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

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서울대학교 대학원
Autographa californica nucleopolyhedrovirusAutographa californica nucleopolyhedrovirusbAcGOZABacgozapBactrusPbactrusBactrusBactrus곤충세포Insect cellsmultiplicity of infectionMultiplicity of infection파밤나방Plutella xylostella배추좀나방Spodoptera exigua
Thesis (master`s)--서울대학교 대학원 :농생명공학부,2004.
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.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Food and Animal Biotechnology (식품·동물생명공학부)Theses (Master's Degree_식품·동물생명공학부)
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