New T - ultra family conantokins, coded polynucleotide and application

A conotoxin peptide and polynucleotide technology, applied in the biological field

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 research on the T-superfamily conotoxins pro

Method used

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  • New T - ultra family conantokins, coded polynucleotide and application
  • New T - ultra family conantokins, coded polynucleotide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Cloning of T-superfamily conotoxin gene

[0079] 1 Extraction of total RNA from conus snails

[0080] The marble cone snails (Conus marmoreus Linnaeus), signal cone snails (Conus littertus Linnaeus), brocade cone snails (Conustextile Linnaeus), barrel-shaped cone snails (Conus betulinus Linnaeus) and wormwood cone snails collected from the coasts of Hainan Island and the Xisha Islands (Conus lividus Hwass) live cone snails of 5 species were 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.

[0081]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 ...

Embodiment 2

[0104] Example 2 Synthesis of T-superfamily conotoxins BeB34M (SEQ ID NO: 2) and LiC33M (SEQ ID NO: 4)

[0105] According to the amino acid sequences of conotoxin peptides BeB34M and LiC33M, these two peptides were artificially synthesized by Fmoc method. The specific method is as follows.

[0106] The above two conotoxin peptides were synthesized on an 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. T...

Embodiment 3

[0107] Example 3 Conotoxin BeB34M and LiC33M linear peptide oxidative folding

[0108] The purified reduced linear peptides BeB34M and LiC33M were oxidatively folded at room temperature for 24 h, respectively. The oxidative folding buffer contained 1 mM reduced (GSH) and 0.5 mM oxidized (GSSG) glutathione (pH 7.5). Oxidized folded peptides were purified using a liquid chromatography system (Bio-Rad). The mixture after the oxidation reaction was loaded onto a preparative chromatography column (4.6×250 mm, SinochromODS-BP) at a flow rate of 1 ml / min, and eluted with 0.1% TFA until all the reaction solution was eluted completely. Solvent A was 0.1% TFA in acetonitrile and solvent B was 0.1% TFA in water. Gradient elution (10% → 100% A, 90% → 0% B, 30 minutes) can purify 96% of the active peptide after oxidative folding.

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Abstract

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

Description

technical field [0001] The invention relates to a new T-superfamily Conotoxin peptide (T-superfamily Conotoxin, T-CTX), its encoding polynucleotide and its use, belonging to the field of biotechnology. Background technique [0002] Conus, a carnivorous mollusc, lives on coral reefs, rocks, and sandy bottoms in the intertidal and subtidal shallow waters of tropical oceans. A few are distributed at water depths ranging from tens of meters to about 200 meters, and usually have beautiful shells. . There are poisonous glands and arrow-shaped radula inside the mouth of the cone snail. The syringe-like and harpoon-like radula can inject toxins into the prey. It is extremely toxic and can cause convulsions, tremors, paralysis, and death in animals. We collectively call the small peptide animal neuropeptide toxins secreted by cone snails as Conotoxin (CTX) to anesthetize prey. [0003] According to their feeding habits, cone snails can be roughly divided into three types: pisicvoro...

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 罗素兰张本长孙东亭陈心
Owner HAINAN UNIVERSITY
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