μ-type conotoxin peptide [Ser 17 ]-dR-μ-CnIIIC and its applications

Abstract

This invention discloses μ-type conotoxin peptide [Ser 17 ]-dR-μ-CnIIIC and its applications. The μ-type conotoxin peptide [Ser] provided by this invention 17 ]-dR-μ-CnIIIC, whose amino acid sequence is shown in SEQ ID NO.1. μ-type conotoxin peptide [Ser 17 Compared to wild-type μ-CnIIIC, dR-μ-CnIIIC exhibits 20-fold increased animal activity, with its IC50... 50 It contains 10 times more conotin than wild-type conotin. Furthermore, [Ser...] 17 The amino acid sequence of ]-dR-μ-CnIIIC is reduced by one position compared to wild-type μ-CnIIIC, which can further reduce the synthesis cost. The [Ser] of this invention... 17 ]-dR-μ-CnIIIC expands the application of μ-type conotoxin peptides in the biomedical field.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry and molecular biology, and particularly to μ-conotoxin peptide [Ser 17 ]-dR-μ-CnIIIC and its applications. Background Technology

[0002] Cone snail toxins are secreted by venom glands in the venom ducts and venom sacs of the marine gastropod mollusc Conus. They are cocktail-like toxins composed of many single toxic peptides. The main components are some active polypeptide compounds with high specificity to different ion channels and nerve receptors. Most of them are composed of 10 to 40 amino acid residues and are rich in two or three pairs of disulfide bonds. They are the smallest nucleic acid-encoded animal neurotoxin peptides discovered and also the small peptides with the highest disulfide bond density. They can act on various types and subtypes of ion channels and receptors.

[0003] However, the stability and bioactivity of natural cone snail toxins are low. Due to their complex structure and the presence of multiple disulfide bonds, chemical synthesis of cone snail toxins is challenging and costly for large-scale production and purification. Therefore, there is an urgent need for a method to improve the bioactivity and efficacy of natural cone snails while reducing production costs. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a μ-type conotoxin peptide [Ser 17 ]-dR-μ-CnIIIC and its applications. This invention aims to provide a conotoxin peptide with higher activity than wild-type conotoxin, which can be used to specifically block the Nav1.4 channel, reduce muscle contraction, reduce or eliminate the formation of wrinkles on the human skin surface, and can also be used for muscle relaxation and analgesia.

[0005] This invention is the first to discover that replacing the 17th amino acid of wild-type cone snail toxin (μ-CnIIIC) with serine (Ser), while simultaneously deleting the first amino acid at the N-terminus of the original sequence and replacing the second amino acid with D-arginine (d-Arg), greatly enhances the activity of μ-CnIIIC. The modified cone snail toxin peptide is named [Ser 17 ]-dR-μ-CnIIIC.

[0006] The μ-type conotoxin peptide [Ser] provided by this invention 17 The amino acid sequence of ]-dR-μ-CnIIIC is shown in SEQ ID NO.1; the amino acid sequence contains three pairs of disulfide bonds, which are located in Cys 2 -Cys 14 Cys 3 -Cys 20 and Cys 9-Cys 21 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 [Ser 17 ]-dR-μ-CnIIIC.

[0008] The present invention also provides a nucleic acid comprising the aforementioned polynucleotide and a nucleotide encoding a purification tag.

[0009] The present invention also provides a carrier comprising the polynucleotide or the nucleic acid.

[0010] The present invention also provides a host cell comprising, according to, the polynucleotide, the nucleic acid, or the vector.

[0011] The present invention also provides a composition comprising the aforementioned μ-type conotoxin peptide [Ser 17 ]-dR-μ-CnIIIC; the μ-type conotoxin peptide [Ser 17 The effective dose of ]-dR-μ-CnIIIC is 0.4–50 μg / kg. Specifically, [Ser 17 When used for muscle relaxation and analgesia in humans, the effective dose of ]-dR-μ-CnIIIC is 0.4–2 μg / kg, and when used for muscle relaxation and analgesia in animals such as mice, the effective dose is 10–50 μg / kg.

[0012] Furthermore, the dosage form of the composition is any one of tablets, capsules, pills, solutions, absorbents, and ointments.

[0013] This invention also provides the μ-type conotoxin peptide [Ser 17 The use of any one of ]-dR-μ-CnIIIC, the polynucleotide, the nucleic acid, the carrier, the host cell, or the composition in the preparation of a medicament for the treatment or prevention of diseases related to sodium ion channels.

