 5.62% (P＜0.05) of control group.The injection experiment of dorsal part striate nucleus group shows that Tat-D1R-CT-S421 makes PKD1 be down to 45.98 ± 17.69% (P＜0.05) of control group to the phosphorylation degree of D1R-CT.The active study of behaviour experiment of Cocaine induced movement shows, Tat-D1R-CT-S421A compares with control group, 20-50min after the Cocaine injection, the locomotor activity amplitude that Tat-D1R-CT-S421 can make Cocaine bring out weakens 55.46 ± 12.50% (P＜0.05) to control group.And this study of behaviour restraining effect is to transduce to realize by the cytoplasmic membrane location and the downstream signal that suppress D1R.The experiment of SK-N-MC clone shows, Tat-D1R-CT-S421 can significantly reduce the cytoplasmic membrane location of D1R and the phosphorylation degree of downstream signaling molecule extracellular signal-regulated kinase (extracellular signal-regulated kinase is hereinafter to be referred as ERK).The injection experiment of dorsal part striate nucleus group shows, Tat-D1R-CT-S421 can make the cytoplasmic membrane location of D1R be down to 64.80 ± 12.95% of control group, and the phosphorylation degree of its downstream signaling molecule ERK and transcription factor cyclisation AMP response element binding protein (cAMP response elements binding protein is hereinafter to be referred as CREB) and the protein level of c fos immediate early gene significantly reduce.
Above result shows that Tat-D1R-CT-S421 is by the phosphorylation degree of interference PKD1 for 421 Serines of D1R C-terminal, and then the cytoplasmic membrane location of inhibition D1R and downstream signal transduction, then suppresses the high locomotor activity that Cocaine brings out.Therefore, polypeptide of the present invention and encoding gene thereof have broad application prospects aspect the high locomotor activity that Cocaine brings out suppressing.
Abbreviation: D1 Dopamine Receptors (D1R); Cocaine (cocaine); Extracellular signal-regulated kinase (extracellular signal-regulated kinase, ERK); Phosphorylation ERK (phosphorylated ERK, pERK); Cyclisation AMP response element binding protein (cyclic AMP response element binding protein, CREB); Phosphorylation CREB (phosphorylated CREB, pCREB); C fos immediate early gene; Dopamine HCL (dopamine, DA).
Description of drawings
Fig. 1 is that PKD1 is to the evaluation of D1R phosphorylation site
Fig. 2 is that Tat-D1R-CT-S421 is on the impact of 421 Serines of PKD1 phosphorylation D1R C-terminal
Fig. 3 is that Tat-D1R-CT-S421 is on the impact of 421 serine phosphorylations of D1R-CT
Fig. 4 is that Tat-D1R-CT-S421 is on the impact of Cocaine induced movement activity
Fig. 5 is that Tat-D1R-CT-S421 weakens D1R in the location of cytoplasmic membrane
Fig. 6 is that Tat-D1R-CT-S421 suppresses the transduction of D1R downstream signal
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The evaluation of the terminal concrete phosphorylation site of embodiment 1, PKD1 (protein kinase D1) phosphorylation D1RC
One, is connected with the preparation of the albumen of GST label
446 amino acid of I type Dopamine Receptors (hereinafter to be referred as D1R) total length, belong to typical g protein coupled receptor, have seven times classical cross-film structures, therefore with the segmentation of D1 acceptor, be defined as respectively N-terminal, the first membrane spaning domain, ring in the first born of the same parents, the second membrane spaning domain, first born of the same parents' outer shroud, the 3rd membrane spaning domain, ring in the second born of the same parents, the 4th membrane spaning domain, second born of the same parents' outer shroud, the 5th membrane spaning domain, ring in the three categories of overseas Chinese, the 6th membrane spaning domain, three categories of overseas Chinese's outer shroud, the seven-transmembrane structural domain, (Fig. 1 a) for C-terminal.
The aminoacid sequence of I type Dopamine Receptors is the sequence 1 in the sequence table, and its nucleotides sequence is classified sequence 6 as.
Use following peptide section: D1R-CT-WT (shown in the sequence 1 in the sequence table from 5 ' terminal 329-446 amino acids in the present embodiment, the D1R albumen n end plays 329-446 amino acids peptide section), D1R-CT-S421A (with the 421st the peptide section that mutant serine is L-Ala of D1R-CT-WT), the irrelevant sudden change of D1R-CT-is (with the peptide section after the sudden change of the amino-acid residue beyond the D1R-CT421 position, sequence 4), D1R-IL3-WT (sequence 1 in the sequence table is from 5 ' terminal 212-268 amino acids), D1R-IL3-S229A (be L-Ala with the 229th mutant serine of D1R-IL3-WT), D1R-IL3-S256A (be L-Ala with the 256th mutant serine of D1R-IL3-WT), D1R-IL3-S229A+S256A (be L-Ala with the 229th Serine and the 256th Serine simultaneous mutation of D1R-IL3-WT) adds the GST label during experiment before each peptide section.
Being prepared as follows of several albumen with the GST label: make up prokaryotic expression carrier and then be prepared.
