Polypeptide LcTx-83 as well as pharmaceutical composition and application thereof

Abstract

The invention relates to the technical field of polypeptide drug research and development. Specifically disclosed are a polypeptide LcTx-83, and a pharmaceutical composition and use thereof. According to the invention, the polypeptide LcTx-83 derived from Shangspider is found for the first time, and the polypeptide LcTx-83 comprises the following polypeptides A1 or A2: A1 is a polypeptide with an amino acid sequence of SEQ ID NO.1; the A2 amino acid sequence is a polypeptide which is obtained by substituting and / or deleting and / or adding a plurality of amino acid residues to the amino acid sequence of SEQ ID NO.1, has 90% or more identity with the SEQ ID NO.1, and has the same function as the A1; according to the present invention, the N-type voltage-gated calcium channel Cav2.2 current inhibition and animal pain model research are performed on the polypeptide, and the research shows that the polypeptide has significant inhibition effect on Cav2.2, has advantages of no addiction and significant analgesic effect, and can be used as the candidate molecule of the substitute drug or the auxiliary drug of the existing analgesic.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drug research and development, and specifically relates to polypeptide LcTx-83, a pharmaceutical composition thereof and uses thereof. Background Art

[0002] Pain is an unpleasant feeling and emotional experience caused by tissue damage or potential tissue damage. It is also a protective defense response of the body to harmful stimuli. Based on the inferred pain mechanism, it is mainly divided into nociceptive pain and non-nociceptive pain, among which non-nociceptive pain is further divided into neuropathic pain and psychological pain. Neuropathic pain is a common chronic pain that seriously affects the quality of life of patients. According to statistics, about 8% of the world's population suffers from it. The current clinical treatment of neuropathic pain mainly includes drug therapy and minimally invasive interventional therapy (Yu Xinren et al. Journal of Tongji University (Medical Edition), 2023, 44(02): 271-277.).

[0003] Although there are some effective drugs for treating neuropathic pain in clinical practice. For example, first-line treatment drugs: tricyclic antidepressants, serotonin norepinephrine reuptake inhibitors and gabapentinoids; second-line treatment drugs: 5% lidocaine patch and 8% capsaicin patch and non-opioid central analgesic tramadol; third-line treatment drugs: opioids and botulinum toxin type A. So far, there are still some patients who are insensitive to the above drugs, and the analgesics approved for clinical use are quite limited. Since the above analgesics were developed a long time ago and most of them are multi-target, most of the existing clinical analgesics have serious side effects, such as headache, drowsiness, addiction, etc. (MURNION B P. Aust Prescr, 2018, 41 (3): 60-63.). Therefore, developing highly specific drugs for a single target will greatly reduce the side effects of the drugs.

[0004] Voltage-gated calcium channels (VGCCs) are the main pathways for depolarization-mediated calcium ions to enter neuronal cells, and can regulate neurotransmitter release, secretion, excitation-contraction coupling, intracellular gene expression, and material metabolism. VGCC is a membrane protein complex composed of multiple subunits. According to the structural and functional characteristics of VGCC and the different sensitivity to blockers, it is divided into five types: L-type, N-type, P / Q-type, R-type, and T-type, and the N-type is the main target of existing calcium channel analgesics (Zamponi GW, et al. Pharmacol Rev, 2015, 67 (4): 821-870.). Cav2.2 is an N-type voltage-gated Ca 2+The channel is mainly expressed in the presynaptic neuron terminals throughout the central and peripheral nervous systems and is essential for the release of neurotransmitters. There are two existing inhibitor drugs targeting Cav2.2: ziconotide and leconotide, which have shown very effective analgesic effects in clinical practice, and there will be no tolerance problems with long-term administration. However, the above two drugs are both pore blockers of Cav2.2, and there are still problems of off-target and large side effects. Existing studies have shown that the specificity of inhibitors targeting the non-pore region of Cav2.2 is significantly higher than that of inhibitors targeting its pore region. Therefore, the development of inhibitors targeting different domains of Cav2.2 (non-pore domains) can be developed into highly effective analgesic drugs, greatly reducing the off-target effects and side effects of drugs targeting this target (Yu Ruihe et al. Pharmaceutical Progress, 2020, 44(01): 56-65; Gao, S., et al. Nature, 2021, 596, 143-147; De Logu, F., et al. Handb Exp Pharmacol, 2019, 260, 161-186.).

