Alpha-conotoxin peptide TxIB/Txd4, as well as pharmaceutical composition and use thereof

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

Active Publication Date: 2014-02-12
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 abov

Method used

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  • Alpha-conotoxin peptide TxIB/Txd4, as well as pharmaceutical composition and use thereof
  • Alpha-conotoxin peptide TxIB/Txd4, as well as pharmaceutical composition and use thereof
  • Alpha-conotoxin peptide TxIB/Txd4, as well as pharmaceutical composition and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Cloning and sequence analysis of α-conotoxin TxIB / Txd4 gene

[0100] 1. Extraction of Genomic DNA from Conus snail

[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 venomous glands. For specific operations, refer to the kit instruction manual. Dissolve the extracted total genomic DNA of Conus snails 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. Measure the OD260, OD280 value and OD260 / OD280 ratio of the DNA solution with a nucleic acid protein analyzer, and calculate the concentration of DNA (μg ml -1 ), pu...

Embodiment 2

[0153] Example 2: Synthesis of α-conotoxin TxIB and TxIB(G)

[0154] According to the amino acid sequences of α-conotoxin TxIB and TxIB(G) mature peptides (SEQ ID NO: 1 and 2, the C-terminals are both amidated), Fmoc method was used to artificially synthesize TxIB and TxIB(G) linear peptides (Figure 2 ). The specific method is as follows:

[0155] 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 TxIB and TxIB (G) is protected with Trt (S-trityl), and the -SH of the 2nd and 4th cysteine ​​is protected with Acm(S- acetamidomethyl) pair protection. 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 sy...

Embodiment 3

[0160] Example 3: α-conotoxin TxIB and TxIB(G) specifically block α6 / α3β2 β3 nAChR assay

[0161] References (Azam L, Yoshikami D, McIntosh JM.Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A].J Biol Chem.2008;283(17):11625 -32.), and the in vitro transcription kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)) instruction manual, to prepare various rat neurotype nAChRs subtypes, α3β2, α6 / α3β2β3 (ie α6β2* -nAChRs), α6 / α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7), human α6 / α3β2β3, and mouse muscle nAChRs (α1β1δε) cRNA, the concentration is measured by OD value under UV260nm . Xenopus laevis (Xenopus la aveis) oocytes (frog eggs) were collected by dissection, and cRNA was injected into the frog eggs, and the injected amount of each subunit was 5 ng cRNA. Inject 0.5-2.5 ng DNA per subunit of muscle nAChR. Frog eggs were cultured in ND-96. cRNA was injected wi...

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Abstract

The invention belongs to the field of biochemistry and molecular biology and relates to a new alpha-conotoxin peptide, as well as a pharmaceutical composition, a preparation method and a use thereof. The invention further relates a propeptide, a nucleic acid construct, an expression vector, transformed cells and a fusion protein of the conotoxin peptide. The invention further relates to a method for blocking an acetylcholine receptor and a pharmaceutical use of the conotoxin peptide. The alpha-conotoxin peptide provided by the invention can specifically block the acetylcholine receptor (nAChRs (nicotinic acetylcholine receptors)) (such as alpha 6/alpha 3 beta 2 beta 3 nAChR), further has the activity in treating addiction, neuropathic pain, Parkinson's disease, dementia, schizophrenia, depression, cancers and the like and has great application prospects in preparation of medicaments for quitting smoking and drugs and relieving pain, the medicaments for treating related mental disorders and the 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 a new α-conotoxin peptide Tx I B / Txd4, its pharmaceutical composition, its preparation method and application. The present invention also relates to the propeptide of the conotoxin peptide, its nucleic acid construct, its expression vector and transformed cell, and its fusion protein. The present invention also relates to a method for blocking acetylcholine receptors and the pharmaceutical use of the conotoxin peptide. Background technique [0002] Nicotinic acetylcholine receptors (nAChRs) are ubiquitous cell membrane proteins with important physiological functions and clinical research significance in the animal kingdom. Acetylcholine receptors. nAChRs are allosteric membrane proteins on cell membranes that mediate numerous physiological functions of the central and peripheral nervous systems, including learning, memory, response, analgesia, and motor control. ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K14/435C12N15/12C12N15/63C12N1/21C12N1/19C12N5/10C07K19/00A61K38/10A61K38/17A61P25/04A61P25/16A61P25/18A61P25/24A61P25/28A61P25/30A61P29/00A61P35/00G01N33/68C07K1/06C07K1/04
Inventor 罗素兰长孙东亭吴勇朱晓鹏胡远艳J·迈克尔·麦金托什
Owner HAINAN UNIVERSITY
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