Baculovirus gene expression is the most popular method to make target protein in cultured insect cells. To fast determine the generation of recombinant virus in cultured cells, donor plasmid of pFastBacI was modified by introducing egfp cassette. In the modified vector, egfp cassette was under the control of ie1 promoter, and target gene cassette was under the control of polyhedron promoter. To evaluate the convenience of the genetically modified donor plasmid used in eukaryotic expression, ns1 gene from Bombyx mori bidensovirus was ligated into the donor plasmid to generate recombinant plasmid pFastBacI-[P(ie1)-egfp-sv40]-[P(polh)-ns1-sv40]. Then the plasmid was transformed into DH10B competent cells containing Bm-Bacmid vector to produce the final recombinant Bm-Bacmid with the help of transposase. The resulting recombinant Bm-Bacmid was transfected into BmN cells to generate recombinant virus, which was easily and rapidly judged by green fluorescent signal observed in BmN cells. After infection for 96 h, the BmN cells were harvested and the total protein extracted from the infected BmN cells was subjected to Western blotting analysis. The result showed that a specific protein band about 36 kDa was detected, indicating that NS1 protein was successfully expressed in the BmN cells. In conclusion, the expression of NS1 protein with the modified expression system is useful for further research on the function of NS1 protein.
Animals ; Baculoviridae ; genetics ; Bombyx ; virology ; Cell Line ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; Plasmids ; Promoter Regions, Genetic ; Transfection ; Viral Nonstructural Proteins ; biosynthesis ; genetics