Polypeptides and their uses

Abstract

The present invention relates to polypeptides and their uses. Provided is the use of polypeptides in preparing drugs for treating and / or preventing PTSD, depression or anxiety, providing a treatment strategy for PTSD, depression or anxiety. The present invention also relates to polypeptides, complexes, nucleic acid molecules, expression vectors and host cells.

Description

Technical Field

[0001] The present invention relates to the field of drug research and development, and in particular to the medical use of polypeptides. Background Art

[0002] Posttraumatic stress disorder (PTSD) refers to a delayed and persistent mental disorder caused by an individual experiencing, witnessing or encountering one or more actual deaths involving oneself or others, or threats of death, or serious injuries, or threats to physical integrity. Studies have shown that PTSD is triggered by a failed response to stress during trauma, so it will bring about a series of changes, including hormonal, neurochemical and brain structural defects. It is hypothesized that cortisol dysregulation during trauma may delay the action of norepinephrine on synapses in the peripheral and central nervous systems, affecting the memory consolidation of the event. In contrast, adrenergic activation contributes to learning under low cortisol levels. In addition, the physical and chemical dysregulation of PTSD affects serotonergic and dopaminergic mechanisms.

[0003] In order to provide richer and better treatment strategies and better elucidate the pathogenesis, there is still a strong need to explore and discover the pathogenesis and treatment targets of PTSD. Summary of the invention

[0004] To achieve the above objectives, the inventors conducted research and found that the D2 receptor in the dopamine (DA) receptor and the 5-HT2CR in the 5-hydroxytryptamine (5-HT) receptor act as dimers. Further results showed that the dimer can be used as a target for the treatment of PTSD, thereby completing the present invention.

[0005] Specifically, DA receptors are receptors located in the body that act through their corresponding membrane receptors. There are five types of DA receptors: D1, D2, D3, D4, and D5. D2 receptors (D2R) are widely expressed in the brain.

[0006] 5-HT receptors are a group of G protein-coupled receptors and ligand-gated ion channels that appear in the central nervous system and peripheral nervous system. 5HT receptors can be divided into seven subfamilies: 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7. 5-HT2 receptors have three subtypes: A, B, and C, namely, three receptor proteins: 5-HT2A, 5-HT2B, and 5-HT2C. Among them, 5-HT2CR is widely expressed throughout the brain, including limbic-mesolimbic cortex and striatal regions, such as VTA, NAc, PFC, amygdala, hippocampus, dorsal striatum, mainly presynaptic localization.

[0007] Based on the recognition of the existence of D2R and 5-HT2CR dimers, the inventors conducted further studies and found that D2R, for example, can interact with 5-HT2CR through its K226-L240 region (hereinafter referred to as KL peptide, the sequence is shown in SEQ ID NO: 1). Subsequently, animal models were used to verify that interfering with the interaction of the D2R / 5-HT2CR complex by KL peptide can play a role in treating PTSD.

[0008] Accordingly, in a first aspect, the present invention provides a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1. In addition, the present invention also provides a use of the polypeptide of the present invention in preparing a medicament for treating and / or preventing PTSD.

[0009] In some embodiments, the polypeptide of the invention may be 15 to 30 aa in length, for example, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 26 aa, 27 aa, 28 aa or 29 aa.

[0010] In some embodiments, the polypeptide of the present invention may be derived from D2R. The D2R from which the polypeptide is derived may be queried through a biological information library (such as Genbank, EMI, DDBJ, etc.), and the sequence of the polypeptide may be confirmed on this basis. For example, in the case where the polypeptide is derived from D2R (Genbank accession number: CAB56463), in addition to the sequence shown in SEQ ID NO: 1 (i.e., positions 226 to 240 of the amino acid sequence in CAB56463), the polypeptide may also include one or more additional amino acid residues; for example, the sequence of the polypeptide may be as shown in positions 225 to 240 in CAB56463, in which case it has 16 amino acid residues.

[0011] In some embodiments, the D2R is of primate origin.

[0012] In a specific embodiment, the D2R is of human origin.

[0013] In some embodiments, the polypeptide of the present invention consists of up to 30 consecutive amino acid residues derived from human D2R and contains the sequence shown in SEQ ID NO:1.

[0014] In an exemplary embodiment, the polypeptide of the invention consists of up to 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16 consecutive amino acid residues derived from human D2R and contains the sequence shown in SEQ ID NO:1.

[0015] In a specific embodiment, the amino acid sequence of D2R derived from human is shown as SEQ ID NO:2.

