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] The present invention relates to the technical field of molecular biology, and in particular to a μ-type conotoxin peptide, a polynucleotide encoding the peptide and applications thereof. Background Art

[0002] Conotoxins are a set of very sophisticated neuropharmacological weapons formed by cone snails (Conus) during the long evolution process, used for predation and defense. Most of them are composed of 12 to 46 amino acid residues, most of which contain two or three pairs of disulfide bonds. They mainly act on various ion channels on the cell membrane and receptors of neurotransmitters and kinins, blocking or enhancing the transmission of nerve excitation signals, and have neurotoxicity. μ-conotoxin (μ-CnIIIC) is a conotoxin peptide composed of 22 amino acid residues among conotoxins, and is an effective antagonist of voltage-gated Nav1.4 sodium channel.

[0003] The hydrophilicity and membrane permeability of natural μ-conotoxin peptides are not high, and their solubility in the body is low, which is not conducive to the absorption and distribution of μ-CnIIIC peptides. They are easy to accumulate in non-target tissues, and have high potential toxicity and side effects. In addition, natural μ-CnIIIC peptides may be degraded by various enzymes in the body, 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] In view of the defects in the prior art, the present invention proposes a μ-conotoxin peptide, a polynucleotide encoding the same and its application. The present invention provides a μ-conotoxin peptide with higher activity than wild-type μ-conotoxin, which can specifically block the Nav1.4 channel, reduce muscle contraction, reduce or eliminate the formation of wrinkles on the surface of human skin, and can be used for muscle relief and analgesia.

[0005] The present invention discovers for the first time that the activity of μ-CnIIIC can be improved by extending the sequence of wild-type μ-conotoxin (μ-CnIIIC), adding a 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).

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

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

[0008] The present invention also provides a nucleic acid construct, which comprises the polynucleotide.

[0009] The present invention also provides an expression vector, which comprises the nucleic acid construct.

[0010] The present invention also provides a transformed cell, comprising the nucleic acid construct or the expression vector according to claim 4.

[0011] The present invention also provides the use of the μ-type conotoxin peptide in preparing a drug for treating or preventing diseases related to sodium ion channels.

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

[0013] The present invention also provides application of the μ-type conotoxin peptide in preparing medicine for treating or preventing pain.

[0014] The present invention also provides application of the μ-type conotoxin peptide in preparing anesthetic drugs.

[0015] The present invention also provides a pharmaceutical composition, which comprises the μ-type conotoxin peptide.

[0016] In some embodiments, the dosage form of the pharmaceutical composition is any one of a tablet, a capsule, a pill, a solution, an absorbent, and an ointment.

[0017] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0018] 1. The present invention provides a novel μ-type conotoxin peptide [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC, compared with the wild type, [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC increased its animal activity by more than 20 times, and its IC 50 It is 25.3 times that of wild-type conoin, which reduces the dosage required for use, reduces the toxicity and side effects caused by the drug, and reduces production costs.

[0019] 2. [Lys 2 ,Ser 17 The amino acid sequence composition of ]-dK,dR-μ-CnIIIC has an increased number of basic amino acids compared to the wild-type μ-CnIIIC, which can further improve the hydrophilicity and membrane permeability of the sequence, increase its solubility in the body, and reach the target cells or tissues more accurately.

[0020] 3. [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC retains the function of the wild-type μ-CnIIIC peptide and can specifically block the Nav1.4 channel, reducing the excitability and contractility of muscles.

[0021] 4.[Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC can effectively reduce muscle activity by specifically blocking the Nav1.4 channel, thereby inhibiting the formation of wrinkles and can be used as a non-invasive anti-wrinkle treatment.