GST-tag cellular lysate liquid: 10mM PMSF/1 * PI/1 * PBS;
GST-tag elution buffer:10mM reduced glutathion (Roche), 50mM Tris-Cl (pH8.0)
Table 1, primer are to sequence table
Be connected with respectively the preparation of the albumen of GST label:
1) amplification of the encoding gene of each albumen: cDNA (extracts RNA with male SD rat (Department Of Medicine, Peking University's Laboratory Animal Science section) striatum (concrete extracting method as described later), reverse transcription obtains cDNA) be template, respectively take sequence shown in the table 1 as primer, carry out pcr amplification, obtain respectively the PCR product of following polypeptide fragment, the PCR product is carried out sequence verification, and the result product sequence is correct:
The nucleotides sequence of D1R-CT-WT is classified in the sequence table sequence 6 as from 5 ' terminal 985-1338 position Nucleotide; Aminoacid sequence is that sequence 1 is from 5 ' terminal 329-446 amino acids in the sequence table, and the primer of employing is that primer in the table 1 is to 2.
The nucleotides sequence of D1R-IL3-WT is classified in the sequence table sequence 6 as from 5 ' terminal 634-804 position Nucleotide; Aminoacid sequence is that sequence 1 is from 5 ' terminal 212-268 amino acids in the sequence table, and the primer of employing is that primer in the table 1 is to 1.
2) structure of point mutation plasmid vector
Adopt Stratagene QuikchangeR XL site-directed mutagenesis kit, USA, CAT# 200517-5 carries out point mutation, and concrete steps are as follows:
The pcr amplification reaction system:
5×PrimeSTAR buffer 10μl
dNTP mix 2μl
D1R-CT-WT/D1R-IL3-WT 100ng
DMSO 2.5μl
5’primer(100ng/μl) 1.25μl
3’primer(100ng/μl) 1.25μl
PrimeSTAR HS polymerase 1μl
Add ddH
2O to 50 μ l
Reaction conditions:
Obtain 4 kinds of PCR products:
The primer that D1R-CT-S421A adopts is that primer is to 3 in the table 1, and template is D1R-CT-WT;
The primer that D1R-IL3-S229A adopts is that primer is to 4 in the table 1, and template is D1R-IL3-WT;
The primer that D1R-IL3-S256A adopts is that primer is to 5 in the table 1, and template is D1R-IL3-WT;
The primer that D1R-IL3-S229A+S256A adopts be in the table 1 primer to 4 and primer to 5, template is D1R-IL3-WT.
With above-mentioned 4 kinds of PCR products, digestion.In above-mentioned PCR reaction solution, add 1 μ l DpnI, flick mixing, 37 ℃ of 1-2h; Conversion, bed board, picking mono-clonal, order-checking identify that the result is:
The aminoacid sequence of D1R-CT-S421A is L-Ala for the 421st mutant serine of aminoacid sequence with D1R-CT-WT, and the primer of employing is that primer is to 3 in the table 1, and template is D1R-CT-WT;
The aminoacid sequence of D1R-IL3-S229A is L-Ala for the 229th mutant serine of aminoacid sequence with D1R-CT-WT, and the primer of employing is that primer is to 4 in the table 1, and template is D1R-IL3-WT;
The aminoacid sequence of D1R-IL3-S256A is L-Ala for the 256th mutant serine of aminoacid sequence with D1R-IL3-WT, and the primer of employing is that primer is to 5 in the table 1, and template is D1R-IL3-WT;
The aminoacid sequence of D1R-IL3-S229A+S256A is for being L-Ala with the 229th Serine of aminoacid sequence and the 256th Serine simultaneous mutation of D1R-IL3-WT, the primer of employing be in the table 1 primer to 4 and primer to 5, template is D1R-IL3-WT.
The other aminoacid sequence sequence 4 of the PCR product among the acquisition-individual clone also, the irrelevant sudden change of called after GST-D1R-CT-(unrelated mutation).
But above-mentioned sequence is synthetic all.
3) contain the preparation of the recombinant bacterium of " encoding gene of each peptide section "
The PCR product D 1R-CT-WT of each above-mentioned peptide species, D1R-IL3-WT, D1R-CT-S421A, D1R-IL3-S229A, D1R-IL3-S256A, D1R-IL3-S229A+S256A, the irrelevant sudden change of GST-D1R-CT-is inserted PGEX-5X-1 carrier (Clontech company through BamHI and XhoI double digestion respectively, Cat# 27-4584-01), the recombinant vectors pGEX-5X-1-D1R-CT that is contained respectively " encoding gene that is connected with each peptide section of GST label ", pGEX-5X-1-D1R-IL3, pGEX-5X-1-D1R-S421A, pGEX-5X-1-D1R-S229A, pGEX-5X-1-D1R-S256A, pGEX-5X-1-D1R-S229A+S256A, the irrelevant sudden change of pGEX-5X-1-GST-D1R-CT-; Respectively recombinant vectors is imported BL21 (DE3) bacterium, the recombinant bacterium that is contained respectively " encoding gene that is connected with each peptide section of GST label ": BL21 (DE3)/pGEX-5X-1-D1R-CT, BL21 (DE3)/pGEX-5X-1-D1R-IL3, BL21 (DE3)/pGEX-5X-1-D1R-S421A, BL21 (DE3)/pGEX-5X-1-D1R-S229A, BL21 (DE3)/pGEX-5X-1-D1R-S256A, BL21 (DE3)/pGEX-5X-1-D1R-S229A+S256A, the irrelevant sudden change of BL21 (DE3)/pGEX-5X-1-GST-D1R-CT-confirms that through order-checking Insert Fragment is correct.
