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Novel conus P-superfamily toxin sequence, preparation method and uses thereof

A technology of cono snail and sequence is applied to the new signal cono snail M-superfamily toxin sequence, preparation and application fields, which can solve the problems of in vitro expression of rare cono toxin genes and the like

Inactive Publication Date: 2008-10-15
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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  • Novel conus P-superfamily toxin sequence, preparation method and uses thereof
  • Novel conus P-superfamily toxin sequence, preparation method and uses thereof
  • Novel conus P-superfamily toxin sequence, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Construction and identification of signal cono venom cDNA library

[0057] Extraction of total RNA and synthesis of cDNA: Isolate the venomous tubes of the signal cone snail in the South China Sea, and extract the total RNA of the virulent tubes according to the instructions of Gibco BRL's TRIZOL LS reagent. Take 1 μg signal cono venom total RNA with SMART IIIolignuclotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3') and CDSIII / 3'PCR primer (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.

[0058] Construction and Identification of Signaling Cono Virus Tube cDNA Library: Extraction of cDNA For ligation reactions, plate after transformation. Single clones were picked from the plate for preservation and a certain number of single clones were randomly picked for sequencing and bioinformatics analysis.

Embodiment 2

[0059] Example 2: Sequence Analysis of Conotoxin Gene Lt16.1

[0060] The plasmid and sequence of the South China Sea signal cone neurotoxin gene Lt16.1 are from the EST sequence cloned from the South China Sea signal cone venom venom cDNA library. The Nanhai signal cono neurotoxin gene Lt16.1 encodes a precursor peptide consisting of 83 amino acid residues, and the mature peptide of the precursor peptide consists of 29 amino acid residues.

[0061] Using BLAST N and BLAST X (http: / / www.ncbi.nlm.nih.gov / BLAST) algorithms to search for homology of the tested nucleotide and derived protein sequences in existing public databases such as GenBank. Potential reading frames (ORFs) were mapped using SEQTOOLS 8.0. Sequence homology comparisons were performed using CLUSTAL X software. Protein signal peptide sequence prediction is performed through the website http: / / www.cbs.dtu.dk / services / SignalP. The molecular weight, theoretical isoelectric point, charge distribution and stability...

Embodiment 3

[0062] Embodiment 3: Construction of signal Cono M-superfamily toxin gene Lt16.1 expression plasmid

[0063] The Lt16.1 gene sequenced by the signal cono venom cDNA library, according to the amino acid sequence of its encoded mature protein, combined with the multiple cloning site of the pMAL-p2X plasmid, designed the amplification primers for the Lt16.1 gene fragment, as shown below . An Xmn I restriction site (GAA GGA TTT) was introduced into the upstream primer, and a HindIII restriction site (AAG CTT) and a double stop codon (TTA TTA) were introduced into the downstream primer. Perform PCR amplification with the above two primers to amplify the nucleotide sequence encoding the mature protein in the Lt16.1 gene. After the PCR product is digested with Xmn I and HindIII and connected to the pMAL-p2X vector, the mal E gene, protease Factor Xa recognition site sequence, nucleotide sequence of Lt16.1 gene encoding mature protein and terminator TTA TAA were fused together to for...

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Abstract

The invention discloses a new gene Lt16.1 of M-superfamily conotoxins of signal conus collected form South China Sea, a polypeptide sequence 1t16a coded by the same, a method for preparing the same and application of the polypeptide in the researche of neurobiology and ion channel as well as in the development of analgesics. The invention provides a new gene Lt16.1 of M-superfamily conotoxins found in a poison canal of signal conus from South China Sea by means of constructing cDNA library and clones a full-length gene by RT-PCR method. The amino acid sequence of the precursor of the polypeptide 1t16a is shown in a sequence list as and 1t; 400 and gt;2 sequence. The amino acid sequence of a mature peptide is shown the sequence list as &1t; 400 and gt; 3 sequence. The recombinant conotoxin 1t16a can obviously block part of sodium channels, provided the concentration is below 0.1um so as to reduce a peak current of sodium channels. The recombinant conotoxin 1t16a has analgesic effect on central nervous system by the mouse hot plate experiment.

Description

technical field [0001] The present invention relates to a Chinese South China Sea signal cone M-superfamily toxin gene Lt16.1 and its encoded polypeptide sequence lt16a and the preparation technology of the polypeptide, and the use of the toxin in neurobiological research, ion channel drugs and analgesia Applications in Drug Development. Background technique [0002] Cono snails belong to the mollusk phylum Gastropoda Conidae. Most of them live in shallow waters of tropical oceans, and a few live in deep waters with a water depth of several meters to more than 200 meters. They are named for their conical or taro-shaped shapes. Conus snails are relatively young organisms. Fossil records prove that the genus Conus first appeared in the Eocene (Eocene). The first large-scale speciation of snails. The second large-scale radiation of cone snails began in the Miocene (Miocene) and basically lasted until now. The formation of each cone snail species is accompanied by the evoluti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/435A61K38/17A61P9/06A61P9/10A61P25/04A61P25/08A61B5/04
Inventor 徐安龙周茂军王磊任政华刘君梁吴赟
Owner SUN YAT SEN UNIV
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