[0022] 5.[Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC is a sodium channel blocking compound that 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The μ-type conotoxin peptide [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC synthesis process diagram;

[0025] Figure 2 This is a chromatogram of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0026] Figure 3 This is the mass spectrum of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0027] Figure 4 The μ-type conotoxin peptide [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC chromatogram;

[0028] Figure 5 The μ-type conotoxin peptide [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC mass spectrum;

[0029] Figure 6 Concentration-effect relationship diagram of wild-type μ-conotoxin μ-CnIIIC on the inhibition of Nav1.4 resting and semi-inactivated sodium currents in Example 2 of the present invention;

[0030] Figure 7 The μ-type conotoxin polypeptide [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC concentration-effect relationship diagram for the inhibition of Nav1.4 resting and semi-inactivated sodium currents. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0032] The term "μ-CnIIIC" in the present invention refers to the wild-type μ-conotoxin;

[0033] “[Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC" means that the first pyroglutamic acid at the N-terminus of the wild-type μ-conotoxin is deleted, the second amino acid is replaced by D-arginine, and the 17th and 6th amino acids are replaced by L-type polar uncharged amino acids, forming a new highly active cono peptide with a sequence length reduced by 1 position compared with the wild-type μ-conotoxin;

[0034] The Chinese meaning of the English abbreviation:

[0035] “dR” refers to D-arginine;

[0036] “S” refers to serine;

[0037] “Y” refers to tyrosine;

[0038] “DCM” means dichloromethane;

[0039] “DIC” refers to N,N-diisopropylcarbodiimide;

[0040] "DMF" means N,N-dimethylformamide;

[0041] “HPLC” means high performance liquid chromatography;

[0042] “MeOH” means methanol;

[0043] “MTBE” means methyl tert-butyl ether;

[0044] "Oxyma" means ethyl 2-oximecyanoacetate;

[0045] “TFA” means trifluoroacetic acid;

[0046] "Fmoc" means 9-fluorenylmethoxycarbonyl;

[0047] “MS” refers to mass spectrometry;

[0048] The present invention first discovered that the sequence of the wild-type μ-conotoxin (μ-CnIIIC) was extended, a D-arginine (d-Arg) was added to the C-terminus, and the 17th amino acid was replaced with serine (Ser), the 1st amino acid was replaced with D-lysine (d-Lys), and the 2nd amino acid was replaced with the basic amino acid lysine (Lys), resulting in highly active μ-CnIIIC. The present invention completes the synthesis of analogs through solid phase peptide synthesis technology, completes the formation of disulfide bonds through liquid phase oxidation, and finally obtains a novel μ-conotoxin peptide [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC, the synthesis process is as follows Figure 1 shown.

[0049] Reagent K: lysis solution, prepared according to the volume ratio of TFA:phenol:water:thioanisole:ethanedithiol=82.5:5:5:5:2.5.

[0050] Wild-type μ-CnIIIC: Sichuan Jisheng Biotechnology Co., Ltd., batch number: 2023041201-3.

[0051] Example 1 μ-type conotoxin peptide [Lys 2 ,Ser 17 Synthesis of ]-dK, dR-μ-CnIIIC

[0052] (1) Preparation of Fmoc-d-Arg(Pbf)-Rink AM resin

[0053] ① Weigh 1.500g (0.96mmol) of Rink Amide AM resin (containing 1% DVB cross-linker, 100-200 mesh, 0.64mmol / g) and add it to a 60mL peptide solid phase reactor, add 15mL of DCM solution to the reactor, set the shaking speed to 550r / min, shake for 45min, drain the solution, add DMF solution to wash the resin twice, the washing solvent volume is 15mL / time, the washing time is 3min / time, and the shaking speed is 500r / min.

[0054] ② After washing, drain the solvent, add 15mL of 20% piperidine / DMF solution to the reactor resin to remove the resin Fmoc protecting group, shake at 500r / min and 25℃ for 5min, and drain the solution; then add 15mL of 20% piperidine / DMF solution to the resin again, shake at 500r / min and 25℃ for 15min, and drain the solution. Wash the resin with DMF solution (15mL / 3min / time) for 5 times.

