Construction of recombinant Bombyx mori Nucleopolyhedrovirus by zero background Tn7 transposition

Abstract:

Abstract: [Objective] In order to construct a recombinant Bombyx mori Nucleopolyhedrovirus by Tn7-mediated transposition in Escherichia coli efficiently, a new zero background transposition system was developed. [Method] The new system consisted of a conditional replication donor vector pRADM and an attTn7 site blocked E. coli containing BmNPV-Bacmid. The donor transposon vector pRADM with the replication origin derived from R6Kγ required the factor π encoded by the pir gene to propagate in host cells. Another conditional replication plasmid pBlockA was constructed to block the attTn7 site in host E. coli genome. [Results] Compared with the original vector with ColE1 origin, the transposition efficiency increased from 5.7% to 66% when using conditional replication vector pRADM transposition into original BmDH10Bac. The attTn7 site blocked strain BmDH10Bac△Tn7 resulted in a significant increase from 5.7% to 23% in the efficacy of generating recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10Bac△Tn7 with pRADM resulted in 100% white colonies. [Conclusion] This highly efficient and zero background transposition system provides a simple and rapid way of construction of recombinant BmNPV to express target genes or produce gene-delivery virus particles in silkworm.

Key words:Keywords: conditional replication origin; zero background transposition; recombinant Bombyx mori Nucleopolyhedrovirus