Development of a bovine adenovirus type 3-based expression vector.
We constructed a non-defective bovine adenovirus type 3 recombinant (BAd3-Luc) containing the firefly luciferase gene inserted in the early region 3 (E3) of the BAd3 genome. Deletion of a 696 bp XhoI-NcoI E3 segment and insertion of the luciferase gene in E3 was confirmed by Southern blot analyses. Luciferase was expressed in Madin-Darby bovine kidney cells infected with BAd3-Luc as measured by enzymic assays and Western blotting. Analyses of luciferase expression in the presence or absence of 1-beta-D-arabinofuranosylcytosine indicated that approximately 70-75% of luciferase expression was dependent on viral DNA replication, suggesting that transcription of the gene was at least partially under the control of a late promoter. Although yields of infectious virus for BAd3-Luc were approximately 10-fold lower than for wild-type virus, replication of the vector was still relatively efficient. In a Western blot experiment, anti-luciferase antibody reacted with a 62 kDa protein which is of the same molecular mass as the purified firefly luciferase polypeptide. Luciferase was also expressed in the 293 cell line infected with BAd3-Luc for at least 6 days post-infection as monitored by luciferase assays. Based on these observations we suggest that BAd-based expression vectors should have excellent potential for the development of recombinant vaccines for cattle and may also be suitable as vectors for gene transfer into human cells.
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
S K
SK
Mittal
L
L
Prevec
F L
FL
Graham
L A
LA
Babiuk
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle / Q000821:virology*; D002460:Cell Line; D015316:Genetic Therapy; D005822:Genetic Vectors; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D008156:Luciferases