[0014] Furthermore, the sodium ion channel-related diseases are any one of epilepsy, arrhythmia, muscle paralysis, tonic-clonic syndrome, and autism spectrum disorder.

[0015] This invention also provides the μ-type conotoxin peptide [Ser 17 The use of any one of ]-dR-μ-CnIIIC, the polynucleotide, the nucleic acid, the vector, the host cell, or the composition in any of the following:

[0016] (i) Use in the preparation of medicines for the treatment or prevention of pain;

[0017] (ii) Application in the preparation of anesthetic drugs.

[0018] Furthermore, the pain is migraine, acute pain, persistent pain, chronic pain, neuropathic pain, or nociceptive pain.

[0019] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0020] 1. The μ-type cone snail toxin peptide [Ser] provided by this invention 17 ]-dR-μ-CnIIIC, compared to wild-type μ-CnIIIC, showed a 20-fold increase in animal activity, with its IC50... 50 is 10 times that of wild-type conoside. 17 The amino acid sequence of ]-dR-μ-CnIIIC is reduced by one position compared to wild-type μ-CnIIIC, which can further reduce the synthesis cost.

[0021] 2. The μ-type cone snail toxin peptide [Ser] of the present invention 17 ]-dR-μ-CnIIIC can specifically block Nav1.4 channels, reduce muscle contraction, reduce or eliminate the formation of wrinkles on the surface of human skin, and can also be used for muscle relaxation and analgesia, and has an anesthetic effect. Attached Figure Description

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The μ-type conotoxin peptide [Ser] of the present invention 17 Schematic diagram of the synthesis process of ]-dR-μ-CnIIIC;

[0024] Figure 2 This is the chromatogram of wild-type μ-cono toxin μ-CnIIIC in Example 1 of the present invention;

[0025] Figure 3 This is the mass spectrum of wild-type μ-cono toxin μ-CnIIIC in Example 1 of the present invention;

[0026] Figure 4 In Example 1 of this invention, the μ-type conotoxin peptide [Ser] 17 Chromatogram of ]-dR-μ-CnIIIC;

[0027] Figure 5 In Example 1 of this invention, the μ-type conotoxin peptide [Ser] 17 Mass spectrum of ]-dR-μ-CnIIIC.

[0028] 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;

[0029] Figure 7 In Example 2 of this invention, μ-type conotoxin peptide [Ser] 17 Concentration-effect relationship of ]-dR-μ-CnIIIC on the inhibition of sodium current in resting and semi-inactive states of Nav1.4. Detailed Implementation

[0030] 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.

[0031] In this invention, the term "μ-CnIIIC" refers to wild-type μ-conotoxin;

[0032] “[Ser 17 "-dR-μ-CnIIIC" refers to a new, highly active conotoxin formed by deleting the first pyroglutamic acid at the N-terminus of wild-type murotoxin, replacing the second amino acid with a D-type basic amino acid, and replacing the 17th amino acid with an L-type polar uncharged amino acid, resulting in a sequence length that is one position shorter than that of wild-type murotoxin.

[0033] The Chinese meaning of the English abbreviation of this invention:

[0034] “d-Arg(dR)” refers to D-arginine.

[0035] “S” refers to serine;

[0036] "DCM" refers to dichloromethane;

[0037] "DIC" refers to N,N-diisopropylcarbodiimide;

[0038] "DMF" refers to N,N-dimethylformamide;

[0039] "HPLC" refers to High Performance Liquid Chromatography;

[0040] “MeOH” refers to methanol;

[0041] "MTBE" refers to methyl tert-butyl ether;

[0042] “Oxyma” refers to ethyl 2-oxime cyanoacetate;

[0043] "TFA" refers to trifluoroacetic acid;

[0044] "Fmoc" refers to 9-fluorenemethyloxycarbonyl.

[0045] "Fmoc-Cys(Trt)-OH" refers to 9-fluorenylmethoxycarbonyl-cysteine-hydroxyphenylcarboxylic acid, where "Fmoc" refers to 9-fluorenylmethoxycarbonyl, "Cys" refers to cysteine, "Trt" refers to triphenylmethyl, and "OH" refers to hydroxyl.

[0046] "MS" refers to mass spectrometry.

[0047] IC 50 "" refers to the half-inhibitory concentration (WIC) of the tested drug formulation.

[0048] Reagent K: lysis buffer, prepared according to the volume ratio of TFA: phenol: water: anisole: ethylene dithiol = 82.5: 5: 5: 5: 2.5.

