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Conotoxin MVII A and Trx fusion protein and its expression and application

A fusion protein and conotoxin technology, applied in the direction of peptide/protein components, DNA/RNA fragments, medical preparations containing active ingredients, etc., can solve the problems of unstable quality, difficult process, low yield, etc., and achieve purification Convenience, simple process, large output effect

Inactive Publication Date: 2004-04-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, ω-conotoxin MVIIA can be obtained by chemical synthesis, but it requires more than 20 steps of reaction, the yield is very low, and the process is very difficult, because there are 6 cysteines (Cys) in the molecule that need to be accurately paired to form two Sulfur bonds, unstable quality, and high cost

Method used

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  • Conotoxin MVII A and Trx fusion protein and its expression and application
  • Conotoxin MVII A and Trx fusion protein and its expression and application
  • Conotoxin MVII A and Trx fusion protein and its expression and application

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Experimental program
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Embodiment T

[0026] A kind of expression and application of embodiment Trx-CTX MVIIA fusion protein

[0027] 1. Materials and methods

[0028] 1.1 Strains and plasmids

[0029] Escherichia coli DH5α, Escherichia coli BL21(DE3), and prokaryotic expression vector pET-32a(+) were all provided by Hangzhou Jiuyuan Gene Engineering Co., Ltd. Research Institute.

[0030] 1.2 Tool enzymes and reagents

[0031] Endonucleases Kpn I and XbaI, T4 polynucleotide kinase, IPTG and protein standard molecular weight are all products of MBI Company; endonuclease EcoR I is a product of Biolab Company; T4 DNA ligase was purchased from Promega Company; CTX DNA fragments were purchased from Shanghai Sheng The resin used for purification was the product of Pharmacia Biotech; the standard molecular weight of DNA was purchased from Shanghai Boya Biotechnology Company; the commonly used chemical reagents were domestic analytical reagents.

[0032] 1.3 Instruments and equipment

[0033] The small high-speed refr...

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Abstract

The present invention provides one kind of conotoxin MVII A and Trx fusion protein and its expression and application. Trx-CTX MVII A fusion protein is first cloned and expressed in colibacillus and then chromatographicaly purified, reduced and oxidized to obtain active Trx-CTX MVII A fusion protein. The obtained fusion protein may be applied in preparing analgetic for late stage cancer and AIDS and that for post-operation pain, burns, etc. and may be also applied in preparing medicine for cerebral ischemia and cerebral demage owing to its nerve protecting function. The present invention can obtain fusion protein with great molecular weight and relatively long half time, and may be applied in medicine development directly or as intermediate to be enzyme incised to obtain active omiga-conotoxin MVII A for further medicine development.

Description

technical field [0001] The invention belongs to genetic engineering and relates to chemically synthesized polypeptides, in particular to the fusion protein of ω-conotoxin MVIIA gene and thioredoxin (Trx) and its expression and application in medicine. Background technique [0002] Conotoxin (CTX) is a small peptide neurotoxin with specific biological activity secreted by the marine mollusk cone snail. Generally, it contains 10-30 amino acids, and most of them are rich in disulfide bonds. According to their targets and pharmacological activities, they can be divided into α, ω, μ and other families [1] . ω-CTX MVIIA is a voltage sensitive calcium channel (Voltagesensitive calcium channel, VSCC) blocker, which mainly acts on N-type VSCC in nerve tissue. It consists of 25 amino acids, containing 6 Cys residues, forming three pairs of disulfide bonds. Non-clinical studies have proved that it has dual effects. On the one hand, it blocks pain transmission and has an analgesic ef...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61P9/10A61P25/00A61P25/04A61P29/00C07K19/00C12N15/62
Inventor 詹金彪陈小颖
Owner ZHEJIANG UNIV
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