Estudo da toxicidade de proteínas (Cry) recombinantes de Bacillus thuringiensis, utilizando o sistema de expressão baseado em baculovírus e células de inseto

AUTOR(ES)

Raimundo Wagner de Souza Aguiar

DATA DE PUBLICAÇÃO

2007

RESUMO

The cry1Ca, cry2Ab and cry10Aa genes from different Brazilian strains of Bacillus thuringiensis were amplified by PCR, cloned into a plasmid cloning vector and sequenced. Sequence analysis showed high identity to previous known cry genes. The genes were removed from the cloning vector and introduced into a transfer vector (pSynXIVVI+X3) for the construction of recombinant baculoviruses by homologous recombination. The recombinant viruses were purified by serial dilution in 96 well plates and used to infect Trichoplusia ni (BTI-Tn5B1-4) insect cells and Spodoptera frugiperda larvae. Transcritptional analysis of the cry2Ab and cry10Aa genes was carried out by RT-PCR, using mRNA extracted from infected insect cells (72 h p.i.), in order to confirm the presence of the gene specific transcript. Recombinant virus (vSyncry1Ca, vSyncry2Ab and vSyncry10Aa) infected insect extracts (96 h p.i.) were used for the purificaton of crystals, made of recombinant proteins, by ultracentrifugation. In SDS-PAGE, the insect extracts showed polypeptides of approximately 65, 65 and 74 kDa, corresponding, respectively to the sizes of the proteins Cry1Ca Cry2Ab e Cry10Aa. These same crystals were analysed by light and electron microscopy and showed the shape of big cuboidal crystals. Furthermore, the crystals preparations were toxic to second instar S. frugiperda and Anticarsia gemmatalis larvae, with a CL of 114,44 and 19,49 ng/mL, respectively (from 50 vSynCry1Ca-infected insect extracts), to second instar S. frugiperda, with a CL de 50 3,40 g/mL (from vSynCry2Ab-infected insect extracts) and neonate A. grandis larvae, of 7,12 g /mL (from vSynCry10Aa-infected insect extracts). This work with a CL 50 showed that recombinant Cry proteíns are similar to their natural couterpart, showing the high toxicity to different insect pests, which demonstrate the utility of the baculovirus expression system for the study of Cry proteins.

ASSUNTO(S)

anticarsia gemmatalis antonomus grandis biologia molecular spodoptera frugiperda proteínas cry bacillus thurinigiensis

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