[0049] Cell lysate: HAM'S / F-12 medium containing 10% fetal bovine serum and 10 μg / mL Blasticidin S.

[0050] Extracellular fluid: 137mM NaCl, 4mM KCl, 1mM MgCl2·6H2O, 2mM CaCl2·2H2O, 10mM D-Glucose, 10mM HEPES, pH adjusted to 7.4 with NaOH;

[0051] Intracellular fluid: 50mM CsCl, 60mM CsF, 10mM HEPES, 20mM EGTA, 10mM NaCl, CsOH adjusted to pH 7.2.

[0052] Wild-type μ-CnIIIC: Sichuan Jisheng Biopharmaceutical Co., Ltd., batch number: 2023041201-3.

[0053] Cell lines:

[0054] CHO cell line, Nav1.4 (SCN4A, gene information: NM_000334).

[0055] Example 1 μ-type conotoxin peptide [Ser 17 Synthesis of ]-dR-μ-CnIIIC

[0056] (1) Preparation of Fmoc-Cys(Trt)-Rink MBHA resin

[0057] ① Weigh 1.500 g (1.005 mmol) of Rink Amide MBHA resin (containing 1% DVB crosslinking agent, 100-200 mesh, 0.67 mmol / g) and add it to a 60 mL peptide solid-phase reactor. Then 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. Wash the resin twice with DMF solution, with a washing solvent volume of 15 mL / wash and a washing time of 3 min / wash. The shaking speed of the shaker is 500 r / min.

[0058] 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 rpm and 25°C for 5 min, then drain the solution. Next, add another 15 mL of 20% piperidine / DMF solution to the resin and shake at 500 rpm and 25°C for 15 min, then drain the solution. Wash the resin 5 times with DMF solution (15 mL / 3 min / wash).

[0059] ③ Weigh 1.766g Fmoc-Cys(Trt)-OH (3eq, 3.015mmol) and 0.428g Oxyma (3eq, 3.015mmol) into a 50mL beaker, add 10mL DMF solution to dissolve, add 0.467mL condensing agent DIC (3eq, 3.015mmol) to the amino acid solution to activate the reaction for 5min, then add it to the above-mentioned deprotected resin, and shake at 500r / min and 25℃ for 1h; after the reaction is completed, wash the resin 5 times with DMF solution (15mL / 3min / time).

[0060] ④ Peptide chain elongation

[0061] 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 5.490 g of peptide resin.

[0062] ⑤ Pyrolysis

[0063] Weigh 5.490 g of the dried resin obtained in step ④ above. Add freshly prepared and pre-cooled K reagent lysis buffer at a ratio of 10 mL lysis buffer per gram of peptide resin. React at 300 rpm and 25 °C 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 °C to constant weight. Finally, 2.335 g of white solid crude peptide is obtained, with a yield of 99.6%.

[0064] ⑥ Cycloning

[0065] 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 8.2, 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.

[0066] ⑦ Preparative HPLC is used for peptide purification.

[0067] The cyclization reaction solution from step ⑥ above was directly injected into the sample, and the sample was purified 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.

[0068] Table 1 Purification and elution procedure for crude product cyclization solution

[0069]

[0070] The collected fractions were analyzed by MS and HPLC. The target fractions were combined, freeze-dried, and used in subsequent experiments in Examples 2 and 3. 17 The purification yield of ]-dR-μ-CnIIIC exceeded 36.6%, with an HPLC purity of 98.183%. MS results showed: [M+2H] 2+ =1168.5021, molecular weight correct. Wild-type μ-CnIIIC chromatogram as 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 [Ser 17 The chromatogram of ]-dR-μ-CnIIIC is as follows Figure 4 As shown, μ-type conotoxin peptide [Ser 17 The mass spectrum of ]-dR-μ-CnIIIC is as follows Figure 5As shown.

[0071] The amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO.2. [Ser] prepared in Example 1 17 The amino acid sequence of ]-dR-μ-CnIIIC is shown in SEQ ID NO.1.

[0072] Example 2: Manual patch-clamp technique for detecting the effect of compounds on the Nav1.4 channel current.

[0073] Wild-type μ-CnIIIC can effectively block Nav1.4 ion channels, as determined by patch-clamp technique [Ser 17 The effect of ]-dR-μ-CnIIIC on the current of Nav1.4 ion channels was investigated to demonstrate the target and activity of the designed sequence.

[0074] 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, 4mM KCl, 1mM MgCl2·6H2O, 2mM CaCl2·2H2O, 10mM D-Glucose, 10mM HEPES, and NaOH to adjust the pH to 7.4.

