Tat-D2R-IL3-S365 polypeptide and application thereof in inhibition of cocaine-induced high movement activity

Abstract

The invention discloses a Tat-D2R-IL3-S365 polypeptide and an application of the Tat-D2R-IL3-S365 polypeptide in inhibition of high movement activity induced by cocaine. The polypeptide is a1) or a2) or a3) or a4): a1) a polypeptide composed of an amino acid sequence shown as a sequence 3 in a sequence table; a2) a fusion peptide obtained by connecting a tag to the N end or / and C end of the polypeptide limited by a1); a3) a polypeptide which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues on the polypeptide defined by a1) or a2) and has the same function as the polypeptide defined by a1) or a2); and a4) a protein which has 90% identity with the polypeptide limited by a1) or a2) or a3) and has the same function as the polypeptide limited by a1) or a2) or a3). Experiments show that the Tat-D2R-IL3-S365 polypeptide can inhibit high movement activity induced by cocaine by inhibiting downstream signal transduction of D2R, and has important significance in reducing behavioral sensitivity of an organism to cocaine stimulation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Tat-D2R-IL3-S365 polypeptide and an application thereof in inhibiting high motor activity induced by cocaine. Background Art

[0002] Cocaine addiction is a serious problem in the world today. Especially in developed countries, people's increased wealth and spiritual needs have made cocaine injection and smoking more rampant. Although only 6-18% of people who inject and smoke cocaine can develop cocaine addiction, once people become addicted to cocaine, their quality of life will spiral downward, and it is difficult for addicts to quit their dependence on cocaine.

[0003] The hypothesis about cocaine addiction is mostly based on the dopamine hypothesis: cocaine can block the reabsorption of dopamine in the synaptic cleft by the presynaptic dopamine transporter of dopaminergic neurons, thereby increasing the dopamine content in the synaptic cleft, and then acting on the postsynaptic dopamine receptors to regulate the signaling pathway mediated by them, thereby promoting the formation of cocaine addiction. Dopamine receptors can be divided into two subclasses: D1-like receptors (D1R and D5R) and D2-like receptors (D2R, D3R and D4R).

[0004] D2R is highly expressed in the striatum, nucleus accumbens, and olfactory tubercle, while its expression is relatively low in the hippocampus, amygdala, hypothalamus, and cortex. i / o To inhibit adenylate cyclase and calcium ion pathways, activate inhibitory G proteins, thereby reducing cAMP levels and inhibiting PKA activity. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide for inhibiting cocaine-induced high locomotor activity and its application.

[0006] In a first aspect, the present invention protects a polypeptide.

[0007] The polypeptide protected by the present invention is the following a1) or a2) or a3) or a4):

[0008] a1) a polypeptide consisting of the amino acid sequence shown in Sequence 3 in the sequence listing;

[0009] a2) a fusion peptide obtained by connecting a tag to the N-terminus and / or C-terminus of the polypeptide defined in a1);

[0010] a3) a polypeptide having the same function as the polypeptide defined in a1) or a2) after one or more amino acid residues are substituted and / or deleted and / or added;

[0011] a4) A polypeptide having 90% identity with the polypeptide defined in a1) or a2) or a3) and having the same function.

[0012] In the fusion peptide described in a2) above, the tag refers to a polypeptide or protein that is fused and expressed together with the target polypeptide using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target polypeptide. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0013] In the polypeptide described in a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 5 amino acid residues, or a substitution and / or deletion and / or addition of no more than 4 amino acid residues, or a substitution and / or deletion and / or addition of no more than 3 amino acid residues, or a substitution and / or deletion and / or addition of no more than 2 amino acid residues, or a substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0014] In the polypeptide described in a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of the identity (%) can be obtained. The identity includes amino acid sequences having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in Sequence 3 of the present invention.

[0015] The polypeptide described in a1) or a2) or a3) or a4) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0016] In a second aspect, the present invention protects the gene encoding the above polypeptide.

