Novel insecticidal toxins derived from bacillus thuringiensis insecticidal crystal proteins
A technology of Bacillus aureus and toxins, which can be applied in the directions of biocides, biocides, antibody mimics/supports, etc., and can solve problems such as the lack of effective insect control agents.
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Embodiment 1
[0121] Example 1: Expression and purification of H04 toxin fragments
[0122] The truncated form of the H04 hybrid toxin gene is cloned into an expression vector such as pBluescript SK-, a Bacillus shuttle vector, or pET 21b(+) for overexpression in E. coli, the truncated form of the H04 hybrid toxin gene. The short form, as described in De Maagd et al., Appl.Environ.Microbiol.62(5):1537-1543 (1996), encodes a Bt toxin consisting essentially of domains I and II of Cry1Ab and domain III of Cry1C . Cells were grown in LB medium containing 50 μg / ml ampicillin at 37°C on a shaker (250 rpm) for 24 to 48 hours. Cells were collected by centrifugation at 7,000 rpm for 10 min. The resulting pellets were sonicated with a Bronson sonicator for 2 minutes and 30 seconds with a pulse interval of 2 seconds. All sonicated objects were examined under a microscope. The soluble fraction was removed by centrifugation at 10,000 rpm for 10 min. The resulting crystalline protein-containing pell...
Embodiment 2
[0123] Example 2: Bioassay
[0124] LC50 values were determined for fall armyworm, pink bollworm, tobacco armyworm, and European cornborer using purified truncated H04 proteins, e.g., as described above. prepared in Example 1. The result is as follows:
[0125]LC50 fall armyworm 133ng / cm 2
[0126] LC50 red bollworm (pink bollworm) 691ng / cm 2
[0127] LC50 Tobacco budworm 299ng / cm 2
[0128] LC50 European cornborer 31ng / cm 2
Embodiment 3
[0129] Example 3: Synthetic H04 Gene Construct
[0130] The synthetic nucleotide sequence encoding the H04 toxin portion was provided through the University of Wisconsin GCG program suite using the maize biased codon table (Murray et al., Nucl Acids Res. 17:477-498, 1989, incorporated herein by reference). "Backtranslation" program, backtranslating the amino acid sequence of the H04 hybrid toxin (domains I and II of Cry1Ab) as described in De Maagd et al., Appl. Environ. Microbiol. 62(5): 1537-1543 (1996). and Cry1C domain III). Preferably, the most frequently used maize codons are applied for each amino acid as described in WO93 / 07278.
[0131] The synthetic nucleotide sequence encoding the H04 toxin moiety can be constructed from several fragments. Each fragment was constructed by hybridization of 10 pairs of oligomers of 60-75 nucleotides in length representing the two strands of the gene. Consecutive pairs of oligonucleotides overlap by about 15 nucleotides to facilitat...
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