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Conotoxin alphaD-GeXXA gene, and polypeptide and applications thereof

A technology of conotoxin and cono snail, applied in the field of biochemistry, can solve problems such as addiction and cardiac side effects, and achieve the effect of improving the ability to inhibit acetylcholine receptors and having good application prospects

Active Publication Date: 2017-02-01
TONGJI UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used non-selective nAChR agonists such as nicotine, although they can alleviate the symptoms of the above neurological diseases, they have strong side effects on the heart and gastrointestinal tract, and are addictive

Method used

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  • Conotoxin alphaD-GeXXA gene, and polypeptide and applications thereof
  • Conotoxin alphaD-GeXXA gene, and polypeptide and applications thereof
  • Conotoxin alphaD-GeXXA gene, and polypeptide and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the crude poison extraction of natural conotoxin αD-GeXXA

[0050] Natural conotoxin αD-GeXXA is extracted from Conus generalis collected in the South China Sea, and its crude poison extraction method comprises the following steps:

[0051] (1), separate the venom tube tissue of the live conus snail, and place it in a petri dish;

[0052] (2) Use surgical scissors to cut the venom tube into pieces (this operation is carried out on ice), add 25mL 0.1% TFA (pure water contains 0.1% TFA in mass fraction), and cut the venom tube in the petri dish Rinse into a 100mL small beaker;

[0053] (3), place the small beaker on ice and magnetically stir for 30 minutes, then centrifuge at 4°C and 12000g for 15min, collect the supernatant in a 500mL freeze-drying bottle, and use 25mL of 20% acetonitrile (containing 0.1 %TFA) was extracted once, then centrifuged at 4°C and 12000g for 15min, the supernatant was collected and combined into a 500mL freeze-drying bottle, the...

Embodiment 2

[0055] Embodiment 2: Separation and purification of natural conotoxin αD-GeXXA

[0056] The separation and purification method of natural conotoxin αD-GeXXA comprises the following steps: take the crude poison sample frozen at -20°C, centrifuge at 4°C and 12000g for 15min to remove the precipitate, take the supernatant and use high performance liquid chromatography Instrument, hydrophobic reverse phase column ZORBAX 300SB-C18 (9.4 × 250mm) separation. The reagents and procedures used in the separation and purification were set as follows: Buffer A was prepared from 0.1% TFA and pure water, Buffer B was prepared from 0.1% TFA and pure acetonitrile, the detection wavelengths were 214nm and 280nm, and the concentration of Buffer B changed from 5% to 5% within 50 minutes. to 55%, the fractions were pooled and lyophilized. Afterwards, the components with higher abundance will be further purified by an analytical ZORBAX 300SB-C18 (5.0×250mm) reverse-phase column to obtain pure samp...

Embodiment 3

[0057] Embodiment 3: the preparation of the complete cDNA sequence of natural conotoxin αD-GeXXA

[0058] The preparation method of the complete cDNA sequence of the natural conotoxin αD-GeXXA is as follows: take out the venom tube of the general conus snail from the -80 ℃ refrigerator, quickly put it into a mortar pre-cooled by liquid nitrogen, and carefully grind it in liquid nitrogen into a powder form, during which liquid nitrogen was continuously added; then the total RNA was extracted according to the method recommended by the Trizol Total RNA Isolation Reagent Kit, and then the complete cDNA sequence of GeXXA was cloned by 3'-RACE and 5'-RACE. The specific steps of the complete cDNA sequence cloning method are as follows: Take two 0.5mL EP tubes (treated with DEPC water), add 8 μl of RNase-free water, then add 2 μl of extracted total RNA, add 1 μl of universal primer AP, in Superscript Under the action of II reverse transcriptase, the first-strand cDNA was synthesized. ...

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Abstract

The present invention provides a conotoxin alphaD-GeXXA gene, and a polypeptide and applications thereof, wherein the conotoxin alphaD-GeXXA gene is derived from South China Sea Strategoconus generalis, and the encoded conotoxin alphaD-GeXXA mature peptide can spontaneously form a homodimer so as to non-competitively inhibit acetylcholine receptors, such that the peptide has good application prospects in preparation of drugs or reagents for inhibition of acetylcholine receptors.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to a gene, polypeptide and application of conotoxin αD-GeXXA. Background technique [0002] Conotoxins are small peptides secreted in the venom tubes of cono snails, molluscs living in shallow water in tropical or subtropical oceans. They can quickly and specifically act on the nervous system, and have shown important clinical application value. . [0003] The expression sequence (propeptide) of conotoxins generally includes a signal peptide. The signal peptide is relatively conservative in evolution. According to its sequence, the currently known conotoxins are divided into 27 superfamilies, including A-, B1-, B2-, B3-, C-, D-, E-, F -,G-,H-,I 1-,I2-,I3-,J-,K-,L-,M-,N-,O1-,O2-,O3-,P-,S-,T- ,V-,Y-(Kaas,Q.,R.Yu,A.H.Jin,S.Dutertre and D.J.Craik,2012:ConoServer:updated content,knowledge,and discovery tools in the conopeptide database.Nucleic acids research,40,D325 -330) and Q-su...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P25/00A61P25/28A61P25/08A61P25/18A61P25/16A61P21/00A61P21/04A61P25/24A61P9/12A61P3/06A61P9/06A61P29/00A61P11/06A61P37/08A61P3/10A61P31/22A61P35/00A61P25/30
Inventor 王春光丁建平大卫·亚当戚正武徐少琼张天龙希瓦·N·康佩拉闫梦迪吕爱平汪燕芳邵晓霞
Owner TONGJI UNIV
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