[0005] Spider venom contains a large number of neurotoxins. Although a large number of peptide inhibitors acting on different domains of human Cav2.1, Cav2.3 or invertebrate Cav have been reported, there are few reports of peptide inhibitors acting on Cav2.2 (Dong, M. et al. Biochemistry, 2018, 57, 6349-6355). Summary of the invention

[0006] The purpose of the present invention is to provide a polypeptide LcTx-83, a pharmaceutical composition and use thereof, in view of the above-mentioned deficiencies in the prior art. The polypeptide LcTx-83 can specifically inhibit the N-type voltage-gated calcium channel Cav2.2, and thus has a strong analgesic activity, and can significantly shorten formalin-induced pain, that is, shorten the paw licking time of mice. The Cav2.2 inhibitor can be used to prepare drugs for the treatment of painful diseases, and has the advantages of being non-addictive and having a significant analgesic effect.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first object of the present invention is to provide a polypeptide LcTx-83, wherein the polypeptide LcTx-83 comprises the following polypeptides A1 or A2: the A1 amino acid sequence is a polypeptide of SEQ ID NO.1; the A2 amino acid sequence is a polypeptide of SEQ ID NO.1 obtained by substitution and / or deletion and / or addition of several amino acid residues, which has an identity of 90% or more with SEQ ID NO.1 and has the same function as A1.

[0009] The second object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned polypeptide LcTx-83.

[0010] Furthermore, the nucleic acid molecule includes the nucleic acid molecule shown in a1, a2 or a3 below:

[0011] a1 coding region includes the nucleic acid molecule of SEQ ID NO.2;

[0012] a2 The nucleotide sequence is a nucleic acid molecule of SEQ ID NO.2;

[0013] a3 has 90% or more identity with the nucleotide sequence defined by a1 or a2, and encodes the nucleic acid molecule as described above.

[0014] The third object of the present invention is to provide a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0015] The fourth object of the present invention is to provide the above-mentioned recombinant expression cell including the above-mentioned recombinant vector.

[0016] The fifth object of the present invention is to provide a method for preparing the above-mentioned polypeptide LcTx-83, comprising the following steps: linearizing the recombinant expression vector of the polypeptide LcTx-83 through the enzyme cutting site; introducing the linearized recombinant expression vector into a host cell to obtain a recombinant expression cell, culturing the recombinant expression cell, and obtaining the polypeptide LcTx-83 from the culture.

[0017] The sixth object of the present invention is to provide a pharmaceutical composition, which comprises the above-mentioned polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

[0018] In the pharmaceutical composition, the amount of the polypeptide LcTx-83 or its pharmaceutically acceptable salt can be a therapeutically effective amount.

[0019] The pharmaceutical excipients can be excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipients can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0020] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0021] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0022] The seventh object of the present invention is to provide a use of the above-mentioned polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof in the preparation of an N-type voltage-gated calcium channel Cav2.2 inhibitor.

[0023] The eighth object of the present invention is to provide the use of the above-mentioned polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diseases associated with the N-type voltage-gated calcium channel Cav2.2.

[0024] The diseases related to the N-type voltage-gated calcium channel Cav2.2 described in the present invention include pain diseases.

[0025] The ninth object of the present invention is the use of the above-mentioned polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof in the preparation of a drug, characterized in that the drug is used to treat pain diseases, and the pain diseases are preferably neuropathic pain diseases, acute pain diseases, intractable diseases, and severe pain diseases.

[0026] The term "pharmaceutically acceptable" as used in the present invention refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0027] The term "pharmaceutically acceptable salt" refers to a conventional salt-forming reaction in the art, for example, a salt is formed by a chemical reaction between a base and an acid, such as: NH3+H2SO4→(NH4)2SO4.

