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Method for preparing recombinant scorpion neurotoxin LqhIT2 protein

A protein and protein technology, applied in the biological field, can solve problems affecting LqhIT2 activity determination, insecticidal spectrum analysis application, etc., and achieve the effects of mass production, cost reduction, and prevention of degradation

Active Publication Date: 2017-05-24
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the high-efficiency expression and purification of active LqhIT2 recombinant protein using the Pichia pastoris expression system, which greatly affects the determination of LqhIT2 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 LqhIT2 protein at a high level and to prepare the biologically active insect neurotoxin LqhIT2 protein rapidly and at low cost

Method used

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  • Method for preparing recombinant scorpion neurotoxin LqhIT2 protein
  • Method for preparing recombinant scorpion neurotoxin LqhIT2 protein
  • Method for preparing recombinant scorpion neurotoxin LqhIT2 protein

Examples

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Embodiment 1

[0036] This example provides an optimized artificially synthesized LqhIT2 gene with a 6×His tag at the 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 LqhIT2 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 cannot find homologous genes in databases such as NCBI.

[0037] The C-terminal DNA of the optimized LqhIT2 neurotoxin with a 6×His tag was connected to the secreted expression vector pPICZαA of Pichia pastoris to obtain the recombinant vector, and then the lithium chloride transformation method provided by the Invitrogen company...

Embodiment 2

[0039] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0040] S1: Construction of expression vector and transformation: The optimized artificially synthesized DNA with a 6×His tag at the C-terminal of Example 1 was connected to the secreted expression vector pPICZαA of Pichia pastoris to obtain the recombinant vector pPICZαA-LqhIT2, and the vector was constructed Such as figure 1 as shown, figure 1 A schematic diagram is constructed for the eukaryotic expression vector pPICZαA-LqhIT2 in the embodiment of the present invention. The main vector construction steps are preferably as follows:

[0041] (1) use X ho I and X the b Ⅰ Digest the plasmid containing the synthesized LqhIT2 gene with double enzymes to obtain the target fragment. The reaction system is as follows (the endonuclease and buffer used are purchased from Dalian TAKARA Company):

[0042] Contains synthetic wxya Gene plasmid 15 μL

[0043] 5 μL ...

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Abstract

The invention discloses a method for efficiently preparing recombinant active scorpion insect neurotoxin LqhIT2. The method mainly comprises a step (1) of optimizing and synthesizing LqhIT2 genes with C- ends provided with 6*His labels according to amino acid sequences of mature LqhIT2 toxins and characteristics of a pichia pastoris expression system and constructing pichia pastoris secretory expression vectors; a step (2) of converting recombinant vectors into a pichia pastoris host, and through screening, obtaining yeast transformants in high-level secretory expression; a step (3) of expressing recombinant scorpion insect neurotoxin LqhIT2 proteins in the pichia pastoris host, and quickly obtaining the recombinant scorpion insect neurotoxin LqhIT2 proteins with the purity over 98% through a Ni2+ affinity chromatography method, wherein the purified recombinant proteins have very strong poisonous activity on insect cells. The method is modified based on conventional expression of insect neurotoxin LqhIT2 through prokaryotic host escherichia coli, the method of using eukaryotic host pichia pastoris to express the insect neurotoxin LqhIT2 is explored, and the yeast transformants in high-level secretory expression are screened.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of a scorpion neurotoxin LqhIT2 optimized gene and its recombinant protein. Background technique [0002] Insect neurotoxins are a kind of polypeptide neurotoxins that only act on the nervous system of insects and have poisonous killing effects. They mainly come from the venom secreted by the venom glands of scorpions, spiders, mites, wasps, ants and other animals. So far, nearly a hundred insect neurotoxins have been isolated, purified and named, mainly from spiders, and secondly from spiders. The scorpion insect neurotoxin LqhIT2 is a species of yellow scorpion from Israel Leiurus quinquestriatus The insect long-chain neurotoxin isolated from the venom of hebraeus (Ehrenberg). The mature toxin consists of 61 amino acids and contains 4 pairs of disulfide bonds in the chain. The action site is the sodium ion channel of the insect. The toxin has ...

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
CPCC07K14/43522C12N15/815C12N2800/102
Inventor 李洪波夏玉先
Owner HUAIHUA UNIV
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