Pain-relief active polypeptide screening method

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 analgesic active polypeptides and belongs to the field of genetic engineering. Background technique [0002] Conotoxins are a type of active peptide toxin secreted by the marine mollusk Conus for self-defense and predatory use. The molecular weight 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 types of conotoxins with variable primary structures, but the signal peptide and leader peptide sequences are relatively conserved. Conotoxins can specifically act on various (potassium, sodium, calcium, etc.) ion channels in the body and 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, neuroprotection,...

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

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