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South China Sea conotoxin encoding sequence and application thereof

A technology of conotoxin and coding sequence, applied in the direction of application, non-central analgesic, medical preparation containing active ingredients, etc.

Inactive Publication Date: 2016-08-31
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 encoding sequence and application thereof
  • South China Sea conotoxin encoding sequence and application thereof
  • South China Sea conotoxin encoding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: High-throughput sequencing of venom tubes of Taro cereus

[0050] Extraction of total RNA and synthesis of cDNA: Isolate the venomous tubes of Taro cerevisiae snails from South China Sea, and extract the total RNA from the venomous tubes according to the instruction manual of TRIZOL LS reagent from Gibco BRL company.

Embodiment 2

[0051] Example 2: Sequence Analysis of Conotoxin Gene Cq1.1

[0052] The plasmid and sequence of Cq1.1 neurotoxin gene Cq1.1 from Cono cereus in South China Sea were obtained from the venom c sequence of Cono cereus in South China Sea. The precursor peptide of the Cq1.1 neurotoxin gene Cq1.1 of the South China sea snail encodes 83 amino acid residues, and the mature peptide encodes 29 amino acid residues.

[0053] 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. The cetin sequence prediction was performed through the website http: / / www.cbs.dtu.dk / services / SignalP. The molecular weight, theoretical isoelectric point, charge distribution and stability parameters of the protein were completed by Pro...

Embodiment 3

[0055] Example 3: Synthesis of Cono snail O-superfamily toxin mature peptide Cq1.1

[0056] (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.

[0057] (2) After 2 hours, wash the resin with DMF, then drain it, repeat this four times, and drain the resin for use.

[0058] (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.

[0059] (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.

[0060] (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 resin. Af...

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Abstract

The invention discloses a South China Sea conotoxin encoding sequence and an application thereof, and concretely relates to a new South China Sea Calamiconus quercinus O-superfamily toxin gene Cq1.1, a polypeptide sequence Cq1.1 encoded by the new South China Sea Calamiconus quercinus O-superfamily toxin gene Cq1.1, a preparation method of the polypeptide, and applications of the polypeptide in neurobiology research, ion channel researches and pain easing drug exploitation. The new O-superfamily toxin gene Cq1.1 is found from the venom ducts of South China Sea Calamiconus quercinus through constructing a cDNA library, and Cq1.1 is obtained by using a high flux sequencing method. Mouse hot plate experiments and zebra fish intramuscular injection experiments of the recombinant conotoxin Cq1.1 prepared in the invention find that the recombinant conotoxin Cq1.1 has central analgesia and nerve anesthesia effects, so the recombinant conotoxin Cq1.1 can be used in classification identification of ion channel type and subtype or research exploitation of tool drugs, and preparation of pain easing drugs as a probe.

Description

technical field [0001] The present invention relates to a Chinese South China Sea conus snail neurotoxin coding sequence and its application, in particular to a Chinese South China Sea yellow taro snail O-superfamily toxin gene Cq1.1 and its encoded polypeptide sequence Cq1.1 and the preparation technology of the polypeptide , and the application of this toxin in neurobiology research, ion channel drug and analgesic 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. [0003] Cono snails have strong natural evolution ability. There are 706 species of cone snails registered in the world, including hundreds of species in a single genus, which makes the cone snail the most successful evolutionary creature among marine inv...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C07K1/06A61K38/17A61P29/00
CPCC07K14/43504A61K38/00
Inventor 陈尚武张晗吴赟王磊黄盛丰
Owner RES INST OF SUN YAT SEN UNIV & SHENZHEN
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