Mu-type conotoxin peptide, polynucleotide coded by mu-type conotoxin peptide and application of mu-type conotoxin peptide

Abstract

The invention discloses a mu-type conotoxin peptide, polynucleotide encoded by the mu-type conotoxin peptide and application of the mu-type conotoxin peptide. The amino acid sequence of the mu-type conotoxin peptide is shown as SEQ ID NO. 1. The invention provides a mu-type conotoxin peptide with higher biological activity than wild-type mu-type conotoxin, which can specifically block a Nav1.4 channel and reduce muscle contraction so as to be helpful for preventing and reducing wrinkles, and also can provide local anesthesia and reduce muscle activity, thereby playing an important role in the fields of beauty and medical treatment.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a μ-type cone snail toxin peptide, the polynucleotide it encodes, and its applications. Background Technology

[0002] Conotoxins are a sophisticated set of neuropharmacological weapons developed by cone snails (Conus) over a long period of evolution, used for predation and defense. Most are composed of 12-46 amino acid residues, containing two or three pairs of disulfide bonds. They primarily act on various ion channels on the cell membrane, as well as receptors for neurotransmitters and kinins, blocking or enhancing the transmission of nerve excitation signals, thus exhibiting neurotoxicity. μ-conotoxin (μ-CnIIIC) is a 22-amino acid peptide from cone snails and is an effective antagonist of voltage-gated Nav1.4 sodium channels.

[0003] Natural μ-conotoxin peptides have low hydrophilicity and membrane permeability, resulting in low solubility in vivo. This hinders the absorption and distribution of μ-CnIIIC peptides, leading to their easy accumulation in non-target tissues and potentially high toxicity and side effects. Furthermore, natural μ-CnIIIC peptides may be degraded by various enzymes in vivo, reducing their effective concentration and biological activity. Higher doses are often required to achieve the desired effect, resulting in high raw material and production costs. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a μ-conotoxin peptide, its encoded polynucleotide, and its applications. This invention provides a μ-conotoxin peptide with higher activity than wild-type μ-conotoxin, capable of specifically blocking the Nav1.4 channel, reducing muscle contraction, reducing or eliminating wrinkles on human skin, and used for muscle relaxation and analgesia.

[0005] This invention is the first to discover that elongating the sequence of wild-type μ-conotoxin (μ-CnIIIC) by adding one D-arginine (d-Arg) at the C-terminus, replacing the 17th amino acid with serine (Ser), the 1st amino acid with D-lysine (d-Lys), and the 2nd amino acid with lysine (Lys) can enhance the activity of μ-CnIIIC.

[0006] This invention provides a μ-type conotoxin peptide, named [Lys] 2 Ser 17 ]-dK,dR-μ-CnIIIC. The amino acid sequence of the μ-type cone snail venom peptide is shown in SEQ ID NO.1. The amino acid sequence contains three pairs of disulfide bonds, which are located at Cys 3 -Cys 15 Cys4 -Cys 21 and Cys 5 -Cys 22 The position is identical to the disulfide bond structure of the natural μ-type conotoxin peptide.

[0007] The present invention also provides a polynucleotide encoding the aforementioned μ-conotoxin peptide.

[0008] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide.

[0009] The present invention also provides an expression vector comprising the aforementioned nucleic acid construct.

[0010] The present invention also provides a transformed cell comprising the nucleic acid construct described herein or the expression vector of claim 4.

[0011] The present invention also provides the use of the aforementioned μ-conotoxin peptide in the preparation of drugs for the treatment or prevention of diseases related to sodium ion channels.

[0012] In some embodiments, the sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, tonic-clonic syndrome, or autism spectrum disorder.

[0013] The present invention also provides the use of the aforementioned μ-type cone snail toxin peptide in the preparation of drugs for treating or preventing pain.

[0014] The present invention also provides the application of the aforementioned μ-type cone snail toxin peptide in the preparation of anesthetic drugs.

[0015] The present invention also provides a pharmaceutical composition comprising the aforementioned μ-type cone snail toxin peptide.

[0016] In some embodiments, the dosage form of the pharmaceutical composition is any one of tablets, capsules, pills, solutions, absorbents, and ointments.