[0028] The salt may be a basic salt, an acidic salt, or a neutral salt. Basic salts produce hydroxide ions in water while acidic salts produce hydronium ions.

[0029] The polypeptide LcTx-83 can form a salt of the polypeptide LcTx-83 of the present invention with a cation or anion, respectively, between anionic groups or cationic groups. These groups can be located in the peptide part of the polypeptide LcTx-83 of the present invention.

[0030] The anionic group of the polypeptide LcTx-83 of the present invention may include a free carboxyl group of the peptide portion. The peptide portion generally includes a free carboxyl group at the C-terminus.

[0031] The cationic groups of the peptide moiety are not limited in the present invention and include the free amino group at the N-terminus (if present) and any free amino groups of internal basic amino acid residues (such as Arg and Lys).

[0032] In a specific embodiment, the analogs of the polypeptide LcTx-83 of the present invention are basic salts. These salts can be formed, for example, between anionic groups of the peptide part and sodium or potassium cations.

[0033] In another specific embodiment, the analogs of the polypeptide LcTx-83 of the invention are acidic salts. These salts can be formed, for example, between cationic groups of the peptide moiety and chloride or acetate anions.

[0034] It is also possible to react a free carboxylic acid group with an alcohol or phenol to form esters of the derivatives of the invention. The ester formation may involve the free carboxyl group at the C-terminus of the peptide and / or any free carboxyl group in the side chain.

[0035] Amides of the derivatives of the invention can also be formed by reacting a free carboxylic acid group with an amine or substituted amine, or by reacting a free or substituted amino group with a carboxylic acid. Amide formation can involve the free carboxyl group at the C-terminus of the peptide, any free carboxyl group in the side chain, the free amino group at the N-terminus of the peptide and / or any free or substituted amino group of the peptide in the peptide and / or side chain.

[0036] In a specific embodiment, the polypeptide LcTx-83 is in the form of a pharmaceutically acceptable salt. In another specific embodiment, the polypeptide LcTx-83 is in the form of a pharmaceutically acceptable amide, preferably with an amide group at the C-terminus of the peptide. In yet another specific embodiment, the polypeptide LcTx-83 is in the form of a pharmaceutically acceptable ester.

[0037] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0038] (1) The present invention uses the desert wolf spider (Lycosa sp.) as the research object. Through transcriptome sequencing of venom gland samples, data analysis and peptide selection, the desert wolf spider-derived polypeptide LcTx-83 and its encoding gene were discovered for the first time. The polypeptide has a significant inhibitory effect on the N-type voltage-gated calcium channel Cav2.2.

[0039] (2) The present invention constructs a recombinant plasmid and a recombinant cell to express the polypeptide LcTx-83 with an amino acid sequence of SEQ ID NO.1, which has a molecular weight of 9366.62 Da, is composed of 83 amino acid residues, and has 7 pairs of disulfide bonds.

[0040] (3) The present invention studies the inhibition of N-type voltage-gated calcium channel Cav2.2 current and animal pain model by the polypeptide LcTx-83. The research results show that the polypeptide LcTx-83 has obvious concentration-dependent inhibition on the N-type voltage-gated calcium channel Cav2.2, and its half-inhibitory concentration is about 16.9±0.25nM. The animal pain model verifies that the polypeptide LcTx-83 has obvious analgesic effect on formalin-induced acute pain and late pain, that is, it shortens the licking time of mice. The polypeptide LcTx-8 can be used to prepare drugs to treat painful diseases, and has the advantages of being non-addictive and having obvious analgesic effect.

