摘要
The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.
The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.
