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New alpha - conantokins, coded polynucleotide and application

A conotoxin peptide and polynucleotide technology, applied to medical preparations containing active ingredients, peptides, peptide sources, etc.

Inactive Publication Date: 2006-07-05
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The vast majority of α-CTX (more than 99%) have not been discovered and studied

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Cloning of embodiment 1 conotoxin gene

[0075] 1 Extraction of total RNA from conus snails

[0076] Live conus lividus Hwass, Conus littertus Linnaeus and C.textile Linnaeus collected from Hainan Island, Xisha Islands and other coastal areas are used as materials. Total RNA was extracted with a small column centrifugal tissue / cell total RNA extraction kit (Shanghai Huasun Bioengineering Co., Ltd.) or a Total RNA Isolation System kit (Promega) according to the operation manual. Let's take the RNA extraction kit produced by Shanghai Huasun as an example.

[0077] First, freeze the cone venom gland or poisonous tube with liquid nitrogen and grind it into powder, and transfer it to a 1.5mL centrifuge tube. Add 500 μL TCL solution (RNA lysate, Shanghai Huasun) to the sample, mix well and centrifuge at 12000 rpm for 3 minutes → transfer the supernatant and add 250 μL 75% ethanol to the supernatant and mix thoroughly → transfer to the RNA adsorption column and centrifuge at...

Embodiment 2

[0107] The synthesis of embodiment 2 conotoxin LC22M, LeD2M

[0108] According to the amino acid sequences of conotoxin mature peptides LC22M and LeD2M, these two peptides were artificially synthesized by Fmoc method. The specific method is as follows.

[0109] Two conotoxin peptides, LC22M and LeD2M, were synthesized on the ABI Prism 433a peptide synthesizer by using the Fmoc method in the solid-phase synthesis method. The side chain protecting groups of Fmoc amino acids are: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys). Using Fmoc HOBT DCC method, Rink resin and Fmoc amino acids, The synthesis steps were carried out referring to the synthesis manual of the instrument. In order to complete the reaction, the piperidine deprotection and coupling time were appropriately extended, and double coupling was used for difficult amino acids. The crude linear peptide was recovered and purified with a 250*4.6mm, Hypersil ODS-2 column. The linear peptide had a purit...

Embodiment 3

[0110] Example 3 Conotoxin LC22M, LeD2M linear peptide oxidative folding

[0111] 10μM fully reduced LC22M, LeD2M linear peptides in folding buffer (100mM NH 4 HCO 3 (pH 8.0), 23-25°C) and folded with stirring for 24-48h. The oxidized folded peptide was then purified by liquid chromatography (Bio-Rad) system.

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PUM

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Abstract

This invention describes a new alpha-CTX, its encoding polynucleotide, the construction units and transformation cells having this encoding polynucleotide, and the reconstruction process for producing the said alpha-CTX. This invention also describes the artificial synthesis and applications of the said alpha-CTX.

Description

technical field [0001] The present invention relates to a new α-conotoxin peptide, a polynucleotide encoding the peptide, a construct and a host cell containing the polynucleotide, and a method for artificially synthesizing and recombining the peptide. The present invention also relates to the use of said conotoxin peptide. Background technique [0002] Conus is a carnivorous mollusk belonging to the gastropod subclass Conus. There are about 500 species of cone snails on the earth, and there are about 50-200 different conotoxins (CTX) in each cone snail venom, and the toxin peptides of different cone snail species are different from each other. There are at least tens of thousands of conotoxins that have the special function of regulating various ion channels. CTX can distinguish different subtypes of various ion channels, and is widely used in neuropharmacology research, and as diagnostic reagents and therapeutic drugs. Therefore, the cone snail venom is called the "marin...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K19/00C12N15/12C12N15/63C12N15/64A61K38/17A01N37/00A61P33/10A61P35/00
Inventor 罗素兰长孙东亭张本权娅茹
Owner HAINAN UNIVERSITY
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