Preparative Purification and Bioassay of Bt Toxin from CrylAb Transgenic Rice

A method of extracting and purifying CrylAb protein(Bt toxin) from CrylAb transgenic rice was established. Most of the Bt toxin present in the tissue of CrylAb transgenic rice was extracted effectively with a solution of 50 mmoi/LNa2CO3 and NaHCO3. The crude protein containing Bt toxin was obtained after the pretreatment of CrylAb transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCI buffer 1.0 mmol/L EDTA, pH=8.0) mixed with 0. 1, 0. 3, 0.5 and 0.8 mol/L NaCI in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.