Conotoxin, polypeptide composition as well as preparation method and application thereof

A technology of polypeptide composition and cone snail, which is applied in the field of cosmetics preparation, can solve problems such as serious adverse reactions and normal membrane electrical signal disorder, and achieve the effect of low production cost and strong anti-wrinkle function

Inactive Publication Date: 2021-10-26
四川丽妍工坊生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (1) Excessive inhibition of skeletal muscle sodium channel Nav1.4 leads to facial muscle stiffness and paralysis; (2) Disturbance of no

Method used

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  • Conotoxin, polypeptide composition as well as preparation method and application thereof
  • Conotoxin, polypeptide composition as well as preparation method and application thereof
  • Conotoxin, polypeptide composition as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] This embodiment provides a kind of conus polypeptide (PIIIA2), the primary structure of its complete amino acid sequence is:

[0062] N-Asn-Gly-Ile-Cys-Cys-Gly-Phe-Pro-Arg-Ser-Cys-Gly-Ser-Arg-Asn-Cys-Lys-Pro-His-Arg-Cys-Cys–C, is a A tightly folded small peptide containing three pairs of disulfide bonds.

[0063] The preparation method is as follows:

[0064] (1) According to the amino acid sequence designed above, the crude polypeptide was synthesized by solid-phase synthesis (entrusted to Jill Biochemical Synthesis);

[0065] (2) Refolding of linear polypeptide advanced structure. The glutathione redox method is used for renaturation of the synthesized linear peptide, which specifically includes the following steps: dissolve 10 mg of the linear peptide synthesized in step (1) in 100 ml, pH=8.0 containing 5 mMGSH, 0.5 mM GSSG, 0.1 M Tris HCl , in a 0.1M NaCl solution, placed in a 25°C incubator for 24 hours to obtain a refolding solution, and the refolding effect wa...

experiment example 1

[0072] This experimental example tests the dispersibility of the skin anti-wrinkle polypeptide PIIIA2 prepared in Example 1 in lipid substances.

[0073] Take the determination of the homogeneity and dispersion of polypeptide PIIIA2 in lanolin as an example. Take 1000g of lanolin and 1000mg of PIIIA2 polypeptide, and mix them evenly with a SHW / R mobile high-shear emulsifier at room temperature at a stirring speed of 120rpm / min for 30 minutes. After mixing, aliquot into 5ml tubes. The theoretical content of PIIIA2 under this condition is 1mg / g. Take and pack into 20 tubes, accurately weigh an appropriate amount of 1g of the mixture (approximately equivalent to PIIIA2 0.1mg 10ml) into a measuring bottle, add 20% ethanol solution to dissolve (ultrasound to dissolve if necessary) and dilute to the mark, accurately measure 2ml with Folin Phenol was used for protein quantification.

[0074] The experimental results show that the average content of PIIIA2 in 20 samples is 0.90±0.0...

experiment example 2

[0076] This experimental example tests the electrophysiological function of the skin anti-wrinkle polypeptide PIIIA2 prepared in Example 1.

[0077] The recording mode of the voltage-gated sodium ion channel is an extracellular high sodium system, and the extracellular fluid is (mM): 140 NaCl, 3KCl, 1 MgCl 2 ,1 CaCl 2 , 10 HEPES NaOH to adjust the pH to 7.3; intracellular fluid (mM): 140 CsF, 1 EGTA, 10NaCl, 3 KCl, 10 MgCl 2 CsOH to adjust the pH to 7.3. The recording method of the sodium ion channel is clamping voltage 80mV 20ms; test voltage 10mV 50ms; clamping voltage 80mV 20ms, and this recording is repeated continuously until the current is stable. Before the cells transiently expressing Nav1.4 were placed in the 8-tube tube of the RSC-200 (BioLogic) rapid perfusion dosing system, the Bathsolution channel was opened, and 100 nM wild-type PIIIA (synthesized by Jill Biochemical) was given by switching different channels. ), 100 nM mutant PIIIA2.

[0078] Result referen...

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Abstract

The invention discloses a conotoxin, a polypeptide composition as well as a preparation method and application thereof, and relates to the technical field of cosmetic preparation. The conotoxin has a sequence as shown in SEQ ID NO. 1. Amino acid residue mutation is performed on wild mu-conotoxin PIIIA, so that the retardation effect of the mu-conotoxin on a sodium channel is moderately weakened, and meanwhile, the stability is kept under acidic and alkaline conditions, and the isoelectric point stability and the target specificity are kept. The novel conotoxin provided by the invention has the characteristics of stability, low production cost and the like. The mutated conotoxin or salt thereof keeps a relatively strong anti-wrinkle function, and the mutated conotoxin can avoid facial muscle stiffness and paralysis caused by excessive inhibition of a skeletal muscle sodium channel Nav1.4, and meanwhile, the risk of inhibiting the myocardial sodium channel and a brain sodium channel is also remarkably reduced.

Description

technical field [0001] The invention relates to the technical field of cosmetic preparation, in particular to a cone snail polypeptide, a polypeptide composition and a preparation method and application thereof. Background technique [0002] With the development of the economy and society and the improvement of living standards, the public's demand for health services is increasing day by day, and the consumption investment in the direction of medical beauty is strong. In terms of revenue, China has become the world's second largest medical beauty service market in 2018, with a revenue of US$18.379 billion, accounting for about 13.5% of the global medical beauty service market. At present, the medical beauty industry widely uses Botox for skin anti-wrinkle. Its anti-wrinkle mechanism is to reduce the dynamic expression lines caused by muscle stretching by blocking the signal transmission between nerves and muscles. However, not everyone can achieve the effect of wrinkle re...

Claims

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Application Information

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IPC IPC(8): C07K14/435A61K38/17A61P17/00A61K8/64A61K8/9794A61K8/67A61Q19/08
CPCC07K14/43504A61P17/00A61K8/64A61K8/9794A61K8/673A61K8/676A61K8/678A61Q19/08A61K2800/5922A61K38/00
Inventor 何阳
Owner 四川丽妍工坊生物科技有限公司
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