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Rapid disruption of Bombyx mori nucleopolyhedrovirus orf60 by red recombination system / 生物工程学报
Chinese Journal of Biotechnology ; (12): 801-805, 2007.
Article in Chinese | WPRIM | ID: wpr-327944
ABSTRACT
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.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Physiology / Bombyx / Viral Proteins / Virology / Gene Expression / Open Reading Frames / Bacteriophage lambda / Nucleopolyhedroviruses / Electroporation / Recombinases Limits: Animals Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2007 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Physiology / Bombyx / Viral Proteins / Virology / Gene Expression / Open Reading Frames / Bacteriophage lambda / Nucleopolyhedroviruses / Electroporation / Recombinases Limits: Animals Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2007 Type: Article