A modified conopeptide, its preparation method and application

Abstract

The present invention provides a modified conotoxin polypeptide, a preparation method and an application thereof, belonging to the technical field of cosmetics preparation. On the basis of the wild-type μ-conotoxin polypeptide PIIIA, the present invention modifies amino acid residues, moderately weakens the blocking effect of the μ-conotoxin polypeptide on sodium channels, and at the same time takes into account maintaining stability under acidic and alkaline conditions, maintaining stable isoelectric point and target specificity. The modified conotoxin polypeptide provided by the present invention has the characteristics of stability and low production cost. After modification, the conotoxin polypeptide still maintains a strong anti-wrinkle function. By using the modified conotoxin polypeptide, it is possible to avoid facial muscle stiffness and paralysis caused by excessive inhibition of the skeletal muscle sodium channel Nav1.4. At the same time, the risk of inhibiting cardiac sodium channels and brain sodium channels is also significantly reduced, and it is easy to penetrate the membrane and be absorbed.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics preparation, and particularly relates to a modified conotoxin polypeptide, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, Botulinum toxin (Botox) is widely used in the medical aesthetics industry for skin anti-wrinkle. Its anti-wrinkle mechanism is to block the signal transmission between nerves and muscles, reducing dynamic expression lines caused by muscle traction. However, not everyone can achieve the anti-wrinkle effect after injecting Botox, and there may also be adverse reactions such as facial muscle paralysis, muscle atrophy, and dysphagia. Therefore, the transformation and synthesis of new peptide-based anti-wrinkle products have broad application prospects.

[0003] Conotoxin is a group of bioactive polypeptides isolated and purified from the venom of the genus Conus, also known as conopeptide. Most conotoxins consist of 10 - 40 amino acid residues, contain one or more disulfide bonds, and have the advantages of small molecular weight, specific action target, and strong activity. They can specifically act on voltage-gated ion channels and ligand-gated ion channels, are important molecular probes for ion channel research, and are also templates for developing new drugs.

[0004] According to the pharmacological action targets of conotoxin, it can be divided into pharmacological families such as α, μ, ω, κ, δ, ψ, σ, ρ, γ, vasopressin, convulsant, and sleep peptide. Among them, μ-conotoxin can specifically block voltage-gated sodium channels, thereby inhibiting the generation of action potentials. The disulfide bond backbone connection mode of μ-conotoxin is "C1-C4, C2-C5, C3-C6". Currently, more than 20 kinds of μ-conotoxins have been isolated. They mainly act on the skeletal muscle sodium channel Nav1.4, and can also act on the brain sodium channel Nav1.2, the cardiac muscle sodium channel Nav1.5, and the pain-related sodium channels Nav1.7 and Nav1.8.

[0005] The significant effect of μ-conotoxin on the skeletal muscle sodium channel Nav1.4 can exert a very potent muscle relaxation effect, that is, a rapid anti-wrinkle effect. Currently, there is a commercialized conotoxin anti-wrinkle polypeptide - XEP-018. It can be applied in various anti-aging personal care products. For example, in eye care, it can significantly remove eye wrinkles and crow's feet at the corners of the eyes; in facial care, it can significantly remove the wrinkles on the forehead caused by muscle contraction.

[0006] Currently, when the existing μ-conotoxin polypeptides are used in products such as drugs and cosmetics, the following defects exist:

[0007] (1) Excessive inhibition of the skeletal muscle sodium channel Nav1.4 leads to facial muscle stiffness and paralysis;

[0008] (2) Serious adverse reactions are caused by the disorder of normal membrane electrical signals resulting from the inhibition of skeletal muscle and cardiac muscle sodium channels. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide a modified conotoxin polypeptide, its preparation method and application. The modified conotoxin polypeptide of the present invention can avoid facial muscle stiffness and paralysis caused by excessive inhibition of skeletal muscle sodium channel Nav1.4. At the same time, it also significantly reduces the risk of inhibiting cardiac muscle sodium channels and brain sodium channels.

[0010] The present invention provides a modified conotoxin polypeptide, and the amino acid sequence of the modified conotoxin polypeptide is shown in SEQ ID No.1.

