CN113549139A - Conus polypeptide, polypeptide composition, preparation method and application thereof - Google Patents

Abstract

The invention discloses a conus polypeptide, a polypeptide composition, a preparation method and an application thereof, and relates to the technical field of cosmetic preparation. The conus polypeptide has a sequence shown as SEQ ID NO. 1. Amino acid residue mutation is carried out on wild mu-Conotoxin PIIIA, so that the blocking effect of the mu-Conotoxin PIIIA on a sodium channel is weakened moderately, and meanwhile, the stability is kept under acidic and alkaline conditions, the stability of isoelectric points and the specificity of targets are kept. The novel conus polypeptide provided by the invention has the characteristics of stability, low production cost and the like. The mutated conus polypeptide or the salt thereof keeps stronger anti-wrinkle function, facial muscle stiffness paralysis caused by excessive inhibition on a skeletal muscle sodium channel Nav1.4 can be avoided by adopting the mutated conus polypeptide, and meanwhile, the risk of inhibition on a myocardial sodium channel and a brain sodium channel is also obviously reduced.

Description

Conus polypeptide, polypeptide composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of cosmetic preparation, and particularly relates to a conus polypeptide, a polypeptide composition, and a preparation method and application thereof.

Background

With the development of economic society and the improvement of living standard, the public demand for health services is increasing day by day, wherein the consumption investment in the medical and aesthetic directions is strong. In terms of income, china has become the second largest medical beauty service market worldwide in 2018, and the income reaches $ 183.79 billion, which accounts for about 13.5% of the market share of the global medical beauty service market. Currently, botulinum (Botox) is widely used in the medical and American industries for anti-wrinkle of skin. The anti-wrinkle mechanism is to reduce dynamic expression lines caused by muscle traction by blocking signal transmission between nerves and muscles. However, not all people can achieve the wrinkle removing effect after the injection of the Botox, and adverse reactions such as facial muscle paralysis and weakness, muscle atrophy, dysphagia and the like can be caused. Therefore, the modified and synthesized novel peptide wrinkle-removing product has wide application prospect.

Conotoxin is a group of bioactive polypeptides separated and purified from venom of Conus, also called conotoxin. The conotoxin is mostly composed of 10 to 40 amino acid residues, contains one or more disulfide bonds, and has the advantages of small molecular mass, specific action target, strong activity and the like. They can specifically act on voltage-gated ion channels and ligand-gated ion channels, are important molecular probes for neurobiology research, and are also lead molecules for developing novel drugs.

According to the pharmacological action target of the conotoxin, the conotoxin can be divided into pharmacological families such as alpha, mu, omega, kappa, delta, psi, sigma, rho, gamma, vasopressin, convulsant, sleeping peptide and the like. Wherein the mu-conotoxin can specifically block a voltage-gated sodium ion channel, thereby inhibiting the generation of action potential. The disulfide bond skeleton connection mode of mu-conotoxins is 'C1-C4, C2-C5 and C3-C6', more than 20 kinds of mu-conotoxins are isolated at present, and the mu-conotoxins mainly act on a skeletal muscle sodium channel Nav1.4(IC50 ═ 50nM), and also act on a brain sodium channel Nav1.2(IC50 ═ 690nM), a cardiac muscle sodium channel Nav1.5(IC50 ═ 10 mu M) and a pain sensation related sodium channel (Nav1.7 and Nav1.8).

Based on the remarkable effect of mu-conotoxin on the skeletal muscle sodium channel Nav1.4, it can be concluded that such polypeptides can provide a very potent muscle relaxant effect, i.e., provide a rapid and immediate anti-wrinkle effect. At present, commercial conus anti-wrinkle polypeptide XEP-018 exists. In various anti-aging personal care products, for example: eye nursing, eye wrinkles and canthus fishtail lines can be obviously removed; facial care can obviously remove the wrinkles on the forehead caused by muscle contraction.

