Conotoxin mutant polypeptide lv1c-aa and its application and preparation method

A technology of lv1c-aa and conotoxin, which is applied to molecular probes and analgesic drugs, polypeptide compounds, conotoxin mutant lv1c-AA and the field of preparation thereof, which can solve the problem that conotoxin resources are not yet available. research and development issues

Active Publication Date: 2020-03-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even so, there are still about 99% of conotoxin resources that have not been researched and developed

Method used

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  • Conotoxin mutant polypeptide lv1c-aa and its application and preparation method
  • Conotoxin mutant polypeptide lv1c-aa and its application and preparation method
  • Conotoxin mutant polypeptide lv1c-aa and its application and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Solid-phase synthesis and separation and purification of the conotoxin lv1c mutant lv1c-AA, that is, the preparation method of the conotoxin mutant polypeptide lv1c-AA, which is prepared in the following manner:

[0027] Step 1: Put a certain weight of Wang resin into the synthesis column, wash it with dimethylformamide (N, N-dimethylformamide, DMF) for 3 times, then add 3-4ml of DMF, and feed nitrogen to make Wang resin in the solution Soak and turn up and down for 20-40 minutes, blow dry with nitrogen. Here, the Rink resin Rink can be set between 0.1g and 0.3g. Here, 0.2g is used to load the synthesis column, and the Rink resin is the RinkAmide resin (RinkAmide).

[0028] Step 2: Add 2-3ml of 20% piperidine to remove the protection of 9-fluorenylmethoxycarbonyl (Fmoc), and react with nitrogen gas for 10-20 minutes; then rinse with DMF for 3 times; repeat the deprotection once, and react with nitrogen gas for 10 minutes -20 minutes, rinse with DMF 4 times, ...

Embodiment 2

[0039] Example 2: Conotoxin mutant lv1c-AA has better analgesic activity than conotoxin lv1c and MVIIA.

[0040] MVIIA is a clinical analgesic drug, and its Chinese name is Qikao polypeptide; lv1c: is the name of the polypeptide before the mutation.

[0041] The analgesic activity of lv1c-AA was determined by the acetic acid writhing method. Kunming mice weighed about 18 g, half male and half male, and randomly divided into groups of 8 mice. The intracerebroventricular injection concentration was 5 μg / μl, and each mouse was injected with 10 μl. At intervals of 1.5h, 3h, and 6h, 0.7% acetic acid was injected intraperitoneally, 20 μl per mouse. The number of writhing times of the mice within 15 min was recorded, and the average value was taken for each group. And compare the significance of writhing frequency difference with normal saline control group. The result is as figure 2 shown. Experiments showed that the analgesic activity of lv1c and its mutant lv1c-AA was super...

Embodiment 3

[0042] Example 3: Inhibitory effect of lv1c-AA on acetylcholine current in hippocampal neurons

[0043] Newborn wistar rats within 24 hours of birth were decapitated, their brains were removed, meninges were separated, and hippocampus were removed, and hippocampal neurons were isolated and cultured by conventional enzymatic hydrolysis for power supply physiological research.

[0044] The hippocampal neurons cultured for 8-9 days were rinsed twice with extracellular solution, and 2ml of extracellular solution was added for experiment. The recording electrode is pulled by the two-step method of the electrode puller, and then thermally polished by the electrode polisher before use, so that the diameter of the tip of the microelectrode is about 1 μm, and the impedance after filling the electrode liquid is 4-5MΩ. The caliber of the administration electrode used to record the current of the acetylcholine receptor is about 3-4M, and the inside is filled with 1mM ACh (prepared from ex...

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Abstract

The invention relates to conotoxin mutant polypeptide, and application of the polypeptide to preparation of a molecular probe for neurobiology research and pain easing medicine. The conotoxin mutant polypeptide is obtained by replacing mature conotoxin lv1c into alanine by ninth-bit arginine and eleventh-bit lysine, and is named as lv1c-AA; the amino acid sequence of the conotoxin mutant polypeptide is shown as a sequence table. The cone shell polypeptide lv1c-AA is obtained by a chemical synthesis method; the polypeptide has the more prominent pain easing effect than lv1c, and has the activity of inhibiting a neuron pattern acetylcholine receptor; important values are realized in pain easing medicine development; good application prospects are also realized for inhibiting addiction symptoms caused by morphine misuse.

Description

technical field [0001] The present invention relates to a polypeptide compound, in particular to a conotoxin mutant lv1c-AA and its preparation method, as well as the application of the polypeptide in the preparation of molecular probes and analgesic drugs for neurobiology research, belonging to biological pharmaceutical field. Background technique [0002] Marine biotoxins are a hotspot in the research of new marine drugs. Among all animal toxins, conotoxin, a polypeptide toxin from the venom of marine mollusc cone snails, is by far the most diverse representative. There are about 500-700 species of cone snails in the world, and each species of cone snails contains as many as 1,000 peptide toxins with different sequence structures, and the toxins contained by any two cone snails rarely overlap. Therefore, it is estimated that there are more than 500,000 types of conotoxins. According to the statistics of the Conoserver website, as of 2010, 4136 conotoxin amino acid seque...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/16C07K1/06C07K1/04A61K38/17A61P29/00A61P25/04
CPCA61K38/00A61P25/04A61P29/00C07K14/43504Y02P20/141
Inventor 王磊苑广伟
Owner SOUTH CHINA AGRI UNIV
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