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

Abstract

The invention discloses a mu-type conotoxin peptide as well as polynucleotide coded 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 novel mu-type conotoxin peptide [Ser17, Tyr5]-dR-mu-CnIIIC, the animal activity of the [Ser17, Tyr5]-dR-mu-CnIIIC is improved by more than 10 times compared with that of a wild-type mu-CnIIIC, meanwhile, the amino acid sequence composition of the [Ser17, Tyr5]-dR-mu-CnIIIC is reduced by one site compared with that of the wild-type mu-CnIIIC, and the synthesis cost can be further reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a μ-type conotoxin peptide and a polynucleotide encoding the same and uses thereof. Background Art

[0002] Calcium channels are widely distributed transmembrane proteins that are mainly present in excitatory cells and regulate the extracellular calcium ions (Ca 2 +) enter the cytoplasm. They are in various Ca 2 + plays an important role in physiological activities that depend on voltage, such as the release of hormones and neurotransmitters, muscle contraction, and cell signaling. According to their sensitivity to voltage, calcium channels can be divided into low voltage-activated calcium channels (LVA) or T-type calcium channels (Cav3.x) and high voltage-activated calcium channels (HVA). HVA calcium channels are further divided into L-type (Cav1.x), P / Q-type (Cav2.1), N-type (Cav2.2), and R-type (Cav2.3). Cav1.x channels are mainly involved in muscle contraction and cell secretion, while Cav2.x channels are involved in neurotransmitter release and neuronal excitation regulation. Calcium channels are composed of α subunits with main functions and β, γ, and δ subunits with auxiliary functions.

[0003] Cone snails are poisonous animals widely distributed in tropical oceans, and their venom contains rich peptide ion channel active substances. μ-conotoxin (μ-CnIIIC) can specifically block voltage-sensitive calcium ion channels and participate in the regulation of Nav1.4 sodium channel-related diseases, such as pain syndrome, arrhythmia, and muscle paralysis. It is an important natural resource for the development of peptide drugs. However, the μ-CnIIIC peptide has the problems of low activity and high production cost. In addition, its amino acid sequence contains three disulfide bonds, which increase the difficulty of peptide synthesis. During the production process, it is necessary to ensure that these disulfide bonds are correctly connected to maintain the conformation and activity of the peptide while controlling costs. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a μ-conotoxin peptide and a polynucleotide encoding the same and uses thereof. The present invention aims to provide a μ-conotoxin peptide (named [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC), which can specifically block the Nav1.4 channel and reduce the contraction of mouse muscles. 17 ,Tyr 5 ]-dR-μ-CnIIIC can be used for muscle relaxation and analgesia. In addition, it can also be used to reduce or eliminate the formation of wrinkles on the surface of human skin.

[0005] The present invention provides a μ-type conotoxin peptide, the amino acid sequence of which is shown in SEQ ID NO.1, the amino acid sequence contains three pairs of disulfide bonds, the disulfide bonds are located at Cys 2 -Cys 14 、Cys 3 -Cys 20 and Cys 9 -Cys 21 The position is the same as the disulfide bond structure of the natural μ-type conotoxin peptide.

[0006] The present invention is the first to discover that replacing the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) with serine (Ser), the 5th amino acid with tyrosine (Tyr), and simultaneously deleting the 1st amino acid at the N-terminus of the original sequence and replacing the 2nd amino acid with D-arginine (d-Arg) can further improve the activity of μ-CnIIIC.

[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 and one or more control sequences operably linked thereto and capable of directing the production of the peptide in an expression host.

[0009] The present invention also provides a transformed cell, wherein the nucleic acid construct is transformed.

[0010] The present invention also provides a pharmaceutical composition, which comprises the μ-type conotoxin peptide and a pharmaceutically acceptable carrier.

[0011] 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.

[0012] 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.

[0013] In some embodiments, the sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

[0014] The present invention also provides application of the μ-type conotoxin peptide in preparing medicine for treating or preventing pain.

[0015] The present invention also provides application of the μ-type conotoxin peptide in preparing anesthetic drugs.

[0016] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0017] (1) The present invention provides a novel μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC, compared to wild-type μ-CnIIIC, [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC animal activity increased by more than 10 times, while [Ser 17 ,Tyr 5 The amino acid sequence composition of ]-dR-μ-CnIIIC is reduced by 1 position compared with the wild-type μ-CnIIIC, which can further reduce the synthesis cost of conotoxin peptide.