[0017] The coding gene of the above polypeptide protected by the present invention is a DNA molecule of any one of the following b1)-b2):

[0018] b1) the DNA molecule shown in Sequence 4 in the sequence listing;

[0019] b2) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in b1) and encodes a polypeptide with the same function.

[0020] In a third aspect, the present invention protects a recombinant vector, a recombinant bacterium, an expression cassette or a transgenic cell line containing the above-mentioned encoding gene.

[0021] In a fourth aspect, the present invention protects new uses of the above-mentioned polypeptide, the above-mentioned encoding gene or the above-mentioned recombinant vector, recombinant bacteria, expression cassette or transgenic cell line.

[0022] The present invention protects the use of the above polypeptide, the above encoding gene or the above recombinant vector, recombinant bacteria, expression cassette or transgenic cell line in the preparation of a product for inhibiting the high motor activity of humans or animals induced by addictive drugs.

[0023] In a fifth aspect, the present invention protects a product for inhibiting the hyperlocomotor activity of humans or animals induced by addictive drugs.

[0024] The active ingredient of the product for inhibiting the high motor activity of humans or animals induced by addictive drugs protected by the present invention is the above polypeptide, the above encoding gene or the above recombinant vector, recombinant bacteria, expression cassette or transgenic cell line.

[0025] In any of the above-mentioned applications or products, the addictive drug may be cocaine.

[0026] In any of the above-mentioned uses or products, the animal may be a rat.

[0027] In any of the above-mentioned applications or products, the high exercise activity is reflected by increasing the exercise distance value.

[0028] The inhibition of the high motor activity of humans or animals induced by addictive drugs can be achieved by inhibiting the D2R downstream signaling pathway, specifically by inhibiting the phosphorylation of ERK protein. The amino acid sequence of the ERK protein is shown in Sequence 7.

[0029] In any of the above-mentioned applications or products, the product may be a medicine.

[0030] The present invention first identifies the specific phosphorylation site of the third intracellular loop of D2R phosphorylated by PKD1 (protein kinase D1), and then based on the site, selects 14 amino acid residues including the PKD1 phosphorylation preference sequence to construct a fusion peptide Tat-D2R-IL3-S365 with membrane-penetrating ability. Further experimental verification shows that the fusion peptide Tat-D2R-IL3-S365 can inhibit the high motor activity induced by cocaine by inhibiting the downstream signal transduction of D2R. The present invention finds through experiments that the fusion peptide Tat-D2R-IL3-S365 can interfere with the phosphorylation degree of PKD1 on the serine at position 365 of the third intracellular loop of D2R, thereby inhibiting the downstream signal transduction of D2R, and then inhibiting the high motor activity induced by cocaine. Therefore, the fusion peptide Tat-D2R-IL3-S365 of the present invention and its encoding gene have broad application prospects in inhibiting the high motor activity induced by cocaine. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 To identify the phosphorylation sites of D2R by PKD1.

[0032] Figure 2 Conservation analysis of human and mouse amino acid sequences.

[0033] Figure 3 The sequences of Tat-D2R-IL3-S365 and Tat-D2R-IL3-S365A and their effects on D2R downstream signal transduction.

[0034] Figure 4 Effects of Tat-D2R-IL3-S365 and Tat-D2R-IL3-S365A on cocaine-induced hyperlocomotor activity. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0036] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0037] Abbreviations used in the present invention include: type II dopamine receptor (D2R); cocaine; extracellular signal-regulated kinase (ERK); phosphorylated ERK (pERK).

