Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the beta-galactosidase gene.
We have constructed a transfer vector (pAgGal) containing the beta-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The beta-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. Beta-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed beta-galactosidase protein was also shown to be biologically active by a beta-galactosidase assay.
Ribeiro
B M
BM
Departamento de Biologia Celular, Universidade de Brasília-UnB, DF, Brazil.
Gatti
C D
CD
Costa
M H
MH
Moscardi
F
F
Maruniak
J E
JE
Possee
R D
RD
Zanotto
P M
PM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006160:Guanosine Triphosphate / Q000235:genetics; D007814:Larva; D007915:Lepidoptera; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000472:pathogenicity; D000077183:Occlusion Body Matrix Proteins; D011401:Promoter Regions, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D000077183:Occlusion Body Matrix Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D006160:Guanosine Triphosphate; D001616:beta-Galactosidase