A high efficient method of constructing recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus based on zero-background Tn7-mediated transposition in Escherichia coli.
A high efficient way for generation of recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus by Tn7-mediated transposition in Escherichia coli was performed. The new system consists of a conditional replication donor vector pRCDM and an attTn7 site blocked E. coli containing BmNPV-Bacmid. The donor vector contains a replication origin derived from R6Kgamma, which propagated only in host cells with pir gene expression decreased in the transposition background greatly. Compared with original vector derived from pUC, the transposition efficiency increased from 5.7 to 66% ( approximately 10 fold) when using conditional replication vector pRCDM transposition into original BmDH10Bac. A further effort to decrease the transposition background was made by blocking the attTn7 site in host E. coli genome. The resulting attTn7 occupied BmDH10BacDeltaTn7 resulted in a significant increase from 5.7 to 23% ( approximately 4 fold) in the efficacy of generate recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10BacDeltaTn7 with pRCDM resulted typically in 100% white colonies, and it indicated that a zero transposition background was accomplished. This high efficient and zero background transposition system provides a new simple and rapid method for construction of recombinant BmNPV used to express target genes or produce gene-delivery virus particles in silkworm.
Sun
Jing Chen
JC
College of Animal Science, South China Agricultural University, Guangzhou 510642, PR China.
Zhang
Er Hui
EH
Yao
Lun Guang
LG
Zhang
Hong Ling
HL
Jin
Peng Fei
PF
D000818:Animals; D012831:Bombyx / Q000821:virology*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements