Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Two kinds of conotoxin peptides, their preparation method and application

A conotoxin peptide and linear peptide technology, applied in the field of biomedicine, to achieve high safety, high lipophilicity, and easy artificial synthesis

Active Publication Date: 2021-05-25
SHENZHEN HUADA GENE INST
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are relatively few polypeptide calcium channel blocker drugs that have been developed so far. For example, Ziconotide is a type of N-type calcium channel blocker used to treat refractory chronic pain.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Two kinds of conotoxin peptides, their preparation method and application
  • Two kinds of conotoxin peptides, their preparation method and application
  • Two kinds of conotoxin peptides, their preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Extraction and identification of two conotoxin peptides ω-CPTx-btl1 and ω-CPTx-btl2 of the present invention

[0038] 1. Cono venom extraction and reductive alkylation

[0039] The shells of 4 barrel-shaped cone snails (Conus beulinus) produced in Hainan were smashed, and the venom tubes were dissected to collect the cone snail venom. The protein concentration in the venom was determined to be 6.48 mg / ml by Bradford method. The total protein amount was taken as 0.5 mg, and DTT with a final concentration of 1 mM was added, and reacted at 56°C for 1 h. After reduction and cooling to room temperature, IAM with a final concentration of 55 mM was added and reacted in a dark room at room temperature for 45 min.

[0040] 2. Toxin peptide enrichment

[0041] The above-mentioned treated conotoxin polypeptides were enriched with polypeptides from conotoxin venoms on a Strata-X C18 column. Strata-X C18 enrichment operation was performed according to standard procedure...

Embodiment 2

[0049] Embodiment 2 The chemical synthesis of conotoxin peptide ω-CPTx-btl1 and ω-CPTx-btl2 of the present invention

[0050] Conotoxin linear peptides with sequences shown in SEQ ID NO: 1 or 2 were synthesized by fluorenylmethoxycarbonyl (Fmoc) solid-phase chemical synthesis method (completed by Shanghai Jier Biochemical Synthesis Co., Ltd.).

[0051] Chemically synthesized peptides are refolded using the glutathione oxidation refolding method, namely:

[0052] The peptide was dissolved in 0.1M Tris-HCl, 0.1M NaCl, 5mM GSH, 0.5mMGSSG, pH 7.4 solution at a mass volume ratio of 1:10, and reacted at 25°C for 24-48h. The renaturation effect was detected by MALDI-TOF-MS.

Embodiment 3

[0053] Example 3 Inhibitory activity of conotoxin peptides ω-CPTx-btl1 and ω-CPTx-btl2 of the present invention on calcium ion channels

[0054] The patch clamp technique was used to detect the inhibitory activity of the conotoxin peptides ω-CPTx-btl1 and ω-CPTx-btl2 of the present invention on calcium ion channels, respectively. Specifically, the refolded conotoxin peptides ω-CPTx-btl1 and ω-CPTx-btl2 prepared in Example 2 were weighed respectively, and the conotoxin peptides ω-CPTx-btl1 or ω-CPTx-btl1 were detected by the whole cell patch clamp method. Effects of ω-CPTx-btl2 on ion channels in rat dorsal root ganglion (DRG) cells. Verapamil was used as a positive control.

[0055] Intracellular and extracellular fluid configuration in patch clamp:

[0056] Extracellular fluid: 140mM NaCl, 4mM KCl, 1mM MgCl 2 , 2mM CaCl 2 , 5mM D-glucose monohydrate (D-Glucose monohydrate), 10mM HEPES (pH=7.4); intracellular fluid: 20mM KCl, 110mM potassium aspartate (KAspartic), 1mM MgCl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Two new conotoxin peptides ω-CPTx-btl1 and ω-CPTx-btl2 are provided, the amino acid sequences of which are shown in SEQ ID NO: 1 or 2 respectively, and polynucleotides encoding the two peptides are also provided , the construct, expression vector and transformed cell containing the polynucleotide and the preparation method of the two peptides, the conotoxin peptide can be used for the treatment of diseases related to calcium ion channels.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, specifically, two new conotoxin peptides ω-CPTx-btl1 and ω-CPTx-btl2, polynucleotides encoding these two peptides, and a construct containing the polynucleotides , expression vector and host cell, the natural extraction and artificial synthesis method of the peptide, and the medical use of the peptide. Background technique [0002] Calcium ion channels are transmembrane proteins widely distributed in excitable cells, regulating extracellular Ca 2+ Enter the cytoplasm and participate in a series of Ca 2+ Dependent physiological activities, such as hormone and neurotransmitter release, muscle contraction and cell signal transduction, etc. According to the sensitivity to voltage, it 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 include L-type (Cav1. x), P / Q-ty...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/00C12N15/00A61K38/10A61P9/00
CPCA61K38/00C07K14/43504
Inventor 刘杰林志龙闻博童婷莫芬
Owner SHENZHEN HUADA GENE INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com