Method for preparing analgesic polypeptide from silkworms
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A silkworm and active polypeptide technology, applied in the field of genetic engineering, can solve the problems of high cost of conotoxins, inability to solve C-terminal amidation, complex modification, etc.
Inactive Publication Date: 2016-01-20
JIANGSU UNIV
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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 l
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Embodiment 1
[0020] Example 1: Conotoxins ω- Synthesis of MVIIA Gene and Construction of Recombinant BmNPV
[0021] Bombyx mori nuclear polyhedrosis virus (BmNPV) poh , gp64 , VP39 Based on the analysis of codon usage frequency of highly expressed genes (GenBank: L33180), the codons of the conotoxin ω-MVIIA gene (GenBank: FJ959111) were optimized (see figure 1 ), the sequence of the codon-optimized ω-MVIIA gene is SEQ ID NO.1, and the sequence of the ω-MVIIA precursor protein encoded by it is SEQ ID NO.2.
[0022] The ω-MVIIA gene (synthesized by Shanghai Jierui Biotechnology Co., Ltd.) was synthesized by chemical methods, and the Bam HI / Hind After III double digestion, the donor plasmid pFastBacDual (purchased from Invitrogen) that had been cut with the same restriction enzyme was connected to form the recombinant plasmid pFBD-MVIIA. Using the primer pair P1 / P2 (SEQIDNO.3; SEQIDNO.4) to amplify the green fluorescent protein (EGFP) gene, the Kpn I / xho After double enzy...
Embodiment 2
[0024] Example 2: Expression and analgesic activity analysis of recombinant ω-MVIIA
[0025] Extracted vBm by liposome method MⅦA / EGFP The silkworm cells cultured in vitro were transfected with DNA, and after 5 days of culture, green fluorescence could be observed under a fluorescent microscope. Collect the cell culture, take the supernatant after centrifugation, transfer it to the silkworm cell cultured in vitro again, inoculate and amplify the virus repeatedly, and a strong fluorescent signal can be observed under a fluorescent microscope. Determination of recombinant virus vBm with green fluorescence by endpoint dilution MVIIA / EGFP The titer of the BV particles was 1.6 x 10 7 pfu / mL.
[0026] Appropriately dilute the high-titer recombinant virus solution, inject the 5th instar silkworm with 200pfu BV particles, and after 5 days of infection, the recombinant virus vBm MⅦA / EGFP The body surface of the infected silkworm showed strong green fluorescence, while the ω-MVIIA...
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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,...
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