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Preparation method for spider neurotoxin TX4(6-1) recombinant protein

A TX4, A-TX4 technology, applied in the biological field, can solve problems such as affecting the activity measurement, and achieve the effect of realizing mass production, preventing degradation and reducing load

Inactive Publication Date: 2017-05-31
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no method for efficiently expressing Tx4(6-1) recombinant protein using eukaryotic expression system, which greatly affects the determination of Tx4(6-1) activity, insecticidal spectrum analysis and its role in resisting agricultural pests. Therefore, it is very necessary to develop a method of using Pichia pastoris to secrete and express a large amount of Tx4(6-1) protein at a high level and to prepare biologically active insect neurotoxin Tx4(6-1) protein rapidly and at low cost.

Method used

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  • Preparation method for spider neurotoxin TX4(6-1) recombinant protein
  • Preparation method for spider neurotoxin TX4(6-1) recombinant protein

Examples

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Embodiment 1

[0038] This example provides an optimized artificially synthesized TX4(6-1) gene with a 6×His tag at its C-terminus. 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 the synthesis of TX4(6-1) neurotoxin, and then optimizes and synthesizes the optimized sequence according to the expression characteristics of the toxin gene and Pichia pastoris codons DNA. The NCBI database can only find the natural protein sequence (GenBank accession number 2108421A) of Tx4(6-1) without its corresponding nucleic acid sequence, so the optimized synthetic Tx4(6-1) Genes have no homologous DNA sequences.

[0039] The artificially synthesized DNA with a 6×His tag at the C-terminal was respectively connected to the secreted expression ...

Embodiment 2

[0041] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0042] 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-TX4(6- 1), the vector is constructed as figure 1 as shown, figure 1 A schematic diagram was constructed for the eukaryotic expression vector pPICZαA-TX4(6-1) in the example of the present invention. The main vector construction steps are preferably as follows:

[0043] (1) use X ho I and X the b Ⅰ Digest the plasmid containing the synthesized TX4(6-1) 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):

[0044] Plasmid containing synthetic TX4(6-1) gene 15 μL

[0045] 5 μL o...

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Abstract

The invention discloses a preparation method for a spider neurotoxin TX4(6-1) recombinant protein. The preparation method comprises the following steps: (1) according to the amino acid sequence of TX4(6-1) mature toxin and the feature of a pichia pastoris expression system, optimizing and synthesizing gene of TX4(61-) of which the C-end is provided with a 6XHis label and constructing a pichia pastoris secretory expression carrier; (2) converting a recombinant carrier to a pichia pastoris host, screening to obtain a yeast converter of high-level secretory expression; and (3) expressing a recombinant spider insect neurotoxin TX4(6-1) protein in the yeast host, to obtain the recombinant spider insect neurotoxin TX4(6-1) protein of which the purity is more than 95% rapidly through a nickel affinity chromatography method, wherein the protein has the remarkable toxicity to silkworms. According to the preparation method, a large number of the stable recombinant spider insect neurotoxin TX4(6-1) proteins can be rapidly obtained. The preparation method has important value for the insect resistant field, specifically for cultivating insect resistant plant varieties, and the preparation method 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 spider insect neurotoxin TX4(6-1) 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 killing effects. They mainly come from the venom secreted by the venom glands of spiders, 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 spider neurotoxin gene TX4(6-1) is an insect-specific neurotoxin derived from the Brazilian wandering spider (Phoneutria nigriventer). important potential value, but the content of natural insect neurotoxins in the venom glands is low and difficult to extract, and often only a very small amount of pure products can be obtained, which brings great diffic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C12N1/19C07K14/435C07K1/34C12R1/84
CPCC07K14/43518C12N15/815C12N2800/102
Inventor 李洪波夏玉先蒋鹰
Owner HUAIHUA UNIV
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