[0055] ③ Weigh 1.245g Fmoc-Arg(Pbf)-OH (2.0eq, 1.92mmol) and 0.465g Oxyma (2eq, 1.92mmol) into a 50mL beaker, add 10mL DMF solution to dissolve, add 0.297mL condensing agent DIC (2eq, 1.92mmol) to the amino acid solution for activation reaction for 5min, then add to the above deprotected resin, shake at 500r / min and 25℃ for 1h; after the reaction is completed, wash the resin 5 times with DMF solution (15mL / 3min / time).

[0056] ④ Peptide chain extension

[0057] According to the sequence composition, steps ② and ③ were repeated until the last amino acid was coupled. The Fmoc protecting group was removed and the resin was washed 5 times with DMF solution (15 mL / 3 min / time). The resin was then washed alternately according to the following procedure: DCM × 5 times (15 mL / 3 min / time), MeOH × 5 times (15 mL / 3 min / time). The resin was finally in a shrunk state and placed in a vacuum drying oven at 25 ° C to constant weight. Finally, 7.111 g of peptide resin was obtained with a yield of 98.5%.

[0058] ⑤ Cracking

[0059] Weigh 7.111g of the dry resin obtained in the above process ④, add the pre-prepared pre-cooled K reagent lysis solution according to the ratio of 15mL lysis solution per gram of peptide resin, and shake at 300r / min 25℃ to avoid light for 3h. After the reaction is completed, the lysis solution is slowly added dropwise to the pre-cooled MTBE solution according to the ratio of lysis solution / methyl tert-butyl ether = 1:10 (v / v), and a white precipitate is generated. Then centrifuge at 500rpm / min, discard the supernatant, add new MTBE solution, shake, centrifuge, discard the supernatant, repeat the above centrifugation process 5 times, collect the sludge-like white precipitate, vacuum dry at 25℃ to constant weight, and finally obtain 2.339g of white solid crude peptide with a yield of 94.0%.

[0060] ⑥Cyclization

[0061] Weigh 0.100 g of the crude white solid peptide obtained in step ⑥ above, add 100 mL of sodium hydrogen phosphate / guanidine hydrochloride buffer solution, adjust the pH to 7.66, stir and react at room temperature for 24 h, and monitor the reaction progress by HPLC. After the reaction is completed, HPLC purification can be performed directly.

[0062] ⑦Preparative HPLC for peptide purification

[0063] The cyclization reaction liquid of the above process ⑥ was directly injected, and the sample purification was completed according to the gradient elution program in Table 1, wherein mobile phase A: 80% acetonitrile / water (containing 0.1% TFA), mobile phase B: water (containing 0.1% TFA); detection wavelength: 220nm; flow rate: 10mL / min; chromatographic column specifications: 20×250mm, 10μm, 120A.

[0064] Table 1 Purification and elution procedures of crude cyclization solution

[0065]

[0066] The fractions were collected for MS and HPLC analysis, and the target fractions were combined and freeze-dried to obtain the final product [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC totaled 39.9 mg, yield 39.9%, HPLC purity 98.629%, MS results showed: [M+2H] 2+ =1297.0859, the molecular weight is correct. The sample obtained above will be used for the experiments of Examples 2 and 3. The chromatogram of wild type μ-CnIIIC is shown in Figure 2 As shown, the mass spectrum of wild-type μ-CnIIIC is shown Figure 3 As shown. μ-type conotoxin peptide [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC chromatogram is shown in Figure 4As shown, μ-conotoxin peptide [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC mass spectrum is shown in Figure 5 shown.

[0067] [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC amino acid sequence is shown in SEQ ID NO.1, and the amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO.2.

