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Omega-conotoxin M VII A mutant and its preparation and uses

A conotoxin, GST-CTXMVIIA technology, applied in the field of genetic engineering, can solve the problems of high cost, low yield, unstable quality, etc., and achieve the effect of low cost, stable quality and simple operation

Inactive Publication Date: 2006-12-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the ω-conotoxin M VIIA that has been on the market is obtained by chemical synthesis. Since there are 6 cysteines 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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  • Omega-conotoxin M VII A mutant and its preparation and uses
  • Omega-conotoxin M VII A mutant and its preparation and uses
  • Omega-conotoxin M VII A mutant and its preparation and uses

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Experimental program
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Effect test

Embodiment 1

[0036] 1 Materials and methods

[0037] 1.1 Materials and reagents

[0038]The pGEX-2T / CTX M VIIA plasmid was constructed by our laboratory; isopropylthio-β-D-galactoside (IPTG) and standard molecular weight markers, and reduced glutathione were purchased from Shanghai Sangon Bioengineering Company; Glutathione- Agarose and Sephacryl S-100HR were purchased from Parmacia Company; thrombin (Thrombin) was purchased from Parmacia Company; Escherichia coli BL21 (DE) strain was purchased from Sigma Company; other reagents were purchased from various domestic companies.

[0039] 1.2 Method

[0040] 1.2.1 Induced expression of GST-CTX M VIIA fusion protein

[0041] The GST-CTX M VIIA plasmid was introduced into Escherichia coli BL21(DE), and BL21(DE) empty bacteria and BL21(DE) containing the plasmid without the target gene were used as negative and positive controls. The clones with successful recombination were picked and inoculated into LB medium containing ampicillin, shaken ov...

Embodiment 2

[0077] For the mass spectrometric identification of genetically recombined CTX M VIIA mutants, we lyophilized the purified CTX M VIIA mutant collection and sent them to Shanghai Institute of Biochemical Cells, Chinese Academy of Sciences for molecular weight identification by MALDI-TOF-MS. The results prove that the molecular weight of the CTX M VIIA mutant with two N-terminal redundant amino acid residues Gly and Ser is 2783, which is consistent with the theoretical value; the results can be found in Figure 12 .

Embodiment 3

[0079] The rat Hot-tail flick was used to measure the analgesic activity of CTX M VIIA mutants. Healthy male Sprague-Dawley rats, weighing 250±20g, were anesthetized with 10% chloral hydrate, implanted in the lateral ventricle, injected with penicillin daily, and carried out pharmacological tests after one week of feeding. Adjust the constant temperature water bath to 53±0.5°C, and measure the pain area for half an hour after administration. The normal mouse pain domain is generally 2s, and the pain domain is 5s for complete analgesia. 30 qualified animals were selected, randomly divided into 6 groups, administered through cannula, and the CTX M VIIA mutant proved to have a strong analgesic effect by the rat hot tail method experiment (see Figure 13 ). Figure 13 Middle: PBS is the normal saline group; Morphine (L) is the low dose morphine group (25 μg / kg); Morphine (H) is the high dose morphine group (250 μg / kg); GST-CTX is the GST-CTX M VIIA fusion protein group (25 μg / k...

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Abstract

The invention relates the Omega-M VII A(CTX M VIIA) mutant, possessing SEQ ID NO:1 amino acid sequence and adopting glutathione S- transferase(GST) fusion expression method. The method comprises the following steps: on the base of pGEX-2T / CTX M VIIA pronucleus expression plasmid, using IPTG to induce and express GST-CTX M VIIA fusion protein in coli bacillus, purifying fusion protein with GST affinity chromatography, using thromboses to produce CTX M VIIA mutant, separating and purifying product; opening the disulfide bridge of mutant with reducing agent, then carrying out dialysis with PBS, carrying out air oxidation, and getting final product. The invention has the advantages of simple operation, stable quality, and low cost.

Description

technical field [0001] The invention belongs to genetic engineering, and relates to an omega-conotoxin M VIIA (CTX M VIIA) mutant, its fusion expression and production method, and its application in the preparation of analgesic drugs. Background technique [0002] Conotoxin (CTX) is a class of biologically active peptide toxins obtained from the marine gastropod mollusk (Conus), and it is estimated that there are more than 50,000 species. 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, they play an important role in neuropharmacology research and disease treatment. [0003] The ω-type CTX is an important research direction of conotoxins in recent years, and has broad prospects for drug development. Wherein, ω-conotoxin M VIIA (CTX M VIIA) is an inhibitor of voltage-sensitive calcium ion channel, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P29/00C12N15/12C12N1/21
Inventor 詹金彪夏铮王峰徐小蓉黄建松
Owner ZHEJIANG UNIV