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Conotoxin and biological preparation method and application thereof

A conotoxin and biological technology, applied in the field of biological preparation of conotoxin, can solve the problems of inability to process and remove the N-terminal signal peptide sequence, unable to solve the problem of C-terminal amidation, small conotoxin molecules, etc. , to achieve the effect of large-scale production, complete post-processing and low production cost

Inactive Publication Date: 2013-01-16
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there have been no literature reports on the in vitro expression of conotoxins with high activity. The possible reasons are as follows: 1. In the prokaryotic expression system, the N-terminal signal peptide sequence cannot be processed and removed, and the problem of C-terminal amidation cannot be solved; 2. , Conotoxin molecules are small, and there are many basic amino acids, so it is difficult to form a specific active conformation

Method used

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  • Conotoxin and biological preparation method and application thereof
  • Conotoxin and biological preparation method and application thereof
  • Conotoxin and biological preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Optimization of conotoxin αACT gene and its expression in silkworm

[0029] 1. Cloning and expression of conotoxin αACT gene

[0030] 1.1. Preparation of relevant solutions and media (Joseph et al., Molecular Cloning Experiment Guide, Third Edition, 2002; Osper et al., Refined Molecular Biology Guide, 1998)

[0031] Solution I: 50mmol / L glucose, 25mmol / L Tris-HCl (pH8.0), 10mmol / LEDTA.

[0032] Solution II: 0.2mol / L NaOH, 1% SDS (prepared and used immediately).

[0033] Solution III: 100mL system, 80mL 5mol / L potassium acetate, 12mL glacial acetic acid, 8mL ddH2O.

[0034] TAE (50×): 242g Tris base, 57.1mL glacial acetic acid, 100mL 0.5mol / LEDTA (pH8.0), dilute to 1000mL with sterile water.

[0035] TER solution: Pancreatic RNAse (RNAse A) was dissolved in 10mM Tris-HCl, 15mMNaCl, made into a 10mg / mL stock solution and stored at -20°C, then diluted with 1×TE buffer to a 20µg / mL working solution, kept at 4°C save.

[0036] PPt Buffer: 22 mL of isopropanol;...

experiment example 1

[0132] Experimental example 1 Evaluation of the analgesic activity of conotoxin αACT1

[0133] The modified conotoxin αACT1 product expressed in silkworm in Example 1 was evaluated for analgesic activity (preliminary experiments showed that the analgesic treatment was carried out with the conotoxin αACT gene expression product not optimized and transformed in Example 1, Its dosage should be 5-10 times higher than the transformed conotoxin αACT1 gene product to have the same analgesic effect; pain activity evaluation).

[0134] According to the pathological pain that peripheral nerve injury causes to be preserved to the greatest extent, the standard of simplicity of operation, reliability, repeatability, effectiveness and practicability, has selected classic formaldehyde acute pain model, evaluates conotoxin αACT1 (embodiment 1 Analgesic activity of conotoxin αACT1 product expressed in silkworm.

[0135] Firstly, formaldehyde (2.5%) acute inflammatory pain model was establish...

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Abstract

The invention discloses conotoxin and a biological preparation method and application thereof. The preparation method includes the steps of firstly, cloning conotoxin gene to an expression cassette of a baculovirus carrier to obtain a recombination transfer expression carrier; secondly, subjecting the recombination transfer expression carrier and baculovirus DNA (deoxyribonucleic acid) to co-transfection so as to obtain recombination baculovirus; and thirdly, infecting insect hosts or insect cells with the recombination baculovirus, culturing expression conotoxin of the infected insect hosts, and collecting and purifying the expression products. The invention further provides optimized conotoxin gene which is evidently improved in expression efficiency in insect hosts compared with original gene. Bioactive conotoxin is expressed efficiently and safely in individual insect bioreactor by the aid of baculovirus expression systems, safety is quite high, and expression products can be directly applied to analgesia drugs and the like. The biological preparation method is high in expression efficiency, complete in post-processing, fine in bioactivity, low in production cost, available for scale production and the like.

Description

technical field [0001] The present invention relates to a preparation method of conotoxin, in particular to a biological preparation method of conotoxin and the products prepared by the biological method and their medical use in analgesia, nerve repair or withdrawal of drug addiction, It belongs to the field of biological preparation of conotoxin. Background technique [0002] Marine toxins is an emerging research field in the past two decades, one of its important progress is the discovery of some extremely toxic small peptide toxins, among which the conotoxin secreted by the mollusk conus is the most important The most important class of neurotoxins is the smallest nucleic acid-encoded animal neurotoxin peptide found so far, and it is also a small peptide with the highest disulfide bond density. Conotoxin has the characteristics of novel chemical structure, strong biological activity and high target selectivity, and has become a powerful tool in pharmacology and neuroscie...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/866C12N5/10C07K14/435A61K38/17A61P25/04A61P25/00A61P25/30
Inventor 张志芳李轶女易咏竹刘兴健郑学星边大勇王国增江峰钟鲁龙
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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