α-conotoxin peptide txic, its pharmaceutical composition, its preparation method and use

A composition and drug technology, applied in the fields of biochemistry and molecular biology, can solve the problems that α-CTX has not yet been discovered and studied

Active Publication Date: 2017-05-17
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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-CTXs that have been discovered and studied so far, or have clinical application value, The vast majority of α-CTX has not been discovered and studied

Method used

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  • α-conotoxin peptide txic, its pharmaceutical composition, its preparation method and use
  • α-conotoxin peptide txic, its pharmaceutical composition, its preparation method and use
  • α-conotoxin peptide txic, its pharmaceutical composition, its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Cloning and sequence analysis of the conotoxin gene

[0107] 1. Extraction of Cono Genomic DNA

[0108] The living organisms of Conus lividus Hwass and C. textile Linnaeus collected from the coasts of Hainan Island and Paracel Islands were used as materials and stored at -80°C for later use. First dissected the venom glands of the cono snails and weighed them. Then use a marine animal genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd., China) to extract venom gland genomic DNA. For specific operations, refer to the kit instructions. The steps are briefly described as follows: cut less than 30mg of venom gland tissue, put it into a centrifuge tube containing 200ul GA buffer (the formula is not provided in the instructions) and 40ul (10mg / ml) RNaseA, vortex for 15s; add 20ul proteinase K ( 20mg / ml) solution, vortex and mix in 56℃ water bath for 1.5h, until the tissue is completely dissolved; add 200ul GB buffer (the formul...

Embodiment 2

[0128] Example 2: Synthesis of α-conotoxin LvIA and TxIC

[0129] According to the amino acid sequences (SEQ ID NO: 3 and SEQ ID NO: 6) of the mature peptides of α-conotoxin LvIA and TxIC, these two polypeptides were artificially synthesized by the Fmoc method. The specific method is as follows.

[0130] 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 protecting groups, and the first and third cysteine ​​(Cys) -SH are used Trt (S-trityl) protection, the 2nd and 4th cysteine ​​-SH with Acm (S-acetamidomethyl) pairwise cross protection. The synthesis steps are: using the Fmoc and FastMoc methods in the solid phase synthesis method, two conotoxin linear peptides, LvIA and TxIC, are synthesized on the ABI Prism433a peptide synthesizer. 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 DC...

Embodiment 3

[0132] Example 3: Experiment of α-conotoxin LvIA to specifically block nAChRs

[0133] Refer to Azam L, Yoshikami D, McIntosh JM. Amino acid residues that conferhigh selectivity of the alpha6 nicotinic acetylcholine receptor subunit toalpha-conotoxin MII[S4A,E11A,L15A]. J Biol Chem. 2008Apr25;283(17):11625-32. The method in Epub2008Feb25, as well as the in vitro transcription kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)) instructions to prepare various rat neuronal nAChRs subtypes (α3β2, α6 / α3β2β3, α6 / α3β4, α9α10, α4β2 , Α4β4, α2β2, α2β4, α7), and mouse muscle-type nAChRs (α1β1δε) cRNA, the concentration of which is measured by the OD value under UV260nm. The Xenopus laveis oocytes (frog eggs) were dissected and collected, and cRNA was injected into the frog eggs. The injection volume of each subunit was 5ng cRNA. Each subunit of intramuscular nAChR was injected with 0.5–2.5ng DNA. Frog eggs are cultured in ND-96. CRNA was injected within 1 to 2 days...

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Abstract

The invention belongs to the fields of biochemistry and molecular biology, and relates to alpha-conotoxin TxIC, a medicinal composition thereof, a preparation method thereof and an application. The invention further relates to a propeptide of conotoxin, a nucleic acid construct thereof, an expression vector thereof and a transformed cell, and a fused protein thereof. The invention further relates to a method for blocking an acetylcholine receptor and a pharmaceutical use of the conotoxin. The alpha-conotoxin disclosed by the invention can be used for specifically blocking the acetylcholine receptor (nAChRs) (such as alpha3beta2nAChR), has high analgesic activity and addiction withdrawal activity, and has a good application prospect on the aspect of preparation of analgesics, smoking addiction withdrawal medicaments or drug addiction withdrawal medicaments and the like.

Description

[0001] The present invention is a divisional application of the parent case with the application number 201110198846.7. The filing date of the parent case is July 15, 2011. The title of the invention is "α-conotoxin peptide, its pharmaceutical composition, its preparation method and use ". Technical field [0002] The invention belongs to the fields 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 cells, and its fusion protein. The invention also relates to a method for blocking acetylcholine receptors and the pharmaceutical use of the conotoxin peptide. Background technique [0003] Conotoxin (conopeptide, conotoxin, CTX) is mostly composed of 7-50 amino acid residues and is rich in cysteine ​​(Cys) neuropeptide toxin. Conotoxin (peptid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K14/435C12N15/12C12N15/63C12N5/10A61K38/10A61K38/17A61P25/04A61P25/16A61P25/28A61P25/34A61P25/36A61P29/00A61P3/06A61P13/12C07K1/06C07K1/04
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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