[0017] In summary, compared with the prior art, the present invention achieves the following technical effects: 1. This invention provides a novel μ-type cone snail venom peptide [Lys] 2 Ser 17 ]-dK,dR-μ-CnIIIC, compared to the wild type, [Lys 2 Ser 17 The animal activity of ]-dK,dR-μ-CnIIIC was increased by more than 20 times, and its IC50 was significantly higher. 50 It is 25.3 times more potent than wild-type conospirin, reducing the dosage required for use, lowering drug toxicity and side effects, and reducing production costs.

[0018] 2. The [Lys] of the present invention 2 Ser 17 Compared to wild-type μ-CnIIIC, the amino acid sequence of ]-dK,dR-μ-CnIIIC increases the number of basic amino acids, which can further improve the hydrophilicity and membrane permeability of the sequence, increase its solubility in organisms, and allow it to reach target cells or tissues more precisely.

[0019] 3.[Lys 2 Ser 17 ]-dK,dR-μ-CnIIIC retains the function of wild-type μ-CnIIIC peptide, specifically blocking Nav1.4 channels and reducing muscle excitability and contractile ability.

[0020] 4. [Lys 2 Ser 17 ]-dK,dR-μ-CnIIIC can effectively reduce muscle activity and thus inhibit wrinkle formation by specifically blocking the Nav1.4 channel, and can be used as a non-invasive anti-wrinkle treatment.

[0021] 5. [Lys] 2 Ser 17 ]-dK,dR-μ-CnIIIC, as a compound that blocks sodium ion channels, can reduce nerve conduction, thereby producing a local anesthetic effect, and can be used to relieve discomfort and pain caused by medical or cosmetic procedures. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The μ-type cone snail venom peptide [Lys] disclosed in this invention 2 Ser 17 Schematic diagram of the synthesis process of ]-dK,dR-μ-CnIIIC; Figure 2 This is the chromatogram of wild-type μ-cono toxin μ-CnIIIC in Example 1 of the present invention; Figure 3 This is the mass spectrum of wild-type μ-cono toxin μ-CnIIIC in Example 1 of the present invention; Figure 4 In Example 1 of this invention, μ-type conotoxin peptide [Lys] 2 Ser17 Chromatogram of ]-dK,dR-μ-CnIIIC; Figure 5 μ-type conotoxin peptide [Lys] in Example 1 of this invention 2 Ser 17 Mass spectrum of ]-dK,dR-μ-CnIIIC; Figure 6 Concentration-effect relationship of wild-type μ-conotoxin μ-CnIIIC on the inhibition of sodium current in resting and semi-inactive Nav1.4 in Example 2 of this invention; Figure 7 In Example 2 of this invention, the μ-type cone snail toxin polypeptide [Lys] 2 Ser 17 Concentration-effect relationship of ]-dK,dR-μ-CnIIIC on the inhibition of sodium current in resting and semi-inactive states of Nav1.4. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] In this invention, the term "μ-CnIIIC" refers to wild-type μ-conotoxin; “[Lys 2 Ser 17 "]-dK,dR-μ-CnIIIC" refers to a new, highly active conotoxin peptide formed by deleting pyroglutamic acid at the first position of the N-terminus of wild-type conotoxin, replacing the second amino acid with D-type arginine, and replacing the 17th and 6th amino acids with L-type polar uncharged amino acids, resulting in a sequence length that is one position shorter than that of wild-type conotoxin. The Chinese meaning of English abbreviations: “dR” refers to D-arginine; “S” refers to serine; “Y” refers to tyrosine; "DCM" refers to dichloromethane; "DIC" refers to N,N-diisopropylcarbodiimide; "DMF" refers to N,N-dimethylformamide; "HPLC" refers to High Performance Liquid Chromatography; “MeOH” refers to methanol; "MTBE" refers to methyl tert-butyl ether; “Oxyma” refers to ethyl 2-oxime cyanoacetate; "TFA" refers to trifluoroacetic acid; "Fmoc" refers to 9-fluorenemethyloxycarbonyl. "MS" refers to mass spectrometry. This invention is the first to discover that by elongating the sequence of wild-type cone snail toxin (μ-CnIIIC), adding one D-arginine (d-Arg) at the C-terminus, and simultaneously replacing the 17th amino acid with serine (Ser), the 1st amino acid with D-lysine (d-Lys), and the 2nd amino acid with the basic amino acid lysine (Lys), highly active μ-CnIIIC is produced. This invention achieves the synthesis of analogs using solid-phase peptide synthesis technology, completes disulfide bond formation through liquid-phase oxidation, and finally obtains novel μ-cone snail toxin peptide [Lys] through high-performance liquid chromatography purification and lyophilization. 2 Ser 17 The synthesis process for ]-dK, dR-μ-CnIIIC is as follows: Figure 1 As shown.

