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Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb

A technology of crystal fusion protein and Bacillus aureus, applied in the field of genetic engineering, can solve problems such as the risk of utilization and the difficulty of predicting the threat to the environment of exogenous gene transgenic crops

Inactive Publication Date: 2013-07-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Genetically modified crops meet human needs for food production and economic benefits, but due to the impact of exogenous genes on crop physiological metabolism and the threat of genetically modified crops to the environment is difficult to predict, their use also has risks, and effective ways to reduce this risk It is to evaluate the safety of foreign proteins expressed by transgenic crops

Method used

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  • Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb
  • Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb
  • Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Codon optimization and total gene synthesis of Bacillus thuringiensis crystal protein Bt Cry3Bb gene

[0042] According to the wild-type Bacillus thuringiensis crystal protein Bt Cry3Bb gene sequence (GenBank: 202-2157 nucleotides of M89794.1) in Bacillus thuringiensis, it is suitable for Escherichia coli after design and repeated verification The expressed optimized Bacillus thuringiensis crystal protein Bt Cry3Bb gene sequence, that is, the wild-type Bacillus thuringiensis crystal protein Bt Cry3Bb gene sequence is transformed into Escherichia coli preferred (high frequency usage) codon optimization sequence, thereby improving the expression level of Bacillus thuringiensis crystal protein Bt Cry3Bb in the E. coli culture environment.

[0043] According to the above method, the optimized Bacillus thuringiensis crystal protein Bt Cry3Bb gene designed according to Escherichia coli preference was finally obtained, named N-Cry3Bb gene, and its gene sequence was ...

Embodiment 2

[0044] Embodiment 2, prokaryotic expression of Bacillus thuringiensis crystal fusion protein Bt Cry3Bb

[0045] 1. Construction of prokaryotic recombinant expression vector pET28a-3Bb

[0046] In order to construct the expression vector, the recognition sequences of restriction sites EcoRI and XhoI were respectively added to the two ends of the optimized Bacillus thuringiensis crystal protein Bt Cry3Bb gene (N-Cry3Bb gene) obtained in Example 1, and the "Seq. 103-2070 bits". The sequence is artificially synthesized, and the DNA fragment shown in the sequence is connected with the pGOV4 cloning vector to obtain a recombinant plasmid. The recombinant plasmid whose DNA fragment shown in "position 103-2070 of sequence 1" was inserted into the pGOV4 cloning vector by sequencing was named pGOV4-3Bb.

[0047] The recombinant plasmid pGOV4-3Bb constructed above was double-digested with restriction endonucleases EcoRI and XhoI to recover the target gene fragment; the resulting target...

Embodiment 3

[0071] The activity determination of the Bacillus thuringiensis crystal fusion protein Bt Cry3Bb obtained in embodiment 3 and embodiment 2

[0072] Get the Bacillus thuringiensis crystal fusion protein Bt Cry3Bb obtained in Example 2, dilute with 0.1% (volume ratio) Triton-100 aqueous solution, be made into the dilution of 5 dilutions, make with 0.1% (volume ratio) Triton aqueous solution simultaneously In contrast, clean cabbage leaves slightly smaller than the bottom of the petri dish were immersed in the diluent for 10 seconds, then dried naturally, and placed in a petri dish with water-soaked filter paper at the bottom, and 10 second-instar Tenebrio molitor larvae were inserted into each dish. Incubate at room temperature. After 72 hours, count the death of Tenebrio molitor larvae (lightly touch the end of the abdomen of the larvae with a pen, if the head cannot swing or crawl forward, it is regarded as dead), and accordingly the POLO software is used to calculate the thur...

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Abstract

The invention discloses a preparation method of bacillus thuringiensis crystal fusion protein Bt Cry3Bb. The method relates to a gene of bacillus thuringiensis crystal protein Bt Cry3Bb, and the gene is subjected to codon optimization and is specifically shown in the sequence 1 in the sequence table. The preparation method of fusion protein Bt Cry3Bb of the bacillus thuringiensis crystal comprises the following steps of: 1) introducing a recombinant vector which contains DNA (Deoxyribonucleic Acid) fragment of the 109-2064th in the sequence 1 or in the whole sequence 1, so as to obtain recombinant escherichia coli; and 2) culturing the recombinant escherichia coli, carrying out inducible expression, collecting and splitting the thallus so as to obtain the bacillus thuringiensis crystal fusion protein Bt Cry3Bb (sequence 2). In the escherichia coli, the bacillus thuringiensis crystal protein Bt Cry3Bb gene (N-Cry3Bb gene) which is subjected to codon optimization shows that the protein activity of the obtained protein is not affected by the codon optimization.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for preparing a Bacillus thuringiensis crystal fusion protein, in particular to a method for preparing a Bacillus thuringiensis crystal fusion protein Bt Cry3Bb. Background technique [0002] Bacillus thuringiensis (Bt) will produce a parasporal crystal during the formation of spores. The main component of this crystal is a protein with insecticidal activity, which is called insecticidal crystal protein (ICP, Insecticidal Crystal Protein) ) or Bt toxin. Bt toxin can poison insects such as Lepidoptera, Diptera and Coleoptera, and is currently the most widely used biological insecticide in the world. So far, nearly 180 Bt poisonous protein genes have been cloned and sequenced, Cry3Bb is one of them, and its main target pests are Coleoptera. [0003] Genetically modified crops meet human needs for food production and economic benefits, but due to the impact of e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C12N15/63C12N5/10C12N1/21C07K14/325A01N47/44A01P7/04C12N15/70C12R1/07
Inventor 王保民张亮郭素琴谭桂玉曹振何丽珊张瑞张威
Owner CHINA AGRI UNIV