Empty carrier pGEX-5X-1 is changed in BL21 (DE3) bacterium, obtain BL21 (DE3)/pGEX-5X-1, through order-checking, do not contain sequence 6.
4) fermentation recombinant bacterium
Preparation is connected with each peptide section of GST label and is connected with the pearl of this albumen: the recombinant bacterium (BL21 (DE3)/pGEX-5X-1-D1R-CT that gets respectively various above-mentioned containing " encoding gene that is connected with each peptide section of GST label ", BL21 (DE3)/pGEX-5X-1-D1R-IL3, BL21 (DE3)/pGEX-5X-1-D1R-S421A, BL21 (DE3)/pGEX-5X-1-D1R-S229A, BL21 (DE3)/pGEX-5X-1-D1R-S256A), BL21 (DE3)/pGEX-5X-1-D1R-S229A+S256A, the irrelevant recombinant bacterium BL21 (DE3) that suddenlys change and contain empty carrier of BL21 (DE3)/pGEX-5X-1-GST-D1R-CT-/pGEX-5X-1 shakes bacterium and spends the night; Get above-mentioned bacterium liquid and add in the fresh culture in the ratio of finally inducing volume 1/10,37 ℃ of 250rpm shake bacterium 3h; Adding final concentration is that 0.5mM IPTG induces, and 30 ℃ of 250rpm continue to shake bacterium 2h; 4 ℃ 3,000rpm * 15min is centrifugal, abandons supernatant liquor, collects bacterial sediment; Every milliliter of bacterium liquid adds 100 μ l (GST-tag cellular lysate liquid: the resuspended thalline of lysate of 10mM PMSF/1 * PI/1 * PBS), ultrasonication cell 40W 6s * 3 times; Add 10%Triton X-100, making its final concentration is 1%, 4 ℃ of suspendible 1h; 4 ℃ 12,000rpm * 20min is centrifugal, gets supernatant liquor; Add 15-20 μ l pearl (Glutathione Sepharose in the supernatant liquor
TM4fast flow, Amersham Pharmacia, Cat# 17-5132-01), 4 ℃ of suspendible 2h; 1 * PBS cleans pearl 3 times, and 4 ℃ 2,000rpm * 2min is centrifugal, removes non-specific binding, and is for subsequent use.
Obtain respectively being connected with Protein G ST-D1R-CT-WT, Protein G ST-D1R-IL3-WT, Protein G ST-D1R-CT-S421A, Protein G ST-D1R-IL3-S229A, Protein G ST-D1R-IL3-S256A, Protein G ST-D1R-IL3-S229A+S256A, the pearl of the irrelevant sudden change of protein D 1R-CT-, the pearl of Protein G ST.
Two, vitro kinase phosphorylation experiment
With the pearl that is connected with Protein G ST of purifying, the pearl of Protein G ST-D1R-CT-WT, the pearl (WT) of Protein G ST-D1R-IL3-WT, add GST label protein elution buffer, 4 ℃ of wash-outs spend the night.Second day with its 2,000rpm * 2min is centrifugal, draws supernatant liquor, tries not to be drawn onto pearl.Draw the part supernatant liquor, carry out the SDS-PAGE electrophoresis, determine expressing quantity and kinases system application of sample amount.The protein content of draw determining, and to be diluted to volume with GST label protein elution buffer be 5 μ l, join contain 25-30 μ l contain γ-
32P-ATP (Beijing Fu Rui bio-engineering corporation, Cat# PN101,5 μ Ci/ samples), DTT (the prosperous bio tech ltd of Beijing ancient cooking vessel state, Cat# 08910202), purifying PKD1 albumen (upstate company, Cat#14-508) kinase reaction damping fluid (Tris-HCl (pH7.6) 30mM, MgCl
210mM, DTT 0.1mM, [γ-
32P] ATP 10 μ Ci/50 μ l) among, 30 ℃ of water-bath 30min.After the water-bath, place immediately 5min on ice, then 2,000rpm * 2min is centrifugal.Add an amount of 6 * SDS-PAGE electrophoresis sample-loading buffer, the SDS-PAGE electrophoresis; When treating electrophoresis forward position indicator to the gel bottom, stop electrophoresis is removed concentrated glue, and separation gel is put into gel-colored liquid (methyl alcohol 500mL, Glacial acetic acid 100ml, Coomassie blue R-250 2.5g, ddH
2O 400ml) in, room temperature on horizontal shaking table (25 ℃) dyeing 40-60min; Gel-colored liquid is reclaimed, be changed to destainer (methyl alcohol 500ml, Glacial acetic acid 100ml, ddH
2O 400ml), continue room temperature decolouring on horizontal shaking table, every 15min changes destainer one time, decolour clear to the target protein band till; Destainer is reclaimed, and (10% dehydrated alcohol, 4% glycerine is used ddH to be changed to dried glue
2O is settled to 100ml), continue the dried glue 20min of room temperature on horizontal shaking table, and simultaneously dried glued membrane is infiltrated in dried glue; Gel is taken out from dried glue, with dried glued membrane that its two sides parcel is smooth, wherein cannot entrained air bubbles, and be fixed, under the room temperature more than the air-dry 8h; The related dried glued membrane of gel is taken off, be attached in the magazine, and in the darkroom, cover the X-ray sensitive film thereon, be placed on-80 ℃ of refrigerators to suitable time, afterwards radioautograph in the darkroom.
