GST fusion expression of conotoxin MVII A gene and its use

A conotoxin and gene technology, which can be applied to DNA/RNA fragments, medical preparations containing active ingredients, and hybrid peptides, etc., can solve the problems of unstable quality, difficult process and high cost, and achieve convenient purification and simple process. , the effect of low cost

Inactive Publication Date: 2005-12-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, ω-conotoxin MVII A can be obtained by chemical synthesis, but because there are 6 cysteines (Cys) in the molecule that need to be accurately paired to form a disulfide bond, more than 20 steps of reaction are required, and the yield is very low. The process is very difficult, the quality is unstable, and the cost is high

Method used

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  • GST fusion expression of conotoxin MVII A gene and its use
  • GST fusion expression of conotoxin MVII A gene and its use
  • GST fusion expression of conotoxin MVII A gene and its use

Examples

Experimental program
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Effect test

Embodiment 1

[0036] A kind of expression of embodiment 1 conotoxin MVIIA gene

[0037] Materials and Methods

[0038] 1.1 Materials and reagents

[0039]The pGEX-2T plasmid and Escherichia coli BL21 strains were stored in our laboratory; CTX gene synthesis was completed by Shanghai Sangong Bioengineering Company; T4 polynucleotide kinase (TPK), T4 DNA ligase, restriction enzyme, isopropyl sulfide Substituted-β-D-galactoside (IPTG), medium molecular weight Marker, and reduced glutathione were purchased from Shanghai Sangon Bioengineering Company; Glutathione-Sepharose 4B was purchased from Parmacia Company; other reagents were purchased from various domestic companies.

[0040] 1.2 Method

[0041] 1.2.1 Chemical synthesis of the target gene

[0042] Because the length of the CTX MVII A polypeptide gene is shorter, the synthesis is more convenient and quick. The full sequence of CTX MVII A was synthesized by Shanghai Sangon Bioengineering Co., Ltd., a restriction site for BamH I was intr...

Embodiment 2

[0090] Because the conotoxin MVIIA can block the N-type voltage-sensitive calcium channel of nerve cells, thereby inhibiting the transmission of pain signals, it has a wide range of analgesic effects. It can be used for acute and persistent pain, such as analgesia for patients with advanced cancer, AIDS, acute pain after surgery, burns, biliary colic patients, refractory neuralgia, etc. It can replace morphine analgesics. We use electroplate analgesia (internationally recognized model), and its effect has been proved. For the method see 1.2.6, for the result see Figure 4 , the results showed that it has high analgesic activity. It is 1000 times greater than the analgesic effect of morphine (effective dose 7.5mg / kg) reported in the anthology. Candidate as an alternative to addictive analgesics such as morphine.

Embodiment 3

[0092] In addition, conotoxin also has a neuroprotective effect. When cerebral ischemia or mechanical trauma occurs, because conotoxin MVI IA can block the N-type voltage-sensitive calcium channel of nerve cells, thereby inhibiting the stimulation of neurotransmitters. The release of cytoplasm and the entry of pathogenic calcium ions into nerve cells may be used as a protective agent for brain injury, which can be used for the development of protective drugs for cerebral ischemia or mechanical trauma [11-13] .

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Abstract

The invention provides a GST fusion expression of the conotoxin MVII A gene and its application. The GST-CTX MVII A recombinant fusion protein is cloned and expressed in Escherichia coli, and purified by GST affinity chromatography to obtain purified GST-CTX MVII A fusion protein. The fusion protein obtained by the present invention can be used in the preparation of intractable pain medicines for treating advanced cancer and AIDS patients, and can also be used in the preparation of analgesics for treating acute pain after surgery, burns, biliary colic patients, refractory neuralgia, etc. Because of its neuroprotective effect, it can also be used in the preparation of therapeutic drugs for cerebral ischemia and brain damage. The technique of the present invention is simple, stable in quality, and cost is lower, and the fusion protein molecular weight that obtains is larger, and the half-life (t 1 / 2 ) is longer and can be used as an intermediate to obtain active ω-conotoxin MVII A small peptide through enzyme digestion, which can be directly used for drug development.

Description

technical field [0001] The invention belongs to genetic engineering and relates to chemically synthesized polypeptides, in particular to the GST fusion expression of ω-conotoxin MVII A gene and its application in medicine. Background technique [0002] Conotoxin (CTX) is a class of biologically active peptide toxins obtained from the marine gastropod mollusk (Conus), and there are more than 50,000 species so far. [1] . These polypeptides have novel structures and unique functions. According to its target site, it can be divided into α-, ω-, μ-, δ- and other types. Because they can selectively act on different ion channels and their subtypes [2] , play an important role in the identification of ion channel subtypes, disease treatment and neurobiological research. [0003] ω-type CTX is an important research direction of conotoxins in recent years, and has broad prospects for drug development [3] . Among them, ω-conotoxin MVII A (CTX MVII A) is an inhibitor of voltage-se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/16A61P1/16A61P17/02A61P25/00A61P25/04A61P29/00C07K19/00C12N15/62C12N15/70
Inventor 詹金彪陈永对
Owner ZHEJIANG UNIV
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