Chinese South China sea signal conus neurotoxin gene Lt3.1 and uses thereof

A neurotoxin, amino acid technology, applied in nervous system diseases, non-central analgesics, medical preparations containing active ingredients, etc.

Inactive Publication Date: 2008-07-16
SUN YAT SEN UNIV
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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. The signal cone snail in the South China Sea is an insectivorous cone snail. There is no report on its toxin research in the world.

Method used

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  • Chinese South China sea signal conus neurotoxin gene Lt3.1 and uses thereof
  • Chinese South China sea signal conus neurotoxin gene Lt3.1 and uses thereof
  • Chinese South China sea signal conus neurotoxin gene Lt3.1 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Construction and identification of signal cono venom tube cDNA library:

[0045] 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 CDS III / 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.

[0046] Construction and identification of the cDNA library of the signal cono virulence tube: 1.5 μl of the cDNA was used for the ligation reaction, and plated 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

[0047] Embodiment 2: Crude poison extraction of signal cone snails:

[0048] Freshly isolated venom tubes were placed in Petri dishes and crude venom was obtained in three different ways. 1) Extrusion method. Pinch one end of the venom sac and squeeze the slurry out of the venom tube. 2) Homogenization method. After dissecting the poisonous tube and poisonous sac, put it in a beaker in ice bath, and crush it with a homogenizer. 3) Liquid nitrogen grinding method. Add liquid nitrogen to the mortar to grind the poison tube and poison foam into powder. The venoms obtained by the three methods were respectively extracted in 1.1% acetic acid buffer solution, and the supernatant was collected by repeated centrifugation, and immediately applied to gel chromatography or stored at -20°C for freezing.

Embodiment 3

[0049] Embodiment 3: Gel chromatography separation and purification

[0050] The crude poison extracted in Example 2 was preliminarily separated with a Sephadex G-25 chromatographic column (balanced with 1.1% acetic acid solution) at a flow rate of 1 ml / min. The chromatogram of the signal cone snail crude virus separated by Sephadex G25 is shown in Figure 1. Collect each elution peak, then use SDS-PAGE electrophoresis to determine the approximate molecular weight of the components contained in each peak, and the results are shown in Figure 2.

[0051] The collected peaks were adjusted to pH 8.8 with Tris-HCl, and then loaded onto Q sepharose highperformance anion-exchange columns (20×2.6 cm, pre-balanced with 50 mM Tris-HCl solution of pH 8.8) for ion-exchange chromatography, The breakthrough peak was collected simultaneously; the flow rate was 1 ml / min. Gradient elution was carried out with 0-1M NaCl solution, and each elution peak was collected. Among them, the ion-exchan...

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Abstract

The invention relates to a novel M-superfamily toxin gene Lt3.1 of Conus litteratus, the coding peptide sequence lt3a and the application of the peptide in preparing the tool drugs and the analgesic drugs for the Neurobiological Study. The gene is cloned from the Conus litteratus venom tube by means of constructing a cDNA library.

Description

technical field [0001] The present invention relates to a Chinese South China Sea signal cone snail M-superfamily toxin gene Lt3.1 and its encoded polypeptide sequence lt3a and the preparation technology of the polypeptide, and the use of the toxin in neurobiology research, ion channel drug and analgesic drug development in the 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. 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 evol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K7/08A61K38/17A61K38/10A61P43/00A61P25/04A61P29/00G01N33/50
Inventor 徐安龙曾夏芸王磊皮灿辉任政华刘君梁孙丹丹周茂军
Owner SUN YAT SEN UNIV
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