[0038] Example 1. Identification of the specific phosphorylation site of the third intracellular loop of D2R phosphorylated by PKD1 (protein kinase D1) I. Preparation of proteins with His tags

[0039] The amino acid sequence of type II dopamine receptor (hereinafter referred to as D2R) is sequence 1 in the sequence table, with a total length of 444 amino acids. It is a typical G protein-coupled receptor with a classic seven-transmembrane structure. Therefore, the D2 receptor is segmented and defined as the N-terminus, the first transmembrane domain, the first intracellular loop, the second transmembrane domain, the first extracellular loop, the third transmembrane domain, the second intracellular loop, the fourth transmembrane domain, the second extracellular loop, the fifth transmembrane domain, the third intracellular loop, the sixth transmembrane domain, the third extracellular loop, the seventh transmembrane domain and the C-terminus.

[0040] The peptide segments used in this example are as follows:

[0041] D2R-IL3: the peptide segment represented by amino acids 213 to 436 from the 5' end of sequence 1 in the sequence listing (the peptide segment represented by amino acids 213 to 436 from the N-terminus of the D2R protein);

[0042] D2R-IL3-S365A: peptide obtained by mutating serine 365 of D2R-IL3 to alanine;

[0043] D2R-IL3-T225A: peptide obtained by mutating the threonine at position 225 of D2R-IL3 to alanine;

[0044] D2R-IL3-T245A: The peptide obtained by mutating the threonine at position 245 of D2R-IL3 to alanine.

[0045] The amino acid mutation sites described in the above D2R-IL3-S365A, D2R-IL3-T225A and D2R-IL3-T245A correspond to the amino acid sequence of the D2R protein shown in SEQ ID NO: 1.

[0046] During the experiment, a His tag was added to the N-terminus of each peptide, and the following peptides were obtained: His-D2R-IL3, His-D2R-IL3-T225A, His-D2R-IL3-T245A, and His-D2R-IL3-S365A.

[0047] The specific preparation methods of each peptide segment with a His tag are as follows:

[0048] 1) Amplification of the D2R-IL3 encoding gene

[0049] Using male SD rat striatum cDNA (RNA extracted and cDNA obtained by reverse transcription) as a template, PCR amplification was performed using the sequence shown in primer pair 1 in Table 1 as a primer to obtain a PCR product. The PCR product was sequenced and verified. The result showed that the product sequence was correct. The PCR product was the D2R-IL3 encoding gene. The D2R-IL3 encoding gene sequence was nucleotides 637-1038 from the 5' end of sequence 2 in the sequence table.

[0050] Table 1. Primer pair sequences

[0051]

[0052] 2) Obtaining the genes encoding D2R-IL3-T225A, D2R-IL3-T245A and D2R-IL3-S365A

[0053] Using the PCR product (D2R-IL3) obtained in step 1) as a template, PCR amplification was performed using the Stratagene QuikchangeR XL site-directed mutagenesis kit (USA, CAT#200517-5) and the sequences shown in primer pairs 2, 3 and 4 in Table 1 as primers to cause point mutations in the D2R-IL3 coding gene, thereby obtaining the coding genes of D2R-IL3-T225A, D2R-IL3-T245A and D2R-IL3-S365A, respectively.

[0054] The primers used for D2R-IL3-T225A were primer pair 2 in Table 1, and the template was D2R-IL3.

[0055] The primers used for D2R-IL3-T245A were primer pair 3 in Table 1, and the template was D2R-IL3.

[0056] The primers used for D2R-IL3-S365A were primer pair 4 in Table 1, and the template was D2R-IL3.

[0057] The PCR amplification reaction system was as follows: 5× PrimeSTAR buffer 10 μL, dNTP mix 2 μL, D2R-IL3 100 ng, DMSO 2.5 μL, 5'primer (100 ng / μL) 1.25 μL, 3'primer (100 ng / μL) 1.25 μL, PrimeSTAR HS polymerase 1 μL, and ddH2O to 50 μL.

[0058] The PCR amplification reaction conditions were as follows: 95°C for 2 min; 95°C for 50 s, 55°C for 30 s, 68°C for 10 min, 18 cycles; 68°C for 10 min, 4°C pause.