[0018] (2) The μ-type conotoxin peptide of the present invention [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC can specifically block the Nav1.4 channel, reduce muscle contraction, reduce or eliminate the formation of wrinkles on the human skin surface, soothe muscles and relieve pain, and has good anesthetic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 The μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC synthesis process diagram;

[0021] Figure 2 This is a chromatogram of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0022] Figure 3 This is the mass spectrum of the wild-type μ-conotoxin μ-CnIIIC in Example 1 of the present invention;

[0023] Figure 4 The μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC chromatogram;

[0024] Figure 5 The μ-type conotoxin peptide [Ser 17 ,Tyr5 ]-dR-μ-CnIIIC mass spectrum. DETAILED DESCRIPTION

[0025] 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.

[0026] The term "μ-CnIIIC" in the present invention refers to the wild-type μ-conotoxin;

[0027] The Chinese meanings of the English abbreviations are as follows:

[0028] “dR” refers to D-arginine;

[0029] “S” refers to serine;

[0030] “Y” refers to tyrosine;

[0031] “DCM” means dichloromethane;

[0032] “DIC” refers to N,N-diisopropylcarbodiimide;

[0033] "DMF" means N,N-dimethylformamide;

[0034] “HPLC” means high performance liquid chromatography;

[0035] “MeOH” means methanol;

[0036] “MTBE” means methyl tert-butyl ether;

[0037] "Oxyma" means ethyl 2-oximecyanoacetate;

[0038] “TFA” means trifluoroacetic acid;

[0039] "Fmoc" means 9-fluorenylmethoxycarbonyl;

[0040] “MS” refers to mass spectrometry;

[0041] The present invention first discovered that replacing the 17th amino acid of the wild-type μ-conotoxin (μ-CnIIIC) with serine (Ser), the 5th amino acid with tyrosine (Tyr), and deleting the 1st amino acid at the N-terminus of the original sequence and replacing the 2nd amino acid with D-arginine (d-Arg) can further improve the activity of μ-CnIIIC. The analog was synthesized by solid-phase peptide synthesis technology, the disulfide bond was formed by liquid-phase oxidation, and finally purified by high-performance liquid chromatography and freeze-dried to obtain a novel μ-conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC, the synthesis process is as follows Figure 1 shown.

[0042] The μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC amino acid sequence is shown in SEQ ID NO.1, and the μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC amino acid sequence, can be obtained without creative work encoding the μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC nucleotide sequence, thus encoding the μ-type conotoxin peptide [Ser 17 ,Tyr 5 The nucleotide sequence of the μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC nucleotide sequence is inserted into a vector or strain, and fermentation is performed to produce the μ-type conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC, all are within the protection scope of the present invention.

[0043] Wild-type μ-CnIIIC: Sichuan Jisheng Biotechnology Co., Ltd., batch number: 2023041201-3.

[0044] Reagent K: lysis solution, prepared according to the volume ratio of TFA:phenol:water:thioanisole:ethanedithiol=82.5:5:5:5:2.5.

[0045] Example 1 μ-type conotoxin peptide [Ser 17 ,Tyr 5 Synthesis of ]-dR-μ-CnIIIC

[0046] (1) Preparation of Fmoc-Cys(Trt)-Rink MBHA resin

[0047] ① Weigh 1.501g Rink Amide MBHA resin (containing 1% DVB cross-linker, 100-200 mesh, 0.68mmol / g) (1.02mmol) and add it to a 60mL peptide solid phase reactor, add 10mL 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.

[0048] 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.

[0049] ③ Weigh 1.195g Fmoc-Cys(Trt)-OH (2.0eq, 2.04mmol) and 0.290g Oxyma (2eq, 2.04mmol) into a 50mL beaker, add 7mL DMF solution to dissolve, add 0.316mL condensing agent DIC (2eq, 2.04mmol) 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).

[0050] ④ Peptide chain extension

[0051] 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, 6.573 g of peptide resin was obtained with a yield of 94.9%.

[0052] ⑤ Cracking

[0053] Weigh 6.573g 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 2.006g of white solid crude peptide with a yield of 89.9%.

[0054] ⑥Cyclization

[0055] 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 7.96, 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.

[0056] ⑦Preparative HPLC for peptide purification

[0057] 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.

