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South China Sea conotoxin coding sequence as well as preparation method and application of South China Sea conotoxin

A conotoxin technology, applied in the South China Sea conotoxin coding sequence

Inactive Publication Date: 2016-07-20
RES INST OF SUN YAT SEN UNIV & SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, cDNA libraries of various cone snails have been constructed abroad. China’s South China Sea snail is an insectivorous cone snail. There is no high-throughput sequencing method in the world to mine its genes and three-dimensional information about it. Report on Toxin Studies on Disulfide Bonds

Method used

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  • South China Sea conotoxin coding sequence as well as preparation method and application of South China Sea conotoxin
  • South China Sea conotoxin coding sequence as well as preparation method and application of South China Sea conotoxin
  • South China Sea conotoxin coding sequence as well as preparation method and application of South China Sea conotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: High-throughput sequencing of venom tubes of Taro cereus

[0048] Extraction of total RNA and synthesis of cDNA: Isolate the venomous tubes of Taro snails from the South China Sea, and extract the total RNA from the venomous tubes according to the instructions of GibcoBRL's TRIZOLLS reagent.

Embodiment 2

[0049] Example 2: Sequence Analysis of Conotoxin Gene Cq1.0

[0050] The plasmid and sequence of Cq1.0 neurotoxin gene Cq1.0 of Cono cereus snail from South China Sea were obtained from the venom c sequence of Cono cereus snail from South China Sea. The precursor peptide of the Cq1.0 neurotoxin gene Cq1.0 of the South China sea snail encodes 93 amino acid residues, and the mature peptide encodes 28 amino acid residues.

[0051] Using BLASTN and BLASTX (http: / / www.ncbi.nlm.nih.gov / BLAST) algorithms to perform homology searches on the measured nucleotide and derived protein sequences in existing public databases such as GenBank. Potential reading frames (ORFs) were located using SEQTOOLS8.0. Sequence homology comparisons were performed using CLUSTALX software. The cetin sequence prediction was performed through the website http: / / www.cbs.dtu.dk / services / SignalP. Protein molecular weight, theoretical isoelectric point, charge distribution and stability parameters, etc. through...

Embodiment 3

[0053] Example 3: Synthesis of the mature peptide Cq1.0 of Cono snail O-superfamily toxin

[0054] (1) Weigh 1.5 g of chlorine resin (degree of substitution: 0.2 mmol / g) into a 150 ml reactor and soak with 50 ml of DCM.

[0055] (2) After 2 hours, wash the resin with DMF, then drain it, repeat this four times, and drain the resin for use.

[0056] (3) Weigh 192mg of threonine (Thr) in a 10ml centrifuge tube, add 5ml of DCM to dissolve it, then add 1ml of DIEA and shake it for 1min. After the solution is clarified, add it to the reactor, and then put the reaction The device was placed in a shaker at 30°C for reaction.

[0057] (4) After 2 hours, cap the head with a certain amount of methanol (methanol:DIEA:DCM=1:1:2) for half an hour, then wash it with DMF four times, and drain it for use.

[0058] (5) Add a certain amount of 20% piperidine (piperidine / DMF=1:4) into the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the res...

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Abstract

The invention discloses a South China Sea conotoxin coding sequence as well as a preparation method and application of the South China Sea conotoxin. A new O-super family toxin gene Cq1.0 is discovered from the venom canal of South China Sea conotoxin through a method for constructing a cDNA library and is obtained by utilizing a high-throughput sequencing method. A mouse hot plate experiment and a zebrafish muscle injection experiment discover that the conotoxin Cq1.0 has the effects of central analgesia and nerve anesthesia, can be taken as a probe for the classification and the identification of ion channel types and subtypes or for the research and the development of tool drugs and the preparation of analgesic drugs.

Description

technical field [0001] The present invention relates to the field of biomedicine, relates to a Chinese South China Sea conotoxin coding sequence, preparation method and application, relates to a new South China Sea snail O-superfamily toxin sequence, preparation method and application, specifically relates to a Chinese South China sea snail O-superfamily toxin gene Cq1.0 and its encoded polypeptide sequence Cq1.0 and the preparation technology of this polypeptide, and the application of this toxin in neurobiological research, ion channel drug and analgesic drug development application. 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. [0003] Cono snails have strong natural evolution ability. There are 706 species of cone snails reg...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C07K1/06A61K38/17A61P25/04
CPCY02P20/55C07K14/43504A61K38/00
Inventor 张晗陈尚武
Owner RES INST OF SUN YAT SEN UNIV & SHENZHEN
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