[0059] The above-mentioned D2R-IL3-T225A, D2R-IL3-T245A and D2R-IL3-S365A encoding gene sequences can all be artificially synthesized.

[0060] 3) Preparation of recombinant vectors containing "encoding genes for each peptide segment"

[0061] The coding genes of D2R-IL3, D1R-IL3-T225A, D1R-IL3-T245A and D1R-IL3-S365A were inserted into the pET-28a(+) vector to obtain recombinant vectors pET-28a(+)-D2R-IL3, pET-28a(+)-D2R-IL3-T225A, pET-28a(+)-D2R-IL3-T245A and pET-28a(+)-D2R-IL3-S365A, respectively.

[0062] 4) Preparation of recombinant bacteria containing "encoding genes for each peptide segment"

[0063] The recombinant vectors pET-28a(+)-D2R-IL3, pET-28a(+)-D2R-IL3-T225A, pET-28a(+)-D2R-IL3-T245A, and pET-28a(+)-D2R-IL3-S365A were introduced into BL21(DE3) bacteria, respectively, to obtain recombinant bacteria BL21(DE3) / pET-28a(+)-D2R-IL3, BL21(DE3) / pET-28a(+)-D2R-IL3-T225A, BL21(DE3) / pET-28a(+)-D2R-IL3-T245A, and BL21(DE3) / pET-28a(+)-D2R-IL3-S365A, respectively, and sequencing confirmed that the inserted fragments were correct.

[0064] At the same time, BL21(DE3) / pET-28a(+) obtained by transferring the empty vector pET-28a(+) into BL21(DE3) bacteria was used as a control.

[0065] 5) Fermentation recombinant bacteria

[0066] Recombinant bacteria BL21(DE3) / pET-28a(+)-D2R-IL3, BL21(DE3) / pET-28a(+)-D2R-IL3-T225A, BL21(DE3) / pET-28a(+)-D2R-IL3-T245A, BL21(DE3) / pET-28a(+)-D2R-IL3-S365A and recombinant bacteria BL21(DE3) / pET-28a(+) containing an empty vector were shaken overnight; the above bacterial solutions were added to fresh culture medium at a ratio of 1 / 10 of the final induction volume, and the culture was shaken at 37°C and 250 rpm for 3 h; a final concentration of 0.5 mM IPTG induction, shaking at 30℃, 250rpm for 2h; centrifugation at 4℃, 3,000rpm for 15min, discarding the supernatant, collecting the bacterial precipitate; adding 100μL lysis buffer (HIS-tag bacterial lysis buffer: 10mM PMSF / 1×PI / 1×PBS) per ml of bacterial solution to resuspend the bacteria, and ultrasonically disrupt the cells (40W, 6s×3 times); adding 10% Triton X-100 to a final concentration of 1%, suspending at 4℃ for 1h; centrifugation at 4℃, 12,000rpm for 20min, taking the supernatant; adding 15-20μL beads (QIAGEN, Ni-NTA agarose beads) and suspended at 4°C for 2h; the beads were washed 3 times with 1×PBS and centrifuged at 4°C and 2,000rpm for 2min to remove non-specific binding, and beads with proteins His-D2R-IL3, His-D2R-IL3-T225A, His-D2R-IL3-T245A, His-D2R-IL3-S365A, and protein His were obtained respectively.

