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Novel modification-specific signal Conus superfamily toxin and uses thereof

A post-translational modification and conch technology, which is applied to the new signal cono P-superfamily toxin with post-translational modification and its application field, can solve the problems of in vitro expression of rare conotoxin genes and the like

Inactive Publication Date: 2008-11-19
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many conotoxin genes have been cloned, there are few reports on the expression of conotoxin genes in vitro

Method used

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  • Novel modification-specific signal Conus superfamily toxin and uses thereof
  • Novel modification-specific signal Conus superfamily toxin and uses thereof
  • Novel modification-specific signal Conus superfamily toxin and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Separation and purification of signal conotoxin lt9b, identification by mass spectrometry and Edman degradation sequencing

[0034] Place the freshly separated venom tube of the South China Sea signal Cono snail in a petri dish, cut it on ice, and extract it with extraction buffer (100mmol / L NaCl, 50mmol / L Tris-HCl, pH 8.8) for 30 min, 4°C, 12000g centrifugation Collect the supernatant at 30 min, repeat extraction and centrifugation three times, combine the supernatants, separate them by Sephadex-G25 Fine, and detect four absorption peaks at a wavelength of 280 nm (see figure 1 ). The peak II sample separated and collected by Sephadex-G25 Fine, after ion exchange chromatography, detected five absorption peaks at 215nm wavelength (see figure 2 ), separately collected and further separated by reversed-phase high performance liquid chromatography.

[0035] Ion exchange chromatography peak IV was further purified by reversed-phase HPLC, and the spectrum obtained un...

Embodiment 2

[0039] Example 2: The effect of signal conotoxin lt9b on sodium ion channels

[0040] In order to observe the effect of this P-superfamily conotoxin on TTX-sensitive voltage-gated sodium channels, we selected rat DR6 cells as the experimental object. After DR6 cells form a whole-cell recording mode, under voltage clamp conditions, the cell membrane potential is clamped at -80mV, and then given a single pulse stimulation with a duration of 50ms and a depolarization voltage of -10mV to induce TTX-sensitive sodium currents . After applying the toxin, give the same stimulation repeatedly and observe the effect of the toxin on the inward current.

[0041] 500nM lt9b has a significant inhibitory effect on TTX-sensitive sodium current, and the maximum inhibition rate is 49.60±2.61% (n=3). After lt9b partially inhibited the sodium current, the shape of the current was consistent with the shape of the control current, indicating that they did not significantly change the dynamic characteri...

Embodiment 3

[0043] Example 3: Experimental method of central analgesic effect on physiological model

[0044] Basic scheme: mouse hot plate method

[0045] Select 100 NIH mice, weighing 20←2g, all females. Before the experiment, the mice were screened. The mice were placed on a hot plate pain meter heated to 55°C in advance, and the time from when the mouse was put on the hot plate to the appearance of hind feet licking was recorded with a stopwatch as the mouse’s pain threshold. Twice, the mice whose pain threshold did not exceed 20s were selected as qualified candidates for formal experiments. The mice after screening were randomly divided into 4 groups, namely the blank control group and the low, medium and high dose groups of lt9b samples, with 10 mice in each group. At the beginning of the experiment, intraperitoneal injection was performed. The low, medium and high dose groups of lt9b were given 0.07 mg / kg, 0.14 mg / kg, and 0.28 mg / kg, respectively. The blank control group was given an e...

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Abstract

The invention relates to a novel South China Sea conus littertus Linnaeus P-superfamily toxin polypeptide sequence 1t9b with post-translational modification, and an application of the polypeptide to the neurobiological study, ionic channel study and analgesic development. The invention obtains the 1t9b from the toxic pipe of the South China Sea signal conus through a plurality of separation and purification methods, and the amino acid sequence of the 1t9b is showed by the andlt;400andgt;1 sequence in a sequence table. The conus toxin 1t9b has an obvious inhibiting action on a TTX sensitive type sodium current in a density of 500nM. A mouse hot plate experiment also discloses that the conus toxin 1t9b has a central analgesia action. Thus, the conus toxin 1t9b can serve as a probe which is applicable to the classification and determination of ion channel types and hypotypes or to the research and development of instrumental medicines and the preparation of analgesics.

Description

Technical field [0001] The present invention relates to a new South China Sea signal Cono P-superfamily toxin lt9b with post-translational modification, and the application of the toxin in neurobiological research, ion channel medicine and analgesic medicine development. Background technique [0002] Cono snails belong to the Conidae family (Conidae) of the mollusc phylum Gastropoda. Most of them live in shallow waters of tropical oceans, and a few are found in deep waters ranging from a few meters to more than 200 meters. They are named for their conical or taro-shaped appearance. Cono snails are relatively young creatures. The fossil record proves that Cono snails first appeared in the Eocene. The disappearance of the marine predatory mollusk ammonite that was extinct at the same time as the terrestrial dinosaurs in the Mesozoic Era has objectively promoted the taro. The first large-scale speciation of snails. The second large-scale radiation of snails began in the Miocene and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/17A61P9/06A61P9/10A61P25/04A61P25/08A61B5/04
Inventor 徐安龙周茂军王磊任政华刘君梁吴赟
Owner SUN YAT SEN UNIV
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