3 repetitions are established in experiment.The result can find out shown in Fig. 1 b, Fig. 1 c, and GST empty carrier albumen can not be by the PKD1 phosphorylation, and GST-D1R-IL3-WT and GST-D1R-CT-WT all can be by the PKD1 phosphorylations.
Three, the related locus with D1R-IL3 and D1R-CT suddenlys change, and carries out the experiment of vitro kinase phosphorylation again
Experimental technique is identical with method in above-mentioned two, and different is, and used pearl is as follows: the pearl that is connected with GST albumen, GST-D1R-IL3-WT (WT), GST-D1R-IL3-S229A (S229A), GST-D1R-IL3-S256A (S256A), GST-D1R-IL3-S229A+256A (S229A+256A), GST-D1R-CT-WT (S229A+256A), the irrelevant sudden change of GST-D1R-CT-S421A, D1R-CT-.
3 repetitions are established in experiment.The result shows, GST-D1R-IL3-WT, GST-D1R-IL3-S256A all can be by the PKD1 phosphorylations, and GST, GST-D1R-IL3-S229A, GST-D1R-IL3-S229A+S256A all can not be by the PKD1 phosphorylation, the phosphorylation site (Fig. 1 c) that 229 Serines that D1R-IL3 is described are PKD1, but this site has sported L-Ala (Fig. 2) in the human sequence, so ensuing experiment is not considered; The irrelevant sudden change of GST-D1R-CT-WT, GST-D1R-CT-can be by the PKD1 phosphorylation, GST, GST-D1R-CT-S421A then can not be by PKD1 phosphorylation (Fig. 1 b), 421 phosphorylation sites that Serine is PKD1 of D1R-CT are described, and this site is conservative.
Comprehensive two, three result shows 421 Serines that PKD1 can phosphorylation D1R C-terminal.
The application of the high locomotor activity that the acquisition of embodiment 2, Tat-D1R-CT-S421 and inhibition Cocaine thereof bring out
One, the acquisition of Tat-D1R-CT-S421
It is one of cross-film delivery vector that TAT (transcriptional activator protein) wears the film peptide, can be with the polypeptide covalently bound with it, protein and DNA equimolecular cross-film transfered cell, even can pass through hemato encephalic barrier, transduction efficiency is high and to the cell not damaged, is used widely in fields such as cytobiology, gene therapeutics and pharmacy.According to 421 Serines of phosphorylation site of PKD1 among the D1R-CT, choose and comprise PKD1 phosphorylation Preference order at 14 interior amino-acid residues, make up and have the fusogenic peptide of wearing the film ability---Tat-D1R-CT-S421.Be L-Ala with 421 mutant serines in the target sequence, namely Tat-D1R-CT-S421A in contrast.
The aminoacid sequence of Tat-D1R-CT-S421 is the sequence 2 in the sequence table, worn film sequence (RKKRRQRRR) and comprised 421 Serines by TAT and form at 14 interior amino-acid residues (sequence 1 in the sequence table is from N-terminal 412-425 amino acids), its nucleotides sequence is classified sequence 5 as.Tat-D1R-CT-S421 is synthetic by the biochemical (Shanghai) Co., Ltd. of gill, and its molecular weight is 2815.44.(Fig. 3 a)
The aminoacid sequence of Tat-D1R-CT-S421A is that biochemical (Shanghai) Co., Ltd. is synthetic by gill shown in the sequence 3 (the 19th mutant serine that is about to sequence 2 in the sequence table is L-Ala) in the sequence table, and its molecular weight is 2799.44.(Fig. 3 a)
Two, Tat-D1R-CT-S421A suppresses the application of the high locomotor activity that Cocaine brings out
1, the experiment of vitro kinase phosphorylation detects Tat-D1R-CT-S421 to the impact of PKD1 phosphorylation D1R-CT
Vitro kinase phosphorylation experimental procedure is identical with method among the embodiment 1, and different is, has added concentration and be Tat-D1R-CT-S421 or the control peptide Tat-D1R-CT-S421A of 5 μ M or 10 μ M in the kinase reaction damping fluid, and is specific as follows:
Experimental group (being denoted as Tat-D1R-CT-S421): in the kinase reaction damping fluid, add simultaneously 5 μ L GST-D1R-CT-WT elutriants and Tat-D1R-CT-S421 (concentration of Tat-D1R-CT-S421 in the kinase reaction damping fluid is 5 μ M or 10 μ M), test.
Control group (being denoted as Tat-D1R-CT-S421A): in the kinase reaction damping fluid, add simultaneously 5 μ L GST-D1R-CT-WT elutriants and Tat-D1R-CT-S421A (concentration of Tat-D1R-CT-S421A in the kinase reaction damping fluid is 5 μ M or 10 μ M), test.