At present, the existing mu-conus polypeptide has the following defects when being used for products such as medicines, cosmetics and the like:

(1) excessive inhibition of the skeletal muscle sodium channel, Nav1.4, results in stiff paralysis of the facial muscles; (2) the serious adverse reaction is generated on the disturbance of normal membrane electric signals caused by the inhibition of skeletal muscle and myocardial sodium channels.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a conus polypeptide, a polypeptide composition, a preparation method and an application thereof to solve the technical problems.

The invention is realized by the following steps:

the invention provides a conus polypeptide, which has a sequence shown as SEQ ID NO. 1.

The conus polypeptide provided by the invention is a compact folding small peptide containing three pairs of disulfide bonds, and contains a compact CS alpha beta motif (cysteine stabilized alpha beta motif) consisting of an alpha helix, a beta hairpin and a plurality of corners, wherein Arg and Lys are key amino acids. The primary structure of the complete amino acid sequence is as follows:

N-Asn-Gly-Ile-Cys-Cys-Gly-Phe-Pro-Arg-Ser-Cys-Gly-Ser-Arg-Asn-Cys-Lys-Pro-His-Arg-Cys-Cys-C。

the conus polypeptide is obtained by carrying out amino acid residue mutation on wild mu-conus polypeptide (mu-Conotoxin) PIIIA, moderately weakens the blocking effect of the mu-conus polypeptide on a sodium channel, and simultaneously keeps stable under acidic and alkaline conditions, and keeps stable isoelectric points and specific targets.

The inventor finds that a plurality of pairs of amino acid residues of the wild mu-conus polypeptide can directly interact with a skeletal muscle sodium channel Nav1.4, and the inventor carries out mutation on a plurality of positively charged amino acid residues of the mu-conus polypeptide, so that the activity of the mu-conus polypeptide is reduced, the function of partially inhibiting the skeletal muscle Nav1.4 channel is still maintained, necessary nerve impulse conduction is retained, and the effect of moderately relaxing muscles is achieved.

Specifically, the inventor simultaneously transforms 2 nd arginine R and 12 th arginine R which are selected from wild mu-conus polypeptide and have interaction with a skeletal muscle Nav1.4 channel into glycine G; meanwhile, in order to keep stable isoelectric point and specific target, 1 st and 15 th glutamine Q is transformed into asparagine N, 3 rd leucine is transformed into isoleucine, and 17 th lysine K is transformed into arginine R, so that the designed and transformed polypeptide PIIIA2 (namely the conus polypeptide in the invention) is obtained.

The polypeptide PIIIA2 shown in SEQ ID NO.1 has a sequence length of 22 amino acids, an isoelectric point of 8.98 and a relative molecular mass of 2398.81Da, while the isoelectric point of wild type PIIIA (wild type mu-conotoxin polypeptide) is 9.49 and the molecular mass is 2597.13 Da.

The invention also provides an application of the conus polypeptide or the salt thereof in preparing a product for preventing and/or treating wrinkles, wherein the conus polypeptide has a sequence shown as SEQ ID NO. 1.

The inventor finds that the mutated conus polypeptide or the salt thereof keeps stronger anti-wrinkle function, the mutated conus polypeptide can avoid facial muscle stiffness paralysis caused by over-inhibition of a skeletal muscle sodium channel Nav1.4, and meanwhile, the risk of inhibition of a myocardial sodium channel and a brain sodium channel is also obviously reduced.

In a preferred embodiment of the present invention, the product is a pharmaceutical or cosmetic product. The cosmetic is a chemical industrial product or a fine chemical product which is applied to any part of the surface of a human body, such as skin, hair, nails, lips and teeth, by smearing, spraying or the like, so as to achieve the purposes of cleaning, maintaining, beautifying, decorating and changing the appearance, or correcting the odor of the human body and keeping a good state.

Salts of conotoxin include, but are not limited to, acetate, citrate, and the like.

The invention also provides an application of the conus polypeptide or the salt thereof in preparing a sodium ion channel inhibition product, wherein the conus polypeptide has a sequence shown as SEQ ID NO. 1.