[0026] Reagent K: lysis buffer, prepared according to the volume ratio of TFA: phenol: water: anisole: ethylene dithiol = 82.5: 5: 5: 5: 2.5.

[0027] Wild-type μ-CnIIIC: Sichuan Jisheng Biopharmaceutical Co., Ltd., batch number: 2023041201-3.

[0028] Example 1 μ-type conotoxin peptide [Lys] 2 Ser 17 Synthesis of ]-dK, dR-μ-CnIIIC (1) Preparation of Fmoc-d-Arg(Pbf)-Rink AM resin ① Weigh 1.500 g (0.96 mmol) of Rink Amide AM resin (containing 1% DVB crosslinking agent, 100~200 mesh, 0.64 mmol / g) and add it to a 60 mL peptide solid-phase reactor. Add 15 mL of DCM solution to the reactor. Set the shaking speed of the shaker to 550 r / min and shake for 45 min. Drain the solution and add DMF solution to wash the resin twice. The washing solvent volume is 15 mL / wash and the washing time is 3 min / wash. The shaking speed of the shaker is 500 r / min.

[0029] ② After washing, drain the solvent and add 15 mL of 20% piperidine / DMF solution to the resin in the reactor to remove the Fmoc protecting group. Shake at 500 r / min and 25℃ for 5 min, then drain the solution. Next, add another 15 mL of 20% piperidine / DMF solution to the resin and shake at 500 r / min and 25℃ for 15 min, then drain the solution. Wash the resin 5 times with DMF solution (15 mL / 3 min / wash).

[0030] ③ Weigh 1.245 g Fmoc-Arg(Pbf)-OH (2.0 eq, 1.92 mmol) and 0.465 g Oxyma (2 eq, 1.92 mmol) into a 50 mL beaker, add 10 mL DMF solution to dissolve, add 0.297 mL condensing agent DIC (2 eq, 1.92 mmol) to the amino acid solution to activate the reaction for 5 min, then add it to the above-mentioned deprotected resin, and shake at 500 r / min and 25℃ for 1 h; after the reaction is completed, wash the resin 5 times with DMF solution (15 mL / 3 min / time).

[0031] ④ Peptide chain elongation Following the sequence composition, steps ② and ③ were repeated until the last amino acid coupling was completed. The Fmoc protecting group was removed, and the resin was washed 5 times with DMF solution (15 mL / 3 min / time). Then, the resin was washed alternately with the following procedure: DCM × 5 times (15 mL / 3 min / time), MeOH × 5 times (15 mL / 3 min / time), until the resin reached a shrunken state. The resin was then placed in a vacuum drying oven and dried at 25°C to constant weight, yielding 7.111 g of peptide resin, with a yield of 98.5%.

[0032] ⑤ Pyrolysis Weigh 7.111 g of the dried resin obtained in step ④ above. Add freshly prepared and pre-cooled K reagent lysis buffer at a ratio of 15 mL lysis buffer per gram of peptide resin. React at 300 r / min, 25℃, and in the dark for 3 h. After the reaction is complete, slowly add the lysis buffer to pre-cooled MTBE solution at a ratio of 1:10 (v / v) of lysis buffer / methyl tert-butyl ether. A white precipitate forms. Centrifuge at 500 rpm, discard the supernatant, add fresh MTBE solution, shake, centrifuge, discard the supernatant, and repeat the centrifugation process 5 times. Collect the sludge-like white precipitate, and vacuum dry at 25℃ to constant weight. Finally, 2.339 g of white solid crude peptide is obtained, with a yield of 94.0%.