[0016] As used herein, the term "treat" refers to causing a desired or beneficial effect in a patient, which may include reducing the frequency or severity of one or more symptoms of a disease, or suppressing or inhibiting further development of the disease, condition or disorder.

[0017] As used herein, the term "prevention" refers to preventing or delaying the occurrence of a disease, or preventing the appearance of clinical or subclinical symptoms thereof.

[0018] In addition, the inventors found through further research that the polypeptide of the present invention is also useful for depression and anxiety.

[0019] Accordingly, the use of the polypeptide of the present invention in preparing a drug for treating and / or preventing depression is provided. At the same time, the use of the polypeptide of the present invention in preparing a drug for treating and / or preventing anxiety is provided.

[0020] As used herein, "depression" refers to a predominantly low mood that is out of proportion to one's situation and can range from melancholy to grief and even stupor.

[0021] As used herein, "anxiety disorder" refers to a neurotic disorder characterized by anxious feelings.

[0022] The polypeptide of the present invention plays an anti-PTSD role through a new pathological mechanism (destroying the D2R / 5-HT2CR complex), and has good clinical development value. In addition, the polypeptide of the present invention can also achieve anti-depression and anti-anxiety effects.

[0023] It is worth noting that, as shown in Examples 4 and 5 below, and Figure 6 , 10 As demonstrated in Figures 1 and 11, the polypeptides of the present invention disrupt the interaction of the D2R / 5-HT2CR complex, but have no significant effect on the expression of DA or 5-HT. In other words, the polypeptides of the present invention act by specifically disrupting the interaction of the D2R / 5-HT2CR complex.

[0024] In a second aspect, the present invention provides a nucleic acid molecule encoding a polypeptide of the present invention.

[0025] In an alternative embodiment, the nucleic acid molecule of the invention is the reverse complement of a polypeptide encoding sequence of the invention.

[0026] In an exemplary embodiment, the nucleic acid molecule of the present invention is shown as SEQ ID NO:4.

[0027] In addition, the invention also provides the use of the nucleic acid molecule of the invention in the preparation of a medicament for treating and / or preventing PTSD, depression and / or anxiety.

[0028] The nucleic acid molecules of the present invention can be used to produce the polypeptides of the present invention. Depending on the expression system used, those skilled in the art can appropriately adjust the sequence of the nucleic acid molecule (eg, based on the codon preference of the selected expression system).

[0029] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention. In addition, the present invention also provides the use of the expression vector of the present invention in the preparation of a drug for treating and / or preventing PTSD, depression and / or anxiety.

[0030] Nucleic acid molecules of the present invention can be inserted into expression vectors using a variety of known methods. For example, nucleic acid molecules can be inserted into appropriate restriction endonuclease sites. Standard techniques for cloning, separation, amplification and purification, and enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, etc., and various separation techniques are known and commonly used by those skilled in the art.

[0031] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule or expression vector of the present invention. In addition, the present invention also provides the use of the host cell of the present invention in the preparation of a drug for treating and / or preventing PTSD, depression and / or anxiety.

[0032] Can use multiple expression system, as the expression vector and host cell in prokaryotic expression system and eukaryotic expression system to produce polypeptide of the present invention.Next, take mammalian expression system as example and illustrate, host cell can comprise the COS-7 cell line of monkey kidney fibroblast and other cell lines that can express compatible vector, as C127, 3T3, CHO, Hela and BHK cell line.Mammalian expression vector should comprise replication origin, suitable promoter and enhancer and any necessary ribosome binding site, polyadenylation site, splicing donor and acceptor site, transcription termination sequence and 5 ' flanking non-transcribed sequence.Dna sequence dna that is derived from for example SV40 splicing and polyadenylation site can be used to provide required non-transcriptional genetic element.Can be by the multiple methods that those skilled in the art are familiar with, include but not limited to for example calcium phosphate transfection, DEAE-dextran-mediated transfection or electroporation, expression vector is introduced into host cell.

[0033] In a fifth aspect, the present invention provides a complex comprising a polypeptide of the present invention and a (transport) carrier connected thereto for permeating the blood-brain barrier. The complex of the present invention is intended to make the polypeptide of the present invention more suitable for delivery to the human or animal body to treat and / or prevent PTSD, depression and / or anxiety.