In one embodiment, the sodium ion channel is a Nav1.4 channel.

In a preferred embodiment of the present invention, the product is a pharmaceutical or cosmetic product.

The invention also provides a polypeptide composition which comprises the conus polypeptide, cosmetic auxiliary materials and other cosmetic raw materials.

In a preferred embodiment of the present invention, the cosmetic auxiliary material is at least one selected from the following materials:

humectant, emulsifier, mineral oil, vegetable oil, thickener, pH regulator and antiseptic.

In one embodiment, the humectant is selected from at least one of the following: glycerin, polyalcohol, sodium hyaluronate, ceramide, trehalose, polysorbate-30 and an amino acid humectant. The glycerin may be used as a moisturizing agent or an antioxidant.

In one embodiment, the emulsifier is selected from lanolin. In other embodiments, the emulsifier may be selected from polyglyceryl-10 stearate, octyl methicone, dimethicone, polymethylsilsesquioxane, babassu seed oil, phytosterol oleate, and the like.

The thickening agent is selected from at least one of the following substances: carbomer, hydroxyethyl cellulose and xanthan gum. In other embodiments, the thickener may also be selected from hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymers.

The pH regulator is selected from at least one of the following substances: citric acid, sodium citrate, lactic acid, sodium lactate, triethanolamine, and arginine.

The preservative is selected from at least one of the following substances: 1, 2-hexanediol, p-hydroxyacetophenone (menthone), and ethylhexyl glycerol.

In a preferred embodiment of the application of the invention, the other cosmetic raw materials are selected from at least one of the following substances: polypeptide substances, plant extracts, cytokines and vitamins.

In one embodiment, the polypeptide substance is selected from at least one of the following substances: carnosine, pentapeptide-3, glutathione, anserine and snake meat peptide.

The plant extract is selected from at least one of the following substances: oat kernel extract and dendrobium officinale extract.

The vitamins are selected from at least one of the following substances: vitamin B3, vitamin C and vitamin E.

The cytokine is selected from at least one of the following substances: epidermal Growth Factor (EGF) and nerve growth factor (VGF). In other embodiments, the cytokine may also be selected from other proteins or polypeptides with defined functions and structures.

The proportion of the cosmetic auxiliary materials and the cosmetic raw materials can be combined in a self-adaptive manner according to the needs.

The invention also provides a preparation method of the conus polypeptide, which comprises the following steps: the conus polypeptide is synthesized by adopting a solid-phase synthesis method or a prokaryotic recombinant expression method.

In a preferred embodiment of the invention, when a solid-phase synthesis method is adopted, crude conus polypeptide is synthesized, and then the crude polypeptide is subjected to renaturation folding of a high-level structure;

preferably, the renaturation folding is renaturation by a glutathione redox method;

preferably, the preparation method further comprises purifying the renatured and folded polypeptide;

preferably, the purification is a desalting purification by HPLC reverse phase column chromatography.

When the conus polypeptide is synthesized by adopting a prokaryotic recombinant expression method, a prokaryotic expression plasmid needs to be constructed firstly, and then is expressed in escherichia coli, and then is subjected to subsequent separation and purification.

The invention has the following beneficial effects:

the invention carries out amino acid residue mutation on the wild mu-Conotoxin PIIIA, moderately weakens the blocking effect of the mu-Conotoxin PIIIA on a sodium channel, simultaneously keeps stable under acidic and alkaline conditions, and keeps stable isoelectric points and specific target spots. The novel conus polypeptide provided by the invention has the characteristics of stability, low production cost and the like. The mutated conus polypeptide or the salt thereof keeps stronger anti-wrinkle function, facial muscle stiffness paralysis caused by excessive inhibition on a skeletal muscle sodium channel Nav1.4 can be avoided by adopting the mutated conus polypeptide, and meanwhile, the risk of inhibition on a myocardial sodium channel and a brain sodium channel is also obviously reduced.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a structural simulation diagram of conus polypeptide PIIIA and skeletal muscle sodium channel Nav1.4;

FIG. 2 is a diagram showing the results of functional assays for wild-type and mutant polypeptides;

FIG. 3 is a graph showing the anti-wrinkle effect of the polypeptide PIIIA2 provided by the present invention.