[0033] ⑥ Cycloning Weigh 0.100 g of the white solid crude peptide obtained in step ⑥ above, add 100 mL of disodium hydrogen phosphate / guanidine hydrochloride buffer solution, adjust the pH to 7.66, and stir the mixture at room temperature for 24 h. Monitor the reaction progress by HPLC. After the reaction is complete, it can be directly purified by HPLC.

[0034] ⑦ Preparative HPLC is used for peptide purification. The cyclization reaction solution from step ⑥ above was directly injected into the sample, and the sample purification was completed according to the gradient elution program in Table 1. The mobile phase A was 80% acetonitrile / water (containing 0.1% TFA), and the mobile phase B was water (containing 0.1% TFA). The detection wavelength was 220 nm, the flow rate was 10 mL / min, and the column specifications were 20×250 mm, 10 µm, and 120 A.

[0035] Table 1 Purification and elution procedure for crude product cyclization solution

[0036] The collected fractions were analyzed by MS and HPLC, the target fractions were combined, and the final product was obtained by freeze drying. 2 Ser 17 The total yield of 39.9 mg of dK and dR-μ-CnIIIC was 39.9%, with an HPLC purity of 98.629%. MS results showed [M+2H] 2+ =1297.0859, molecular weight correct. The sample obtained above will be used in experiments in Examples 2 and 3. The chromatogram of wild-type μ-CnIIIC is shown below. Figure 2 As shown, the mass spectrum of wild-type μ-CnIIIC is as follows: Figure 3 As shown. μ-type conotoxin peptide [Lys] 2 Ser 17 The chromatogram of ]-dK, dR-μ-CnIIIC is as follows Figure 4 As shown, μ-type conotoxin peptide [Lys] 2 Ser 17 The mass spectrum of ]-dK, dR-μ-CnIIIC is as follows Figure 5 As shown.

[0037] [Lys] prepared in Example 1 2 Ser 17 The amino acid sequence of ]-dK,dR-μ-CnIIIC is shown in SEQ ID NO.1, and the amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO.2.

[0038] Example 2: Manual patch-clamp technique for detecting the effect of peptides on Nav1.4 channel current Wild-type μ-CnIIIC can effectively block the Nav1.4 ion channel. To demonstrate the target and activity of the designed sequence, patch-clamp technique was used to measure [Lys] 2 Ser 17 The effect of dK, dR-μ-CnIIIC on the Nav1.4 ion channel current is shown in the detailed test procedure below: The intracellular and extracellular fluids used in this embodiment have an ionic composition close to that of "cell physiology". The extracellular fluid components are: 137mM NaCl, 4 mM KCl, 1 mM MgCl2•6H2O, 2 mM CaCl2•2H2O, 10 mM D-Glucose, 10 mM HEPES, and NaOH to adjust the pH to 7.4. The intracellular fluid composition was: 50 mM CsCl, 60 mM CsF, 10 mM HEPES, 20 mM EGTA, 10 mM NaCl, and CsOH adjusted to pH 7.2.

[0039] In this embodiment, the CHO cell line Nav1.4 (SCN4A, gene information: NM_000334) stably expressing the Nav1.4 channel was used. Before the electrophysiological experiments, the Nav1.4 cell line should be maintained at less than 70% of its maximum density during the logarithmic growth phase. All reagents were preheated to 37°C before use. The old culture medium was discarded from a 6 cm culture dish, 1 mL of PBS was added, the dish was gently shaken to rinse the bottom, and then removed. 1 mL of trypsin was then added, and the dish was gently shaken to cover all cells. The cells were cultured at 37°C for 2–3 minutes, and then gently pipetted to suspend the adherent cells. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The cell concentration was adjusted to 2 × 10⁻⁶ cells / min. 3 / mL, 500 µL of cell solution was seeded into cell spreads in 24-well plates, and patch-clamp assays were performed after the cells adhered well.

[0040] In this embodiment, the sample concentration was 10 mM. The test solution was prepared using deionized water, and the concentrations were 1000 nM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM and 1 nM.

