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Plasmid for efficiently expressing polypeptide toxin as well as preparation method and application of plasmid

A high-efficiency expression and toxin technology, applied in the field of biomedicine, can solve the problems of less polypeptides and high cost, and achieve the effect of improving soluble expression, high-yield expression and good expression effect.

Pending Publication Date: 2022-03-01
成都佩德生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The amount of peptides that can be obtained directly from animal venom by traditional methods or through chemical synthesis is small, and for peptides with disulfide bonds, the cost is too high due to the cumbersome renaturation process

Method used

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  • Plasmid for efficiently expressing polypeptide toxin as well as preparation method and application of plasmid
  • Plasmid for efficiently expressing polypeptide toxin as well as preparation method and application of plasmid
  • Plasmid for efficiently expressing polypeptide toxin as well as preparation method and application of plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of pET-26b-DsbC expression plasmid

[0035] Insert the DsbC gene with the signal peptide removed into the N-terminal cloning site of the pET-26b vector to obtain the pET-26b-DsbC expression plasmid, the structure of which is shown in figure 1 As shown, it contains T7 promoter, Lac operator, ribosome binding site, DsbC coding region, pelB / ompT, MSC, His coding region, TEV site and T7 terminator.

[0036] The applicant found in the previous research that the polypeptide toxin ω-LGTX-F2 was expressed prokaryotically using the vector pET-32a, and its protein was in the form of an inclusion body, which made it impossible to obtain the recombinant protein by normal means. The polypeptide has 4 pairs of disulfide bonds and needs specific folding processing to form the correct conformation and be successfully expressed. Given the multifaceted properties of its polypeptide toxin, it was investigated whether it could be successfully produced by secretory expression,...

Embodiment 2

[0038] Using the pET-26b-DsbC expression plasmid prepared in Example 1 to prepare spider toxin ω-LGTX-F2 by means of genetic engineering

[0039] (1) Preparation of plasmid expressing ω-LGTX-F2

[0040] According to the codon usage preference of Escherichia coli, the expressed gene series was optimized. In order to enable the TEV enzyme to effectively cut the recombinant protein to remove the fusion tag, a specific recognition coding sequence ENLYFQG was introduced at the N-terminus of ω-LGTX-F2.

[0041] The ω-LGTX-F2 gene (Shanghai Bioengineering Co., Ltd.) was synthesized by chemical method, and then ligated into the same prokaryotic expression vector pET-26b-DsbC (purchased from Novagen) with the same restriction enzyme digestion and added DsbC tag after double digestion with NcoI and XhoI. , DNA sequencing of recombinant plasmids.

[0042] (2) Introduce the plasmid expressing the spider toxin ω-LGTX-F2 into the host expression system

[0043] The successfully constructed ...

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Abstract

The plasmid for efficiently expressing the polypeptide toxin provided by the invention is obtained by cloning a coding gene of the polypeptide toxin into a plasmid vector pET-26b-DsbC, the plasmid vector pET-26b-DsbC carries His, pelB / ompT and a DsbC tag, the DsbC tag and a pelB / ompT signal peptide jointly act, polypeptide to be expressed is transferred into periplasm which is more beneficial to protein folding and disulfide bond formation, and the expression efficiency of the polypeptide toxin is improved. The soluble expression of the polypeptide toxin is improved, the problem of no expression or low expression quantity of the polypeptide toxin is solved, and the high-yield expression of the polypeptide toxin is realized. The invention further provides a preparation method of the plasmid, the plasmid for expressing the spider toxin is prepared, then the gene engineering bacteria are used for culturing and expressing the spider toxin, the prepared spider toxin is high in yield and low in production cost, and the preparation method has huge application prospects in the field of polypeptide toxin production.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a plasmid for highly expressing a polypeptide toxin, a preparation method and application thereof. Background technique [0002] Animal polypeptide toxins are currently a hot spot in medical research, not only as tool reagents to study the physiological effects of molecular targets (such as ion channels and receptors), but also to use polypeptide toxins as drugs to study or treat human diseases. LGTX-F2 is derived from the tarantula spp., and its wild type and mutants can effectively promote the enzyme activity of blood coagulation factors FXa, FXIIa, thrombin and Kallikrein, and accelerate blood coagulation. The amount of peptides that can be obtained directly from animal venom by traditional methods or by chemical synthesis is small, and for peptides with disulfide bonds, the tedious renaturation process is required, resulting in high costs. Contents of the invention [...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N15/12C07K14/435C07K1/22C12P21/06
CPCC12N15/70C12N15/66C07K14/43518C12P21/06C12N2800/101C12N2800/22
Inventor 谷陟欣
Owner 成都佩德生物医药有限公司
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