[0067] 2. In vitro kinase phosphorylation assay

[0068] Add His-tagged protein elution buffer to the purified beads with proteins His-D2R-IL3, His-D2R-IL3-T225A, His-D2R-IL3-T245A, His-D2R-IL3-S365A, and protein His, and elute overnight at 4°C. Centrifuge at 2,000rpm for 2min the next day, and aspirate the supernatant, trying not to aspirate the beads. Aspirate part of the supernatant and perform SDS-PAGE electrophoresis to determine the protein expression level and the amount of kinase system loading. Aspirate the determined amount of protein and dilute it to a volume of 5μL with His-tagged protein elution buffer, and add it to a 25-30μL γ- 32P-ATP (Beijing Furui Bioengineering Co., Ltd., Cat#PN101, 5 μCi / sample), DTT (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Cat#08910202), purified PKD1 protein (Upstate, Cat#14-508) in kinase reaction buffer (Tris-HCl (pH 7.6) 30 mM, MgCl2 10 mM, DTT 0.1 mM, [γ- 32 P]ATP 10μCi / 50μL), and place in a 30℃ water bath for 30min. After the water bath, place on ice for 5min immediately, and then centrifuge at 2,000rpm for 2min. Add an appropriate amount of 6×SDS-PAGE electrophoresis loading buffer, and perform SDS-PAGE electrophoresis; when the electrophoresis front indicator reaches the bottom of the gel, stop the electrophoresis, remove the concentrated gel, and place the separation gel in a gel staining solution (500mL of methanol, 100mL of glacial acetic acid, 2.5g of Coomassie blue R-250, 400mL of ddH2O), and stain on a horizontal shaker at room temperature (25℃) for 40-60min; recover the gel staining solution and replace it with a destaining solution (500mL of methanol, 100mL of glacial acetic acid, 400mL of ddH2O). 400mL), continue to decolorize on a horizontal shaker at room temperature, change the decolorizing solution every 15 minutes, and decolorize until the target protein band is clear; recover the decolorizing solution and change it to a dry gel solution (10% anhydrous ethanol, 4% glycerol, fixed to 100mL with ddH2O), continue to dry the gel at room temperature on a horizontal shaker for 20 minutes, and at the same time, soak the dry gel film in the dry gel solution; take the gel out of the dry gel solution, wrap it flat on both sides with a dry gel film, without any bubbles, and fix it, and air-dry it at room temperature for more than 8 hours; remove the gel with the dry gel film, stick it in a dark box, cover it with an X-ray sensitive film in a dark room, place it in a -80℃ refrigerator for a suitable time, and then perform radioautoradiography in a dark room.

[0069] The experimental results are as follows Figure 1 As shown in the figure, it can be seen that His-D2R-IL3, His-D2R-IL3-T225A, and His-D2R-IL3-T245A can be phosphorylated by PKD1, but His-D2R-IL3-S365A cannot be phosphorylated by PKD1. This indicates that serine 365 of the third intracellular loop of D2R is the phosphorylation site of PKD1, and this site is conserved ( Figure 2 ). This indicates that PKD1 can phosphorylate serine 365 of the third intracellular loop of D2R.

[0070] Example 2: Acquisition of Tat-D2R-IL3-S365 and its effect on D2R downstream signaling pathway

[0071] 1. Acquisition of Tat-D2R-IL3-S365

[0072] TAT (transcriptional activator protein) transmembrane peptide is one of the transmembrane delivery vectors. It can introduce molecules such as polypeptides, proteins and DNA covalently linked to it into cells across the membrane, and can even pass through the blood-brain barrier. It has high transduction efficiency and no damage to cells. It is widely used in cell biology, gene therapy and pharmacy. According to the 365th serine of the phosphorylation site of PKD1 in D2R-IL3, 14 amino acid residues including the PKD1 phosphorylation preference sequence were selected to construct a fusion peptide with transmembrane ability, Tat-D2R-IL3-S365. The 365th serine in the target sequence was mutated to alanine, that is, Tat-D2R-IL3-S365A, as a control.

[0073] The amino acid sequence of Tat-D2R-IL3-S365 is sequence 3 in the sequence list, which consists of the TAT transmembrane sequence (RKKRRQRRR) and 14 amino acid residues including serine at position 365, and its coding gene sequence is sequence 4. Tat-D2R-IL3-S365 was synthesized by GenScript Biotech (Shanghai) Co., Ltd., and its molecular weight is 3054.65.