[0041] In this embodiment, the whole-cell patch-clamp recording experiment used Patchmaster software to acquire and store Nav1.4 sodium current data on a computer via an EPC-10 amplifier. The specific testing procedure is as follows: 1) First, use forceps to remove the cell slide from the cell culture dish, add extracellular fluid, and place it in the bath on the stage of an inverted microscope. Then, use a P-1000 microelectrode puller to pull a glass microtube, fill the microtube (recording electrode) with intracellular fluid to 1 / 3 volume, and place it in the electrode holder. Use a motorized micromanipulator (Scientifica-Double1U) to bring the recording electrode into contact with the cell surface. After the sealing resistance between the recording electrode and the cell membrane is >1 GΩ, apply negative pressure to rupture the membrane to form a whole-cell recording mode. After the membrane rupture is stable, compensate for the membrane capacitance (Cs) and series resistance (Rs).

[0042] 2) Stimulation procedure: ① A square wave pulse was applied with a clamping voltage of -120 mV, ranging from -120 to -10 mV, with a step voltage of 10 mV and a duration of 8000 ms. The voltage was then stepped back to -10 mV for 30 ms, and finally returned to -120 mV. A graph was plotted with membrane potential on the x-axis and relative current I / Imax on the y-axis. The Boltzmann equation I / Imax = 1 / {1 + exp[(V - V1 / 2) / k]} was used for fitting, yielding the steady-state inactivation curve (V1 / 2). 1 / 2 (where k is the conditional pulse voltage when half of the channels are inactive, and k is the slope factor).

[0043] ② The clamping voltage was -120 mV, depolarized to 0 mV for 40 ms to stimulate the resting current of the sodium channel, and then stepped to the conditional pulse voltage of the V1 / 2 channel when half of the channel is inactive for 8000 ms. The voltage was then repolarized to -120 mV for 30 ms, depolarized to 0 mV for 40 ms to stimulate the half-inactive current of the sodium channel, and finally returned to -120 mV. The current was recorded every 20 s.

[0044] 3) At room temperature, record the Nav1.4 sodium channel current before drug administration. After the control current value reaches steady state, i.e., after the most recent four consecutive current recording lines coincide, use the cumulative drug administration method to add negative (extracellular fluid) and seven drug concentrations (from low to high) in sequence.

[0045] 4) Data Analysis The raw data Nav1.4 current peak value was extracted from PatchMaster software. The formula for calculating the current suppression rate is as follows: Peak current suppression rate = (1 - Peak current compound / Peak current vehicle). The mean and standard error are calculated for each concentration. The concentration-effect relationship is obtained by fitting the Hill equation: I = Imax. {1 / [1+(C 1 / 2 / [C])h]},where[C] represents the drug concentration, C 1 / 2 The half-maximal inhibitory concentration (IC50) 50 h is the Hill coefficient, and the analysis and statistics were completed using GraphpadPrism 8.0.2 software.

[0046] Using tetrodotoxin as a positive control, the half-maximal inhibitory concentration (IC50) of the compound on the Nav1.4 ion channel was determined using manual patch-clamp technique. 50 The concentration-effect relationship between wild-type μ-CnIIIC and the inhibition of Nav1.4 sodium current in resting and semi-inactive states, fitted by the Hill equation, is shown in the figure below. Figure 6 [Lys] 2 Ser 17 The concentration-effect relationship between ]-dK, dR-μ-CnIIIC and the inhibition of Nav1.4 sodium current in resting and semi-inactive states is shown in the figure. Figure 7 .

[0047] from Figures 6-7 The results show that, compared with wild-type μ-CnIIIC, [Lys 2 Ser 17 ]-dK,dR-μ-CnIIIC can specifically activate Nav1.4 ion channels, and its resting state IC 50 The concentration was 7.746 nM, which is 25.3 times that of wild-type μ-CnIIIC; the IC50 in the semi-inactivated state was... 50 The concentration was 7.0342 nM, which is 25.7 times that of wild-type μ-CnIIIC in the semi-inactive state, indicating that [Lys 2 Ser 17 ]-dK,dR-μ-CnIIIC exhibits stronger inhibitory activity against Nav1.4, further demonstrating that [Lys 2 Ser 17 ]-dK,dR-μ-CnIIIC exhibits higher activity and potency compared to wild-type μ-CnIIIC.

[0048] Example 3 Wild-type μ-CnIIIC and [Lys 2 Ser 17 Bioactivity assay of ]-dK, dR-μ-CnIIIC The mice used in the experiments of this invention were adult male Kunming mice, which were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Sciences. The animal experiments were approved by the Ethics Committee of Lanzhou Peptide Valley Research Institute.