3 repetitions are established in experiment.The result is shown in Fig. 3 b, and Tat-D1R-CT-S421 significantly suppresses PKD1 to the phosphorylation of D1R-CT.The result shows: Tat-D1R-CT-S421 makes PKD1 significantly reduce the phosphorylation degree of D1R-CT.Above result show Tat-D1R-CT-S421 can competitive inhibition PKD1 to the phosphorylation of D1R-CT, but can not block its phosphorylation fully.
2, the injection Tat-D1R-CT-S421 of dorsal part striate nucleus group experiment detects Tat-D1R-CT-S421 to the impact (the injection experiment of dorsal part striate nucleus group) of 421 serine phosphorylations of D1R-CT
1) modeling
Healthy male SD rat (available from Department Of Medicine, Peking University's Laboratory Animal Science section), initial body weight 180-200g, section provides by Department Of Medicine, Peking University's Laboratory Animal Science.Rat all places in the plastics casing of place mat sawdust, 4 raisings of every cage.Give natural illumination, freely drink water and diet.By the time rat body weight reaches 250-350g, can test.
Male SD rat is through 10% chloral hydrate anesthesia, and head is shaved hair, plugs the ear bar, its head is fixed on the stereotaxic instrument, successively through Iodophor and 75% ethanol disinfection; Touch the skull first half and do otch, and cut off subcutaneous fascia; Dip 3%H with disinfecting cotton swab
2O
2Solution, calcination subcutis and muscle expose skull surface, until see each sutura sign above the skull clearly, determine bregma, and the tissue of calcination is cleaned out; The registration arm of stereotaxic instrument is aimed at bregma, read its coordinate data; Use the bregma coordinate Calculation to go out bilateral dorsal part striatum coordinate, registration arm is moved to the corresponding position, carry out mark in skull surface; Use the rigidity syringe needle to hole in the position of skull surface mark, stop boring when breaking through sense feeling; With the used outer tube of the intubate of making in advance vertically insert in the hole of holing, and an inner sleeve of making inserted in the outer tube; Spill a small amount of dental base acrylic resin powder in skull surface, drip subsequently a small amount of seccotine, then dental basse acrylic resin liquid and dental base acrylic resin powder are mixed, spread upon on the powerful glue-line; Injured skin cleaning around the otch is appropriate, and the dental basse acrylic resin liquid mud layer is embedded under the skin, treat that it solidifies; Subcutaneous injection 200,000 a units/penicillin; Rat is placed in the thermal environment, treat that it revives a little in, inner sleeve is pulled out, give sufficient drinking water, and injected continuously penicillin 3-5 days, 200,000 units/pcs/day; After treating that it recovered seven days, test, during injectable drug, the entry needle that is connected with the PE-50 pipe of making is inserted in the outer tube, slow injectable drug, injection volume must not be greater than 3 μ l, and injection speed is 0.2 μ l/min.
2) administration and mensuration
The rat intubate is recovered at first to measure locomotor activity under the 30min base state after seven days, records once motion distance value in per 5 minutes, draws distance-time curve and represents its locomotor activity.Divide two groups according to the foundation motion activity:
Experimental group (being denoted as Tat-D1R-CT-S421): dorsal part striatum intubate injection Tat-D1R-CT-S421, dosage is 15pmol/0.4 μ l, tests totally 7.
Control group (being denoted as Tat-D1R-CT-S421A): dorsal part striatum intubate injection Tat-D1R-CT-S421A, dosage is 15pmol/0.4 μ l, tests totally 8.
Behind the injection 30min, measure the 30min locomotor activity; Behind the 30min, the normal saline solution of abdominal injection 20mg/kg Cocaine (Sigma company, Cat#C5776, the Cocaine final concentration in physiological saline is 10mg/kg) is measured the 1h locomotor activity behind the 20min again.
3) preparation of striatum total protein extract
Above-mentioned 2) in, behind the mensuration 1h locomotor activity rat acute broken end is got brain, separate striatum.
The preparation of striatum total protein extract: rat broken end, separate rapidly striatum, one-sided striatum adds 200 μ l and organizes lysate [Tris-HCl (pH7.5) 50mM, NaCl 250mM, EDTA 10mM, NP-400.5%, Leupeptin10uM, PMSF 1mM, NaF 4mM], tissue homogenizer (Pellet
Motor, Rontes) grind, add 400 μ l lysates, 4 ℃ of suspendible 1h after the grinding fully; 4 ℃ 12,000g * 4min is centrifugal, and the chorista fragment is got supernatant liquor and is striatum total protein extract.
Protein quantification and Western Blot: it is quantitative to organize the total protein extract to carry out BCA, gets the sample of identical total protein concentration, adds 6 * SDS-PAGE electrophoresis sample-loading buffer, boil, ice bath 5min is centrifugal; The SDS-PAGE electrophoresis; Transferring film, sealing, primary antibodie pS421-D1R antibody (preparation of Shanghai gill biochemical corp, CAT#A100803-ZJ306, dilution in 1: 500) are hatched, and put 4 ℃ of shaking tables and spend the night; 1 * TBST washes film 5min * 3 time, and two anti-HRP mark goat-anti rabbits two anti-(Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, CAT# ZB 2306,1: 1000 dilutions) hatch room temperature 1h; 1 * TBST washes film 10min * 3 time; The darkroom exposure.
