A method of using silkworm to prepare analgesic polypeptide

Abstract

The invention discloses a method for preparing analgesic polypeptide from silkworms and belongs to the field of gene engineering. The preparation method comprises steps as follows: a conotoxin omega-MVIIA gene sequence is optimized according to BmNPV (Bombyx mori Nucleopolyhedrovirus) codon usage frequency, genes obtained after chemical synthesis are linked to a donor plasmid pFastBacDual and introduced to EGFP (enhanced green fluorescent protein) genes, and a recombinant donor plasmid pFBD-MVIIA / EGFP is established. MVIIA / EGFP is transposed to BmNPV with a Bac-to-Bac method, and a recombinant virus vBmMVIIA / EGFP is formed. DNA of the recombinant virus is transfected to cells of the silkworms, BV particles are obtained and injected into the silkworms at 200 pfu after the titer of the BV particles is tested by the aid of green fluorescence, and blood of the silkworms is collected after five days of transfection. Acetic-acid-induced mouse writhing experiments show that the blood of the silkworms infected with expressed conotoxin omega-MVIIA has remarkable analgesic activity, and the number of the average writhing times is only 3.55 and is remarkably higher than 37.30 in a control group of mice injected with normal saline. A new method is provided for expression of conotoxin omega-MVIIA with analgesic activity, and has potential application value.

Description

technical field [0001] The invention discloses a method for preparing an analgesic active polypeptide by using silkworm, and belongs to the field of genetic engineering. Background technique [0002] Conotoxins are produced by the marine mollusk Cono ( Conus ) is a class of active peptide toxoids secreted 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, up to more than 50,000, and their primary structures are variable, but the sequence of signal peptide and leader peptide is conserved. According to the conserved signal peptide sequence, conotoxins can be divided into: A, M, O, I, P, S, T superfamily (superfamily), and then according to the cysteine ​​skeleton of the toxin in the family, combined with its pharmacological activity , and can be divided into α, μ, ω, δ, Ψ, σ, λ, κ,...

AI Technical Summary

Problems solved by technology

However, the cost of artificially synthesizing conotoxin is very high, and it cannot fully meet the requirements of commercial production as a drug.

Therefore, some scientists have proposed that conotoxin genes can be transformed into microorganisms such as Escherichia coli or yeast for expression. However, the amino acid sequence of conotoxin is short, and it is difficult to isolate and purify later, and the post-translational modification is complicated, so it cannot be expressed in microbial expression systems. To solve the problem of amidation at the C-terminus, highly active recombinant conotoxins cannot be obtained (Zhan et al. A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity. Biochem Biophys Res Commun. 2003, 311: 495-500)

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