[0068] Example 2: Manual patch clamp technique to detect the effect of polypeptide on Nav1.4 channel current

[0069] Wild-type μ-CnIIIC can effectively block the Nav1.4 ion channel. In order to prove the target and activity of the designed sequence, the patch clamp technique was used to measure [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC effects on Nav1.4 ion channel current, the detailed test process is as follows:

[0070] The extracellular and extracellular fluids used in this embodiment have an ion composition close to that of "cell physiology", wherein the extracellular fluid components are: 137 mM NaCl, 4 mM KCl, 1 mM MgCl2·6H2O, 2 mM CaCl2·2H2O, 10 mM D-Glucose, 10 mM HEPES, and NaOH is used to adjust the pH to 7.4;

[0071] The components of the intracellular fluid are: 50 mM CsCl, 60 mM CsF, 10 mM HEPES, 20 mM EGTA, 10 mM NaCl, and pH adjusted to 7.2 with CsOH.

[0072] In this example, a CHO cell line stably expressing the Nav1.4 channel, Nav1.4 (SCN4A, gene information: NM_000334) was used. Before the start of the electrophysiological experiment, the Nav1.4 cell line should be kept within 70% of the maximum density in the logarithmic growth phase. All reagents are preheated to 37°C before use. Discard the spent culture medium from the 6 cm culture dish, add 1 mL of PBS, gently shake the culture dish, rinse the bottom of the dish, and then remove it, then add 1 mL of trypsin, gently shake the culture dish, and cover all the cells. Incubate at 37°C for 2 to 3 minutes, gently blow the cells with a pipette to suspend the adherent cells. Transfer the cell suspension into a bacterial centrifuge tube and centrifuge at 1000 rpm / min for 5 minutes, adjust the cell concentration to 2×10 3 / mL, and 500 μL of cell solution was inoculated on the cell slides of 24-well plates. After the cells were well attached to the wall, the patch clamp test experiment was carried out.

[0073] In this embodiment, the sample configuration concentration is 10mM, and the test solution is prepared using deionized water, and the preparation concentrations are 1000nM, 300nM, 100nM, 30nM, 10nM, 3nM and 1nM, respectively.

[0074] The whole-cell patch clamp recording experiment in this embodiment uses Patchmaster software to collect and store Nav1.4 sodium current data on a computer through an EPC-10 amplifier. The specific test process is as follows:

[0075] 1) First, use tweezers to remove the cell slide from the cell culture dish, add extracellular fluid, and place it in the bath on the inverted microscope stage. Then use the P-1000 microelectrode puller to pull a glass microtube, fill 1 / 3 of the volume of the glass microtube (recording electrode) with intracellular fluid, and place it in the electrode holder; use an electric micromanipulator (Scientifica-Double1U) to contact the recording electrode to the cell surface. After the sealing resistance between the recording electrode and the cell membrane is >1GΩ, negative pressure is applied to break the membrane to form a whole-cell recording mode. After the membrane is stable, the membrane capacitance (Cs) and series resistance (Rs) are compensated.

[0076] 2) Stimulation procedure:

[0077] ① The clamping voltage was -120mV, and a square wave train stimulation of -120 to -10mV, with a step of 10mV and a duration of 8000ms was given, followed by a step to -10mV for 30ms, and finally restored to -120mV. The membrane potential was used as the abscissa and the relative current I / Imax as the ordinate to plot, and the Boltzmann process I / Imax = 1 / {1+exp[(V-V1 / 2) / k]} was used for fitting to obtain the steady-state inactivation curve (V 1 / 2 is the conditional pulse voltage when the channel is half inactivated, and k is the slope factor).

[0078] ② The clamping voltage is -120mV, depolarized to 0mV for 40ms to stimulate the resting current of the sodium channel, and then stepped to the conditional pulse voltage when the inactivation curve V1 / 2 channel is half inactivated, with a time course of 8000ms, repolarized to -120mV for 30ms, and then depolarized to 0mV for 40ms to stimulate the half-inactivated current of the sodium channel, and finally restored to -120mV, and the current was recorded every 20s.