[0011] Preferably, the N-terminus of the modified conotoxin polypeptide is acetylated.

[0012] Preferably, the isoelectric point of the modified conotoxin polypeptide is 9.01; the relative molecular mass of the modified conotoxin polypeptide is 2496.92 Da.

[0013] The present invention also provides a preparation method of the modified conotoxin polypeptide described in the above scheme, which is characterized by including the following steps: synthesizing the modified conotoxin polypeptide by solid-phase synthesis method or prokaryotic recombinant expression method.

[0014] The present invention also provides the application of the modified conotoxin polypeptide described in the above scheme or the modified conotoxin polypeptide prepared by the preparation method in the preparation of anti-wrinkle and / or wrinkle-removing products.

[0015] The present invention also provides the application of the modified conotoxin polypeptide described in the above scheme or the modified conotoxin polypeptide prepared by the preparation method in the preparation of sodium ion channel inhibitor products.

[0016] Preferably, the product includes cosmetics and / or drugs.

[0017] Preferably, the sodium ion channels include one or more of skeletal muscle sodium channels, cardiac muscle sodium channels and brain sodium channels.

[0018] The present invention also provides an anti-wrinkle and / or wrinkle-removing cosmetic, which includes the modified conotoxin polypeptide described in the above scheme or the modified conotoxin polypeptide prepared by the preparation method.

[0019] Preferably, the cosmetic also includes cosmetic adjuvants and / or other cosmetic raw materials; the cosmetic adjuvants include one or more of humectants, emulsifiers, mineral oils, vegetable oils, thickeners, pH regulators and preservatives; the other cosmetic raw materials include one or more of other polypeptide substances, plant extracts, cytokines and vitamins.

[0020] The present invention provides a modified conotoxin polypeptide, and the amino acid sequence of the modified conotoxin polypeptide is shown in SEQ ID No. 1. The modified conotoxin polypeptide of the present invention is based on the wild-type μ-conotoxin PIIIA, and amino acid residues are modified. Specifically, the arginine R at the 2nd and 12th positions, which interact with the skeletal muscle Nav1.4 channel, on the wild-type μ-conotoxin PIIIA is simultaneously modified to alanine A; at the same time, in order to maintain the stability of the isoelectric point and the specificity of the target, the glutamine Q at the 1st and 15th positions is modified to asparagine N, the leucine at the 3rd position is modified to isoleucine, and the lysine K at the 17th position is modified to arginine R, thus obtaining the designed and modified conotoxin polypeptide YULUO19. The present invention modifies amino acid residues on the wild-type μ-conotoxin PIIIA, moderately weakens the blocking effect of the μ-conotoxin on sodium channels, and at the same time takes into account maintaining stability under acidic and alkaline conditions, maintaining the stability of the isoelectric point and the specificity of the target. The modified conotoxin polypeptide provided by the present invention has the characteristics of stability and low production cost. The conotoxin polypeptide after modification and decoration retains a strong anti-wrinkle function. Using the modified conotoxin polypeptide can avoid the over-inhibition of the skeletal muscle sodium channel Nav1.4 leading to facial muscle stiffness and paralysis. At the same time, it also significantly reduces the risk of inhibiting the cardiac sodium channel and the brain sodium channel, and is easy to penetrate the membrane and be absorbed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0022] Figure 1 FIG. is a structural simulation diagram of conotoxin polypeptide PIIIA and skeletal muscle sodium channel Nav1.4;

[0023] Figure 2 FIG. is a functional test result diagram of wild-type and modified polypeptides. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] In order to make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0025] The present invention provides a modified conotoxin polypeptide YULUO19, and the amino acid sequence of the modified conotoxin polypeptide is shown in SEQ ID No.1, specifically: NAICCGFPRSCASRNCRPHRCC.

[0026] The modified conotoxin polypeptide YULUO19 of the present invention is a tightly folded small peptide containing three pairs of disulfide bonds, and contains a tightly packed CSαβ motif (cysteine stablilized αβ motif) composed of one α helix, one β hairpin and several turns. Arg and Lys are key amino acids.

