Chinese south China sea signal taro snail nerve toxin gene Lt3.2 and uses thereof

A neurotoxin, amino acid technology, applied in medical preparations containing active ingredients, animal/human peptides, material testing products, etc.

Inactive Publication Date: 2008-06-25
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 taro snail nerve toxin gene Lt3.2 and uses thereof
  • Chinese south China sea signal taro snail nerve toxin gene Lt3.2 and uses thereof
  • Chinese south China sea signal taro snail nerve toxin gene Lt3.2 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction and identification of a cDNA library of signal cono venom tube:

[0053] Extraction of total RNA and cDNA synthesis: Isolate the South China Sea signal cono tube, and extract the total RNA from the tube according to the TRIZOL LS reagent manual of Gibco BRL. Take 1μg of total RNA from signal conotoxin tube with SMART III olignuclotide (5’-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3’) and CDSIII / 3’ PCR primers (5’-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3') Reverse transcription is performed to synthesize the first strand to obtain 10 μl of cDNA first strand product.

[0054] Construction and identification of cDNA library of signal cono tube: 1.5μl of cDNA was used for ligation reaction, and plated after transformation. Pick a single clone from the plate for conservation and randomly pick a certain number of single clones for sequencing and bioinformatics analysis.

Embodiment 2

[0055] Example 2: Extraction of Crude Toxin from Signal Cono:

[0056] The freshly separated venom tube is placed in a petri dish, and crude venom is 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 poison tube and the poison sac, place them in a beaker in an ice bath and crush them with a homogenizer. 3) Liquid nitrogen milling method. Add liquid nitrogen to the grinding bowl to grind the poison tube and the poison bubble into powder. The venom obtained by the three methods were respectively extracted in 1.1% acetic acid buffer, and the supernatant was collected by repeated centrifugation, and immediately subjected to gel chromatography or placed in -20°C for cryopreservation.

Embodiment 3

[0057] Example 3: Separation and purification by gel chromatography

[0058] The crude poison extracted in Example 2 was preliminarily separated using a Sephadex G-25 chromatography column (equilibrated with a 1.1% acetic acid solution) at a flow rate of 1 ml / min. The chromatographic peak diagram of the crude signal of Cono snails separated by Sephadex G25 is shown in Figure 1. Collect each elution peak, and then use SDS-PAGE electrophoresis to determine the approximate molecular weight of the components contained in each peak. The results are shown in Figure 2.

[0059] Adjust the pH of the collected peaks to 8.8 with Tris-HCl, and then load the samples on a Q sepharose high performance anion exchange column (20×2.6cm, pre-equilibrated with a 50mM Tris-HCl solution of pH 8.8) for ion exchange chromatography. At the same time, the breakthrough peak is collected; the flow rate is 1ml / min. Gradient elution was performed with 0~1M NaCl solution, and each elution peak was collected. A...

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Abstract

The invention relates to a new South China Sea conus M-superfamily toxin gene Lt3.2 and a polypeptide sequence lt3b coded by the same, and the application of the polypeptide in preparing tool medicine for the neurobiological research and analgesic drugs. By constructing cDNA library, the new M-superfamily toxin gene is obtained by cloning the South China Sea conotoxin, the cDNA sequence of Lt3.2 is shown by Sq. (400) 1 in the sequences table. As for the polypeptide (conotoxin lt3b) coded by the gene, the amino acid sequence of the precursor peptide is shown in Sq. (400) 2 of the sequences table, and the amino sequence of the presumed mature peptide is shown in Sq. (400) 3 of the sequences table. The neurotoxin lt3b of the invention can increase the opening current of the sodium channel and has an effect of abirritation. The neurotoxin lt3b can also be used for preparing tool medicine for the neurobiological research and analgesic agent.

Description

Technical field [0001] The present invention relates to a South China Sea signal Cono M-superfamily toxin gene Lt3.2 and its encoded polypeptide sequence lt3b and preparation technology of the polypeptide, and the development of the toxin in neurobiological research, ion channel drugs and analgesic drugs In the application. Background technique [0002] Cono snails belong to the Conidae family (Conidae) of the mollusc phylum Gastropoda. Most of them live in shallow waters of tropical oceans, and a few are found in deep waters ranging from several meters to more than 200 meters. They are named for their conical or taro-shaped appearance. Cono snails are relatively young creatures. The fossil record proves that Cono snails first appeared in the Eocene. The disappearance of the marine predatory mollusk ammonite, which was extinct at the same time as the terrestrial dinosaurs during the Mesozoic era, objectively promoted the taro. The first large-scale speciation of snails. The secon...

Claims

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Application Information

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