[0034] In an exemplary embodiment, the carrier used to permeate the blood-brain barrier can be: HIV-1 Tat protein, insulin, cationized albumin, anti-rat transferrin receptor monoclonal antibody (OX26), human insulin receptor mouse monoclonal antibody (HIRMAb), Penetratin, the transduction domain of Tat protein, Pep-1 peptide, S4 13 -One or more of PV, Magainin 2 and Buforin 2. For example, the TAT transduction domain can be transduced across the membrane into the cell, and its amino acid sequence is YGRKKRRQRRR (as shown in SEQ ID NO: 5).

[0035] The polypeptide of the present invention can be connected to a carrier for permeating the blood-brain barrier by an appropriate connection technology. Exemplary connection technologies can be avidin-biotin technology, spacer arm technology based on polyethylene glycol (PEG), fusion protein technology, etc. For example, when the transduction domain of HIV-1 Tat protein is used as a carrier for permeating the blood-brain barrier, the polypeptide of the present invention can be directly connected to the transduction domain of Tat protein by fusion protein technology.

[0036] In some embodiments, when fusion protein technology is used, a linker can also be used to connect the polypeptide of the present invention to a carrier for permeating the blood-brain barrier. Exemplary linkers can be flexible linkers with glycine, such as G, GSG, GSGGSG, GSGGSGG, GSGGSGGG, GGGGSGGG, GGGGS and SGG, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The immunoblot results of co-immunoprecipitation of mouse hippocampal tissue are shown;

[0038] Figure 2 The immunoblot results of co-immunoprecipitation performed in the SPS mouse model are shown;

[0039] Figure 3 The position of the fragment involved in Example 3 on D2R is shown;

[0040] Figure 4 The immunoblot results of GST pulldown analysis on mouse hippocampal tissue using the fragments in Example 3 are shown;

[0041] Figure 5 The experimental design flow chart of Example 4 is shown;

[0042] Figure 6 The immunoblot results of co-immunoprecipitation of 293T cells treated with polypeptides are shown;

[0043] Figure 7 The immunoblotting results of immunoprecipitation of hippocampal tissues of SPS mice treated with peptides are shown (left); the effect of peptide treatment on the resting level of SPS mice (right);

[0044] Figure 8 The effect of the peptide on the level of corticosterone in the hippocampus of SPS mice is shown;

[0045] Fig. 9 The effects of the peptides on cortisol levels in the hippocampus and cortex of SPS mice are shown;

[0046] Fig.10 The effects of the peptides on DA levels in the hippocampus and cortex of SPS mice are shown;

[0047] Fig.11 The effects of the peptides on 5-HT levels in the hippocampus and cortex of SPS mice are shown;

[0048] Fig.12 The results of OFT, TST, FST and SPT analysis of CRS mice treated with polypeptides in Example 6 are shown; among them, *P<0.05, **P<0.01, ***P<0.001;

[0049] Fig.13 The results of OFT analysis of CRS mice treated with the polypeptide in Example 7 are shown, where *P<0.05, **P<0.01;

[0050] Fig.14 The results of EPM analysis of CRS mice treated with the polypeptide in Example 7 are shown, wherein **P<0.01. DETAILED DESCRIPTION

[0051] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0052] The following will describe the embodiments of the present invention in detail in conjunction with the examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0053] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are conventional products that can be obtained commercially.

[0054] Experimental animals

[0055] Balb / c mice were purchased from Guangdong Medical Experimental Animal Center, China, male, 12-14 weeks old, and housed at 18-22°C, 12 h light / 12 h dark. Food and tap water were freely available throughout the process.

[0056] Adult C57BL / 6J mice (male, 12-14 weeks) were housed at 18-22°C with 12 h light / 12 h dark, with free access to food and tap water throughout the whole procedure.

[0057] Statistical analysis

[0058] Results are presented as mean + sem. Statistical analysis was performed using Graphpad Prism 8 software. One-way analysis of variance (ANOVA) and post hoc comparisons were used to assess potential differences between means. Unless otherwise stated, independent sample t-tests were used throughout the study to compare any given two groups. Unless otherwise stated, the significance level of the test was set at P < 0.05.

[0059] Example 1 Confirmation of D2R / 5-HT2CR complex formation in vivo

[0060] Protein samples (100-500 μg of protein) from hippocampal tissue of Balb / c mice were used for co-immunoprecipitation using mouse monoclonal antibodies against 5-HT2CR and D2R, D2R or 5-HT2CR with 25 μl of protein A / G + agarose beads slurry (Santa Cruz Biotechnology, sc-2001).