[0027] In the present invention, the N-terminus of the modified conotoxin polypeptide is preferably acetylated, and the primary structure of the entire amino acid sequence is:

[0028] N(Ac)-Asn-Ala-Ile-Cys-Cys-Gly-Phe-Pro-Arg-Ser-Cys-Ala-Ser-Arg-Asn-Cys-Arg-Pro-His-Arg-Cys-Cys-C.

[0029] When synthesizing the primary sequence of the modified conotoxin polypeptide of the present invention, acetylation protection is carried out on the N-terminus, which helps to improve the stability of the polypeptide. The modified polypeptide has a better lipid-water partition coefficient and is easy to absorb through the membrane.

[0030] The modified conotoxin polypeptide YULUO19 of the present invention is based on the wild-type μ-conotoxin PIIIA, and amino acid residues are modified, moderately weakening the blocking effect of the μ-conotoxin on sodium channels, while maintaining stability under acidic and alkaline conditions, maintaining the isoelectric point stability and target specificity.

[0031] There are multiple pairs of amino acid residues in the wild-type μ-conotoxin that can directly interact with the skeletal muscle sodium channel Nav1.4. In the present invention, several positively charged amino acid residues of the μ-conotoxin are modified, reducing the activity of the μ-conotoxin, but still maintaining its partial function of inhibiting the skeletal muscle Nav1.4 channel, retaining the necessary nerve impulse conduction, and achieving a moderate muscle relaxation effect.

[0032] Specifically, in the present invention, the arginine R at the 2nd and 12th positions that interact with the skeletal muscle Nav1.4 channel on the wild-type μ-conotoxin PIIIA are simultaneously modified to alanine A; at the same time, in order to maintain the isoelectric point stability and target specificity, the glutamine Q at the 1st and 15th positions is modified to asparagine N, the leucine at the 3rd position is modified to isoleucine, and the lysine K at the 17th position is modified to arginine R, thereby obtaining the designed and modified conotoxin polypeptide YULUO19.

[0033] In the present invention, the modified conotoxin polypeptide YULUO19 has a sequence length of 22 amino acids, an isoelectric point of 9.01, and a relative molecular mass of 2496.92 Da, while the isoelectric point of wild-type PIIIA (wild-type μ-conotoxin polypeptide) is 9.49 and the molecular weight is 2597.13 Da.

[0034] The present invention also provides a method for preparing the modified conotoxin polypeptide described in the above solution, which is characterized by comprising the following steps: synthesizing the modified conotoxin polypeptide by a solid-phase synthesis method or a prokaryotic recombinant expression method.

[0035] When using the solid-phase synthesis method, preferably, the crude polypeptide of the modified conotoxin polypeptide is first synthesized in the present invention, and then the crude polypeptide is subjected to refolding of the higher-order structure.

[0036] In the present invention, the refolding preferably uses the glutathione redox method for refolding.

[0037] After subjecting the crude polypeptide to refolding of the higher-order structure, preferably, the present invention further comprises purifying the refolded polypeptide.

[0038] In the present invention, the purification preferably includes desalting and purification by HPLC reverse-phase column chromatography.

[0039] When synthesizing the conotoxin polypeptide by the prokaryotic recombinant expression method, preferably, a prokaryotic expression plasmid is first constructed in the present invention, expressed in Escherichia coli, and then subjected to subsequent separation and purification.

[0040] The present invention also provides the application of the modified conotoxin polypeptide described in the above solution or the modified conotoxin polypeptide prepared by the preparation method in the preparation of anti-wrinkle and / or wrinkle-removing products.

[0041] The present invention also provides the application of the modified conotoxin polypeptide described in the above solution or the modified conotoxin polypeptide prepared by the preparation method in the preparation of sodium ion channel inhibition products.

[0042] The modified conotoxin polypeptide of the present invention retains a strong anti-wrinkle function. Using the modified conotoxin polypeptide can avoid excessive inhibition of the skeletal muscle sodium channel Nav1.4 resulting in facial muscle stiffness and paralysis. At the same time, the risk of inhibiting the cardiac sodium channel and the brain sodium channel is also significantly reduced.