Detailed Description

In order to make the objects, 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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

The invention aims to provide a polypeptide product for resisting skin wrinkles, which is safe and reliable, has definite curative effect and good stability and is easy to produce through structural modification and design. On the basis of conus polypeptides (mu-Conotoxin PIIIA) with higher side effects, a three-dimensional structure of PIIIA (figure 1A) and a skeletal muscle sodium channel Nav1.4 (figures 1B and C) is simulated by using a structure simulation software Rosetta, a high-affinity compound model is analyzed, direct interaction of a plurality of pairs of PIIIA polypeptides and Nav1.4 channel key amino acid residues is obtained, and the amino acid residues with direct interaction refer to the following table 1:

table 1 list of key amino acid residues for direct interaction.

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

From the analysis of the information in the above table it follows that:

(1) conus polypeptide mu-Conotoxin PIIIA and skeletal muscle sodium channel Nav1.4 may have a plurality of pairs of interactions, and the direct interaction of the amino acids is the basis of the high affinity of the PIIIA on the sodium channel;

(2) by mutating positively charged amino acid residues on the PIIIA, the blocking effect of the PIIIA on a sodium channel can be weakened moderately, and the stability is kept under acidic and alkaline conditions;

(3) arginine R at the 2 nd position and arginine R at the 12 th position which have interaction with a skeletal muscle Nav1.4 channel are selected from wild type PIIIA and are simultaneously transformed into glycine G; meanwhile, in order to keep stable isoelectric point and specific target, 1 st and 15 th glutamine Q is modified into asparagine N, 3 rd leucine is modified into isoleucine, and 17 th lysine K is modified into arginine R, so that the designed and modified polypeptide PIIIA2 is obtained.

Example 1

This example provides a conus polypeptide (PIIIA2), whose primary structure of the complete amino acid sequence is:

N-Asn-Gly-Ile-Cys-Cys-Gly-Phe-Pro-Arg-Ser-Cys-Gly-Ser-Arg-Asn-Cys-Lys-Pro-His-Arg-Cys-Cys-C, which is a tightly folded small peptide containing three pairs of disulfide bonds.

The preparation method comprises the following steps:

(1) synthesizing a crude polypeptide by a solid phase synthesis method based on the designed amino acid sequence (committed Gill Biochemical Synthesis);

(2) the linear polypeptide has high-order structure renaturation folding. The method for renaturating the synthesized linear peptide by a glutathione redox method specifically comprises the following steps: 10mg of the linear peptide synthesized in step (1) was dissolved in 100ml of a solution containing 5mM GSH,0.5mM GSSG,0.1M Tris HCl and 0.1M NaCl at pH 8.0 and left at 25 ℃ in an incubator for 24 hours to obtain a renaturation solution, the renaturation effect was measured by RP-HPLC and the elution peak was collected, and the purity and the renaturation result were measured by mass spectrometry.

(3) Desalting and purifying the renaturation liquid obtained in the step (2) by HPLC reverse phase column chromatography, and identifying the purity of the renaturation liquid until the purity of the polypeptide is not lower than 95%.

HPLC purification and identification method: filtering 10mL renaturation liquid with a 0.22 mu m filter membrane, wherein the mobile phase A is 0.1% trifluoroacetic acid-water, the mobile phase B is 0.1% trifluoroacetic acid-acetonitrile, loading is started after a base line is stable, a chromatographic column is a silica gel alkyl bonded phase C18 column (4.6mm multiplied by 300mm, the size of colloidal particles is 5 mu m, and the size of pore diameter is 100A), and a binary mobile phase gradient elution system is adopted for carrying out linear gradient elution, namely, the content of the mobile phase B in an eluent is increased from 0% to 100% in a linear relation within 200min, the flow rate is 1mL/min, the detection wavelength is 280nm, and the detection is carried out at 25 ℃.