[0061] After precipitation, immunoblot analysis was performed: the denatured (100°C, 10 min) proteins were separated on 8% SDS-PAGE gels and then transferred to nitrocellulose membranes, which were blocked with skim milk in TBST (Tris-buffered saline, 0.1% Tween 20); the membranes were then incubated with primary antibodies at 4°C overnight; finally, HRP-conjugated secondary antibodies were used for 1 hour and the signals were detected using the ECL Super Signal Chemiluminescent Kit (4A Biotech, cat: 4AW011-500), and the bands were analyzed for density using Image Lab software. The results are shown in Figure 2. Figure 1 shown.

[0062] Depend on Figure 1 It can be seen that 5-HT2CR antibody pulls down D2R in hippocampal tissue, confirming the coupling of D2R and 5-HT2CR.

[0063] Example 2 Verification of the pathological significance of the complex for PTSD

[0064] To determine whether this coupling is related to PTSD, we constructed a single prolonged stress (SPS) model:

[0065] The SPS model was established using male Balb / c mice. A modified SPS protocol was used to include a series of stress exposures: restraint stress (in a 50 ml plastic tube with several ventilation holes) for 2 hours, rest for 3 minutes, forced swimming in a transparent glass tank for 12 minutes (~24°C), rest for 15 minutes (dry under a warm lamp), predator (rat) odor exposure for 20 minutes, followed by ether anesthesia (until the mice completely lost consciousness). Afterwards, the mice were transferred back to their home cages with fresh bedding. The control group of mice was housed in groups and did not receive any stress exposure. After the model was established, all mice were housed undisturbed for one week.

[0066] The constructed SPS mouse model was used to study the interaction between D2R and 5-HT2CR in the model according to the immunoprecipitation and immunoblotting methods in Example 1. The results are as follows: Figure 2 shown.

[0067] Depend on Figure 2 It can be seen that significant interaction between D2R and 5-HT2CR was found in the SPS model, which confirmed their dimerization.

[0068] Example 3 Confirmation of the binding sites of the complex

[0069] In order to confirm the mutual binding sites of the D2R / 5-HT2CR complex, the D2R full-length cDNA clone (Genbank accession number: X51645) was first amplified to obtain cDNA fragments of the CT region of D2R (D2R-CT, T428-C443) and the third intracellular loop (IL3) region of D2R (D2R-IL3, K211-Q373). These fragments were subcloned into the BamH1 / EcoR1 or BamH1 / Xho1 site of the pGEX-4T-3 plasmid (Ubc no: VT1255). The initiating methionine residue and the stop codon were incorporated as appropriate. All constructs were resequenced to confirm that the splicing fusion was properly achieved. E. coli BL21 active cells (Kangti Health Life Technology Co., Ltd., No. KTSM104L) were used for expression, and GST fusion protein containing the IL3 region of D2R (GST-D2R-IL3) and GST fusion protein containing the CT region of D2R (GST-D2R-CT) were purified from bacterial lysate. The specific positions of the coding sequences of the CT region and the IL3 region on D2R are as follows: Figure 3 shown.

[0070] Solubilized mouse hippocampal tissue extract (500 μg protein) was diluted with 1×PBS / 1% Triton X-100 and then incubated with 20 μl protein GST resin saturated with GST protein alone or 15 μg GST fusion protein as above at 4°C overnight. The beads were washed 1-8 times with 1×PBS / 1% Triton X-100. The bound proteins were eluted with SDS-PAGE protein loading buffer (YeSen, no: 20315ES05), separated by SDS-PAGE, and immunoblotted with the respective antibodies. The results are shown in Table 1. Figure 4 As shown in A.

[0071] Depend on Figure 4 As shown in Figure A, the IL3 region of D2R can pull down 5-HT2CR.

[0072] Next, in order to further explore the interaction sequences / sites between D2R and 5-HT2CR, we divided the IL3 region into: the KVC (K211-V270) region of D2R, the ES (E271-S321) region of D2R, and the PQ (P322-Q373) region of D2R. The specific locations of these regions on D2R are as follows Figure 3 shown.

[0073] According to the method of this example, pulldown analysis was performed using a GST fusion protein containing the KVC region of D2R (GST-D2R-KVC), a GST fusion protein containing the ES region of D2R (GST-D2R-ES), and a GST fusion protein containing the PQ region of D2R (GST-D2R-PQ). The results of immunoblotting are shown in FIG. Figure 4 As shown in B.