[0041] (4) The present invention obtains a novel N-type voltage-gated calcium channel Cav2.2 inhibitor, which provides a new lead polypeptide molecule for the development of new analgesics, and provides a method reference for other undeveloped toxic animal resources of active polypeptides, and can be used as a candidate molecule for alternative drugs or auxiliary drugs for existing analgesics. The polypeptide LcTx-83 has a strong analgesic effect and is non-addictive. It has great value in solving the addiction and other side effects of current clinical analgesic drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The 3D structure and disulfide bond pairing structure diagram of the polypeptide LcTx-83 provided by the present invention;

[0043] Figure 2 This is a diagram showing the identification results of the recombinant expression plasmid LcTx-83-PET-32a(+) constructed in the present invention;

[0044] Figure 3 This is a SDS-PAGE identification result diagram of the polypeptide LcTx-83 of the present invention; Lane 1: bacterial liquid supernatant; Lane 2: fusion protein LcTx-83; Lane 3: degradation of fusion protein LcTx-83;

[0045] Figure 4 It is a trend diagram of the inhibition of the current of N-type voltage-gated calcium channel Cav2.2 by the concentration of the polypeptide LcTx-83 of the present invention;

[0046] Figure 5 The IC of the polypeptide LcTx-83 of the present invention on the current of N-type voltage-gated calcium channel Cav2.2 50 Graphs;

[0047] Figure 6 This is a graph showing the analgesic activity evaluation results of the polypeptide LcTx-83 of the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0049] Male Kunming mice: Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.

[0050] Cav2.2 plasmid: The vector construction method was carried out according to the literature (Gao, S., Yao, X. & Yan, N. Structure of human Ca(v)2.2 channel blocked by the painkiller ziconotide. Nature 596, 143-147, doi: 10.1038 / s41586-021-03699-6 (2021).).

[0051] Human embryonic kidney cell 293T: Kunming Cell Bank of Type Culture Collection of Chinese Academy of Sciences.

[0052] RNA stabilization solution was purchased from Biosharp Biotechnology Co., Ltd., polypeptide expression vector was purchased from Nanjing GenScript Co., Ltd., TEV enzyme was purchased from Solebao Co., Ltd., BL21 (DE3) was purchased from Quanshijin Co., Ltd., XB-C18 reversed-phase chromatography column was purchased from Nanovitamin Technology Co., Ltd., IPTG, ampicillin, LB medium, imidazole and sodium chloride were all analytical grade reagents purchased from Shanghai Bioengineering Co., Ltd.

[0053] LB medium containing ampicillin resistance: the medium components contain tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, glycerol 4ml / L, and ampicillin resistance 100μg / mL.

[0054] Definition and explanation:

[0055] The term "identity" used in the present invention refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage 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 values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of identity (%) can then be obtained.

[0056] The term "protein with the same function" used in the present invention is the same or similar to the meaning conventionally understood by those skilled in the art, and refers to that the amino acid sequence of a certain fragment is a part of the amino acid sequence of a complete protein or polypeptide, and has the same or similar function or activity as the complete protein or polypeptide. It should be understood by those of ordinary skill in the art that changing a few amino acid residues in certain regions of a polypeptide, such as non-important regions, will not substantially change the biological activity. For example, the sequence obtained by appropriately replacing certain amino acids will not affect its activity (see Watson et al., Molecular Biology of The Gene, 4th edition, 1987, The Benjamin / Cummings Pub. Co. P224). Therefore, those of ordinary skill in the art are able to implement such replacements and ensure that the resulting molecules still have the desired biological activity.

[0057] Example 1

[0058] 1. Preparation of polypeptide LcTx-83:

[0059] (1) Discovery of the peptide LcTx-83 fragment sequence

[0060] The venom glands of the dissected desert wolf spider (Lycosa sp.) were quickly preserved in RNA preservation solution (BL621A, Biosharp) and sent to Beijing Novogene Technology Co., Ltd. to complete transcriptome sequencing using the second-generation sequencing technology. After obtaining the original offline data, the specific analysis method of the toxin peptide was carried out using the technology disclosed in Chinese patents (CN2023101737781, CN 2023104476980). Finally, the coding gene and amino acid sequence of LcTx-83 were obtained. The mature peptide of LcTx-83 contains 83 amino acids (see sequence SEQ ID NO: 1). The 3D structure of LcTx-83 was obtained by homology modeling method using the online software SWISS-MODEL (using A0A482Z9Q3.1.A as template). The specific disulfide bond pairing pattern of LcTx-83 was determined by the 3D structure of homology modeling and the disulfide bond pairing pattern of the homologous polypeptide of LcTx-83 (ID: P81790) in the uniprot database.