(4) Measuring the collected single peak by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF), wherein the molecular weight after renaturation is 2392 Da;

the measurement method is as follows: dissolving the polypeptide purified in the step (4) in deionized water to prepare a 1 mu M solution, mixing 10 mu L of the solution with an equal volume of saturated matrix solution (alpha-cyano-4-hydroxycinnamic acid is dissolved in a 50% acetonitrile solution containing 0.1% trifluoroacetic acid to prepare a saturated solution, centrifuging, and taking supernatant), and then measuring.

(5) The isoelectric point of the purified polypeptide was determined to be 8.98 by isoelectric focusing electrophoresis, and the amino acid sequence structure of the purified polypeptide was determined to be NGICCGFPRSCGSRNCKPHRCC (SEQ ID NO.1) by an automated amino acid sequencer.

Experimental example 1

This experimental example tested the dispersibility of the anti-wrinkle polypeptide PIIIA2 in lipid materials, prepared in example 1.

The uniformity and dispersibility of the polypeptide PIIIA2 in lanolin were determined as an example. Taking 1000g of lanolin and 1000mg of PIIIA2 polypeptide, uniformly mixing the lanolin and the PIIIA2 polypeptide in an SHW/R type mobile high-shear emulsifying machine at room temperature, stirring at the speed of 120rpm/min, and stirring for 30 minutes. After mixing, the mixture was split into 5ml portions. The theoretical content of PIIIA2 under these conditions was 1 mg/g. Taking 20 tubes, accurately weighing appropriate amount of 1g mixture (equivalent to PIIIA20.1mg 10ml) into measuring flask, adding 20% ethanol solution, dissolving (ultrasonic dissolving if necessary) and diluting to scale, accurately weighing 2ml, and quantifying protein with Folin phenol.

The experimental results show that the average content of PIIIA2 in 20 samples is 0.90 +/-0.05 mg/g, and the content range of each sample is within 90% of the theoretical content. The PIIIA2 designed by the invention is shown to have good dispersibility in a fatty environment. Namely, the anti-wrinkle peptide PIIIA2 can be used for developing anti-wrinkle skin care products.

Experimental example 2

This experimental example tested the electrophysiological function of the anti-wrinkle polypeptide PIIIA2 in skin prepared in example 1.

The recording mode of the voltage-gated sodium ion channel is an extracellular high-sodium system, with extracellular fluid (mM):140 NaCl,3 KCl,1 MgCl₂,1 CaCl₂adjusting pH to 7.3 with 10 HEPES NaOH; the intracellular fluid (mM) is 140 CsF,1 EGTA,10 NaCl,3 KCl,10 MgCl₂CsOH adjusted the pH to 7.3. The recording mode of the sodium ion channel is that the clamping voltage is 80mV and 20 ms; the test voltage is 10mV for 50 ms; the voltage is clamped at 80mV for 20ms, and the recording is repeated continuously and circularly until the current is stabilized. The cells transiently expressing Nav1.4 were placed in front of 8-tubes of RSC-200(BioLogic) rapid perfusion dosing system, the Bath solution channel was opened, and 100nM wild-type PIIIA (Biochemical synthesized by Gill) and 100nM mutant PIIIA2 were administered by switching different channels.

Results referring to FIG. 2, it can be seen that wild-type PIIIA completely inhibited Nav1.4 current (FIG. 2A); mutant PIIIA2 partially inhibited the nav1.4 current (fig. 2B), inhibiting by about 90%, and retained about 10% of the muscle current conduction. Meanwhile, PIIIA2 at this concentration was almost inactive against other sodium channels that were responsible for important physiological functions, such as Nav1.2 (FIG. 2C), Nav1.5 (FIG. 2D), Nav1.7 (FIG. 2E) (FIGS. 2C-E); and wild type PIIIA has inhibitory effects (figure 2F-H) on Nav1.2 (figure 2F), Nav1.5 (figure 2G) and Nav1.7 (figure 2H), so that the modified polypeptide ensures target specificity.