[0074] Depend on Figure 4 As shown in B, the KVC region of D2R can pull down 5-HT2CR.

[0075] Furthermore, we divided the KVC region into: the KT (K211-T225) region of D2R, the KL (K226-L240) region of D2R, the KV (K241-V255) region of D2R, and the IV (I256-V270) region of D2R. The specific locations of these regions on D2R are as follows Figure 3 shown.

[0076] According to the method of this example, pulldown analysis was performed using a GST fusion protein containing the KT region of D2R (GST-D2R-KT), a GST fusion protein containing the KL region of D2R (GST-D2R-KL), a GST fusion protein containing the KV region of D2R (GST-D2R-KV), and a GST fusion protein containing the IV region of D2R (GST-D2R-IV). The results of immunoblotting are shown in FIG. Figure 4 As shown in C.

[0077] Depend on Figure 4 As shown in C, D2R can bind to 5-HT2CR in mouse hippocampal tissue through KL peptide (K226-L240), indicating that D2R can interact with 5-HT2CR through the K226-L240 region.

[0078] Example 4 Confirmation of the effect of polypeptides on reducing D2R / 5-HT2CR dimerization

[0079] In order to explore whether uncoupling 5HT2CR and D2R by the competitive peptide KL polypeptide can reverse their dimerization-related effects, the C-terminus of the KL region (K226-L240) of D2R was fused to the N-terminus of the transduction domain of the HIV-1 type Tat protein (as shown in SEQ ID NO:5, hereinafter referred to as TAT) to obtain a fusion protein that can permeate the blood-brain barrier, named TAT-D2R-KL.

[0080] Next, 293T cells were treated with TAT-D2R-KL in the presence of 5-HT2CR and D2R agonists, and TAT treatment was used as a control. Figure 5 As shown, human 293T cell lines were cultured in high glucose modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were maintained in a 37°C incubator with an atmosphere of 95% air and 5% carbon dioxide. When the cells grew to 70% density, 5-HT2CR and D2R plasmids (purchased from Addgene and Vigenebio (Cat#66411 and CH805293)) were transfected at a ratio of 1:1.

[0081] The treated cells were analyzed according to the method in Example 1. The results are as follows Figure 6 As shown. It can be seen that TAT-D2R-KL treatment significantly reduced the interaction between 5-HT2CR and D2R.

[0082] Example 5 Evaluation of the Anti-PTSD Effect of Peptides

[0083] In order to evaluate the anti-PTSD effect of the polypeptide of the present invention, an SPS mouse model was established according to Example 2.

[0084] Fear conditioning, safety learning, and recall tests were performed in two contexts (fear conditioning and safety learning were performed in context A; recall test in context B). The shapes and odors in context A and context B were different. The floor of the chamber was cleaned with 1% acetic acid or 75% ethanol before testing. A camera placed on the top of the experimental test chamber recorded the freezing behavior, and the freezing level was manually calculated based on the recorded video after the experiment was completed. The criterion for freezing behavior was that the mouse was considered to be static if no movement was detected within 2 seconds except for normal breathing. The experimental mice received 4 CS+ and CS- stimulations (30 seconds, 60 dB; CS+: 50ms pips tone (3kHz); CS-: white noise). Fear conditioning experiments were performed on the second day by pairing CS+ with US (2s foot shock, 0.8mA) (3 CS+ / US pairings, inter-trial interval (ITI): 90s). On the third day, mice were tested in context B with 5 or 15 CS+ and CS-, respectively. At the time of safety learning (day 9), SPS mice were treated with TAT-D2R-KL for 1.5 h (ip, 3 nM TAT peptide, 100 μl / 10 g mouse) and then received CS+ and CS- in a pseudo-randomized manner (ITI: 90 s) in context A, where CS+ was paired with US and CS- was never reinforced by foot shock. On day 10, peptide-treated mice were tested again in context B.

[0085] Interestingly, TAT-D2R-KL treatment reduced the level of quiescence in SPS mice, as 5-HT2CR and D2R interaction decreased ( Figure 7 In addition, TAT-D2R-KL significantly reduced elevated corticosterone in the hippocampus of SPS mice ( Figure 8 ) levels. However, TAT-D2R-KL did not significantly change the levels of cortisol ( Fig. 9 )、DA( Fig.10 ) levels, and did not reduce the upregulated 5-HT( Fig.11 ).

[0086] The above results confirm that the polypeptide of the present invention achieves the PTSD effect by uncoupling the D2R / 5-HT2CR complex.

