Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

Abstract

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by nonoccluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Cry4aa and Cry4ba From Bacillus Thuringiensis Subsp. Israelensis Expressed in Insect Cells by Recombinant Baculoviruses Are Toxic to Aedes Aegypti Larvae

“…This region of Cry proteins contains a higher concentration of cysteine residues, which are responsible for disulfide bond formation and are believed by some authors to contribute to crystal formation (Ge et al, 1998). The expression of cry genes in insect cells, such as cry11A, cry1Ab, cry1Ac and cry1Ca, using baculovirus as vectors have been shown in previous works (Pang et al, 1992;Ribeiro & Crook, 1998;Lima et al, 2008). Remarkably, in our work, cytoplasmic Cry4Aa crystals expressed by vAcCry4Aa seem to be two fold bigger than those expressed by vSynCry4Aa.…”

“…The expression of Cry toxins using the heterologous protein expression system based on baculovirus has been reported, as part of efforts to obtain isolated Cry toxins for toxicity studies or to potentialize the pathogenicity of such insect virus to crop pests (Aguiar et al, 2006;Martins et al, 2008;Lima et al, 2008;Merryweather et al, 1990;Pang et al, 1992;Ribeiro & Crook 1993;Ribeiro & Crook 1998;Chang et al, 2003). Baculoviruses are a very diverse group of viruses with double-stranded, circular, supercoiled genomes.…”

A Recombinant Truncated Cry1Ca Protein Is Toxic to Lepidopteran Insects and Forms Large Cuboidal Crystals in Insect Cells

“…2). The formation of crystals by Cry proteins in insect cells infected with recombinant baculoviruses has been reported for the Cry1Ab, Cry1Ac, and Cry11Aa proteins [17,19,20,21]. These recombinant proteins were expressed in high amounts and some formed larger crystals than when expressed in Bt, suggesting that the size of the crystal produced in Bt is limited by the size of the bacteria [21].…”

“…The expression of cry genes in insect cells using BEVs can be an alternative method for the study of single Cry proteins. Some Cry proteins have already been expressed in insect cells using BEVs, such as Cry1Ab, Cry1Ac [20,21] and Cry11Aa [19], showing that the recombinant proteins were biologically similar to their native counterparts, with toxicity towards different insects. However, the pathogenicity of the recombinant baculovirus was not improved.…”

“…Baculoviruses are biological control agents used in agriculture and also one of the most popular vectors (BEVs) for heterologous gene expression in insect cells [14], due to the presence of strong promoters (p10 and polyhedrin promoters) that allow a high level of heterologous protein expression late in infection. BEVs have been used to express full-length and truncated forms of Cry proteins from Bt [17,19,20,21] and fusion of a Cry protein with the main protein of baculovirus occlusion bodies (OBs) [4]. In the present study, we have introduced a truncated version of the cry1Ca gene into the genome of a baculovirus and analyzed the recombinant protein expression in cultured insect cells and insect larvae.…”