From the above results, the inventors have made creative design and modification to provide polypeptides with a moderate function of inhibiting muscle contraction, and without serious side effects caused by the action on other ion channels such as nav1.2, nav1.5, nav1.7, etc.

Experimental example 3

This experimental example tested the anti-wrinkle polypeptide PIIIA2 produced in example 1 for cytotoxicity.

In the experimental example, the toxicity of the anti-wrinkle peptide PIIIA2 on human skin fibroblast HFF-1 is detected by an MTT method.

Human skin fibroblasts, HFF-1, were purchased from Kunming cell banks. Firstly, culturing fibroblasts in DMEM containing 15% fetal calf serum and double antibody (100U/ml each of penicillin and streptomycin), after the cells are full, digesting with 0.25% trypsin, washing twice with the culture medium, resuspending the cells, counting the cells, adding 100 mu l of cell suspension into a 96-well cell culture plate to make the number of cells in each well reach 10⁵And (4) respectively. Adding the anti-wrinkle polypeptide PIIIA2 prepared in example 1, adding sterilized ultrapure water with the same volume to the control group, standing at 37 deg.C and 5% CO₂Culturing in an incubator for 24 h. After the incubation, 20. mu.l of 5mg/ml MTT solution (prepared with cell culture PBS buffer) was added to each well of the 96-well cell culture plate, the incubation was continued for 5 hours, the wells were aspirated by syringe, 100. mu.l DMSO was added to each well, and the purple crystals were completely dissolved by blowing several times with a pipette. The light absorption was then detected using a microplate reader at 490nm and 630nm as the reference wavelength.

Table 2 statistical table of toxicity of the anti-wrinkle peptide PIIIA2 on HFF-1 cells.

PIIIA2 concentration (. mu.g/ml)	Cytotoxicity%
		1	0.00
50	1.22±0.2
		200	2.16±0.3

The results are shown in table 2, which indicates that the cytotoxicity of the anti-wrinkle peptide PIIIA2 is only 5.14% when the concentration is 160 mug/ml, and indicates that the anti-wrinkle peptide PIIIA2 has very low cytotoxicity on human skin fibroblasts and does not harm human normal skin cells, thereby being very beneficial to further development and application.

Experimental example 4

This experimental example tested the anti-wrinkle function of the anti-wrinkle polypeptide PIIIA2 prepared in example 1.

This experimental example measured the effect of anti-wrinkle peptide PIIIA2 in skin on wrinkles caused by UVB irradiation. UVB energy applied to the dorsal side of each mouse was controlled by varying the uv exposure time. The Minimal Erythemal Dose (MED) per mouse is about 36mj/cm². PIIIA2(10ng and 100 ng/mouse) was topically applied to the back of each mouse daily for 12 weeks. Initial UVB dose was set at 36mJ/cm²Then increased to 54mJ/cm at weeks 1-4²Increased to 72mJ/cm at weeks 4-7²Increased to 108mJ/cm at weeks 7-10²Finally increases to 122mJ/cm at weeks 10-12². The frequency of UVB irradiation was set three times a week, followed by topical application of vehicle (vehicle in this experimental example selected from lanolin as blank control and uv control) and PIIIA 2.

In this protocol, wrinkles in the back area began to be visually observed around 6 weeks after the start of UVB irradiation (fig. 3). To evaluate the formation of wrinkles after UVB irradiation, each hairless mouse was anesthetized with pentobarbital (50mg/kg body weight) by intraperitoneal injection at 6 and 9 weeks, and the degree of wrinkle formation was evaluated according to the grading criteria (table 3).