[0049] Mice were allowed free access to food and water for one week prior to testing. Intramuscular injection of 20 μL was administered into the right anterior tibial muscle group of mice using a 50 μL microsyringe (30 G needle). An equal volume of physiological saline was injected as a negative control, and wild-type μ-CnIIIC was used as a positive control. Concentration gradients of 25 and 50 μM were established. The activity of the sample was assessed by observing the onset time and duration of drug effect after injection, as well as the behavior of the mice after injection, including toe clenching (inhibition of muscle contraction), leg dragging / paralysis (anesthetic effect), and death behavior.

[0050] The mouse toe-clamping (inhibition of muscle contraction) behavior was analyzed using the mouse toe abduction scoring test (DAS). Mice were suspended by their tails to elicit a characteristic shock response in terms of hind limb extension and abduction. The mice were then injected with saline, wild-type μ-CnIIIC, and different concentrations of μ-conotoxin peptide [Lys] into the right anterior tibial muscle group. 2 Ser 17 After dK, dR-μ-CnIIIC, the degree of toe abduction of the left and right hind limbs was measured as a function of time. The corresponding behaviors were observed according to a 5-point scale (0 indicates normal, 1 indicates only 2 toes (index and middle toes) are together, 2 indicates 3 toes (index, middle, and little toes) are together; 3 indicates 4 toes except the ring toe are together; 4 indicates the maximum reduction in toe abduction and leg extension).

[0051] The dragging / paralysis (anesthetic effect) behavior in mice was observed by injecting the right tibialis anterior muscle group into the mice and then placing them in a new environment to stimulate their exploratory instincts. In the early stages of the dragging / paralysis effect, the mice crawled forward with their lower body close to the ground; in the later stages, the entire body became paralyzed. Toe clenching and dragging / paralysis are two separate behaviors in mice, and they generally occur simultaneously.

[0052] The experimental results are shown in Table 2: Table 2 Wild-type μ-CnIIIC and [Lys] 2 Ser 17 Animal experiment results (50 μM) of ]-dK, dR-μ-CnIIIC

[0053] Note 1: " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated. Table 2 shows that after intramuscular injection of 50 μM wild-type μ-CnIIIC into the hind leg of mice, the mice exhibited toe-clamping behavior, indicating that wild-type μ-CnIIIC at a concentration of 50 μM exhibited inhibitory activity against muscle contraction. However, injection of the same concentration of [Lys...] 2Ser 17 Following [-dK, dR-μ-CnIIIC], mice exhibited lethal behaviors due to short-term overdose. Specifically, mice showed obvious leg dragging and toe-clamping behavior 3 minutes after injection, and became paralyzed and immobile 4 minutes later, eventually dying 8 minutes later. This indicates that [Lys] 2 Ser 17 ]-dK,dR-μ-CnIIIC exhibits higher activity compared to wild-type μ-CnIIIC.

[0054] To further explore [Lys 2 Ser 17 The activity of ]-dK, dR-μ-CnIIIC will [Lys 2 Ser 17 The concentration of ]-dK,dR-μ-CnIIIC was reduced to 25 μM, and the results are shown in Table 3.

[0055] Table 3 [Lys] 2 Ser 17 Animal experiment results (25 μM) of ]-dK, dR-μ-CnIIIC

[0056] Table 3 shows the results of intramuscular injection of 25 μM [Lys] in the hind leg of mice. 2 Ser 17 After 5 minutes of treatment with dK, dR-μ-CnIIIC, mice exhibited significant leg-dragging behavior, which lasted for 16 minutes, ultimately leading to death due to excessive dosage. [Lys] 2 Ser 17 ]-dK,dR-μ-CnIIIC still exhibits activity in inhibiting muscle contraction and paralysis at low concentrations, and shows high activity.