[0058] Table 1 Purification and elution procedures of crude cyclization solution

[0059]

[0060] The fractions were collected for MS and HPLC analysis, and the target fractions were combined and freeze-dried to obtain [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC totaled 39.1 mg, with a yield of 39.1% and a HPLC purity of 98.236%.

[0061] MS results showed: [M+2H] 2+ =1192.5120, the molecular weight is correct. The sample obtained above will be used in the experiment of Example 2. 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 [Ser 17 ,Tyr 5]-dR-μ-CnIIIC chromatogram Figure 4 As shown, μ-conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC mass spectrum Figure 5 shown.

[0062] Example 1 Preparation of [Ser 17 ,Tyr 5 The amino acid sequence of ]-dR-μ-CnIIIC is shown in SEQ ID NO. 1. The amino acid sequence of wild-type μ-CnIIIC is shown in SEQ ID NO. 2.

[0063] Example 2 Wild-type μ-CnIIIC and [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC biological activity assay

[0064] 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 intramuscularly inject 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 experimental setting concentration gradient was 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.

[0065] 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 response of the mice to extend their hind limbs and abduct them. The right tibialis anterior muscle group of mice was injected with physiological saline, wild-type μ-CnIIIC, and different concentrations of μ-conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC, the degree of toe abduction of the left and right hind limbs was measured as a function of time, and the mouse behavior was evaluated according to 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).

[0066] 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.

[0067] The experimental results are shown in Table 2:

[0068] Table 2 Wild-type μ-CnIIIC and [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC mouse experimental results (50μM)

[0069]

[0070] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0071] 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 inhibitory muscle contraction activity at a concentration of 50 μM. 17 ,Tyr 5 ]-dR-μ-CnIIIC, mice showed lethal behaviors caused by overdose in a short period of time. The mice dragged their legs 8 minutes after injection and then collapsed and died 2 minutes later. This indicates that [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC has higher activity than wild-type μ-CnIIIC.

[0072] To further explore [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC activity, [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC concentration was reduced to 25 μM, and the results are shown in Table 3.

[0073] Table 3 [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC Animal Experiment Results (25μM)

[0074]

[0075] The results in Table 3 show that after injection of 25 μM [Ser 17,Tyr 5 ]-dR-μ-CnIIIC 11 minutes later, the mice showed obvious leg-dragging behavior, which lasted for more than 24 hours. 17 ,Tyr 5 ]-dR-μ-CnIIIC still exhibited the activity of inhibiting muscle contraction and paralysis at low concentrations and showed high activity.

[0076] 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.

[0077] Table 4 Animal experimental results of wild-type μ-CnIIIC at concentrations greater than 50 μM

[0078]

[0079] Note: 1. “ / ” means that the mice were asymptomatic after drug injection, so the onset time and duration of the effect could not be calculated;

[0080] The results in Table 4 show that mice injected with equal volumes of saline at different concentrations behaved normally, and mice died only after 500 μM wild-type μ-CnIIIC was injected into the calf muscle. This further illustrates that the μ-conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC has an activity more than 10 times that of wild-type μ-CnIIIC.

[0081] In summary, compared with wild-type μ-CnIIIC, μ-conotoxin peptide [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC has high activity and is increased by at least 10 times, indicating that [Ser 17 ,Tyr 5 ]-dR-μ-CnIIIC has high activity and can specifically block Nav1.4 channels, reduce or inhibit muscle contraction, and has analgesic and anesthetic effects. It can be used to prepare drugs for treating or preventing diseases related to Nav1.4 sodium ion channels, or can be used to prepare cosmetics for eliminating wrinkles, or can be used to prepare anesthetic drugs, or can be used to prepare drugs for treating or preventing pain.

[0082] 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.

[0083]

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, characterized in that Encodes the μ-type conotoxin peptide according to claim 1.

3. A nucleic acid construct, characterized in that The invention comprises the polynucleotide of claim 2, and one or more control sequences operably linked thereto and capable of directing the production of the peptide in an expression host.

4. A transformed cell, characterized in that The nucleic acid construct of claim 3 is transformed.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the μ-type conotoxin peptide according to claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that The dosage form of the pharmaceutical composition is any one of tablets, capsules, pills, solutions, absorbents and ointments.

7. 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.

8. The use according to claim 5, characterized in that: The sodium ion channel-related disease is any one of epilepsy, arrhythmia, muscle paralysis, myotonia, and autism spectrum disorder.

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

10. Use of the μ-conotoxin peptide according to claim 1 in the preparation of anesthetic drugs.

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