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Chinese south China sea signal taro snail nerve toxin gene 1t5.4 and uses thereof

A technology of neurotoxins and amino acids, applied in the fields of application, genetic engineering, nervous system diseases, etc., can solve the problems of few reports on conotoxin research

Inactive Publication Date: 2007-09-26
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are few reports on conotoxin research in my country

Method used

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  • Chinese south China sea signal taro snail nerve toxin gene 1t5.4 and uses thereof
  • Chinese south China sea signal taro snail nerve toxin gene 1t5.4 and uses thereof
  • Chinese south China sea signal taro snail nerve toxin gene 1t5.4 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction of the cDNA library of the signal cono venom tube in the South China Sea and EST sequence analysis

[0038] 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 cone venom total RNA with SMART III olignuclotide (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.

[0039] Construction and identification of cDNA library of signal cone venom tube: take 1.5 μl of cDNA for ligation reaction, 5 μl of reaction system, transform 1 μl of it, take 5 ul of transformation liquid to plate separately, take 300 ul of transformation liquid for shaking total library colonies. The number of single ...

Embodiment 2

[0041] Example 2: Determination and Analysis of Neurotoxin Gene lt5.4 Sequence

[0042] The plasmid and sequence of the South China Sea signal cone snail neurotoxin gene lt5.4 are from 18 highly homologous EST sequences cloned from the cDNA library of the South China Sea signal cone venom tube in Example 1, and the neurotoxin gene lt5.4 is highly expressed Genes account for about 4% of all EST sequences. Taking No. 5 plate D12 clone as an example for bioinformatics analysis, the results show that the length of its cDNA sequence is 523bp. The base sequence was analyzed by the tool software SEQTOOL, and the maximum reading frame was obtained, which was 189 bp in length and encoded a precursor peptide of 62 amino acid residues. Further analysis showed that the amino acid residues 1-20 of the precursor peptide were its signal peptide region, and its 50th residue was arginine, which could be considered as a standard proteolysis signal. Mature peptide fractions are separated. Ther...

Embodiment 3

[0043] Embodiment 3: Separation and purification of neurotoxin lt5d

[0044] Take the signal cone snail tube, cut it into pieces, extract with 50mmol / L Tris-Cl, pH8.6+100mmol / L NaCl, separate with G25Fine (i.d2.6*100cm), elute with extraction buffer, and detect at 280nm ; Collect the Q Sepharose fast flow separation on the second peak, NaCl gradient elution, collect the first peak on the C18 reverse phase column separation, acetonitrile gradient elution, and collect the peak with a retention time of 33.7min. The sequence of the isolated and purified neurotoxin lt5d is DCCPAKLLCCNP, with a molecular weight of 1274.8778 Daltons. The first and third cysteines in the molecule form a pair of disulfide bonds, and the second and fourth cysteines form a pair of disulfide bonds. to disulfide bonds.

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Abstract

The invention discloses a Chinese South China Sea signal Conidae neuropoison gene It 5.4 and coded peptide It5d as well as the application in the instrumental drug and anodyne, which constructs cDNA library to clone T-super of Conidae poison in the poisonous pipe of Chinese South China Sea signal Conidae.

Description

technical field [0001] The invention relates to a Chinese South China Sea signal cono neurotoxin gene lt5.4 and its coded polypeptide lt5d, and the application of the polypeptide in the preparation of a tool drug and analgesic drug for neurobiology research. Background technique [0002] Cono snails, also known as cone snails, are the general term for animals in the family Molluscs, Gastropoda, Prebranchia, and Conidae. The shell is conical or spindle-shaped, and there are often various patterns on the surface. The shell mouth is narrow and long, and the shell is too small to cover the shell mouth. There are about 500 species in the world, and more than 100 species in my country. They are distributed in the shallow waters of tropical seas, a few in the coastal areas of Guangdong and Guangxi, and the northern part of Hainan Island, and some species extend to the East China Sea. Although there are many types of cone snails, with great differences in individual size and food...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12A61K38/17A61P25/00A61P25/04
Inventor 徐安龙刘君梁王磊陈尚武
Owner SUN YAT SEN UNIV
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