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.
Artículo
en Chino
| 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.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Fisiología
/
Bombyx
/
Proteínas Virales
/
Virología
/
Expresión Génica
/
Sistemas de Lectura Abierta
/
Bacteriófago lambda
/
Nucleopoliedrovirus
/
Electroporación
/
Recombinasas
Límite:
Animales
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2007
Tipo del documento:
Artículo
Similares
MEDLINE
...
LILACS
LIS