Pain-relief active polypeptide screening method

A technology of active polypeptide and screening method, applied in the field of genetic engineering, can solve the problems of C-terminal amidation, complicated modification, limited application of ω-MVIIA, etc.

Inactive Publication Date: 2015-10-14
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex structure of conotoxin, it is difficult to obtain ω-MVIIA by chemical synthesis, and the cost is very high, which cannot fully meet the requirements of commercial production as a drug.
At the same time, some scientists have proposed that the gene of conotoxin can be transformed into microorganisms such as Escherichia coli or yeast for expression (Zhan J, Chen X, Wang C, Qiu J, Ma F, Wang K, and Zheng S. A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity. Biochemical and Biophysical Research Communication. 2003, 311: 495-500.), but the amino acid sequence of conotoxin is short, it is difficult to isolate and purify later, and the post-translational modification is complex. The problem of C-terminal amidation cannot be solved in the microbial expression system, and highly active recombinant conotoxin cannot be obtained
[0005]Due to the above factors, the clinical application of ω-MVIIA is greatly limited

Method used

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  • Pain-relief active polypeptide screening method
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  • Pain-relief active polypeptide screening method

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Experimental program
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Effect test

Embodiment 1

[0024] 1. Discovery of ω-MVIIA knottins by means of bioinformatics

[0025] Conotoxin ω-MVIIA belongs to the knottin family. Knottin, which has the same disulfide bond skeleton as ω-MVIIA, is a polypeptide with potential analgesic activity. In this regard, the present invention conducts bioinformatics analysis on the existing 2193 members in the knottin database (the KNOTTIN database, http: / / knottin.cbs.cnrs.fr), and finds that 1574 members have the same The disulfide bond backbone (see figure 1), in the present invention, this kind of polypeptide is called ω-MVIIA-like knottin (ω-MVIIA-like knottin). Most of these ω-MVIIA knottins were derived from spiders (775) and cone snails (530), and quite a few were from insects (59), plants (27) and viruses (53). ω-MVIIA knottins from spiders and cone snails are usually neurotoxic, and there are few functional studies on ω-MVIIA knottins from other sources. The functions of insect ω-MVIIA knottins have been defined to involve ph...

Embodiment 2

[0038] 1. Synthesis and identification of the Musca domestica phenol oxidase inhibitor MdPOI gene

[0039] According to the protein sequence of the housefly phenol oxidase inhibitor MdPOI (SEQ ID NO.4, GenBank: P81765), its gene sequence was deduced, and the codons of the MdPOI gene were optimized according to the codon usage frequency of Escherichia coli. The sequence of the MdPOI gene is SEQ ID NO.3.

[0040] The MdPOI gene was synthesized by chemical method (Shanghai Jierui Biotechnology Co., Ltd.), after BamHI / HindIII double enzyme digestion, it was connected into the prokaryotic expression vector pET-32a that had been digested by the same enzyme, and the recombinant plasmid pET-32a-MdPOI was verified by DNA sequencing (See figure 2 B).

[0041] 2. Expression and purification of recombinant MdPOI

[0042] The correct recombinant plasmid pET-32a-MdPOI was introduced into the competent cells of Escherichia coli Origami (DE3) strain by heat shock method, and the genet...

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Abstract

The invention relates to a pain-relief active polypeptide screening method and belongs to the field of gene engineering. The screening method specifically comprises the following steps: firstly, deriving coding DNA sequences of GsGUR and MdPOI according to amino acid sequences of GsGUR and MdPOI belonging to omega-MVIIA knottin and optimizing codon of GsGUR gene and MdPOI gene; after an optimized related gene is synthesized, connecting to a prokaryotic expression vector, respectively introducing obtained recombinant plasmids to escherichia coli Origami (DE3) strains, respectively inoculating obtained gene engineering strains to LB culture media containing ampicillin, performing shake culture, and after IPTG induction, generating soluble recombinant protein; and after separation and purification, obtaining recombinant active polypeptide GsGUR and MdPOI. According to the invention, the fact that recombinant GsGUR and MdPOI have pain-relief activity is firstly proved through a mouse acetic acid twisting method, sources of pain-relief polypeptide is widened, a new thought and a new technological means are provided for screening of pain-relief polypeptide, high potential for extracting natural pain-relief active polypeptide from related plants and insects in an industrialized manner is realized and requirements for drug research and development and clinical application are met.

Description

technical field [0001] The invention relates to a screening method for an analgesic active polypeptide, which belongs to the field of genetic engineering. Background technique [0002] Conotoxins are a class of active peptide toxins secreted by the marine mollusk Conus for self-defense and predation. The molecular mass of conotoxins is small, usually composed of 10-30 amino acid residues, most of which are rich in cysteine, and have a highly conserved disulfide bond skeleton. There are many kinds of conotoxins and their primary structures are variable, but the sequence of signal peptide and leader peptide is relatively conservative. Conotoxins can specifically act on various (potassium, sodium, calcium, etc.) ion channels in the body, various neurotransmitters and hormone receptors on the cell membrane, thereby interfering with signal transmission in cells or nerves. Therefore, it has broad application prospects in the treatment of chronic pain, acute pain, epilepsy, neuro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02A61K38/17A61K38/16A61P29/02
Inventor 何华纲朱姗颖楚鹰李云亮马海乐
Owner JIANGSU UNIV
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