[0075] The intracellular fluid composition was: 50mM CsCl, 60mM CsF, 10mM HEPES, 20mM EGTA, 10mM NaCl, and CsOH adjusted to pH 7.2.

[0076] In this embodiment, a CHO cell line stably expressing the Nav1.4 channel was used, Nav1.4 (SCN4A, gene information: NM_000334).

[0077] Before the electrophysiological experiments, the Nav1.4 cell line was maintained at less than 70% of its maximum density during the logarithmic growth phase. All reagents were preheated to 37°C before use. The spent culture medium was discarded from a 6cm culture dish, 1mL of PBS was added, the dish was gently shaken to rinse the bottom, and then removed. 1mL of trypsin was then added, and the dish was gently shaken to cover all cells. The cells were incubated 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 × 10⁶ cells / min. 3 Cells / mL were seeded into 500 μL of cell culture in 24-well cell slides. After the cells adhered well, patch-clamp assays were performed.

[0078] The sample was prepared at a concentration of 10 mM. Test solutions were prepared using deionized water at concentrations of 1000 nM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM, and 1 nM.

[0079] Whole-cell patch-clamp recording experiments were performed using Patchmaster software. Nav1.4 sodium current data were acquired and stored on a computer via an EPC-10 amplifier. The specific testing procedure is as follows:

[0080] 1) First, use forceps to remove the cell slide from the cell culture dish, add extracellular fluid, and place it in a 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 stabilizes, compensate for the membrane capacitance (Cs) and series resistance (Rs).

[0081] 2) Stimulation procedure:

[0082] ① With a clamping voltage of -120mV, a square wave train stimulation of -120 to -10mV, stepping 10mV for 8000ms, was applied, followed by a step to -10mV for 30ms, and finally returned to -120mV. 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 to obtain the steady-state deactivation curve (V1 / 2). 1 / 2 (where k is the conditional pulse voltage when half of the channels are deactivated).

[0083] ② The clamping voltage is -120mV, depolarized to 0mV for 40ms, stimulating the resting current of the sodium channel. Then, it steps to the conditional pulse voltage of the inactivation curve V1 / 2 when the channel is half inactivated for 8000ms. It is then repolarized to -120mV for 30ms, depolarized to 0mV for 40ms, stimulating the half-inactivated current of the sodium channel. Finally, it returns to -120mV. The current is recorded every 20s.

[0084] 3) At room temperature, record the Nav1.4 sodium channel current before drug administration. After the control current value reaches steady state (i.e., the four most recent consecutive current recording lines coincide), use the cumulative drug administration method to sequentially add negative (extracellular fluid) and seven drug concentrations (from low to high).

[0085] 4) Data Analysis

[0086] The raw data Nav1.4 current peak value was extracted from PatchMaster software. The formula for calculating the current suppression rate is as follows:

[0087] Peak current suppression rate = ((1-Peak current compound / Peak current vehicle), calculate the mean and standard error for each concentration, and 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 Graphpad Prism 8.0.2 software.

[0088] 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. Figure 6 [Ser 17 The concentration-effect relationship between ]-dR-μ-CnIIIC and the inhibition of sodium current in the resting and semi-inactive states of Nav1.4 is shown in the figure. Figure 7 .

[0089] from Figures 6-7 As can be seen, compared with the half-maximal inhibitory concentration (50% MIC) of wild-type μ-CnIIIC for the Nav1.4 ion channel at rest (196.33 nM), [Ser 17 ]-dR-μ-CnIIIC can specifically activate Nav1.4 ion channels, and its resting state IC50... 50 The concentration was 19.168 nM, which is 10.2 times that of wild-type μ-CnIIIC. The IC50 in the semi-inactive state... 50 The concentration was 17.800 nM, which is 10.1 times that of wild-type μ-CnIIIC in the semi-inactive state, indicating that [Ser 17 ]-dR-μ-CnIIIC exhibits stronger inhibitory activity against Nav1.4 than wild-type μ-CnIIIC.

[0090] Example 3 Wild-type μ-CnIIIC and [Ser 17 Bioactivity assay of ]-dR-μ-CnIIIC

[0091] 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.

[0092] 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 (30G needle). An equal volume of physiological saline was injected as a negative control, and wild-type μ-CnIIIC was used as a positive control. Multiple concentration gradients were established: 25 μM and 50 μM. 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.

