Alpha-conotoxin peptide, and medical composition, preparation method and purpose thereof

A technology of medicine and use, applied in the direction of peptide preparation method, drug combination, botanical equipment and method, etc., can solve the problems that α-CTX has not been discovered and researched

Active Publication Date: 2013-01-16
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, it is estimated that there are at least 1,000 different α-CTXs (α-conotoxins) in the venom of Conus, and there are only a dozen kinds of α-

Method used

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  • Alpha-conotoxin peptide, and medical composition, preparation method and purpose thereof
  • Alpha-conotoxin peptide, and medical composition, preparation method and purpose thereof
  • Alpha-conotoxin peptide, and medical composition, preparation method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1: Cloning and sequence analysis of the conotoxin gene

[0108] 1. Extraction of Cono snail genomic DNA

[0109]Conus lividus Hwass and 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. The steps are briefly described as follows: cut less than 30mg of venomous gland tissue, put it into a centrifuge tube containing 200ul GA buffer (the formula is not provided in the manual) and 40ul (10mg / ml) RNaseA, vortex for 15s; add 20ul proteinase K ( 20mg / ml) solution, vortexed and mixed in a 56°C water bath for 1.5h until the tissue was completely dissolved; add 200ul GB buffer (the formula is...

Embodiment 2

[0129] Embodiment 2: the synthesis of α-conotoxin LvIA, TxIC

[0130] According to the amino acid sequences (SEQ ID NO: 3 and SEQ ID NO: 6) of the α-conotoxin mature peptides LvIA and TxIC, the two peptides were artificially synthesized by using the Fmoc method. The specific method is as follows.

[0131]The resin peptides of LvIA and TxIC are artificially synthesized by Fmoc chemical method. Except for cysteine, the other amino acids use standard side chain protection groups, of which the -SH of the first and third cysteine ​​(Cys) is used Trt (S-trityl) protection, the second and fourth cysteine ​​-SH with Acm (S-acetamidomethyl) paired cross-protection. The synthesis steps are as follows: two conotoxin linear peptides, LvIA and TxIC, were synthesized on an ABI Prism 433a peptide synthesizer by using the Fmoc and FastMoc methods 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)...

Embodiment 3

[0133] Example 3: Experiment of α-conotoxin LvIA specifically blocking α3β2 nAChRs

[0134] Refer to 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 Apr 25; 283(17): 11625-32. The method in Epub 2008 Feb 25, 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 , α6 / α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7), and the cRNA of mouse muscle nAChRs (α1β1δε), the concentration was measured by the OD value under UV 260nm. Xenopus laveis 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 within 1-2 da...

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Abstract

The invention belongs to the fields of biochemistry and molecular biology, relates to an alpha-conotoxin peptide, and a medical composition, a preparation method and a purpose thereof, also relates to a propeptide of the conotoxin peptide, a nucleic acid construct, an expression vector, a transformed cell and a fusion protein, and also relates to a method for blocking acetylcholine receptors and a pharmaceutical purpose of the conotoxin peptide. The alpha-conotoxin peptide can specifically block the acetylcholine receptors (nAChRs) (such as alpha3beta2 nAChR), has high analgesic activity and addiction withdrawal activity and has a good application prospect in the aspects of preparing analgesics, smoking cessation or drug treatment medicines and the like.

Description

technical field [0001] The invention belongs to the field of biochemistry and molecular biology, and relates to an α-conotoxin peptide, 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] Most conotoxins (conopeptide, conotoxin, CTX) are composed of 7-50 amino acid residues and are rich in cysteine ​​(Cys) neuropeptide toxins. Conotoxins (peptides) can be divided into α, ω, μ, δ and other subtypes according to their receptor targets. α-CTX can specifically act on acetylcholine (nAChR) receptors at nerve terminals (Livet BG, Sandall DW, Keays D, Down J, Gayler KR, Satkunanathan N, Khalil Z. Therapeutic applications of ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K14/435C12N15/12C12N15/63C12N1/21C12N1/19C07K19/00A61K38/10A61K38/17A61P25/04A61P25/16A61P25/28A61P25/34A61P25/36A61P29/00G01N33/68C07K1/06C07K1/04C12N15/11
CPCY02P20/55
Inventor 罗素兰长孙东亭吴勇胡远艳朱晓鹏陈琴保罗・F・阿莱温奥德J・迈克尔・麦克托什
Owner HAINAN UNIVERSITY
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