[0087] Example 6: Using a drug evaluation model to evaluate the antidepressant ability of a polypeptide

[0088] To further evaluate the ability of peptides as antidepressant drugs, CRS and CMS mouse models were constructed as follows:

[0089] CRS:

[0090] C57BL / 6J mice were horizontally fixed for 6 h per day (10:00 to 16:00) in an acrylic cylindrical flat-bottomed head-first restrainer (25×90 mm) for 2 weeks. The filter has several slots to firmly restrain the mouse according to the size of each mouse and inhibit body movement of the limbs without causing pain. After being restrained, the mice were immediately returned to their home cages. Non-restrained mice (controls) remained in their home cages when the CRS procedure was not performed, and both control mice and CRS mice had no access to food and water during CRS exposure.

[0091] CMS:

[0092] C57BL / 6J mice were subjected to various stresses including: restraint (4 hours), cage tilt (45 times, 12 hours each), light-dark cycle reversal (once), flashing (12 hours), and dirty cage (2 times, 14 hours each). This regimen lasted for 6 weeks. Control mice were not subjected to the corresponding stress.

[0093] Mice were treated with TAT and TAT-D2R-KL (all treatments were single intraperitoneal administration, 3 nmol / g); open field test (OFT), forced swimming test (FST), tail suspension test (TST) and SPT (sugar preference test) were performed 1 hour after treatment.

[0094] The test method is as follows:

[0095] OFT (Total Distance for Test): Mice were acclimated to the experimental environment for 1 h and placed in a 45×45×30 cm chamber. A 5-min video was recorded to observe the locomotor activity of the mice. The total distance covered by the mice was measured and analyzed and expressed in millimeters.

[0096] FST: The mice were placed in a plexiglass cylinder (70 cm in height and 30 cm in diameter) filled with water (water temperature 23 ± 1 °C), with the water height above 30 cm. The mice were videotaped for 5 minutes and analyzed, and the duration of immobility was recorded. Mice were defined as immobility when they floated motionlessly in the water or raised their nose above the water surface; horizontal movement across the cylinder was defined as swimming; and vertical movement against the cylinder wall was defined as climbing.

[0097] TST: Mice were suspended with tape 40 cm above the floor in a rectangular compartment (length 55 cm × width 20 cm × depth 11.5 cm). Video was recorded for 5 minutes and the duration of immobility was recorded. EthoVisionXT software was used for recording and analysis.

[0098] SPT (Sugar Preference Test): It was conducted using a two-bottle free choice paradigm. Mice were habituated to 1% sucrose solution for 3 days and randomly divided into groups. To assess their individual sucrose intake, mice were deprived of water and food for 24 h for 3 days. On the second day, each mouse had free access to two bottles containing sucrose and water. The positions of the water and sucrose-containing bottles were changed after 2.5 h, and the total test time was 5 h. Finally, the volumes of water and sucrose solutions consumed were recorded and calculated as follows (I):

[0099]

[0100] Depend on Fig.12 The analysis results showed that TAT-D2R-KL treatment had no effect on the total motor ability of mice. Compared with TAT-treated mice, TAT-D2R-KL-treated mice had significantly reduced immobility time and significantly enhanced sucrose preference, showing a significant antidepressant effect.

[0101] Example 7: Using a drug evaluation model to evaluate the anti-anxiety ability of a polypeptide

[0102] In order to evaluate the antianxiety-like effect of the polypeptide of the present invention, a CRS mouse model was constructed using the method in Example 6, and mice were treated with TAT and TAT-D2R-KL (the treatment method was a single intraperitoneal administration, 3nmol / g). After 1 hour of treatment, an open field test (OFT) and an elevated plus maze test (EPM) for testing the central zone residence time were performed. The test method is as follows:

[0103] OFT (used to test the time spent in the central area): the open bottom surface was divided into 25 grids of equal area, and the middle 9 grid areas were the central area. One hour after the mice were treated with the peptide, they were placed in the central area and recorded for 5 minutes, and the time the mice spent in the central area was recorded.

[0104] EPM: It consists of two open arms (25cm×8cm) and two closed arms (25cm×8cm), with the intersection as the central area (8cm×8cm), 40cm above the ground. One hour after receiving peptide treatment, the mice were placed in the central area facing the open arms, and their free activities were recorded for 5 minutes. After each animal was tested, the device was wiped with 70% alcohol. The time the mice stayed in the open arms, the time they stayed in the closed arms, and the total distance the mice moved were recorded.

