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α-conotoxin peptide lvia/lvd21, its pharmaceutical composition and use

A technology of use and medicine, applied in the field of biochemistry and molecular biology, can solve problems such as cardiac side effects and addiction

Active Publication Date: 2016-05-18
HAINAN UNIV
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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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  • α-conotoxin peptide lvia/lvd21, its pharmaceutical composition and use
  • α-conotoxin peptide lvia/lvd21, its pharmaceutical composition and use
  • α-conotoxin peptide lvia/lvd21, its pharmaceutical composition and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1: Cloning and sequence analysis of α-conotoxin LvIA / LvD21 gene

[0103] 1. Extraction of Genomic DNA from Cono snails

[0104] Live 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 the Marine Animal Genomic DNA Extraction Kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd., China) to extract the genomic DNA of the poisonous glands. For specific operations, please refer to the kit manual; you can also refer to the literature, Zheng Xiaodong, Gao Bingmiao, Li Baozhu, Peng Chao, Wu Aiyin, Zhu Xiaopeng, Chen Xin, Changsun Dongting, Luo Sulan, Screening of primers for novel α-conotoxin gene cloning, Biotechnology, 2011, 21(4): 40-44.

[0105] Dissolve the extracted venom gland genome total DNA in 100 μLTE, take 5 μL for 1.0% agarose gel electrophoresis, and use λ-EcoT14IdigestDNAMarker as the ...

Embodiment 2

[0138] Embodiment 2: the artificial synthesis of α-conotoxin LvIA / LvD21

[0139] According to the amino acid sequence of the α-conotoxin LvIA / LvD21 mature peptide (SEQ ID NO: 4, C-terminal amidation), the LvIA / LvD21 linear peptide was artificially synthesized by the Fmoc method ( Figure 2B ). The specific method is as follows:

[0140] 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 LvIA / LvD21 is protected with Trt (S-trityl), and the -SH of the 2nd and 4th cysteine ​​is formed 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 ABIPrism433a polypeptide synthesizer. The sid...

Embodiment 3

[0144] Example 3: Experiments of α-conotoxin LvIA / LvD21 blocking various nAChRs

[0145] 参照文献(AzamL,YoshikamiD,McIntoshJM.Aminoacidresiduesthatconferhighselectivityofthealpha6nicotinicacetylcholinereceptorsubunittoalpha-conotoxinMII[S4A,E11A,L15A].JBiolChem.2008;283(17):11625-32.)中的方法,以及体外转录试剂盒(mMessagemMachineinvitrotranscriptionkit(Ambion,Austin, TX)) instructions for preparing various rat neuron nAChRs subtypes (α3β4, α6 / α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7), human α3β4, and mouse muscle nAChRs (α1β1δε) cRNA, its concentration was measured by OD value under UV260nm. Xenopuslaveis oocytes (frog eggs) were collected by dissection, and cRNA was injected into the frog eggs, and the injected amount of each subunit was 5ngcRNA. 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 for voltage-clamp recordings of nAChRs within 1-4 days after injection.

[...

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Abstract

The invention belongs to the fields of the biochemistry and the molecular biology, and relates to a new alpha-conotoxin peptide LvIA / LvD21, and a pharmaceutical composition and a use thereof. The invention also relates to a propeptide of the conotoxin peptide, a nucleic acid construct of the propeptide, an expression vector of the propeptide and a cell transformed by the propeptide, and a fusion protein of the propeptide. The invention further relates to a method for blocking an acetyl choline receptor, and a pharmaceutical use of the conotoxin peptide. The alpha-conotoxin peptide provided by the invention is capable of specifically blocking the acetyl choline receptor (nAChRs) (for example, alpha 3 beta 2 nAChRs), and has the activity of treating the diseases such as neuralgia, habituation, parkinsonism, dementia, schizophrenia and cancer, so that the alpha-conotoxin peptide LvIA / LvD21 has excellent application prospect in the aspects of preparing medicaments for analgesia, smoking cessation and drug rehabilitation, and medicaments for treating related mental diseases and cancers, serving as a neurobiological tool medicament 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 LvIA / LvD21, 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] Conotoxin, Conopeptide, CTX, secreted by the carnivorous mollusk Conus living in the tropical ocean, has the special function of regulating various ion channels, and has been shown to be clinically important. value. 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K7/08C12N15/12C12N15/63C12N1/21C12N1/19C12N5/10C07K19/00A61K38/17A61K38/10A61P25/04A61P25/16A61P25/18A61P25/24A61P25/28A61P25/30A61P29/02A61P35/00G01N33/68C07K1/06C07K1/04
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 UNIV
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