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Production method of toxin Tx4 (6-1) label-free recombinant protein

A protein and recombinant vector technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of low expression, low production, difficult separation and purification, and achieve high biological activity.

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

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Problems solved by technology

[0003] In the prior art, insect neurotoxins are constructed as expression vectors, and then transformed into E. coli BL21(DE3) was expressed, but all of the obtained inclusion bodies were inactive. Through dissolution, denaturation, renaturation and purification under appropriate conditions in vitro, only a trace amount of soluble protein could be obtained from each liter of medium. Low; there is also a method to express the constructed expression vector in yeast, but the expression level is low and separation and purification are difficult
At present, there is no way to efficiently produce Tx4(6-1) recombinant protein by modifying the DNA sequence of Tx4(6-1) and optimizing the expression and purification method of Tx4(6-1), which greatly affects the Tx4(6-1) The analysis and study of insect resistance characteristics and biological safety especially affect its application in insect resistance, so it is necessary to develop a method for insect neurotoxin Tx4(6-1) that can reduce costs, achieve mass production and have biological activity

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  • Production method of toxin Tx4 (6-1) label-free recombinant protein
  • Production method of toxin Tx4 (6-1) label-free recombinant protein
  • Production method of toxin Tx4 (6-1) label-free recombinant protein

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Embodiment 1

[0039] This embodiment provides an optimized artificially synthesized Tx4(6-1) gene, the specific sequence is shown in sequence 1 in the sequence listing, and the protein sequence corresponding to the gene is 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. According to the expression characteristics of Escherichia coli, the optimized DNA was optimized and synthesized. 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.

[0040] Connect the genes before and after the above optimization to the pET series of E. coli expression vectors to obtain recombinant vectors. The above recombinant vectors verified by sequencing were transfor...

Embodiment 2

[0043] This embodiment provides a method for preparing protein, which specifically includes the following steps:

[0044] S1: Gene optimization, construction of prokaryotic expression vector and transformation: artificially synthesize the optimized mature scorpion insect neurotoxin Tx4(6-1) gene, and connect it to the expression vector pET32 to obtain the recombinant expression vector pET32 / Tx4(6-1) , the vector is constructed as figure 1 shown. The main vector construction steps are as follows:

[0045] (1) use B wxya I and H ind Ⅲ double enzyme cut recombinant vector Tx4(6-1) / pUC to obtain the target fragment PCP, the reaction system is as follows (endonucleases and buffers used were purchased from Dalian TAKARA Company):

[0046] carrier Tx4(6-1) / pUC 15 μL

[0047] 10×H buffer 5 μL

[0048] B wxya I 5U

[0049] h ind III 5U

[0050] Sterile water to 50μL

[0051] (2) with B wxya I and H ind Ⅲ Digest pET32 with double enzymes to obtain vector fragme...

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Abstract

The invention relates to a production method of toxin Tx4 (6-1) label-free recombinant protein. Gene provided by the invention is DNA molecule shown by a sequence 1 in a sequence table. The production method includes that (1), soluble high-level fusion expression of the toxin gene in Escherichia coli can be realized by utilizing a gene sequence of the sequence table 1; (2), recombinant protein completely identical with naturally mature Tx4 (6-1) protein in amino acid sequence is obtained by cutting and removing non-target protein from soluble protein in fusion expression; (3), the obtained recombinant protein has high bioactivity. By the production method, DNA sequence of Tx4 (6-1) is optimized, and expression strain and expression vector are screened; a method for efficiently producing active Tx4 (6-1) recombinant protein is provided, thereby being conducive to conducting detained research on insect-resistant characteristic and bio-safety of the toxin protein and of important significance in screening corresponding insect-resistant plants and high-toxicity transgenic biological insecticides.

Description

technical field [0001] The invention relates to the field of biotechnology utilization, in particular to the production of an optimized coding gene and recombinant protein of a spider neurotoxin Tx4(6-1). 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. The spider neurotoxin gene T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K14/435C12R1/19
CPCC07K14/43518C12N15/70C12N2800/101
Inventor 李洪波蒋鹰夏玉先
Owner HUAIHUA UNIV
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