Toxic sequential, its preparation and use

A technology of cono snail and thread pattern, which is applied to the new thread pattern conotoxin sequence, preparation and application fields, can solve the problems of in vitro expression of rare cono toxin genes, and achieve the effects of delaying inactivation and prolonging the opening time.

Inactive Publication Date: 2007-06-20
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many conotoxin genes have been cloned, there are few reports on the expression of conotoxin genes in vitro

Method used

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  • Toxic sequential, its preparation and use
  • Toxic sequential, its preparation and use
  • Toxic sequential, its preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Construction and identification of the lined cono venom cDNA library:

[0039] Extraction of total RNA and synthesis of cDNA: Isolate the venomous tubes of Cono snails, and extract total RNA from the venomous tubes according to the instruction manual of TRIZOL reagent from Gibco BRL Company. Take 1 μg of the total RNA of Cono snail venom tubes, and use SMARTIII olignuclotide (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3′) and CDS III / 3′PCR primers (5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3′) was reverse transcribed to synthesize the first strand to obtain 10 μl of cDNA first strand product. 2μl of the first-strand product was washed with 5’PCR primer (5’-AAGCAGTGGTATCAACGCAGAGT-3’), CDS III / 3’PCR primer (5’-ATTCTAGAGGCCGAGGCGGCCGACATGd(T) 30 N -1 N-3') for second strand amplification. After digesting and purifying the second chain with proteinase K, it was cleaved with Sfi I and ligated into the pcDNA3.0 library vector (purchased from Invitrogen)...

Embodiment 2

[0040] Embodiment 2: Sequence Analysis of Conotoxin S6.8 Gene

[0041] The plasmid and sequence of the conotoxin S6.8 gene were obtained from the gene cluster of strivd4 (QID) in the library of Example 1. Bioinformatics analysis of it showed that its full-length cDNA sequence was 601bp in length, and its reading frame encoded 78 amino acid residues, including a 25-amino acid signal peptide, a 26-amino acid leader peptide and a 27-amino acid mature peptide. Its signal peptide sequence and mature peptide cysteine ​​arrangement (C-C-CC-C-C) are highly conserved with other members of the O-superfamily conotoxins. Amino acid analysis of the leader and mature peptides showed a higher similarity to members of the delta-family conotoxins (see Figure 1). Further analysis showed that the mature peptide encoded by the S6.8 gene of striata conotoxin contained less charged amino acids, and the degree of hydrophobicity was closest to that of conotoxin members of the δ-family. This family...

Embodiment 3

[0042] Embodiment 3: Construction of recombinant conotoxin S6.8 expression plasmid

[0043] According to the amino acid sequence of the mature peptide encoded by the S6.8 gene and the codon preference of Escherichia coli, the template for the PCR reaction was designed and synthesized, and combined with the pTRX plasmid (the applicant constructed and applied for a Chinese patent, the patent name is: a high-efficiency prokaryotic Expression vector, patent number 00124832.4, authorized announcement number 1189565C) multiple cloning site, design the amplification primers of the S6.8 gene fragment, as shown below. A Kpn I restriction site and a ProScission Protease cutting site were introduced into the upstream primer, and a Not I restriction site and a double stop codon were introduced into the downstream primer. After the PCR product is digested with Kpn I and Not I and connected to the pTRX vector, TRX gene, 6 histidine sequences, protease PreScission Protease recognition site s...

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Abstract

Linear conotoxin sequence and its encoded polypeptide sequence S6.8, its production and use are disclosed. The process is carried out by constructing cDNA library and cloning O-super-family conotoxin. It can delay vertebrate cerebral neuron cell membrane sodium-ion channel inactivation and sodium-ion channel opening time. It can be used as probe for ion-channel type and sub-type discrimination or as pilot compound to make research of medicine.

Description

technical field [0001] The invention relates to a new Chinese South China Sea conotoxin gene and its coded polypeptide sequence S6.8, the preparation technology of the polypeptide, and the application of the toxin in neurobiology research and ion channel drug development. Background technique [0002] Cono snails belong to the family Conidae of the gastropod class and inhabit shallow waters of tropical oceans. They are named for their conical or taro-shaped shapes. Cono snails have a strong natural evolutionary ability, including hundreds of species in a single genus, which makes the cone snail the most evolutionarily successful creature among marine invertebrates. The shape of the hunting device and the chemical composition of the venom of the cone snail have also evolved and developed in the natural evolution process to adapt to different habitats and prey objects, forming a very diverse form. Cono snails have a wide range of foods, and are divided into fish-eating cone s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/70C12N1/21A61K38/17A61P9/06A61P25/08A61P25/00
Inventor 徐安龙刘芸王磊董美玲
Owner SUN YAT SEN UNIV
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