[0043] In the present invention, the product preferably includes cosmetics and / or drugs.

[0044] In the present invention, the sodium ion channel preferably includes one or more of the skeletal muscle sodium ion channel, the cardiac sodium ion channel, and the brain sodium ion channel.

[0045] In the present invention, the skeletal muscle sodium channel is preferably the skeletal muscle sodium channel Nav1.4.

[0046] The present invention also provides a wrinkle-resistant and / or wrinkle-removing cosmetic, comprising the modified conotoxin polypeptide described in the above solution or the modified conotoxin polypeptide prepared by the preparation method described above.

[0047] In the present invention, the cosmetic preferably further comprises cosmetic adjuvants and / or other cosmetic raw materials.

[0048] In the present invention, the cosmetic adjuvants preferably include one or more of a humectant, an emulsifier, a mineral oil, a vegetable oil, a thickener, a pH regulator, and a preservative.

[0049] In the present invention, the humectant preferably includes one or more of glycerol, polyhydric alcohol, sodium hyaluronate, ceramide, trehalose, polysorbate-30, and amino acid humectant.

[0050] In the present invention, the glycerol serves as both a humectant and an antioxidant.

[0051] In the present invention, the emulsifier preferably includes one or more of lanolin, polyglyceryl-10 stearate, octyl polymethylsiloxane, polydimethylsiloxane, polymethylsilsesquioxane, babassu oil, and phytosterol oleate.

[0052] In the present invention, the thickener preferably includes one or more of carbomer, hydroxyethyl cellulose, xanthan gum, and acryloyldimethyltaurate / VP copolymer.

[0053] In the present invention, the pH regulator preferably includes one or more of citric acid, sodium citrate, lactic acid, sodium lactate, triethanolamine, and arginine.

[0054] In the present invention, the preservative preferably includes one or more of 1,2-hexanediol, p-hydroxyacetophenone (Freshol), and ethylhexylglycerol.

[0055] In the present invention, the other cosmetic raw materials preferably include one or more of other polypeptide substances, plant extracts, cytokines, and vitamins.

[0056] In the present invention, the other polypeptide substances preferably include one or more of carnosine, pentapeptide-3, glutathione, anserine, and snake meat peptide.

[0057] In the present invention, the plant extract preferably includes oat kernel extract and / or Dendrobium officinale extract. The present invention has no special limitation on the extraction method of oat kernel extract and Dendrobium officinale extract.

[0058] In the present invention, the vitamin preferably includes one or more of vitamin B3, vitamin C, and vitamin E.

[0059] The present invention has no special limitation on the ratio of the cosmetic adjuvants and other cosmetic raw materials, and they can be adaptively combined according to needs.

[0060] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention.

[0061] The purpose of the present invention is to provide a polypeptide product for skin anti-wrinkle that is safe, reliable, has definite curative effects, good stability, and is easy to produce through structural modification and design. Based on the conotoxin polypeptide (μ-Conotoxin PIIIA) with relatively high side effects, the three-dimensional structures of A in Figure 1 PIIIA and BC in the skeletal muscle sodium channel Nav1.4 Figure 1 were simulated using the structural simulation software Rosetta. The high-affinity complex model was analyzed to obtain multiple pairs of direct interactions between the PIIIA polypeptide and the key amino acid residues of the Nav1.4 channel. The directly interacting amino acid residues are shown in Table 1 below:

[0062] Table 1 List of key amino acid residues with direct interactions

[0063] PIIIA R2 R12 R14 K17 R20 Nav1.4 W761 E758 E755 D1241 D1541

[0064] It can be concluded from the information analysis in the above table that:

[0065] (1) There may be multiple pairs of interactions between the conotoxin polypeptide μ-Conotoxin PIIIA and the skeletal muscle sodium channel Nav1.4, and these direct amino acid interactions are the basis for the high affinity of PIIIA on the sodium channel;

[0066] (2) By modifying the positively charged amino acid residues on PIIIA, the blocking effect of PIIIA on the sodium channel can be moderately weakened, and at the same time, stability can be maintained under acidic and alkaline conditions;

[0067] (3) On the wild-type PIIIA, the arginine R at the 2nd and 12th positions that interact with the skeletal Nav1.4 channel were simultaneously modified to glycine G; at the same time, to maintain the stability of the isoelectric point and target specificity, the glutamine Q at the 1st and 15th positions was modified to asparagine N, the leucine at the 3rd position was modified to isoleucine, and the lysine K at the 17th position was modified to arginine R, thus obtaining the designed and modified polypeptide YULUO19.

