Zhu Y, Qi Y, Liu D, Joshua M N, Wang Y
Institute of Virology, Wuhan University, China.
J Virol Methods. 1998 Dec;76(1-2):101-8. doi: 10.1016/s0166-0934(98)00128-1.
A host range expanded recombinant Autographa californica multiple-nucleocapsid nucleopolyhedrosis virus AcMNPV/r2 was obtained by cotransfection of the bacmid DNA from Escherichia coli DH10Bac along with a plasmid pBmH-M containing HindIII M fragment of Bombyx mori nuclear polyhedrosis virus (BmNPV) genomic DNA. A recombinant transposon vector carrying a mutant green fluorescent protein gene (GFP) and a polyhedrin gene was constructed. Transposition was carried out in both E. coli DH10Bac and E. coli DH10BmH, which contains AcMNPV/r2 and a helper plasmid. Recombinant DNAs were transfected into Sf-9 cells to generate recombinant virus AcMNPV/r3 and AcMNPV/r4 respectively. Viral stock of AcMNPV/r4 was then infected into Bombyx mori cells (BmN) and Bombyx mori larvae (silkworm). Analysis shows that GFP was highly expressed in Bombyx mori larvae. This expression system, is practicable therefore for mass production of foreign gene products.
通过将来自大肠杆菌DH10Bac的杆粒DNA与含有家蚕核型多角体病毒（BmNPV）基因组DNA HindIII M片段的质粒pBmH-M共转染，获得了宿主范围扩大的重组苜蓿银纹夜蛾多核衣壳核型多角体病毒AcMNPV/r2。构建了携带突变绿色荧光蛋白基因（GFP）和多角体蛋白基因的重组转座子载体。在大肠杆菌DH10Bac和含有AcMNPV/r2及辅助质粒的大肠杆菌DH10BmH中进行转座。将重组DNA转染到Sf-9细胞中，分别产生重组病毒AcMNPV/r3和AcMNPV/r4。然后将AcMNPV/r4的病毒原液感染家蚕细胞（BmN）和家蚕幼虫（蚕）。分析表明，GFP在家蚕幼虫中高表达。因此，该表达系统可用于大规模生产外源基因产物。