[0105] OFT results are shown in Fig.13 In the experiment, TAT-D2R-KL peptide significantly increased the time mice spent in the central area. The EPM results are shown in Fig.14 In the experiment, the TAT-D2R-KL polypeptide significantly increased the open arm residence time of mice, indicating that the polypeptide of the present invention effectively reduces anxiety. Sequence Listing <110> Shenzhen Chenyang Biotechnology Co., Ltd. <120> Polypeptides and their uses <130> 2202339-I-CP-CYSW <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial sequence <400> 1 Lys Arg Ser Ser Arg Ala Phe Arg Ala His Leu Arg Ala Pro Leu 1 5 10 15 <210> 2 <211> 443 <212> PRT <213> Homo sapiens <400> 2 Met Asp Pro Leu Asn Leu Ser Trp Tyr Asp Asp Asp Leu Glu Arg Gln 1 5 10 15 Asn Trp Ser Arg Pro Phe Asn Gly Ser Asp Gly Lys Ala Asp Arg Pro 20 25 30 His Tyr Asn Tyr Tyr Ala Thr Leu Leu Thr Leu Leu Ile Ala Val Ile 35 40 45 Val Phe Gly Asn Val Leu Val Cys Met Ala Val Ser Arg Glu Lys Ala 50 55 60 Leu Gln Thr Thr Thr Asn Tyr Leu Ile Val Ser Leu Ala Val Ala Asp 65 70 75 80 Leu Leu Val Ala Thr Leu Val Met Pro Trp Val Val Tyr Leu Glu Val 85 90 95 Val Gly Glu Trp Lys Phe Ser Arg Ile His Cys Asp Ile Phe Val Thr 100 105 110 Leu Asp Val Met Met Cys Thr Ala Ser Ile Leu Asn Leu Cys Ala Ile 115 120 125 Ser Ile Asp Arg Tyr Thr Ala Val Ala Met Pro Met Leu Tyr Asn Thr 130 135 140 Arg Tyr Ser Ser Lys Arg Arg Val Thr Val Met Ile Ser Ile Val Trp 145 150 155 160 Val Leu Ser Phe Thr Ile Ser Cys Pro Leu Leu Phe Gly Leu Asn Asn 165 170 175 Ala Asp Gln Asn Glu Cys Ile Ile Ala Asn Pro Ala Phe Val Val Tyr 180 185 190 Ser Ser Ile Val Ser Phe Tyr Val Pro Phe Ile Val Thr Leu Leu Val 195 200 205 Tyr Ile Lys Ile Tyr Ile Val Leu Arg Arg Arg Arg Lys Arg Val Asn 210 215 220 Thr Lys Arg Ser Ser Arg Ala Phe Arg Ala His Leu Arg Ala Pro Leu 225 230 235 240 Lys Gly Asn Cys Thr His Pro Glu Asp Met Lys Leu Cys Thr Val Ile 245 250 255 Met Lys Ser Asn Gly Ser Phe Pro Val Asn Arg Arg Arg Val Glu Ala 260 265 270 Ala Arg Arg Ala Gln Glu Leu Glu Met Glu Met Leu Ser Ser Thr Ser 275 280 285 Pro Pro Glu Arg Thr Arg Tyr Ser Pro Ile Pro Pro Ser His His Gln 290 295 300 Leu Thr Leu Pro Asp Pro Ser His His Gly Leu His Ser Thr Pro Asp 305 310 315 320 Ser Pro Ala Lys Pro Glu Lys Asn Gly His Ala Lys Asp His Pro Lys 325 330 335 Ile Ala Lys Ile Phe Glu Ile Gln Thr Met Pro Asn Gly Lys Thr Arg 340 345 350 Thr Ser Leu Lys Thr Met Ser Arg Arg Lys Leu Ser Gln Gln Lys Glu 355 360 365 Lys Lys Ala Thr Gln Met Leu Ala Ile Val Leu Gly Val Phe Ile Ile 370 375 380 Cys Trp Leu Pro Phe Phe Ile Thr His Ile Leu Asn Ile His Cys Asp 385 390 395 400 Cys Asn Ile Pro Pro Val Leu Tyr Ser Ala Phe Thr Trp Leu Gly Tyr 405 410 415 Val Asn Ser Ala Val Asn Pro Ile Ile Tyr Thr Thr Phe Asn Ile Glu 420 425 430 Phe Arg Lys Ala Phe Leu Lys Ile Leu His Cys 435 440 <210> 3 <211> 1525 <212> DNA <213> Homo sapiens <400> 3 ctggccaccc agtcggtcca ccgccctgat ggatccactg aatctgtcct ggtatgatga 60 tgatctggag aggcagaact ggagccggcc cttcaacggg tcagacggga aggcggacag 120 accccactac aactactatg ccacactgct caccctgctc atcgctgtca tcgtcttcgg 180 caacgtgctg gtgtgcatgg ctgtgtcccg cgagaaggcg ctgcagacca ccaccaacta 240 cctgatcgtc agcctcgcag tggccgacct cctcgtcgcc acactggtca tgccctgggt 300 tgtctacctg gaggtggtag gtgagtggaa attcagcagg attcactgtg acatcttcgt 360 cactctggac gtcatgatgt gcacggcgag catcctgaac ttgtgtgcca tcagcatcga 420 caggtacaca gctgtggcca tgcccatgct gtacaatacg cgctacagct ccaagcgccg 480 ggtcaccgtc atgatctcca tcgtctgggt cctgtccttc accatctcct gcccactcct 540 cttcggactc aataacgcag accagaacga gtgcatcatt gccaacccgg ccttcgtggt 600 ctactcctcc atcgtctcct tctacgtgcc cttcattgtc accctgctgg tctatatcaa 660 gatctacatt gtcctccgca gacgccgcaa gcgagtcaac accaaacgca gcagccgagc 720 tttcagggcc cacctgaggg ctccactaaa gggcaactgt actcaccccg aggacatgaa 780 actctgcacc gttatcatga agtctaatgg gagtttccca gtgaacaggc ggagagtgga 840 ggctgcccgg cgagcccagg agctggagat ggagatgctc tccagcacca gcccacccga 900 gaggacccgg tacagcccca tcccacccag ccaccaccag ctgactctcc ccgacccgtc 960 ccaccatggt ctccacagca ctcccgacag ccccgccaaa ccagagaaga atgggcatgc 1020 caaagaccac cccaagattg ccaagatctt tgagatccag accatgccca atggcaaaac 1080 ccggacctcc ctcaagacca tgagccgtag gaagctctcc cagcagaagg agaagaaagc 1140 cactcagatg ctcgccattg ttctcggcgt gttcatcatc tgctggctgc ccttcttcat 1200 cacacacatc ctgaacatac actgtgactg caacatcccg cctgtcctgt acagcgcctt 1260 cacgtggctg ggctatgtca acagcgccgt gaaccccatc atctacacca ccttcaacat 1320 tgagttccgc aaggccttcc tgaagatcct ccactgctga ctctgctgcc tgcccgcaca 1380 gcagcctgct tcccacctcc ctgcccaggc cggccagcct cacccttgcg aaccgtgagc 1440 aggaaggcct gggtggatcg gcctcctctt caccccggca ggccctgcag tgttcgcttg 1500 gctccatgct cctcactgcc cgcac 1525 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <400> 4 aaacgcagca gccgagcttt cagggcccac ctgagggctc cacta 45 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <400> 5 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10