The grading scoring criteria were as follows: 0 no coarse wrinkles; 2 some shallow and rough wrinkles were observed in the dorsal skin area (Bisset grade 1); 4 light rough wrinkles were observed across the back skin (Bisset grade 2); 6 some deep and long wrinkles were observed on the dorsal skin (Bisset's grade 3).

The results in table 3 show that the anti-wrinkle polypeptide PIIIA designed in example 1 has a good anti-wrinkle effect compared to the control group.

TABLE 3 anti-wrinkle Effect score for anti-wrinkle polypeptide PIIIA2

Group of	Blank control	Ultraviolet contrast	PIIIA2(10ng)	PIIIA2(100ng)
					Wrinkle score at 6 weeks	0.0±0.0	3.93±0.62	1.23±0.21**	2.31±0.34**
Wrinkle score at 9 weeks	0.0±0.0	4.84±0.54	1.44±0.32**	2.62±0.46**

**P<0.05

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Sichuan Liyan workshop Biotechnology Ltd

<120> conus polypeptide, polypeptide composition, preparation method and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> PRT

<213> Artificial sequence

<400> 1

Asn Gly Ile Cys Cys Gly Phe Pro Arg Ser Cys Gly Ser Arg Asn Cys

1 5 10 15

Lys Pro His Arg Cys Cys

20

Claims (10)

1. A conus polypeptide is characterized by having a sequence shown as SEQ ID NO. 1.

2. The application of the conus polypeptide or the salt thereof in preparing a product for preventing and/or treating wrinkles is characterized in that the conus polypeptide has a sequence shown as SEQ ID No. 1.

3. Use according to claim 2, wherein the product is a pharmaceutical or cosmetic product.

4. The application of the conus polypeptide or the salt thereof in preparing a sodium ion channel inhibition product is characterized in that the conus polypeptide has a sequence shown as SEQ ID NO. 1.

5. Use according to claim 4, wherein the product is a pharmaceutical or cosmetic product.

6. A polypeptide composition, which comprises the conus polypeptide of claim 1, cosmetic adjuvants and other cosmetic raw materials.

7. The polypeptide composition of claim 6, wherein the cosmetic adjuvant is selected from at least one of the following:

humectant, emulsifier, mineral oil, vegetable oil, thickener, pH regulator and antiseptic;

preferably, the humectant is selected from at least one of the following: glycerin, polyalcohol, sodium hyaluronate, ceramide, trehalose, polysorbate-30 and an amino acid humectant;

the emulsifier is selected from lanolin;

the thickening agent is selected from at least one of the following substances: carbomer, hydroxyethyl cellulose and xanthan gum;

the pH regulator is selected from at least one of the following substances: citric acid, sodium citrate, lactic acid, sodium lactate, triethanolamine, and arginine;

the preservative is selected from at least one of the following substances: 1, 2-hexanediol, p-hydroxyacetophenone, and ethylhexylglycerin.

8. The polypeptide composition of claim 6, wherein the other cosmetic raw material is selected from at least one of the following: polypeptide substances, plant extracts, cytokines and vitamins;

preferably, the polypeptide substance is selected from at least one of the following substances: carnosine, pentapeptide-3, glutathione, anserine and snake meat peptide;

the plant extract is selected from at least one of the following substances: oat kernel extract, dendrobium officinale extract;

the cytokine is selected from at least one of the following substances: epidermal growth factor and nerve growth factor;

the vitamin is selected from at least one of the following substances: vitamin B3, vitamin C and vitamin E.

9. A method for preparing a conus polypeptide of claim 1, comprising the steps of: the conus polypeptide is synthesized by adopting a solid-phase synthesis method or a prokaryotic recombinant expression method.

10. The preparation method according to claim 9, wherein when the solid phase synthesis method is adopted, a crude conus polypeptide is synthesized, and then the crude polypeptide is subjected to renaturation folding of a high-order structure;

preferably, the renaturation folding is renaturation by a glutathione redox method;

preferably, the preparation method further comprises purifying the renatured and folded polypeptide;

preferably, the purification is desalting purification by HPLC reverse phase column chromatography.

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