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Preparation method of scorpion neurotoxin Bj alpha IT gene and recombinant protein thereof

A gene and protein technology, applied in the field of preparation of scorpion neurotoxin BjαIT recombinant gene and its protein, can solve problems affecting BjαIT activity measurement, insecticidal spectrum analysis application, etc., achieve significant biological activity, reduce cost, and reduce load.

Inactive Publication Date: 2017-04-26
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant reports on the high-efficiency expression and purification of active BjαIT recombinant protein using the Pichia pastoris expression system, which greatly affects the determination of BjαIT activity, insecticidal spectrum analysis and its application in anti-agricultural pests. It is very necessary for Pichia pastoris to secrete and express a large amount of BjαIT protein at a high level and to prepare the biologically active insect neurotoxin BjαIT protein rapidly and at low cost

Method used

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  • Preparation method of scorpion neurotoxin Bj alpha IT gene and recombinant protein thereof
  • Preparation method of scorpion neurotoxin Bj alpha IT gene and recombinant protein thereof
  • Preparation method of scorpion neurotoxin Bj alpha IT gene and recombinant protein thereof

Examples

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Embodiment 1

[0044] This example provides an optimized artificially synthesized BjαIT gene with a 6×His tag at its C-terminal and its yeast transformant. The specific sequence is shown in sequence 1 in the sequence listing, and the protein sequence corresponding to the gene is shown in Shown in sequence 2 in the sequence listing. The sequence before optimization in this example is based on the DNA sequence provided by the NCBI database, which is the natural DNA for synthesizing BjαIT neurotoxin, and then optimizes and synthesizes the optimized DNA according to the expression characteristics of the toxin gene and Pichia pastoris codons. The optimized DNA sequence has no obvious homology with the natural polynucleotide of BjαIT (GenBank accession number AB839884).

[0045] The natural DNA of the BjαIT neurotoxin before optimization and the optimized artificially synthesized DNA with a 6×His tag at the C-terminal were respectively connected to the secretory expression vector pPICZαA of Pichia...

Embodiment 2

[0047] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0048] S1: Construction of expression vector and transformation: The optimized artificially synthesized C-terminal DNA with 6×His tag in Example 1 was connected to Pichia pastoris secretory expression vector pPICZαA to obtain recombinant vector pPICZαA-BjαIT, and vector construction Such as figure 1 as shown, figure 1 A schematic diagram is constructed for the eukaryotic expression vector pPICZαA-BjαIT in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0049] (1) use X ho and x the b The plasmid containing the synthesized BjαIT gene was double-digested to obtain the target fragment. The reaction system was as follows (all endonucleases and buffers used were purchased from Dalian TAKARA Company):

[0050] Contains synthetic BjαIT Gene plasmid 15 μL

[0051] 5 μL of 10×M buffer

[0052] x ho...

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Abstract

The invention discloses a preparation method of a scorpion neurotoxin Bj alpha IT gene and a recombinant protein thereof. The preparation method comprises the following steps that: (1) According to the amino acid sequence of the Bj alpha IT mature toxin and the characteristics of Pichia pastoris expression systems, Bj alpha IT genes with 6*His tags at C- ends are optimally synthesized and Pichia pastoris secretion expression vectors are constructed; (2) the recombinant vectors are transferred into Pichia pastoris hosts, yeast transformants with high level secretion expression are obtained, and recombinant scorpion insect Bj alpha IT proteins with a purity of over 95% are obtained by nickel affinity chromatography. A method for expressing insect neurotoxin Bj alpha IT genes with eukaryotic host Pichia pastoris is disclosed. The recombinant scorpion insect Bj alpha IT protein with high purity is quickly obtained by the nickel affinity chromatography, and the protein performs high bioactivity at both cellular and animal levels. The protein is of great value in the field of insect resistance, especially for breeding varieties of insect resistant plants, and has important significance for improving the yield of crops.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of a scorpion neurotoxin BjαIT recombinant gene and protein thereof. Background technique [0002] Insect neurotoxins are a kind of polypeptide neurotoxins that only act on the nervous system of insects and have poisonous and killing effects. They mainly come from scorpions, and secondly from spiders. The action sites of this kind of toxins that have a specific poisoning effect on insects are various ion channels. The neurotoxins with high insect specificity are mainly long-chain neurotoxins that act on sodium ion channels. Such long-chain neurotoxins Generally composed of 60-70 amino acids, with 2-4 pairs of disulfide bonds in the chain. Neurotoxins from different species have no obvious conservation in amino acid composition, while neurotoxins from the same species have high conservation in primary structure. BjαIT is a species derived from sc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/81C07K1/36C07K1/34C07K1/22C12R1/84
CPCC07K14/43522C12N15/815
Inventor 李洪波夏玉先
Owner HUAIHUA UNIV
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