3 repetitions are established in experiment.
The result can find out that shown in Fig. 3 c Tat-D1R-CT-S421 (Tat-S421) significantly suppresses the phosphorylation of D1R-CT421 position Serine.
The quantitative statistics result shows: Tat-D1R-CT-S421 makes the phosphorylation degree of D1R-CT421 position Serine be down to 45.98 ± 17.69% (control group thinks 100%) (P＜0.05) of control group.Above result shows in the body situation, the phosphorylation that Tat-D1R-CT-S421 can competitive inhibition D1R-CT421 position Serine, but can not block this phosphorylation fully.
3, Tat-D1R-CT-S421 suppresses the effect of the high locomotor activity that Cocaine brings out
The locomotor activity model that the Cocaine acute irritation brings out is used to show that body is for the study of behaviour susceptibility of Cocaine.Body weight is the male SD rat about 250g, and abdominal cavity acute injection 20mg/kg Cocaine normal saline solution is observed the impact of the locomotor activity that the fusogenic peptide pre-treatment brings out for Cocaine, and experiment flow is specifically seen Fig. 4 a.
1) modeling: experimental technique and measure 2 among flow process and the above-mentioned A 1) identical;
2) administration and mensuration
The rat intubate is recovered at first to measure locomotor activity under the 30min base state after seven days, records once motion distance value in per 5 minutes, draws distance-time curve and represents its locomotor activity.Divide two groups according to the foundation motion activity,
Experimental group (being denoted as Tat-D1R-CT-S421): dorsal part striatum intubate injection Tat-D1R-CT-S421, dosage is 15pmol/0.4 μ l, tests, totally 7 rats.
Control group (being denoted as Tat-D1R-CT-S421A): dorsal part striatum intubate injection Tat-D1R-CT-S421A, dosage is 15pmol/0.4 μ l, tests, totally 8 rats.
Behind the injection 30min, measure the 30min locomotor activity; Behind the 30min, the normal saline solution of abdominal injection 20mg/kg Cocaine is measured the 1h locomotor activity behind the 20min again.
The experiment triplicate, results averaged.
The result shown in Fig. 4 b,
Experimental group Cocaine injection 20-50 minute (85-110min of X-coordinate, every group totally 6 numerical value) each point move distance value is: 2279,1462,1371,1258,1097,927mm; 20-50 minute each point move distance value of control group Cocaine injection is: 2336,2201,2657,2323,2174,1692mm, as seen from the figure, the Cocaine injection is after 20 minutes, and locomotor activity sharply increases, and descends afterwards, the locomotor activity value drops to lower level (experimental group 470mm after 1 hour, control group is 460mm, corresponding 140min), but still be higher than basic value.Compare the Tat-D1R-CT-S421A group, the locomotor activity fall of Tat-D1R-CT-S421 group obviously reduces.
Statistics such as Fig. 4 c show that Tat-D1R-CT-S421A compares with control group, 20-50min after the Cocaine injection, and the area under curve that Tat-D1R-CT-S421 brings out Cocaine is 5188mm*min; The locomotor activity amplitude that Tat-D1R-CT-S421A brings out Cocaine is 9355mm*min; Can find out, the locomotor activity amplitude that Tat-D1R-CT-S421 can make Cocaine bring out weakens 55.46 ± 12.50% (be 100% according to control group, experimental group motion distance total value/control group motion distance total value) (P＜0.05) to control group.
Three, Tat-D1R-CT-S421 is on the impact of D1R cytoplasmic membrane location
1, Tat-D1R-CT-S421 suppresses the cytoplasmic membrane location of D1R in the SK-N-MC clone
The cell processing mode is to take by weighing DMEM solid medium (Gibco-BRL, USA, Cat#12800-017) 13.48g at cell culture fluid DMEM[and be dissolved in 800ml ddH
2Among the O, use 3.7g NaHCO
3Adjust pH is settled to 1L to 7.2-7.4.0.22 the degerming of μ m filtering with microporous membrane, 4 ℃ of preservations.Be mixed with before use the perfect medium that contains 10%FBS (Hyclone, USA)] the middle polypeptide solution that adds respective volume, in cell culture incubator, keep former culture condition and continue to cultivate the corresponding time.
Experimental group (being denoted as Tat-D1R-CT-S421): (solvent is the ddH that autoclaving is crossed with 5 μ M Tat-D1R-CT-S421
2O) treatment S K-N-MC cell (Beijing consonance cell resource center, Cat# 3111C0001CCC000263) is 2 hours, then carries out the experiment of biotin labeling surface of cell membrane Protein Detection.
Control group (being denoted as Tat-D1R-CT-S421A): processed cell 2 hours with 5 μ M Tat-D1R-CT-S421A, then carry out the experiment of biotin labeling surface of cell membrane Protein Detection.
Biotin labeling cell surface protein experiment: adopt EZ link-NHS-LC-biotin labelling kit (Pierce, CAT#21335) and
Immobilized NeutrAvidin
TMProtein Plus (Pierce, CAT# 53151).
