A kind of conotoxin variant gmviia and its preparation method and application

A conotoxin and variant technology, applied in the field of genetic engineering, can solve the problems that enterokinase cannot effectively cut, affect the permeability of conotoxin, and affect the analgesic effect, and achieve potential application value, significant analgesic activity, The effect of high analgesic activity

Inactive Publication Date: 2018-12-14
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Trx tag itself has a length of 109 amino acid residues and a large molecular weight. If it is not cut off, it will greatly affect the permeability of conotoxin in the body, thereby affecting the analgesic effect
In theory, the Trx tag can be removed by cleavage of the recombinant protein using enterokinase, but there is a cysteine ​​residue (Cys) at the C-terminus of the native mature ω-MVIIA, and a half after the enterokinase cleavage site (DDDDK) Enterokinase cannot efficiently cleave cystine residues (Cys)

Method used

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  • A kind of conotoxin variant gmviia and its preparation method and application
  • A kind of conotoxin variant gmviia and its preparation method and application
  • A kind of conotoxin variant gmviia and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Synthesis and identification of conotoxin GMVIIA gene

[0026] According to the frequency of E. coli codon usage, the codons of the conotoxin ω-MVIIA gene (GenBank: FJ959111) were optimized. In order to enable enterokinase to effectively cleave the recombinant protein to remove the expression tag, a glycine (Gly) residue was introduced at the N-terminus of the conotoxin ω-MVIIA, named GMVIA. The gene sequence of the designed conotoxin ω-MVIIA variant GMVIIA is SEQ ID NO.1 (in order to introduce a restriction enzyme upstream of GMVIIA Kpn The enzyme cleavage site of I, the coding sequence of enterokinase cleavage site DDDDK), the sequence of the encoded GMVIA protein is SEQ ID NO.2.

[0027] The GMVIA gene was synthesized by chemical method (synthesized by Shanghai Jierui Biotechnology Co., Ltd.), Kpn I / Hind After III double enzyme digestion, it was connected to the prokaryotic expression vector pET-32a (purchased from Novagen) that was digested with the same e...

Embodiment 2

[0029] Example 2: Expression, isolation, purification and cleavage of recombinant conotoxin GMVIIA

[0030] The heat-strike method was used to introduce the correct recombinant plasmids pET-32a-GMVIIA and pET-32a-ω-MVIIA into competent cells of E. coli Origami (DE3) strain (purchased from Novagen) to obtain genetically engineered bacteria Origami-GMVIIA, Origami-ω-MVIIA.

[0031] Inoculate the Origami-GMVIIA and Origami-ω-MVIIA strains into LB medium (containing 100 µg / mL ampicillin), and shake culture at 37°C to OD 600 The value is about 0.8. Cool the cultured cells to 28°C, add IPTG with a final concentration of 1mmol / L, and culture at 28°C for 12 hours with shaking. The cells were collected by centrifugation, frozen and thawed repeatedly at 37°C / -20°C for 5 times, and disrupted by 200w ultrasound. After high-speed centrifugation, the soluble recombinant Trx-GMVIIA and Trx-ω-MVIIA with His tag were separated from the supernatant using nickel ion resin. The purified recombinant ...

Embodiment 3

[0034] Example 3: Analysis of analgesic activity of recombinant conotoxin GMVIIA

[0035] The experimental group was injected with the recombinant conotoxin GMVIIA, Trx-GMVIIA, and Trx-ω-MVIIA solutions obtained in Example 2 at a dosage of 2.0 mg / kg, and the negative control group was injected with physiological saline (0.9% NaCl). Each group was injected with 20 male mice, and the weight of each mouse was 20±0.2 g. 45 minutes after the administration, each mouse was injected with 0.2 mL of 0.6% glacial acetic acid to stimulate writhing behavior, and the writhing movements of each mouse within 20 minutes were counted, and then the experimental data was statistically analyzed. The results showed that the average writhing times of the mice injected with Trx-GMVIIA and Trx-ω-MVIIA solutions were 20.60 and 20.05, respectively, and that of the normal saline control group was 37.4 times. The recombinant proteins Trx-GMVIIA and Trx-ω-MVIIA can be seen Both have significant analgesic ac...

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Abstract

The invention discloses a preparation method of a conotoxin variant GMVIIA with analgesic activity and belongs to the field of gene engineering. The preparation method comprises steps as follows: the gene sequence of the conotoxin variant GMVIIA is optimized according to the use frequency of Escherichia coli codons, a prokaryotic expression vector, namely, a pET-32a vector, is connected after chemical synthesis, then a recombinant plasmid is introduced into an Escherichia coli Origami (DE3) strain, and efficient soluble expression of recombinant protein Trx-GMVIIA is realized after IPTG induction; the recombinant protein Trx-GMVIIA is separated and purified with the nickel ion affinity chromatography; the recombinant protein Trx-GMVIIA is cut with enterokinase to release GMVIIA, Trx labels are removed with the nickel ion affinity chromatography, and the conotoxin variant GMVIIA is obtained. The mouse acetic acid writhing method proves that the prepared conotoxin variant GMVIIA has significant analgesic activity and has potential application value in the analgesic field.

Description

Technical field [0001] The invention discloses a preparation method of a conotoxin variant GMVIA, which belongs to the field of genetic engineering. Background technique [0002] Conotoxin is produced by the marine mollusk Cono ( Conus ) A secreted type of active peptide toxin used for self-defense and predation, usually composed of 10-30 amino acid residues, most of which are rich in cysteine ​​and have a highly conserved disulfide bond backbone. Conotoxin can specifically act on a variety of ion channels in the body (potassium, sodium, calcium, etc.), various neurotransmitters and hormone receptors on cell membranes, thereby interfering with signal transmission in cells or nerves. Therefore, it has broad application prospects in the treatment of chronic pain, acute pain, epilepsy, neuroprotection, cardiovascular disease, mental disorders, movement disorders, spasticity, cancer and stroke. ω-conotoxin MVIIA (ω-MVIIA) is derived from the marine organism Phantom Cono ( Conus mag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C07K14/435A61K38/17A61P29/00
Inventor 朱姗颖吴莺常鹏飞黄海吕增磊何华纲
Owner JIANGSU UNIV
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