[0079] 3) At room temperature, record the Nav1.4 sodium channel current before drug addition. After the control current value reaches a steady state, that is, after the most recent four consecutive current recording lines overlap, use the cumulative drug addition method to add negative (extracellular fluid) and 7 drug concentrations (from low to high) in sequence.

[0080] 4) Data analysis

[0081] The raw data Nav1.4 current peak was extracted from PatchMaster software, and the current inhibition rate was calculated as follows:

[0082] Peak current inhibition rate = ((1-Peak current compound / Peak current vehicle), the mean and standard error were calculated for each concentration, and the concentration-effect relationship was 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 inhibitory concentration (IC 50 ), h is the Hill coefficient, and Graphpad Prism 8.0.2 software was used to perform statistical analysis.

[0083] Using tetrodotoxin as a positive control, manual patch clamp technique was used to detect the half-inhibitory concentration (IC) of the compounds on Nav1.4 ion channels. 50 ), the concentration-effect relationship of wild-type μ-CnIIIC on the inhibition of Nav1.4 resting and semi-inactivated sodium currents is shown in Figure 6 , [Lys 2 ,Ser 17 The concentration-effect relationship of ]-dK, dR-μ-CnIIIC on the inhibition of Nav1.4 resting and semi-inactivated sodium currents is shown in Figure 7 .

[0084] from Figures 6-7 The results show that compared with the wild-type μ-CnIIIC, [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC can specifically activate Nav1.4 ion channels, and its IC 50 is 7.746nM, which is 25.3 times that of wild-type μ-CnIIIC; in the semi-inactive state, IC 50 is 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 has stronger inhibitory activity against Nav1.4, further proving that [Lys2 ,Ser 17 ]-dK,dR-μ-CnIIIC has higher activity and potency than wild-type μ-CnIIIC.

[0085] Example 3 Wild-type μ-CnIIIC and [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC biological activity assay

[0086] The mice used in the experiment of the present invention are adult male Kunming mice, which were purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Sciences, and the animal experiments were approved by the Ethics Committee of the Lanzhou Peptide Valley Research Institute. The mice were free to eat and drink water one week before the test. A 50 μL microsyringe (30G needle) was used to inject intramuscularly into the right tibialis anterior muscle group of the mouse, and the injection volume was 20 μL. An equal volume of saline was injected as a negative control, and the wild type μ-CnIIIC was used as a positive control. The experiment set a concentration gradient of 25 and 50 μM. The activity of the sample was judged by observing the onset time, duration, and behavior of the mice after injection, including toes together (inhibiting muscle contraction), dragging legs / paralysis (anesthetic effect), and death behavior.

[0087] The toe abduction score (DAS) test was used to analyze the toe abduction behavior of mice. The mice were suspended by their tails to stimulate the characteristic startle reaction of extending their hind limbs and abducting them. The right tibialis anterior muscles of the mice were injected with physiological saline, wild-type μ-CnIIIC, and different concentrations of μ-conotoxin peptide [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC, the degree of toe abduction of the left and right hind limbs was measured as a function of time, and whether there was corresponding behavior was observed based on a 5-point scale (0 means normal, 1 means only two toes (index and middle toe) are together, 2 means three toes (index, middle and little toe) are together; 3 means all four toes except the ring toe are together, and 4 means the maximum reduction of toe abduction and leg extension).

[0088] The behavior of leg dragging / paralysis (anesthetic effect) in mice is tested by injecting the drug into the right tibialis anterior muscle group of mice, placing them in a new environment to stimulate their exploratory nature, and observing the activity of the mice; the initial onset of leg dragging / paralysis in mice is characterized by the lower body crawling on the ground, and the whole body lying down in the later stage of the onset. Toes together and leg dragging / paralysis are two behaviors of mice, and leg dragging and toes together usually occur at the same time.