[0074] The amino acid sequence of Tat-D2R-IL3-S365A is shown in Sequence 5 in the sequence table, which consists of a TAT transmembrane sequence (RKKRRQRRR) and 14 amino acid residues including alanine at position 365, and its coding gene sequence is Sequence 6. Tat-D2R-IL3-S365A was synthesized by GenScript Biotech (Shanghai) Co., Ltd., and its molecular weight is 3038.65.

[0075] 2. Tat-D2R-IL3-S365 inhibits D2R downstream signal transduction

[0076] Cocaine acts on the body and can activate D2R by increasing the dopamine concentration in the synaptic site. In order to verify whether Tat-D2R-IL3-S365 can trigger a series of signal transductions downstream of D2R, HEK293 cell lines were treated with Tat-D2R-IL3-S365, and the ERK phosphorylation of the treated cells was detected. It was found that Tat-D2R-IL3-S365 can inhibit the phosphorylation of ERK in HEK293 cell lines, indicating that Tat-D2R-IL3-S365 can inhibit the downstream signal transduction of D2R. The specific experimental steps are as follows:

[0077] 1. Treat cells with peptides. According to the different peptides used, they are divided into experimental group and control group. The treatment method of each group is as follows:

[0078] Experimental group (denoted as Tat-S365): cells were treated with 0.17 μM Tat-D2R-IL3-S365.

[0079] Control group (denoted as Tat-S365A): cells were treated with 0.17 μM Tat-D2R-IL3-S365A.

[0080] 2. After 2 hours of treatment, the total cell protein was extracted. The specific extraction method was as follows: Take the treated HEK293 cell line, use 1×PBS (NaCl 8g, Na2HPO4·12H2O 2.08g, KCl 0.2g, KH2PO4 0.2g dissolved in 800mL ddH2O, adjust the pH to 7.2-7.4, and make up to 1L) to gently wash the cells 3 times, add appropriate amount of lysis buffer (Tris-HCl (pH7.5) 50mM, NaCl250mM, EDTA 10mM, NP-40 0.5%, Leupeptin 10μM, PMSF 1mM, NaF 4mM) to lyse the cells, put on ice for 15min, scrape the cells, centrifuge at 12,000g for 4min, and take the supernatant, which is the total protein extract.

[0081] 3. BCA protein quantification; add 6×SDS-PAGE electrophoresis loading buffer, SDS-PAGE electrophoresis; transfer, block, incubate with primary antibody pERK (Cell Signaling Technology, 1:250 dilution) and ERK (Santa Cruz, 1:500 dilution, amino acid sequence is shown in SEQ ID NO: 7), and secondary antibody HRP-labeled goat anti-rabbit secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., CAT#ZB2306, 1:1000 dilution), room temperature for 1 hour, and expose.

[0082] The results are as follows Figure 3 As shown, the results showed that when cells were treated with 0.17 μM Tat-D2R-IL3-S365 for 2 hours, the phosphorylation level of ERK, a downstream molecule of D2R, was significantly lower than that of the control group. The above results indicate that Tat-D2R-IL3-S365 can inhibit the downstream signal transduction of D2R.

[0083] Example 3: Use of Tat-D2R-IL3-S365 in inhibiting cocaine-induced hyperlocomotor activity

[0084] The locomotor activity model induced by acute cocaine stimulation is used to show the behavioral sensitivity of the body to cocaine. The present invention verifies that Tat-D2R-IL3-S365 can inhibit the high locomotor activity induced by cocaine by constructing the locomotor activity model induced by acute cocaine stimulation. The specific steps are as follows:

[0085] 1. Modeling

[0086] Test materials: Healthy male SD rats with an initial body weight of 180-200g. The rats were placed in a plastic box covered with sawdust, 4 rats per cage. Natural lighting was provided, and drinking water and food were available freely. Experiments were conducted only when the rats weighed 250-350g.

