Cloning, expression and bioassay of Vip3A protein from an indigenous Bacillus thuringiensis isolate.

Abstract

The present study was undertaken to test vip3A protein from Bacillus thuringiensis for use against lepidopteran pests especially Spodoptera litura and whether cloning and expression of the vip3A gene will help in formulating an improved bioinsecticide. Eight isolates that showed amplification of the vip3A gene (675bp) were selected from 150 B. thuringiensis isolates. The isolate BT-EG1 was selected for further studies as the vip3A protein extracts from this isolate was found to cause mortality of Spodoptera litura (LC 50 of 9.09-9.92 µg/ml). The 2.367bp complete CDS was amplified using VCL1 and VCL2 specific primers and cloned into P UC19 at NdeI and XhoI restriction sites. The positive clones were sequence confirmed. The vip3A was the successfully cloned into pET21a (NdeI/XhoI) expression vector and confirmed by restriction digestion and SDS-PAGE. The crude protein extracts obtained after 16h of IPTG induction showed a LC 50 of 5.83 µg/ml against S. litura at 72h. The extracts were however highly toxic to Plutella xylostella (LC 50 of 0.43 µg/ml after 16h of IPTG induction at 48h). The extracts however did not cause mortality of Helicoverpa armigera suggested the requirement of a combination of vip proteins for broad spectrum activity.