Claims

1. Use of a polypeptide in the preparation of a drug for treating PTSD, depression or anxiety, wherein: The polypeptide is derived from the amino acid sequence of human D2R and consists of the sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein The amino acid sequence of human D2R is shown in SEQ ID NO:

2.

3. A polypeptide, which is derived from the amino acid sequence of human D2R and consists of the sequence shown in SEQ ID NO:

1.

4. The polypeptide according to claim 3, wherein The amino acid sequence of human D2R is shown in SEQ ID NO:

2.

5. A complex comprising the polypeptide according to claim 3 or 4 and a carrier for permeating the blood-brain barrier connected thereto, wherein: The carrier for permeating the blood-brain barrier is selected from one or more of the group consisting of HIV-1 Tat protein, insulin, cationized albumin, monoclonal antibody against rat transferrin receptor, mouse monoclonal antibody against human insulin receptor, Penetratin, transduction domain of Tat protein, Pep-1 peptide, S4 13 -PV, Magainin 2 and Buforin 2.

6. A nucleic acid molecule, wherein: a) encoding the polypeptide according to claim 3 or 4 or the complex according to claim 5; b) is the reverse complement of a); or c) as shown in SEQ ID NO:

4.

7. An expression vector comprising the nucleic acid molecule according to claim 6.

8. A host cell comprising the nucleic acid molecule according to claim 6 or the expression vector according to claim 7.

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