[0089] The experimental results are shown in Table 2:

[0090] Table 2 Wild-type μ-CnIIIC and [Lys2 ,Ser 17 ]-dK, dR-μ-CnIIIC Animal Experiment Results (50μM)

[0091]

[0092] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0093] The results in Table 2 show that after the mouse calf muscle was injected with 50 μM concentration of wild-type μ-CnIIIC, the mouse showed toe-closing behavior, indicating that wild-type μ-CnIIIC showed muscle contraction inhibition activity at a concentration of 50 μM. 2 ,Ser 17 ]-dK, dR-μ-CnIIIC, mice showed lethal behaviors caused by overdose in a short period of time. The mice showed obvious leg dragging and toe-pulling behaviors 3 minutes after injection. The mice were paralyzed and motionless 4 minutes after injection, and died 8 minutes later. This indicates that [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC has higher activity than wild-type μ-CnIIIC.

[0094] To further explore [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC activity, [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC concentration was reduced to 25 μM, and the results are shown in Table 3.

[0095] Table 3 [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC Animal Experiment Results (25μM)

[0096]

[0097] The results in Table 3 show that after injection of 25 μM [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC 5 minutes later, the mice showed obvious leg-dragging behavior, which lasted for 16 minutes and eventually died due to excessive dosage. 2 ,Ser 17 ]-dK, dR-μ-CnIIIC still exhibited the activity of inhibiting muscle contraction and paralysis at low concentrations and showed high activity.

[0098] Subsequently, mice were treated with μ-CnIIIC at two concentrations, 100 μM and 500 μM, and the onset time and duration of the drug after injection as well as the toe-closing (inhibiting muscle contraction) behavior of the mice after injection were observed. The results are shown in Table 4.

[0099] Table 4 Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM

[0100]

[0101] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0102] In Table 4, mice injected with equal volumes of saline at each concentration showed normal behavior. The results showed that mice died only after 500 μM wild-type μ-CnIIIC was injected into the calf muscle, further demonstrating that μ-conotoxin peptide [Lys 2 ,Ser 17 ]-dK,dR-μ-CnIIIC has an activity more than 20 times that of wild-type μ-CnIIIC.

[0103] In summary, the present invention extends the sequence of the wild-type μ-conotoxin (μ-CnIIIC), adds a D-arginine (d-Arg) at the C-terminus, replaces the 17th amino acid with serine (Ser), the 1st amino acid with D-lysine (d-Lys), and the 2nd amino acid with lysine (Lys), completes the synthesis of the analogue by solid phase peptide synthesis technology, completes the formation of disulfide bonds by liquid phase oxidation, and finally purifies and freeze-dries by high performance liquid chromatography to obtain a novel μ-conotoxin peptide [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC, the length of the new highly active conotoxin peptide [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC. By patch clamp detection, [Lys 2 ,Ser 17 ]-dK, dR-μ-CnIIIC can specifically block Nav1.4 channels and can be used to prepare drugs for treating or preventing 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, has analgesic and anesthetic effects, and can be used to prepare drugs for treating or preventing pain.

[0104] 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.

[0105] Sequence Listing

[0106] SEQ ID NO.1

[0107] Three characters:

[0108] 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

[0109] Single character:

[0110] k CCNGPKGCSSKWCR S HARCC r -NH2

[0111] SEQ ID NO.2

[0112] Three characters:

[0113] Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2

[0114] Single character: (X represents pyroglutamic acid)

[0115] XGCCNGPKGCSSKWCRDHARCC-NH2

Claims

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

1.

2. A polynucleotide encoding the μ-type conotoxin peptide according to claim 1.

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

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

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

6. Use of the μ-conotoxin peptide according to claim 1 in the preparation of a drug for treating or preventing diseases related to sodium ion channels.

7. The use according to claim 6, characterized in that: The sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

8. Use of the μ-conotoxin peptide according to claim 1 in the preparation of a drug for treating or preventing pain.

9. Use of the μ-conotoxin 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 conotoxin peptide according to claim 1.

Images