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GST fusion expression of conotoxin MVII A gene and its use

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

Inactive Publication Date: 2004-02-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

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
Comparison scheme
Effect test

Embodiment 1

[0026] A kind of expression of embodiment 1 conotoxin MVIIA gene

[0027] Materials and Methods

[0028] 1.1 Materials and reagents

[0029] 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.

[0030] 1.2 Method

[0031] 1.2.1 Chemical synthesis of the target gene

[0032]Because the length of the CTX MVIIA polypeptide gene is shorter, the synthesis is more convenient and quick. The full sequence of CTX MVIIA was synthesized by Shanghai Sangon Bioengineering Co., Ltd., a restriction site for BamH I was introd...

Embodiment 2

[0063] 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

[0065] In addition, conotoxins also have neuroprotective effects. When brain ischemia or mechanical trauma occurs, conotoxin MVIIA can block N-type voltage-sensitive calcium ion channels in nerve cells, thereby inhibiting stimulating neurotransmitters. The release of pathogenic calcium ions 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 cerebral ischemia or mechanical trauma protective drugs [11-13] .

[0066] Some references involved in the present invention 1. Baldomero M, Olivera, Lourdes J, Cruz Conotoxins, in retrospect Toxicon 2001; 39:7-142. Olivera BM, Rivier J, Clark C, et al.Diversity of conus neuropeptides.J Science , 1990, 249: 13383. Olivera BM, Miljanick GP, Ramachandran J, et al. Calcium channel diversity and

[0067] neurotransmitter release: the omega-conotoxins and omega-agatoxins.

[0068] J. Annu. Rew. Biochem, 1994, 63: 823-8674. Sambrook J, Fritsch EF...

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Abstract

The present invention provides the GST fusion expression and application of a kind of conotoxin MVII A gene. GST-CTX MVII A recombinant fusion protein is cloned and expressed in colibacillus, and through GST affinity chromatography purified GST-CTX MVII A fusion protein is obtained. The fusion protein the present invention obtains may be applied in preparing medicine for treating intractable painof cancer and AIDS patient in the late stage, preparing analgesic for treating post-operation acute pain, pain of burn, biliary colic, intractable neuralgia, etc., and preparing medicine with nerve protecting function for treating cerebral ischemia and cerebral damage. The obtained fusion protein has relatively great molecular weight and relatively long half life in body and may be enzyme incisedto obtain active omiga-conotoxin MVII A small peptide for direct medicine 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 MVIIA 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 MVIIA (CTX MVIIA) is an inhibitor of voltage-sensi...

Claims

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Application Information

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Patent Type & Authority Applications(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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