[Rapid disruption of Bombyx mori nucleopolyhedrovirus orf60 by red recombination system].
BmNPV bacmid constructed recently and Red recombinant system were used to rapidly disrupted Bombyx monri nucleopolyhedrovirus (BmNPV) orf60 in Escherichia coli (E. coli) BW25113. BmNPV bacmid isolated from E. coli BmDH10Bac was electroporated into E. coli BW25113, which harbors plasmid pKD46 encoding lamda Red recombinase,to produce E. coli BW25113-Bac, which could be used for gene deletion of BmNPV. A linear fragment was amplified by PCR from plasmid pKD3 (containing a chloramphenicol acetyltransferase gene cat) using a pair of primers with length of 63bp,which had 45 bp homologous to the orf60 gene and 18bp homologous to cat sequences. The linear fragment was electroporated into E. coli BW25113-Bac and homologous recombination occurred between the linear fragment and orf60 with the help of lamda Red recombinase. Three specific primer pairs were used to confirm the replacement of orf60 by cat gene. Western blot analysis showed that orf60 was not expressed in BmN cells infected with knockout bacmid.
Institute of Life Science, Jiangsu University, Zhenjiang 212013, China.
Qiang
Q
Wang
Zhong-Jian
ZJ
Guo
Qin
Q
Yao
Hai-Yan
HY
Wang
Ke-Ping
KP
Chen
D000818:Animals; D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D012831:Bombyx / Q000821:virology*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D005814:Genes, Viral / Q000235:genetics*; D017924:Nucleopolyhedroviruses / Q000201:enzymology / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics / Q000502:physiology; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D045522:Recombinases; D014764:Viral Proteins