[0087] Experimental methods: Male SD rats were anesthetized with 10% chloral hydrate, their heads were shaved, ear bars were inserted, and their heads were fixed on a stereotaxic instrument, which was disinfected with iodine and 75% ethanol in turn; an incision was made on the front half of the skull, and the subcutaneous fascia was cut; a sterile cotton swab was dipped in 3% H2O2 solution, and the subcutaneous tissue and muscle were burned to expose the skull surface until the suture marks on the skull were clearly seen, the anterior font was determined, and the burned tissue was cleaned; the positioning arm of the stereotaxic instrument was aligned with the anterior font, and its coordinate data was read; the coordinates of the bilateral dorsal striatum were calculated using the anterior font coordinates, the positioning arm was moved to the corresponding position, and a mark was made on the skull surface; a rigid syringe needle was used to drill holes at the marked positions on the skull surface, and drilling was stopped when a breakthrough was felt; the outer sleeve for the cannula prepared in advance was vertically inserted into the drilled hole, and the prepared inner sleeve was inserted into the outer sleeve; a small amount of dental tray powder was sprinkled on the skull surface, and then a small amount of Super glue, then mix the denture water and denture powder evenly, and apply it on the super glue layer; clean the damaged skin around the incision properly, and bury the denture cement layer under the skin and wait for it to solidify; inject 200,000 units of penicillin per rat subcutaneously; place the rat in a warm environment, and when it wakes up slightly, remove the inner cannula, give it sufficient food and water, and continuously inject penicillin for 3-5 days, 200,000 units per rat per day; after it recovers for seven days, conduct the experiment. When injecting the drug, insert the prepared injection needle connected with PE-50 tube into the outer cannula, and slowly inject the drug. The volume of the drug on each side is 1.5μL, and the injection speed is 0.25μL / min.

[0088] 2. Drug administration and determination

[0089] 1. Effects of interfering peptides on rat motor activity under basal conditions

[0090] Seven days after the rats recovered from intubation, the motor activity was first measured at the basal state for 30 minutes, and the distance traveled was recorded every 5 minutes. The distance-time curve was drawn to represent the motor activity. The interfering peptide was injected 30 minutes later. The experimental group was injected with the interfering peptide Tat-D2R-IL3-S365, and the control group was injected with the control peptide Tat-D2R-IL3-S365A. The treatment of each group was as follows:

[0091] Experimental group (denoted as Tat-D2R-IL3-S365): Tat-D2R-IL3-S365 was injected into the dorsal striatum with a dose of 5 nmol / 1.5 μL of interfering peptide on each side. A total of 6 rats were included in the experiment.

[0092] Control group (denoted as Tat-D2R-IL3-S365A): Tat-D2R-IL3-S365A was injected into the dorsal striatum with a control peptide dose of 5 nmol / 1.5 μL on each side. A total of 6 rats were included in the experiment.

[0093] 30 min after injection, locomotor activity was measured for 90 min.

[0094] The process diagram and results are as follows Figure 4 As shown in a, the results show that within 30-90 minutes of interfering peptide injection, the motor activity of rats was no different from that of the control group. The average exercise values ​​of the experimental group every 5 minutes were 653cm, 547cm, 344cm, 556cm, 627cm, 897cm, 578cm, 517cm, 689cm, 589cm, 542cm, 598cm, and the average exercise values ​​of the control group every 5 minutes were 904cm, 568cm, 550cm, 653cm, 541cm, 533cm, 544cm, 579cm, 595cm, 597cm, 351cm, 434cm. Statistics showed that the average total distance of the experimental group and the control group was 7140cm and 6855cm, respectively, 30-90 minutes after the interfering peptide injection, a total of 1 hour. There was no difference between the experimental group and the control group.