The bottled biotin of balance is to room temperature, takes by weighing 1mg and is dissolved in 2ml and contains Ca
2+, Mg
2+Among the DPBS, the biotin final concentration is 0.5mg/ml; With the ice-cold Ca that contains
2+, Mg
2+DPBS fine laundering cell 3 times adds an amount of above-mentioned biotin solution (covering at the bottom of the ware), 4 ℃ of 1-2h; Carefully discard biotin solution, with the neutralizer [Ca that contains the 100mM glycine of precooling
2+, Mg
2+DPBS (PH 7.2)] fine laundering cell 3 times, neutralize residual not with protein bound biotin; Add an amount of lysate (lysing cell of 1%Triton X-100/0.1%SDS/1 * PI/1 * PBS), 15min scrapes cell on ice, 12,000g * 4min is centrifugal, gets supernatant, is the total protein extract; The BCA protein quantification; Add 20 μ l pearls (cleaning 3 times with PBS in advance) in every tubulin (200 μ g), 4 ℃ of suspendibles spend the night; 4 ℃ 5,000g * 1min is centrifugal, wash buffer[1%Triton X-100/0.1%SDS/1 * PBS] fine laundering pearl 6 times; Abandon clean supernatant, add 10-15 μ l 2 * SDS-PAGE electrophoresis sample-loading buffer, the SDS-PAGE electrophoresis; Transferring film, sealing, primary antibodie D1R (Santa Cruz, CAT#sc-14001, dilution in 1: 500), two anti-HRP mark goat-anti rabbits two anti-(Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, CAT# ZB 2306,1: 1000 dilutions) hatch, expose.
Experiment repeats 3 times.The result can find out that shown in Fig. 5 a 5 μ M Tat-D1R-CT-S421 processed cell 2 hours, causes that the cytoplasmic membrane location of D1R significantly descends than the Tat-D1R-CT-S421A group.Above presentation of results, Tat-D1R-CT-S421 can suppress the cytoplasmic membrane location of D1R.
2, the injection Tat-D1R-CT-S421 of dorsal part striate nucleus group suppresses the cytoplasmic membrane location of D1R
Consistent described in one the A of experimental technique and embodiment 2, different is: get after the striatum, extract membranin in the tissue; Quantitatively, use primary antibodie to be D1R (Santa Cruz among the Western Blot, CAT#sc-14001, dilution in 1: 500) putting 4 ℃ of shaking tables spends the night, two anti-HRP mark goat-anti rabbits two anti-(Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, CAT# ZB 2306,1: 1000 dilutions) hatch, room temperature 1h is with embodiment 1.
Extract membranin experiment in the tissue: the rat broken end, separate rapidly striatum, one-sided striatum adds 200 μ l and organizes lysate [Tris-HCl (pH7.5) 50mM, NaCl 250mM, EDTA 10mM, NP-40 0.5%, Leupeptin 10uM, PMSF 1mM, NaF 4mM], tissue homogenizer (Pellet
Motor, Rontes) grind, add 400 μ l lysates, 4 ℃ of suspendible 1h after the grinding fully; 4 ℃ 12,000g * 4min is centrifugal, and the chorista fragment is got supernatant and is striatum total protein extract; Supernatant is placed the 2ml centrifuge tube, and 4 ℃ 25,000g * 60min is centrifugal, and supernatant is blotted, and throw out is membranin in the tissue; Throw out adds the lysate of organizing that 100 μ l contain 1%Triton X-100,4 ℃ of suspendible 30min; 4 ℃ of 500g * 10min, sediment separate out is got supernatant and is striatum Membrane protein extraction thing.
Experimental group (being denoted as Tat-D1R-CT-S421): dorsal part striatum intubate injection Tat-D1R-CT-S421, dosage is 15pmol/0.4 μ l, tests.
Control group (being denoted as Tat-D1R-CT-S421A): dorsal part striatum intubate injection Tat-D1R-CT-S421A, dosage is 15pmol/0.4 μ l, tests.
Experiment repeats 3 times.The result is shown in Fig. 5 b, and experimental group is injected Tat-D1R-CT-S421, and the cytoplasmic membrane location that causes D1R is than the significantly decline (Fig. 5 b) of Tat-D1R-CT-S421A group.Above presentation of results, Tat-D1R-CT-S421 can suppress the cytoplasmic membrane location of D1R, thereby suppress the high locomotor activity that Cocaine brings out.
Four, Tat-D1R-CT-S421 suppresses the transduction of D1R downstream signal
Cocaine acts on body, can be by increasing the dopamine concentration at cynapse position, and then activate D1R, cause a series of signal transduction in D1R downstream.Wherein the study of behaviour effect with Cocaine contacts the closer ERK that has, the expression of CREB and c-fos gene etc.
