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.
Mittal
S K
SK
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
Prevec
L
L
Graham
F L
FL
Babiuk
L A
LA
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