[0061] like Figure 1 As shown, the specific pairing patterns of disulfide bonds of polypeptide LcTx-83 are C9-C26, C16-C32, C23-C55, C25-C43, C34-C41, C62-C69, and C73-C79.

[0062] (2) Recombinant expression of peptide LcTx-83

[0063] The amino sequence of the polypeptide LcTx-83 was optimized by online codon optimization software (https: / / www.novopro.cn / tools / codon-optimization.html) (see sequence SEQ ID NO: 2). An enterokinase cleavage site (GACGACGACGACAAG) and a stop codon (TAA) were added to the front end of the optimized nucleotide sequence encoding LcTx-83, and EcoRI / HindIII restriction sites were added to the front and back ends of the above nucleotide sequence, respectively. The above sequence was artificially synthesized (completed by Nanjing GenScript Biotechnology Co., Ltd.) and constructed into the pET32a vector (such as Figure 2 As shown). The constructed LcTx-83-pET32a plasmid was transformed into BL21 (DE3) Escherichia coli, and a single clone was picked and inoculated into LB medium containing ampicillin resistance (ST007, Beyotime), and cultured at 37℃ and 220rpm for 6-8 hours. 300μL was aliquoted and sent to Qingke Biotechnology for sequencing, and the correct sequenced bacterial solution was used for subsequent prokaryotic expression. The correctly sequenced and revived bacterial solution was inoculated into 1L LB medium containing resistance at a ratio of 1:100, and cultured in a shaker at 37℃ and 220rpm. When its OD600nm After the value reaches 0.6-0.8, add IPTG with a final concentration of 1mM to induce polypeptide expression. Induce expression at 28°C, 100rpm, and the induction time is 12-16 hours. After the induction is completed, the bacterial solution is collected by centrifugation, washed twice with ultrapure water, and then centrifuged to remove the supernatant. Resuspend the bacteria with PBS until there is no obvious bacterial precipitation. Pour the bacterial solution into a homogenizer and break it continuously for 3 times to ensure that there is no obvious precipitation in the bacterial solution. Centrifuge and collect the supernatant of the bacterial solution.

[0064] (3) Isolation and purification of peptide LcTx-83

[0065] The supernatant sample of the pretreated bacterial solution was loaded into the balanced nickel column, and the impurities were eluted with 10mM and 30mM imidazole solutions, and the target protein was eluted and collected with 300mM imidazole solution. In the 25mM Tris-HCl (pH8.0) system, 0.2U enterokinase (P4237-1000U, Biyuntian) was added for every 0.1mg Flag fusion protein for enzyme digestion. The enzyme digestion conditions were 25°C and the enzyme digestion lasted for 12-14 hours. After the bacterial cells were broken, the supernatant samples, the samples after nickel column purification, and the samples after enzyme digestion were subjected to SDS-PAGE electrophoresis analysis. After the analysis was completed, the polypeptide samples after enzyme digestion were separated and purified by C18 reverse self-loading column. The specific conditions are as follows: equilibrate the column with 0.1% TFA in 5% acetonitrile water, load the digested sample onto the equilibrated column, desalt and wash with 2 column volumes of 0.1% TFA in 5% acetonitrile water, and elute the target protein with one column volume of 0.1% TFA in 40% acetonitrile water.

