Construction and characterization of an improved recombinant baculovirus producing polyhedra that contain Bacillus thuringiensis Cry1Ac crystal protein

Abstract

For the enhancement of insecticidal activity in wt AcNPV and the reduction of side-effect on environment in recombinant baculovirus, an improved recombinant baculovirus, Autographa californica nucleopolyhedrovirus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) Cry1Ac crystal protein and having a potential of returning to wt AcNPV in several passage, has been constructed and characterized. The toxic domain of Bt cry1Ac gene was inserted between two polyhedrin genes which were cloned in tandem into pBacPAK8 under the control of polyhedrin promoter and the resulted construct, pBactrus was used as transfer vector. By the cotransfection of parental virus, bAcGOZA and pBactrus, the recombinant baculovirus named as Bactrus was constructed. SDS-PAGE and immunoblot analysis of polyhedra produced by Bactrus in insect cells showed that it expressed a 130 kDa of fusion protein and what is more, 30 kDa of polyhedrin resulted from a homologous recombinantion. The Bactrus produced recombinant polyhedra which were nearly similar to those of wt AcNPV. The Insecticidal activity of recombinant polyhedra against fall armyworm (Spodoptera exigua) larvae was similar to that of wt AcNPV, but for the diamondback moth (Plutella xylostella) larvae, it showed a significantly higher pathogenicity in comparison with those of wt AcNPV. In SDS-PAGE, expression level of fusion protein was decreased along serial passage. But the decrease of fusion protein expression was smaller in Sf-9 cells infected with Bactrus passaged at 10 multiplicity of infection (MOI) than that passaged at 1 MOI. Also insecticidal activity of recombinant polyhedra produced by the Bactrus passaged at 1 and 10 MOI against P. xylostella larvae was decreased along serial passage with similar trend to the result in SDS-PAGE. The production of recombinant polyhedra by the Bactrus was examined with Sf-9, Sf-21, Tn5, Se301 and High-Five cells with monolayer and suspension culture system. In monolayer culture, the largest amount of fusion protein was expressed in Se301 cells. However the highest expression of fusion protein in suspension culture was observed in High-Five cells when infected with 5 MOI and the optimal cell density on time of infection was 3.0×105 cells per ml.