Abstract
Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of killing. In this study a truncated form of cry1Ab gene derived from Bacillus thuringinsis (Bt) subsp. aegypti isolate Bt7 was engineered into the genome of the baculovirus Autographa californica multiple nuclearpolyhedrosis wild type virus, in place of the polyhedrin gene by using homologous recombination in Spodoptera frugiperda (Sf) cells between a transfer vector carrying the Bt gene and the wild type virus linearized DNA. Recombinant wild type virus containing the cry1Ab gene was detected as blue occlusion-negative plaques in monolayers of Sf cells grown in the presence of X-Gal. In Sf cells infected with plaque-purified recombinant virus, the cry1Ab gene was expressed to yield a protein of approximately 82-kDa, as determined by immunoblot analysis. The toxicity of the recombinant virus expressing the insecticidal crystal protein (ICP) was compared to that of the wild-type virus. Infected-cell extract was toxic to cotton leaf worm Spodoptera littoralis second instar larvae and the estimated LC50 was 1.7 μg/ml for the recombinant virus compared with that of wild-type virus which was 10 μg/ml.
Highlights
The development of synthetic pesticides since 1940 coupled with the improvement in chemical applications technology dramatically increased the potential for agricultural pest control [1]
These results proved that wild type baculovirus can be used to express and study the properties of the insecticidal Bacillus thuringiensis protein yielding information relevant to an understanding of the molecular biology of this protein as well as to improve the baculovirus insecticidal activity
This protein produced in considerable amounts was biologically active, but as expected, did not precipitate into crystals confirming that the C-terminal part of the Cry1Ab insecticidal crystal protein (ICP) is required for crystal formation [31,32]
Summary
The development of synthetic pesticides since 1940 coupled with the improvement in chemical applications technology dramatically increased the potential for agricultural pest control [1] It did not take long before people began to see the shortcomings of this new technology. Baculoviruses are major insect pathogens and are characterized by the only the N-terminal half, in the same time the truncated proteins do not form inclusions. These insecticidal transgenes included insect hormone genes which disturb the physiological hormonal balance of the insect [15,16] and Bt gene(s). This study aims to introduce a truncated cry1Ab gene from Bacillus thuringiensis into a baculovirus in order to enhance its insecticidal activity
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