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Alpha-conotoxin peptide TxIC/Txd1 and medicinal composition and application thereof

A technology for drugs and uses, applied in the fields of biochemistry and molecular biology, to solve problems such as addiction and cardiac side effects

Active Publication Date: 2014-03-26
HAINAN UNIVERSITY
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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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  • Alpha-conotoxin peptide TxIC/Txd1 and medicinal composition and application thereof
  • Alpha-conotoxin peptide TxIC/Txd1 and medicinal composition and application thereof
  • Alpha-conotoxin peptide TxIC/Txd1 and medicinal composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Gene cloning and sequence analysis of α-conotoxin TxIC / Txd1

[0100] 1. Extraction of Genomic DNA from Cono venom glands

[0101] Live brocade snails (C.textile Linnaeus) collected from the coasts of Hainan Island and Xisha Islands were used as materials, and stored at -80°C for later use. Cono venom glands were first dissected out and weighed. Then use a marine animal genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd., China) to extract the genomic DNA of the poisonous glands, and the specific operation was carried out according to the kit instructions. Genomic DNA of venom glands was obtained.

[0102] Dissolve the extracted venom gland genomic DNA in 100 μL TE, take 5 μL for 1.0% agarose gel electrophoresis, and use λ-EcoT14 I digest DNA Marker as a standard to detect the integrity and size of the obtained DNA. Determination of OD of DNA solution with a nucleic acid protein analyzer 260 、OD 280 value and OD 26...

Embodiment 2

[0136] Embodiment 2: the artificial synthesis of α-conotoxin TxIC

[0137] According to the amino acid sequence of the α-conotoxin TxIC mature peptide (SEQ ID NO: 7, C-terminal amidation), the TxIC linear peptide was artificially synthesized by the Fmoc method ( Figure 2A ). The specific method is as follows:

[0138]The resin peptide is artificially synthesized by Fmoc chemical method, and the resin peptide can be synthesized by a peptide synthesizer or manual synthesis. Except for cysteine, the remaining amino acids use standard side chain protecting groups. The -SH of the 1st and 3rd cysteine ​​(Cys) of TxIC is protected with Trt (S-trityl), and the -SH of the 2nd and 4th cysteine ​​is protected with Acm (S-acetamidomethyl) pairwise . The synthesis steps are as follows: using the Fmoc and FastMoc methods in the solid-phase synthesis method, three isomeric linear peptides were synthesized on an ABI Prism 433a peptide synthesizer. The side chain protecting groups of F...

Embodiment 3

[0142] Example 3: Experiment of α-conotoxin TxIC specifically blocking α3β4nAChR

[0143] References (Azam L, Yoshikami D, McIntosh JM.Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxinMII[S4A,E11A,L15A].J Biol Chem.2008;283(17):11625 -32.) method, and the in vitro transcription kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)) instructions to prepare various rat neurotype nAChRs subtypes (α3β4, α6 / α3β4, α9α10, α4β2 , α4β4, α3β4, α2β2, α2β4, α7), human α3β4, and mouse muscle nAChRs (α1β1δε) cRNA, the concentration was measured by OD value under UV 260nm. Xenopus laveis oocytes (frog eggs) were collected by dissection, and cRNA was injected into the frog eggs, and the injection volume of each subunit was 5 ng cRNA. Each subunit of muscle nAChR was injected with 0.5-2.5ng DNA. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after frog egg collection, and used fo...

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Abstract

The invention belongs to the field of biochemistry and molecular biology, and relates to an alpha-conotoxin peptide TxIC / Txd1 and a medicinal composition and application thereof. The alpha-conotoxin peptide can specifically block an acetyl choline receptor (nAChRs, such as alpha3beta4 nAChR (nicotinic acetylcholine receptor)), has curative activity to habituation, neuralgia, Parkinsonism, dementia, schizophrenia, depression, cancers, fear and the like, and has a good application prospect on the aspect of preparation of smoking cessation, drug treatment and pain relieving medicaments, medicaments relevant to the treatment of mental diseases and cancers, neuroscience tool medicaments and the like.

Description

technical field [0001] The invention belongs to the field of biochemistry and molecular biology, and relates to alpha-conotoxin peptide TxIC / Txd1, its pharmaceutical composition and application. Background technique [0002] Conus is a class of tropical marine carnivorous molluscs belonging to the genus Conus of the gastropod subclass Conus, which secretes polypeptide venom through venom tubes for predation and defense. Conotoxins usually contain 10-46 amino acids, are rich in disulfide bonds, have strong biological activity, and can specifically act on receptors and ion channels on animal cell membranes. In particular, it has high selectivity for voltage-gated or ligand-gated ion channels (including a few G-protein-related receptors, etc.). Conotoxins are divided into different gene families according to the similarity of the endoplasmic reticulum signal peptide sequence and cysteine ​​pattern of their precursor proteins. So far, all known conotoxins can be divided into 18...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K7/08C07K19/00C07K1/06C07K1/20C12N15/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K38/16A61K38/10A61P25/00A61P25/30A61P25/16A61P25/28A61P25/18A61P25/24A61P35/00A61P29/00A61P25/34A61P25/36A01N47/44A01N43/36A01P7/04
CPCA61K38/00C07K14/43504
Inventor 罗素兰长孙东亭胡远艳朱晓鹏吴勇J·迈克尔·麦金托什
Owner HAINAN UNIVERSITY
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