[0093] 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 [Ser] into the right anterior tibial muscle group. 17 After administration of ]-dR-μ-CnIIIC, the degree of toe abduction in the left and right hind limbs was measured as a function of time, and scored according to a 5-point scale (0 = normal to 4 = maximum reduction in toe abduction and leg extension). Mouse dragging / paralysis (anesthetic effect) behavior was observed by injecting the right tibialis anterior muscle group into mice and placing them in a new environment to stimulate their exploratory instincts. In the early stages of the dragging / paralysis effect, mice crawled forward with their lower body close to the ground; in the later stages, their entire body became paralyzed.

[0094] The experimental results are shown in Tables 2-4:

[0095] Table 2 μ-CnIIIC and [Ser 17 Results of mouse experiments treated with dR-μ-CnIIIC (50 μM)

[0096]

[0097] Note 1: " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated.

[0098] 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 [Ser...] 17 Following administration of ]-dR-μ-CnIIIC, mice exhibited lethal behavior due to short-term overdose. Specifically, mice showed obvious leg-dragging behavior 4 minutes after injection, followed by immediate death, indicating that [Ser 17]-dR-μ-CnIIIC exhibits higher activity compared to wild-type μ-CnIIIC.

[0099] To further explore [Ser 17 The activity of ]-dR-μ-CnIIIC will [Ser 17 The concentration of ]-dR-μ-CnIIIC was reduced to 25 μM, and the results are shown in Table 3:

[0100] Table 3 [Ser] 17 Results of mouse experiments treated with dR-μ-CnIIIC (25 μM)

[0101]

[0102] Note 1: " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated.

[0103] Table 3 shows the results: 25 μM concentration [Ser] injected intramuscularly into the hind leg of mice. 17 Following administration of ]-dR-μ-CnIIIC, mice immediately exhibited pronounced leg-dragging behavior, which lasted for 16 minutes, ultimately leading to death due to excessive dosage. [Ser] 17 ]-dR-μ-CnIIIC still exhibits activity in inhibiting muscle contraction and paralysis at low concentrations, and shows high activity.

[0104] Table 4. Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM.

[0105]

[0106] Note 1: " / " indicates that the mice were asymptomatic after the drug injection, so the onset time and duration of action could not be calculated.

[0107] Table 4 shows that mice injected with an equal volume of saline at each concentration appeared normal. Death only occurred after intramuscular injection of 500 μM wild-type μ-CnIIIC into the calf muscles, further demonstrating the efficacy of μ-conotoxin peptide [Ser...]. 17 The activity of ]-dR-μ-CnIIIC is 20 times that of wild-type μ-CnIIIC.

[0108] In summary, μ-conotoxin peptide [Ser 17 The activity of ]-dR-μ-CnIIIC was 20-fold higher than that of wild-type μ-CnIIIC. Patch-clamp results showed that the activity of μ-conotoxin peptide [Ser 17 IC of ]-dR-μ-CnIIIC 50 It is more than 10 times that of the wild type, indicating that [Ser 17]-dR-μ-CnIIIC can specifically block Nav1.4 channels, reduce or inhibit muscle contraction, and has analgesic and anesthetic effects.

[0109] 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.

[0110]

Claims

1. μ-type conotoxin peptide [Ser] 17 ]-dR-μ-CnIIIC, characterized in that, The μ-type conotoxin peptide [Ser] 17 The amino acid sequence of ]-dR-μ-CnIIIC is shown in SEQ ID NO.1; The μ-type conotoxin peptide [Ser] 17 The structure of ]-dR-μ-CnIIIC is as follows: The amino acid sequence contains three pairs of disulfide bonds, which are located in Cys. 2 -Cys 14 Cys 3 -Cys 20 and Cys 9 -Cys 21 Location.

2. A polynucleotide, characterized in that, The polynucleotide encodes the μ-type conotoxin peptide [Ser] of claim 1. 17 ]-dR-μ-CnIIIC.

3. A nucleic acid, characterized in that, The nucleic acid comprises the polynucleotide of claim 2 and the nucleotide encoding the purification tag.

4. A carrier, characterized in that, The vector comprises the polynucleotide of claim 2 or the nucleic acid of claim 3.

5. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 2, the nucleic acid of claim 3, or the vector of claim 4.

6. A composition, characterized in that, The composition comprises the μ-type conotoxin peptide [Ser] as described in claim 1. 17 ]-dR-μ-CnIIIC.

7. The composition according to claim 6, characterized in that, The dosage form of the composition is any one of tablets, capsules, pills, solutions, and ointments.

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