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A preparation method for a crystal fusion protein Cry34B of Bacillus thuringiensis

A technology of crystal fusion protein and thuringiensis spore is applied in the field of genetic engineering, which can solve problems such as environmental safety risks, and achieve the effects of overcoming cumbersome extraction and fast and sensitive regulation.

Inactive Publication Date: 2014-12-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, there are also environmental safety risks, the randomness and uncertainty of exogenous gene introduction, changes in the physiological and biochemical effects of gene expression on crops, threats to the ecological environment, etc. are difficult to predict

Method used

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  • A preparation method for a crystal fusion protein Cry34B of Bacillus thuringiensis
  • A preparation method for a crystal fusion protein Cry34B of Bacillus thuringiensis
  • A preparation method for a crystal fusion protein Cry34B of Bacillus thuringiensis

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Embodiment 1

[0027] Embodiment 1, the preparation method of Bt Cry34B fusion protein

[0028] (1) Optimization of Bt Cry34B gene coding sequence and acquisition of gene fragments

[0029] The Bt Cry34B gene sequence (GenBank: AY536900.1) was codon-optimized, and the optimized sequence is shown as nucleotides 5179-5574 in SEQ ID №1.

[0030] (2) Construction of Bt Cry34B gene cloning vector

[0031] Artificially synthesize the nucleotide sequence shown in the 5173-5580th nucleotide in SEQ ID No.: 1, that is, the restriction site EcoR Ⅰ and Xho Ⅰ sequences are connected to both ends of the target gene.

[0032] The above synthesized nucleotide sequence was ligated into pGOV4 (purchased from Gene Oracle Inc., a gene synthesis company) cloning vector plasmid to obtain a Bt Cry34B gene recombinant plasmid, which was designated as pGOV4-34B. The plasmid was sequenced and verified, and the results showed that the sequence of the foreign gene inserted in the plasmid was shown in nucleotides 5173...

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Abstract

The invention discloses a preparation method for a crystal protein Cry34B of Bacillus thuringiensis. The method comprising the steps of: 1) inserting the DNA sequence shown in SEQ ID No. 1 into a pET28a expression vector to obtain a recombinant expression vector; 2) introducing the recombinant expression vector into E. coli BL21 (DE3), to obtain a recombinant strain; 3) culturing the recombinant strain, inducing for expression, and collecting cells; and lysing the cells to obtain the crystal fusion protein. According to the invention, a genetic engineering method is employed to synthesize the Bt Cry34B gene at home for the first time, and the Bt Cry34B fusion protein is prepared for the first time, laying foundations for further study of detection methods, physiological functions, action mechanism and degradation mechanisms of the protein,.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a method for preparing a fusion protein, in particular to a method for preparing a Bacillus thuringiensis crystal fusion protein. Background technique [0002] Bacillus thuringiensis (Bt) is a Gram-positive bacterium widely distributed in soil. During the formation of its spores, it will form a crystal protein (Cry protein) with insecticidal activity. This protein has high toxicity to various pests such as Lepidoptera, Diptera, Coleoptera, and Hymenoptera, and is the most widely used toxic protein. Because of this feature, the gene encoding Cry protein is considered by biologists as a popular candidate gene for transgenic crops, and several transgenic crop varieties have been transferred to this gene and commercialized, and are widely planted around the world. Bt strains contain a large number of different insecticidal crystal protein coding genes. So far, nearly 180 di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/32C12N15/62C07K19/00C12N15/63C12N5/10C12N1/21C12R1/91
Inventor 王保民张威谭桂玉曹振何丽珊郭素琴张亮张瑞
Owner CHINA AGRI UNIV