[0068] Example 1

[0069] This embodiment provides a modified conotoxin polypeptide (YULUO19), and the primary structure of its complete amino acid sequence is as follows:

[0070] N(Ac)-Asn-Ala-Ile-Cys-Cys-Gly-Phe-Pro-Arg-Ser-Cys-Ala-Ser-Arg-Asn-Cys-Arg-Pro-His-Arg-Cys-Cys-C, which is a tightly folded small peptide containing three pairs of disulfide bonds.

[0071] The preparation method is as follows:

[0072] (1) According to the designed amino acid sequence above, a crude polypeptide is synthesized by solid-phase synthesis method;

[0073] (2) Renaturation folding of the linear polypeptide's higher-level structure. For the synthesized linear peptide, glutathione redox method is used for renaturation, which specifically includes the following steps: Dissolve 10 mg of the linear peptide synthesized in step (1) in 100 ml of a solution containing 5 mM GSH, 0.5 mM GSSG, 0.1 M Tris HCl, and 0.1 M NaCl with pH = 8.0, place it in an incubator at 25 °C for 24 h to obtain a renaturation solution, detect the renaturation effect by RP-HPLC and collect the elution peak, and detect the purity and renaturation result by mass spectrometry.

[0074] (3) The renaturation solution obtained in step (2) is desalted and purified by HPLC reverse-phase column chromatography to identify its purity until the purity of the polypeptide is not less than 95%.

[0075] HPLC purification and identification method: Filter 10 ml of the renaturation solution with a 0.22 μm filter membrane. Mobile phase A is 0.1% trifluoroacetic acid - water, and mobile phase B is 0.1% trifluoroacetic acid - acetonitrile. After the baseline is stable, start loading the sample. The chromatographic column is a silica gel alkyl-bonded phase C18 column (4.6 mm × 300 mm, particle size 5 μm, pore size 100 Å). A binary mobile phase gradient elution system is used for linear gradient elution, that is, within 200 min, the content of mobile phase B in the eluent increases linearly from 0% - 100%, the flow rate is 1 mL / min, the detection wavelength is 280 nm, and the measurement is carried out at 25 °C.

[0076] (4) Determine the collected single peak by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). The molecular weight after renaturation is 2490 Da;

[0077] The measurement method is as follows: Dissolve the polypeptide purified in step (4) in deionized water to prepare a 1 μM solution. Take 10 μL and mix it with an equal volume of saturated matrix solution (dissolve α-cyano-4-hydroxycinnamic acid in a 50% acetonitrile solution containing 0.1% trifluoroacetic acid to make a saturated solution, centrifuge, and take the supernatant), and then measure.

[0078] (5) The isoelectric point of the purified polypeptide was measured by isoelectric focusing electrophoresis to be 9.01, and the amino acid sequence structure of the purified polypeptide was determined by an automatic amino acid sequencer to be NAICCGFPRSCASRNCRPHRCC (SEQ ID NO.1).

[0079] Experimental Example 1

[0080] This experimental example tests the dispersibility of the polypeptide YULUO19 prepared in Example 1 in lipid substances.

[0081] Taking the measurement of the homogeneity and dispersibility of polypeptide YULUO19 in lanolin as an example. Take 1000 g of lanolin and 1000 mg of YULUO19 polypeptide, mix them evenly in a SHW / R type mobile high-shear emulsifying machine at room temperature, with a stirring speed of 120 rpm / min and stir for 30 minutes. After mixing evenly, divide them into 5 ml per tube. Under this condition, the theoretical content of YULUO19 is 1 mg / g. Take 20 tubes of the aliquot, accurately weigh an appropriate amount of 1 g of the mixture (equivalent to about 0.1 mg of YULUO19 in a 10 ml volumetric flask), add 20% ethanol solution to dissolve it (ultrasonic if necessary) and dilute to the scale. Accurately measure 2 ml and perform protein quantification using the Folin phenol method.

