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South China sea conus littertus linnaeus nervotoxin and its coding sequence and use

A neurotoxin and sequence technology, which can be used in nervous system diseases, medical preparations containing active ingredients, animal/human peptides, etc.

Inactive Publication Date: 2006-06-07
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the currently known conotoxins are isolated from fish-eating cone snails, and there are relatively few studies on the venom of insectivorous cone snails.

Method used

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  • South China sea conus littertus linnaeus nervotoxin and its coding sequence and use
  • South China sea conus littertus linnaeus nervotoxin and its coding sequence and use
  • South China sea conus littertus linnaeus nervotoxin and its coding sequence and use

Examples

Experimental program
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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 snail 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 s...

Embodiment 2

[0041] Example 2: Determination and Analysis of Neurotoxin Gene lt14.1 Sequence

[0042] The plasmid and sequence of the South China Sea signal cone snail neurotoxin gene lt14.1 are from 23 highly homologous EST sequences cloned from the cDNA library of the South China Sea signal cone venom venom gene in Example 1, and the neurotoxin gene lt14.1 is highly expressed The gene accounts for about 5% of all EST sequences. The bioinformatics analysis of No. A02 clone of No. 1 plate as an example shows 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 195 bp in length and encoded a precursor peptide of 65 amino acid residues. The precursor peptide was found to be compatible with alpha conotoxin qcl.1 (accession number: gi|46405113|gb|AAS93422.1| ) have homology (Identities=21 / 59 (35%), Positives=33 / 59 (55%)). Further analysis showed that the amino acid residues 1-20 of th...

Embodiment 3

[0043] Example 3: Solid phase chemical synthesis of neurotoxin lt14a

[0044] The solid-phase chemically synthesized neurotoxin lt14a sequence is MCPPLCKPSCTNC* (* indicates amidation modification), the molecular weight is 1391.7 Daltons, the first and third cysteines in the molecule form a pair of disulfide bonds, and the second and third cysteines form a pair of disulfide bonds. Four cysteines form another pair of disulfide bonds. The instrument synthesis strategy was adopted, using the FmocPHOBtPDCC method, Rink resin and Fmoc-amino acid, the coupling agent was DCC-HOBT, and the piperidine was de-Fmoc-based. The synthesis steps were carried out according to the instrument synthesis manual. The composition of the peptide-resin cleavage reagent and the peptide recovery method are the same as the manual synthesis method. The purity of the neurotoxin 1t14a synthesized by solid-phase chemical synthesis (Fig. 4a) was 96.5% by HPLC analysis (Fig. 4a), and its molecular weight was...

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Abstract

The present invention discloses South China Sea Conuslittertus Linnaeus nervotoxin and its coding sequence and use, and is especially one kind of South China Sea Conuslittertus Linnaeus nervotoxin gene 1t14.1 its coded polypeptide 1t14a, and the application of the polypeptide in preparing tool medicine for neurobiology research and analgesic. By means of cDNA library constituting process, the present invention clones new super family member 1t14.1 of conotoxin from South China Sea Conuslittertus Linnaeus poison canal with the DNA sequence as shown in <400>1 of the sequence list. The gene coded polypeptide, nervotoxin 1t14a, has precursor peptide and conjectural mature peptide with the amino acid sequence as shown in <400>2 of the sequence list. The nervotoxin 1t14a of the present invention can block the transmitter transfer between nerve and muscular joint, has analgesic effect and may be used in preparing tool medicine for neurobiology research and analgesic.

Description

technical field [0001] The invention relates to a Chinese South China Sea signal cono neurotoxin gene lt14.1 and its coded polypeptide lt14a, 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 kinds of cone snails with great differences in individual size and foo...

Claims

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Application Information

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IPC IPC(8): C07K14/435A61K38/17A61P25/04
Inventor 徐安龙彭灿王磊陈尚武
Owner SUN YAT SEN UNIV
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