[0066] like Figure 3 As shown, the electrophoresis of the supernatant sample after breaking the bacteria, the sample after nickel column purification and the sample after enzyme digestion, wherein lane 1 in the figure is the supernatant sample after breaking the bacteria, lane 2 is the sample after nickel column purification, and lane 3 is the electrophoresis of the sample after nickel column purification and enzyme digestion, the arrows in lanes 1 and 2 point to the target protein with fusion head, and the bands pointed by the upper and lower arrows in lane 3 are the fusion head and polypeptide LcTx-83 after enzyme digestion, respectively, and M in the figure represents the protein electrophoresis standard. The present invention adopts the prokaryotic expression method, and the target polypeptide is efficiently expressed in the Escherichia coli expression bacteria BL21 through pET32a. After TEV enzyme digestion, SDS-PAGE electrophoresis is used to analyze the expression of the recombinant polypeptide. In lane 3, two obvious fusion protein and target polypeptide bands can be observed, proving that the enzyme digestion is successful.

[0067] Example 2

[0068] Peptide LcTx-83 inhibits N-type voltage-gated calcium channel Cav2.2 model current experiment

[0069] Cav2.2 plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. HEK293T cell lines were placed in 6-well culture plates for culture. When the cells reached 60-80% fusion state under a microscope, transfection reagent was used for cell transfection. 2 μg Cav2.2 plasmid was added to each well. The same amount of blank vector was used as a reference. After 24 hours of cell transfection, the cells were digested with trypsin and transferred to a single small culture dish for culture for 3 hours. A dish of cells was taken out from the incubator and the cell status was observed under a microscope. A piece of the cell state was taken out, and the piece was gently rinsed with extracellular solution balanced at room temperature, and the piece was placed in a culture dish (35×10mm) with 1.5ml of electrode extracellular solution. The patch clamp experiment was carried out at room temperature (20-25°C). The internal solution of the electrode is: CsCl 120mM, HEPES 20mM, EGTA 10mM, Na2-GTP 0.4mM, Mg-ATP 5mM, pH7.2; the external solution of the electrode is: TEA-Cl 160mM, BaCl2 2mM, TTX 200nM, glucose 10mM, HEPES 10mM, pH7.4.

[0070] First, under an inverted microscope, select cells with smooth, complete and clear plasma membranes, strong three-dimensional sense, and uniform cytoplasm. Apply a slight positive pressure so that the tip of the electrode forms a liquid surface convex outward to ensure that the electrode is clean when entering the water. It is best when the resistance measured after the electrode enters the liquid is 4 to 6MΩ. Then compensate and eliminate the potential difference between the liquid inside the electrode and the liquid outside the electrode (liquid junction potential (LJ)). Use a micromanipulation system to keep the electrode close to the cell. When the electrode is close to the cell membrane, the electrode resistance will gradually increase. When it increases by 0.5MΩ, stop moving the electrode to release the positive pressure and apply negative pressure, so that the glass microelectrode is tightly adsorbed on the cell surface, and a gigohm (1GΩ=109Ω) seal is formed between the electrode and the cell membrane. Compensate and eliminate the fast capacitance of the electrode. Clamp the cell membrane potential at -60mv, and apply a short and strong negative pressure to break the small piece of cell membrane clamped by the electrode. Compensate and eliminate the slow capacitance of the cell. After the whole-cell recording mode is formed, the cell potential is clamped at -80mV and stabilized for about 5 minutes to allow the electrode fluid and intracellular components to balance each other, and then a depolarizing pulse is applied to test the current size. The recorded current signal is filtered by the EPC-10 patch clamp amplifier with double filtering at 3kHZ and 10kHZ. The system series resistance is generally compensated between 60% and 80%, and it should always be kept within the range of 5 to 10MΩ during the experiment. The test voltage of the Cav2.2 channel is 0mV and the stimulation time is 200ms.

[0071] The peptide LcTx-83 was prepared into the corresponding dosing concentration with the electrode extracellular solution, and the micro-syringe (IM-5B, Narishige) was used to add the drug under the condition of electrical stimulation induction of the cells. The drug adding tube was made of a core-inserted glass blank (GD-1, Japan), which was drawn in one step, and the tip diameter was 30-50μm. 10μl of drug was added each time. When the drug adding tube was filled with liquid, a slight positive pressure should be applied to prevent the extracellular fluid from being sucked back due to the negative pressure in the tube when the drug adding tube was filled with liquid, thereby diluting the drug concentration. When adding the drug, the visual distance between the tip of the drug adding tube and the experimental cell was about 80-100μm, and the cell and the drug adding tube should be clearly visible in one visual plane of the microscope. The drug addition was completed within 10s to avoid the drug concentration around the cell being reduced due to the prolonged time. The effect of the drug on the current was observed, and the data was recorded in the computer storage location.

