RESUMO
The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p20 gene, pP1B carries p20, and pDP1B contains p20 with cry1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that crylAb was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1:1.4:1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1:1.1:1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B < P1B < DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC50. This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab.
Assuntos
Animais , Bacillus thuringiensis , Genética , Metabolismo , Proteínas de Bactérias , Genética , Metabolismo , Farmacologia , Bioensaio , Métodos , Western Blotting , Eletroporação , Endotoxinas , Genética , Metabolismo , Farmacologia , Proteínas Hemolisinas , Genética , Metabolismo , Farmacologia , Microscopia Eletrônica de Transmissão , Mariposas , Regiões Promotoras Genéticas , GenéticaRESUMO
The vip3 A gene in a size of 2.3 kb amplified from wild-type Bacillus thuringiensis strain S184 by PCR was cloned into pGEM-T Easy vector and its sequence was analysized by DNASTAR. The plasmid pOTP was constructed by inserting vip3A-S184 gene into the expression vector pQE30 and then was transformed into E. coli M15. E. coli M15 cells harbouring the plasmid pOTP were induced with 1 mmol/L IPTG to express 89 kD protein which was confirmed to be Vip3A-S184 by Western blot. Experiments showed that about 19% of Vip3A-S184 proteins were soluble, and others were insoluble proteins and formed inclusion bodies observed by transmission electron microscopy(TEM). The target protein was purified under the native condition and the polyclonal antibody was prepared by immunizing rabbits. The polyclonal antibody was used to detect Vip3A proteins expressed in Bacillus thuringiensis. Bioassay showed that Vip3A-S184 showed a high toxicity against 3 tested insect larvae including Spodoptera exigua, Spodoptera litura and Helicoverpa armigera.