[0082] The experimental results show that the average content of YULUO19 in 20 samples is 0.90 ± 0.05 mg / g, and the content range of each sample is within 90% of the theoretical content. It shows that YULUO19 designed in the present invention has good dispersibility in a lipid environment. That is, the polypeptide YULUO19 can be used for the development of anti-wrinkle skin care products.

[0083] Experimental Example 2

[0084] This experimental example tests the electrophysiological function of the polypeptide YULUO19 prepared in Example 1.

[0085] The recording mode of voltage-gated sodium channels is an extracellular high-sodium system. The extracellular solution is (mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, adjusted to pH 7.3 with NaOH; the intracellular solution is (mM): 140 CsF, 1 EGTA, 10 NaCl, 3 KCl, 10 MgCl2, adjusted to pH 7.3 with CsOH. The recording method of sodium channels is to clamp the voltage at 80 mV for 20 ms; the test voltage is 10 mV for 50 ms; the clamping voltage is 80 mV for 20 ms, and this recording is continuously repeated until the current is stable. Before placing the cells transiently expressing Nav1.4 in front of the 8-well tube of the RSC-200 (BioLogic) rapid perfusion and drug addition system, first open the Bathsolution channel, and by switching different channels, 100 nM wild-type PIIIA and 100 nM modified YULUO19 are respectively given.

[0086] The results are referred to Figure 2 As shown, it can be seen that the wild-type PIIIA completely inhibits the current of Nav1.4 ( Figure 2 A in); the modified YULUO19 partially inhibits the current of Nav1.4 ( Figure 2 B in), with an inhibition of about 90%, and about 10% of muscle current conduction is retained. At the same time, YULUO19 at this concentration has almost no activity on other sodium channels that undertake important physiological functions, such as Nav1.2 ( Figure 2 C in), Nav1.5 ( Figure 2 D in), Nav1.7 ( Figure 2 E in) ( Figure 2 C - E in); while the wild-type PIIIA has inhibitory effects on Nav1.2 ( Figure 2 F in), Nav1.5 ( Figure 2 G in), Nav1.7 ( Figure 2 H in) ( Figure 2 F - H in), so the modified polypeptide ensures target specificity.

[0087] From the above results, it can be seen that through creative design and modification, the inventors made the polypeptide have the function of moderately inhibiting muscle contraction and will not act on other ion channels such as Nav1.2, Nav1.5, and Nav1.7 to produce serious side effects.

[0088] Experimental Example 3

[0089] This experimental example tests the cytotoxicity of the polypeptide YULUO19 prepared in Example 1.

[0090] Specifically, the MTT method is used in this experimental example to detect the toxicity of the polypeptide YULUO19 to human skin fibroblasts HFF-1.

[0091] The human skin fibroblasts HFF-1 were purchased from the Kunming Cell Bank. First, the fibroblasts were cultured in DMEM containing 15% fetal bovine serum and double antibiotics (100 U / ml each of penicillin and streptomycin). After the cells grew to confluence, they were digested with 0.25% trypsin, washed twice with the above medium, and the cells were resuspended. After cell counting, 100 μl of the cell suspension was added to a 96-well cell culture plate so that the number of cells per well reached 10 5 cells. The polypeptide YULUO19 prepared in Example 1 was added, and the control group was added with the same volume of sterilized ultrapure water. It was cultured in an incubator at 37 °C and 5% CO2 for 24 h. After the culture, 20 μl of 5 mg / ml MTT solution (prepared with cell culture PBS buffer) was added to each well of the 96-well cell culture plate, and the culture was continued for 5 h. The liquid in the wells was aspirated with a syringe, 100 μl of DMSO was added to each well, and the pipette was used to blow several times to completely dissolve the purple crystals. Then, a microplate reader was used to detect the light absorption, the measurement wavelength was 490 nm, and the reference wavelength was 630 nm.

