Clonagem e expressão da protease 3C do vírus deformador da asa (deformed wing virus – DWV)

Abstract

Bees are insects of the Hymenoptera order, and the best known representative is the Apis mellifera L., repred on a large scale for the production of honey, bee wax and propolis. In the last years apiculture have been affected by an increase of deleterious events that culminate in loss of bee hives, among these events are the viral infections. The most abundant virus around the world the deformed wing virus (Deformed Wing Virus - DWV), a virus belonging to Picornaviridae family that infects the bees both vertically and horizontally causing death in the early stages of development or deformation of the wings and abdominal shortening among other behavioral and physical characteristics in adults. The Picornaviridae family of viruses are small viruses with genome constituted of simple strand RNA with positive sense and have a single ORF in its genome which encodes a polyprotein, which after translated, is cleaved by the 3C protease contained therein. In other viruses of this family, it was demonstrated that this protease participated in the regulation of functions of the host cells via proteolytic degradation of host proteins, preventing and / or antiviral blocking mechanisms. This study aims, perform the molecular cloning of the sequence were used as tamplote for PCR encoding the 3C protease from DWV virus. Positive DNA samples of DWV virus using specific primers to amplify the region of the viral genome which encodes the 3C protease. It is was added in a fragment of 6 his-tagg for subsequent protein purification. The amplified fragment was cloned into pGEX4T-1 plasmid by circular PCR. The production of recombinant 3C protease has a molecular weight of approximately 10 kDa was induced in E. coli strain BL21 (DE3) pLysS, for about 6 hours of induction conducting tests every 2 hours induction. The results were analyzed by SDS-PAGE.