[0095] 2. Effects of interfering peptides on the motor activity of rats under cocaine stimulation

[0096] Seven days after the rats recovered from intubation, the motor activity was first measured at the basal state for 30 minutes, and the distance traveled was recorded every 5 minutes. The distance-time curve was drawn to represent the motor activity. The experimental group was injected with the interfering peptide Tat-D2R-IL3-S365, and the control group was injected with the control peptide Tat-D2R-IL3-S365A. The treatment of each group was as follows:

[0097] Experimental group (denoted as Tat-D2R-IL3-S365): Tat-D2R-IL3-S365 was injected into the dorsal striatum with a dose of 5 nmol / 1.5 μL of interfering peptide on each side. A total of 8 rats were included in the experiment.

[0098] Control group (denoted as Tat-D2R-IL3-S365A): Tat-D2R-IL3-S365A was injected into the dorsal striatum with a control peptide dose of 5 nmol / 1.5 μL on each side. A total of 8 rats were included in the experiment.

[0099] 30 minutes after the injection, the rats in the experimental group and the control group were given 20 mg / kg of cocaine stimulation, and then the locomotor activity was measured for 90 minutes.

[0100] The process diagram and results are as follows Figure 4 As shown in b, the results showed that cocaine stimulation was given 30 minutes after the interfering peptide was injected. Within 1 hour of cocaine stimulation, the motor activity of the rats in the experimental group was significantly lower than that of the control group. The average motor values ​​of the experimental group every 5 minutes were 885cm, 1135cm, 1008cm, 959cm, 824cm, 819cm, 816cm, 613cm, 543cm, 569cm, 494cm, and 492cm, respectively, and the average motor values ​​of the control group every 5 minutes were 2373, 2144, 1845cm, 1590cm, 1410cm, 1419cm, 1174cm, 1243cm, 1137cm, 922cm, 999cm, and 848cm, respectively. The distance of 1 hour was calculated from 30-90 minutes of interfering peptide injection. The average total distance of the experimental group was 9163cm, and the average total distance of the control group was 17108cm. The motor activity of the rats in the experimental group was also significantly lower than that of the control group (p<0.05).

[0101] The above results indicate that Tat-D2R-IL3-S365 can inhibit the high locomotor activity induced by cocaine by inhibiting the downstream signal transduction of D2R.

[0102] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A polypeptide, which is a1) or a2) or a3) or a4): a1) a polypeptide consisting of the amino acid sequence shown in Sequence 3 in the sequence listing; a2) a fusion peptide obtained by connecting a tag to the N-terminus and / or C-terminus of the polypeptide defined in a1); a3) a polypeptide having the same function as the polypeptide defined in a1) or a2) after one or more amino acid residues are substituted and / or deleted and / or added; a4) A protein having 90% identity with the polypeptide defined in a1) or a2) or a3) and having the same function.

2. A gene encoding the polypeptide according to claim 1.

3. The coding gene according to claim 2, characterized in that: The gene is a DNA molecule of any one of the following b1)-b2): b1) the DNA molecule shown in Sequence 4 in the sequence listing; b2) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in b1) and encodes a polypeptide with the same function.

4. A recombinant vector, recombinant bacteria, expression cassette or transgenic cell line containing the coding gene according to claim 2 or 3.

5. Use of the polypeptide according to claim 1, the encoding gene according to claim 2 or 3, or the recombinant vector, recombinant bacteria, expression cassette or transgenic cell line according to claim 4 in the preparation of a product for inhibiting the hyperlocomotor activity of humans or animals induced by addictive drugs.

6. A product for inhibiting the hyperlocomotor activity of humans or animals induced by addictive drugs, wherein the active ingredient is the polypeptide according to claim 1, the encoding gene according to claim 2 or 3, or the recombinant vector, recombinant bacteria, expression cassette or transgenic cell line according to claim 4.

7. The use according to claim 5 or the product according to claim 6, characterized in that: The addictive drug is cocaine.

8. The use or product according to any one of claims 5 to 7, characterized in that: The animals are rats.

9. The use or product according to any one of claims 5 to 8, characterized in that: The high exercise activity is manifested by an increased exercise distance value.

10. The use or product according to any one of claims 5 to 9, characterized in that: The product described is a drug.

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