[0092] Table 2 Statistical table of the toxicity of polypeptide YULUO19 to HFF-1 cells.

[0093] Concentration of YULUO19 (μg / ml) Cytotoxicity % 1 0.00 50 1.25±0.32 200 2.67±0.48

[0094] As shown in Table 2, it shows that the cytotoxicity of polypeptide YULUO19 at a concentration of 200 μg / ml is only 2.67%, indicating that polypeptide YULUO19 has very low cytotoxicity to human skin fibroblasts and will not cause harm to normal human skin cells. Therefore, it is very conducive to its further development and application.

[0095] Experimental Example 4

[0096] This experimental example tested the anti-wrinkle function of the polypeptide YULUO19 prepared in Example 1.

[0097] This experimental example measured the effect of the skin polypeptide YULUO19 on wrinkles caused by UVB irradiation. The UVB energy irradiated to the dorsal side of each mouse was controlled by changing the UV irradiation time. The minimum erythema dose (MED) of each mouse was about 36 mj / cm 2 . YULUO19 (10 ng and 100 ng / mouse) was topically applied to the back of each mouse every day for 12 weeks. The initial dose of UVB was set at 36 mJ / cm 2 , and then increased to 54 mJ / cm in weeks 1-4 2 , increased to 72 mJ / cm in weeks 4-7 2 , increased to 108 mJ / cm in weeks 7-10 2 , and finally increased to 122 mJ / cm in weeks 10-12 2。The frequency of UVB irradiation was set at three times a week, and then the excipients (blank control and UV control) and YULUO19 were applied topically.

[0098] The grading criteria were as follows: 0, no rough wrinkles; 2, some shallow and rough wrinkles were observed in the dorsal skin area (Bisset grade 1); 4, shallow and rough wrinkles were observed on the entire dorsal skin (Bisset grade 2); 6, some deep and long wrinkles were observed on the dorsal skin (Bisset's grade 3).

[0099] Table 3 Anti-wrinkle effect scores of anti-wrinkle polypeptide YULUO19

[0100] Group Blank control Ultraviolet control YULUO19 (10 ng) YULUO19 (100 ng) Wrinkle score at 6 weeks 0.0±0.0 3.96±0.63 1.16±0.22** 2.42±0.38** Wrinkle score at 9 weeks 0.0±0.0 4.94±0.74 1.47±0.35** 2.68±0.52**

[0101] **P < 0.05.

[0102] Table 3 results showed that, compared with the control group, the anti-wrinkle polypeptide YULUO19 designed in Example 1 had a good anti-wrinkle effect.

[0103] Although the above embodiments have described the present invention in detail, they are only some embodiments of the present invention rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A modified conopeptide, characterized in that, The amino acid sequence of the modified conotoxin polypeptide is shown in SEQ ID No. 1; the N-terminus of the modified conotoxin polypeptide is acetylated.

2. The modified conotoxin polypeptide according to claim 1, wherein, The isoelectric point of the modified conotoxin polypeptide is 9.01; the relative molecular mass of the modified conotoxin polypeptide is 2496.92 Da.

3. The preparation method of the modified conotoxin polypeptide according to claim 1 or 2, characterized in that, It includes the following steps: The modified conotoxin polypeptide is synthesized by solid-phase synthesis or prokaryotic recombinant expression.

4. Use of the modified conotoxin polypeptide according to claim 1 or 2 or the modified conotoxin polypeptide prepared by the preparation method according to claim 3 in the preparation of anti-wrinkle and / or wrinkle-removing products.

5. An anti-wrinkle and / or wrinkle-removing cosmetic, comprising the modified conotoxin polypeptide according to claim 1 or 2 or the modified conotoxin polypeptide prepared by the preparation method according to claim 3.

6. The cosmetic according to claim 5, characterized in that, The cosmetic further comprises cosmetic adjuvants and / or other cosmetic raw materials; the cosmetic adjuvants include one or more of humectants, emulsifiers, mineral oils, vegetable oils, thickeners, pH regulators and preservatives; the other cosmetic raw materials include one or more of other polypeptide substances, plant extracts, cytokines and vitamins.

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