Conus lividus neurotoxin and coding sequence and use thereof

A technology of neurotoxin and wart pickle, which is applied in the field of polypeptide and its coding sequence and application, can solve the problems of relatively little research on insectivorous cone snail venom, and achieve the effect of excellent drug effect and inhibition of transmitter transmission

Inactive Publication Date: 2011-08-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most of the currently known conotoxins are isolated from fish-eating cone snails, and there are relatively few studies on the venom of insectivorous cone snails

Method used

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  • Conus lividus neurotoxin and coding sequence and use thereof
  • Conus lividus neurotoxin and coding sequence and use thereof
  • Conus lividus neurotoxin and coding sequence and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Extraction of total RNA from wart venom tube tissue and cloning of toxin cDNA

[0024] The extraction of total RNA refers to the method of Gibcol BRL company LS reagent instructions were carried out. Taq DNA polymerase, 10×PCRBuffer and dNTP were purchased from Dingguo Company; PCR primers were synthesized by Invitrogen Company; other organic reagents were domestic analytical reagents and were purchased from Guangzhou Chemical Reagent Factory.

[0025] Take live snails, break the snail shell with a masonry hammer to expose the snail meat, carefully and quickly separate the poison tube tissue on ice, weigh it, quickly put it into liquid nitrogen and grind it fully, add 15 times the weight volume of LS reagent (that is, add 15ml to 1g of tissue), fully homogenize in an ice bath, and store in a -80°C refrigerator for extraction of total RNA. Take 50-100mg tissue sample, use 0.75ml Homogenize the LS reagent and let stand at 15-30°C for 5 minutes. Then add ...

Embodiment 2

[0054] Example 2: Determination and Analysis of Neurotoxin Gene lv1.3 Sequence

[0055] The neurotoxin gene lv1.3 was cloned and sequenced from the cDNA library of Cono snail venom tube in South China Sea constructed in Example 1. The neurotoxin gene lv1.3 is a highly expressed gene. The base sequence of the neurotoxin gene lv1.3 was analyzed by using the tool software SEQTOOL, and its maximum reading frame was obtained, with a length of 195bp, encoding a precursor peptide of 65 amino acid residues. Further analysis showed that the amino acid residues 1-21 of the precursor peptide were its signal peptide region, and its 48th residue was arginine, which could be considered as a standard proteolysis signal. Mature peptide fractions are separated. In addition, in most conotoxins, the glycine residue at the end of the precursor peptide is often excised during the post-translational modification process, providing an amidation modification signal for the previous amino acid residu...

Embodiment 3

[0056] Embodiment 3: the solid-phase chemical synthesis of polypeptide lv1c

[0057] The solid-phase chemically synthesized neurotoxin lv1c sequence is GCCSDPPCRHKHQDLC* (* indicates amidation modification), the molecular weight is 1795.4 Daltons, the first and third cysteines in the molecule form a pair of disulfide bonds, and the second and third cysteines form a pair of disulfide bonds. Four cysteines form another pair of disulfide bonds. The instrument synthesis strategy was adopted, using the FmocPHOBtPDCC method, Rink resin and Fmoc-amino acid, the coupling agent was DCC-HOB T, and the piperidine was de-Fmoc-based. The synthesis steps were carried out according to the instrument synthesis manual. The composition of the peptide-resin cleavage reagent and the peptide recovery method are the same as the manual synthesis method. Such as Figure 4 As shown, the HPLC analysis of the solid-phase chemically synthesized polypeptide lv1c identified its purity as 96.5%; Figure ...

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Abstract

The invention provides a Conus lividus neurotoxin gene 1v1.3 and a polypeptide 1v1c coded thereby and use of the polypeptide 1v1c in the preparation of tool medicines for neurobiological study and analgesic medicines. In the invention, a process of constructing a cDNA library is adopted to clone the conotoxin gene 1v1.3 in the toxin canal of Conus lividus, and through analysis on a base sequence with a tool software SEQTOOL, the mature peptide amino acid sequence of the polypeptide 1v1c coded by the Conus lividus neurotoxin gene 1v1.3 is determined to be represented by a sequence 400(2) in a sequence table. The polypeptide 1v1c provided by the invention can block the transmission of a transmitter of neuromuscular junction, and according to a mouse hot-plate method, the polypeptide 1v1c has pain relieving effect and can be used for preparing the tool medicines for neurobiological study and analgesic medicines.

Description

technical field [0001] The present invention relates to a polypeptide and its coding sequence and application, in particular to a cono neurotoxin polypeptide and its coding sequence and application. Background technique [0002] Cono snails, also known as cone snails, are the general term for animals in the family Molluscs, Gastropoda, Prebranchia, and Conidae. Cono snails are a kind of carnivorous marine molluscs, which belong to the gastropod probranchia Conus family in taxonomy. They anesthetize animals through the conotoxin in their poison tubes to help prey. Cono snails can be divided into three types according to their feeding habits. Three types: piscivorous, insectivorous and snail-eating snails, among which the toxins of piscivorous snails are more toxic to humans and mammals. Conotoxin, the active ingredient in its venom, is a small peptide with unique neuropharmacological effects, composed of 7-41 amino acid residues, generally rich in disulfide bonds. According...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P25/04A61P21/00
Inventor 徐安龙强媛媛王磊任政华吴赟冯宇超
Owner SUN YAT SEN UNIV
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