1, Tat-D1R-CT-S421 suppresses the phosphorylation of D1R downstream molecules ERK
1), Tat-D1R-CT-S421 suppresses the phosphorylation of SK-N-MC clone ERK in Dopamine HCL stimulation situation
Experimental technique: get the SK-N-MC clone after the processing, use 1 * PBS (NaCl 8g, Na2HPO412H2O 2.08g, KCl 0.2g, KH2PO4 0.2g is dissolved among the 800ml ddH2O, adjust pH is settled to 1L to 7.2-7.4.) fine laundering cell 3 times, add an amount of lysate (Tris-HCl (pH7.5) 50mM, NaCl 250mM, EDTA 10mM, NP-400.5%, Leupeptin 10 μ M, PMSF 1mM, NaF 4mM) lysing cell, 15min scrapes cell on ice, 12,000g * 4min is centrifugal, gets supernatant, is the total protein extract; The BCA protein quantification; Add 6 * SDS-PAGE electrophoresis sample-loading buffer, the SDS-PAGE electrophoresis; Transferring film, sealing, primary antibodie pERK (Cell Signaling Technology, dilute at 1: 250) and ERK (Santa Cruz, dilution in 1: 500, aminoacid sequence is as sequence 7), two anti-HRP mark goat-anti rabbits, two anti-(Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, CAT# ZB 2306, dilution in 1: 1000) hatches room temperature 1h, exposure.
Experimental group (being denoted as Tat-S421): processed cell 2 hours with 5 μ M Tat-D1R-CT-S421, then (Cat# H8502 is dissolved in ddH to 10 μ M Dopamine HCLs for DA, Sigma
2O) stimulate 10min, extract afterwards total protein of cell.
Control group (being denoted as Tat-S421A): processed cell 2 hours with 5 μ M Tat-D1R-CT-S421A, then 10 μ M Dopamine HCLs stimulate 10min, extract afterwards total protein of cell.
Experiment repeats 3 times.The result is shown in Fig. 6 a and 6b, and 5 μ M Tat-D1R-CT-S421 processed cell 2 hours, caused that the phosphorylation degree of the downstream molecules ERK of D1R significantly reduces than control group.Above presentation of results, Tat-D1R-CT-S421 can suppress the downstream signal of D1R.
2), the injection Tat-D1R-CT-S421 of dorsal part striate nucleus group suppresses the phosphorylation of D1R downstream molecules ERK
Experimental technique is with consistent described in the embodiment 1, and different is: get after the striatum, extract and organize total protein.
Experimental group (being denoted as Tat-S421): dorsal part striatum intubate injection Tat-D1R-CT-S421, dosage is 15pmol/0.4 μ l, tests.
Control group (being denoted as Tat-S421A): dorsal part striatum intubate injection Tat-D1R-CT-S421A, dosage is 15pmol/0.4 μ l, tests.
Experiment repeats 3 times.The result shows, experimental group, and injection Tat-D1R-CT-S421 causes that the phosphorylation degree of D1R downstream molecules ERK is than the significantly decline (Fig. 6 c) of Tat-S421A group.Above presentation of results, in the body situation, Tat-D1R-CT-S421 can suppress the downstream signal of D1R, thereby suppresses the high locomotor activity that Cocaine brings out.
2, the injection Tat-D1R-CT-S421 of dorsal part striate nucleus group suppresses the phosphorylation of D1R downstream molecules CREB and the expression of c fos immediate early gene
Experimental technique is with consistent described in the embodiment 2, and different is: get after the striatum, extract and organize total protein; Quantitatively, the use primary antibodie is respectively pCREB (Santa Cruz, CAT#sc-101663, dilution in 1: 200) among the Western Blot, (aminoacid sequence is as sequence 8 for CREB, available from Santa Cruz, CAT#sc-25785, dilution in 1: 400), c-fos (Santa Cruz, 1: 200 dilution), two anti-HRP mark goat-anti rabbits two resist (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, CAT# ZB 2306,1: 1000 dilutions) hatch room temperature 1h.
Experimental group (being denoted as Tat-S421): dorsal part striatum intubate injection Tat-D1R-CT-S421, dosage is 15pmol/0.4 μ l, tests.
Control group (being denoted as Tat-S421A): dorsal part striatum intubate injection Tat-D1R-CT-S421A, dosage is 15pmol/0.4 μ l, tests.
Experiment repeats 3 times.The result is shown in Fig. 6 c, and experimental group is injected Tat-D1R-CT-S421, causes the phosphorylation degree of D1R downstream molecules CREB and the expression of c-fos, all significantly descends than the Tat-D1R-CT-S421A group.
Above presentation of results, in the body situation, Tat-D1R-CT-S421 can suppress the downstream signal of D1R, thereby suppresses the high locomotor activity that Cocaine brings out.
Claims (6)
1. a peptide species, for as follows a) or b):
A), the polypeptide that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;
B), the polypeptide that is formed from the aminoacid sequence shown in 5 ' the terminal 412-425 amino acids residue by sequence in the sequence table 1.
2. the encoding gene of polypeptide described in the claim 1.
3. encoding gene as claimed in claim 2 is characterized in that: described encoding gene be in the sequence table sequence 6 from the dna molecular of 5 ' terminal 1234-1275 position Nucleotide.
4. the recombinant vectors, recombinant bacterium, expression cassette or the transgenic cell line that contain claim 2 or 3 described encoding genes.
5. the application of polypeptide claimed in claim 1 in the high locomotor activity medicine of the preparation inhibition drug-induced human or animal of habituation;
Described dependence producing drug is Cocaine, and described animal is rat, and described high locomotor activity embodies by improving motion distance value.
6. medicine that suppresses the high locomotor activity of human or animal that Cocaine brings out, its active ingredient is polypeptide claimed in claim 1; Described animal is rat, and described high locomotor activity embodies by improving motion distance value.
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