[0072] The experimental results showed that the peptide LcTx-83 could effectively inhibit more than 95% of Cav2.2 current at 50 nM (e.g. Figure 4 As shown in Figure 2, the inhibition of Cav2.2 by peptide LcTx-83 is concentration-dependent. According to the inhibitory effect of different concentrations of peptide LcTx-83 on Cav2.2 current in the experiment, nonlinear fitting was performed to calculate the half-inhibitory concentration of LcTx-83 on Cav2.2 current to be ~16.9 nM (as shown in Figure 2 Figure 5 shown).

[0073] Example 3

[0074] Study on the effect of peptide LcTx-83 in animal pain model

[0075] Male Kunming mice, weighing 18-22g, were randomly divided into control group, model group and different concentrations of drug administration groups, 5 mice in each group. In a room with an ambient temperature of 25℃, different concentrations of drug administration groups (with saline solution) were intracranial injected with different amounts of peptide LcTx-83. After 60 minutes, 100μL of 5% formalin was drawn by a microsyringe into the palm of the right hind foot of the mouse for subcutaneous injection to induce pain in the model group and different concentrations of drug administration groups, and the control group was subcutaneously injected with an equal amount of saline. The mice were placed in a 10cm×10cm×15cm observation cage for observation, and the licking time of each group of mice was continuously observed and recorded for 30 minutes, of which 0-5 minutes was the acute pain period called phase I; 10-15 minutes was the interval period; 15-30 minutes was the late pain period called phase II.

[0076] The experimental results are as follows Figure 6 As shown, the polypeptide LcTx-83 has a significant analgesic effect on formalin-induced acute pain and late pain, that is, the licking time is shortened, and it is non-addictive.

[0077] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide LcTx-83, characterized in that: The polypeptide LcTx-83 includes the following A1 or A2 polypeptides: A1 has an amino acid sequence of SEQ ID NO.1; A2 has an amino acid sequence of SEQ ID NO.1, which is obtained by replacing and / or deleting and / or adding several amino acid residues, and has an identity of 90% or more with SEQ ID NO.1 and has the same function as A1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide LcTx-83 as claimed in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule includes the nucleic acid molecule shown in a1, a2 or a3 below: a1 coding region includes the nucleic acid molecule of SEQ ID NO.2; a2 The nucleotide sequence is a nucleic acid molecule of SEQ ID NO.2; a3 has 90% or more identity with the nucleotide sequence defined by a1 or a2, and encodes the nucleic acid molecule as described in claim 2.

4. A recombinant vector, characterized in that: Comprising the nucleic acid molecule according to any one of claims 2-3.

5. A recombinant expression cell, characterized in that: Comprising the recombinant vector as described in claim 4.

6. A method for preparing the polypeptide LcTx-83 as claimed in claim 1, characterized in that: The method comprises the following steps: linearizing the recombinant expression vector of the polypeptide LcTx-83 through the enzyme cutting site; introducing the linearized recombinant expression vector into a host cell to obtain a recombinant expression cell; culturing the recombinant expression cell, and obtaining the polypeptide LcTx-83 from the culture.

7. A pharmaceutical composition, characterized in that The invention comprises the polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof as claimed in claim 1, and pharmaceutical excipients.

8. Use of the polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of an N-type voltage-gated calcium channel Cav2.2 inhibitor.

9. Use of the polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a medicament for treating a disease associated with the N-type voltage-gated calcium channel Cav2.

2.

10. Use of the polypeptide LcTx-83 or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a medicament, characterized in that: The medicine is used to treat pain diseases, and the pain diseases are preferably neuropathic pain diseases, acute pain diseases, intractable diseases, and severe pain diseases.

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