A novel recombinant baculovirus overexpressing a Bacillus thuringiensis Cry1Ab toxin enhances insecticidal activity
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Biological Procedures Online
RESEARCH
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A novel recombinant baculovirus overexpressing a Bacillus thuringiensis Cry1Ab toxin enhances insecticidal activity Wael El-Menofy1, Gamal Osman1,2*, Abdulrahman Assaeedi2 and Mohamed Salama3
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. Keywords: Bacillus thuringiensis, Toxin, Baculovirus, Cry 1Ab, LC50
Introduction 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. Pests that had been naturally controlled by predators and parasites began to cause significant damage and became resistant to chemical pesticides [2]. Moreover, chemical pesticides have become expensive and imposed greater hazard to the environment and all living organisms [3]. An alternative strategy for effective control of pests is the use of biological insecticides either by itself or within an integrated pest management program (IPM) [4,5]. The benefit of biological control by viruses, especially baculoviruses has become apparent in recent years. Baculoviruses are major insect pathogens and are characterized by the * Correspondence: [email protected] 1 Agricultural Genetic Engineering Research Institute (AGERI) - ARC, 9 Gamaa St, Giza, Egypt 2 Biology Department, Faculty of Applied Sciences, Umm Al Qura University, Makka 21955, PO Box 715, Kingdom of Saudi Arabia Full list of author information is available at the end of the article
presence of a large circular double stranded DNA genome and enveloped rod shaped virions. Baculoviruses and their use as biological insecticides have been studied for several years [6]. Since baculoviruses are host-specific and do not infect vertebrates or
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