[0057] To further explore and study [Lys] 2 Ser 17 The safety of maintaining the activity of ]-dK,dR-μ-CnIIIC under low concentration conditions, [Lys 2 Ser 17 The concentration of dK, dR-μ-CnIIIC was reduced to 10 μM, and the results of animal experiments are shown in Table 4. Table 4 [Lys] 2 Ser 17 Animal experimental results of ]-dK, dR-μ-CnIIIC (10 μM)

[0058] Table 4 shows the results of intramuscular injection of 10 μM [Lys] in the hind leg of mice. 2 Ser 17 After 10 minutes of treatment with dK, dR-μ-CnIIIC, mice began to exhibit obvious leg-dragging and toe-clamping behaviors, which lasted for more than 300 minutes. [Lys] 2 Ser 17 ]-dK,dR-μ-CnIIIC still exhibited inhibitory activity against muscle contraction and paralysis at a lower dose of 10 μM. Meanwhile, mice remained in good condition during the observation period without significant toxic side effects, indicating that this peptide has good safety while exerting its pharmacological effects.

[0059] Subsequently, mice were treated with μ-CnIIIC at two concentrations of 100 μM and 500 μM. The onset time, duration of action, and toe-clamping (inhibition of muscle contraction) behavior of the mice after injection were observed. The results are shown in Table 5.

[0060] Table 5. Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM.

[0061] Note 1: " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated. Table 5 shows that mice injected with an equal volume of physiological saline at each concentration appeared normal. The results indicate that death only occurred after intramuscular injection of 500 μM wild-type μ-CnIIIC into the hind leg, further demonstrating the efficacy of μ-type conotoxin peptide [Lys]. 2 Ser 17 The activity of ]-dK,dR-μ-CnIIIC is more than 20 times that of wild-type μ-CnIIIC.

[0062] In summary, this invention elongates the sequence of wild-type conotoxin μ-CnIIIC by adding one D-arginine (d-Arg) at the C-terminus, replacing the 17th amino acid with serine (Ser), the 1st amino acid with D-lysine (d-Lys), and the 2nd amino acid with lysine (Lys). The analogue was synthesized using solid-phase peptide synthesis technology, and the disulfide bond was formed via liquid-phase oxidation. Finally, the novel conotoxin peptide [Lys] was obtained through high-performance liquid chromatography purification and lyophilization. 2 Ser 17 ]-dK, dR-μ-CnIIIC, forming a new highly active conotoxin peptide [Lys] that is one position shorter than the wild-type μ-conotoxin sequence. 2 Ser 17]-dK, dR-μ-CnIIIC. Detected by patch clamp, [Lys 2 Ser 17 [Lys]-dK,dR-μ-CnIIIC can specifically block Nav1.4 channels and can be used in the preparation of drugs for the treatment or prevention of diseases related to Nav1.4 sodium ion channels. Mouse experiments have shown that [Lys] 2 Ser 17 ]-dK,dR-μ-CnIIIC can reduce or inhibit muscle contraction, and has analgesic and anesthetic effects. It can be used in the preparation of drugs for the treatment or prevention of pain.

[0063] 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 principles of the present invention should be included within the protection scope of the present invention.

[0064] sequence list SEQ ID NO.1 Three characters: d-Lys-Lys -Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg- Ser -His-Ala-Arg-Cys-Cys- d-Arg -NH2 Single character: kK CCNGPKGCSSKWCR S HARCC r -NH2 SEQ ID NO.2 Three characters: Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2 Single character: (X represents pyroglutamic acid) XGCCNGPKGCSSKWCRDHARCC-NH2

Claims

1. A μ-type conotoxin peptide, characterized in that, The amino acid sequence of the μ-type cone snail toxin peptide is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the μ-type cone snail toxin peptide of claim 1.

3. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the polynucleotide as described in claim 2.

4. An expression carrier, characterized in that, The expression vector comprises the nucleic acid construct according to claim 3.

5. A transformed cell, characterized in that, It comprises the nucleic acid construct of claim 3 or the expression vector of claim 4.

6. The use of the μ-type cone snail toxin peptide according to claim 1 in the preparation of drugs for treating or preventing diseases related to sodium ion channels.

7. The application according to claim 6, characterized in that, The sodium ion channel-related diseases mentioned are any one of epilepsy, arrhythmia, muscle paralysis, tonic-clonic syndrome, and autism spectrum disorder.

8. The use of the μ-type cone snail venom peptide according to claim 1 in the preparation of drugs for treating or preventing pain.

9. The use of the μ-type cone snail venom peptide according to claim 1 in the preparation of anesthetic drugs.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the μ-type cone snail toxin peptide according to claim 1.

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