1 research outputs found
Construction of bovine adenovirus type 3 E1 and E3, substitution plasmids and the sequencing analysis of DNA pol and pTP /
The relative ease to concentrate and purify adenoviruses, their well
characterized mid-sized genome, and the ability to delete non-essential regions from
their genome to accommodate foreign gene, made adenoviruses a suitable candidate
for the construction of vectors. The use of adenoviral vectors in gene therapy,
vaccination, and as a general vector system for expressing foreign genes have been
documented for some time.
In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector
carrying the kanamycin resistant gene, a dominant selectable marker with useful
applications in studying vectored gene expression in mammalian cells. To accomplish
the objective of this study, more information about BAV3 DNA sequences was required
in order to make the manipulation of the virus genome accessible. Therefore,
sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the
determined sequences revealed the primary structure of important viral gene products
coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative
analysis of these proteins with their counterparts from human and non human
adenoviruses revealed important insights as to the evolutionary lineage of BAV3.
In order to insert the kanamycin resistance gene in either E1 or E3, it was
necessary to delete BAV3 sequences to accommodate the foreign gene so as not to
exceed the limit of the packaging capacity of the virus. To construct a recombinant
BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle
vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the
deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome
and was expected to generate BAV3 recombinants that can be grown and propagated
in cells that can complement the missing E1 functions.
To construct a similar shuttle vector for E3 deletion, DNA sequences extending
from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been
cloned into a plasmid vector. The deleted region corresponds to those that have been
shown to be non-essential for viral replication in cell culture. The resulting plasmid was
used to construct another recombinant plasmid with BAV3 DNA sequences extending
from 37.1% to 100% and with a deletion of E3 sequences that were replaced by
kanamycin resistance gene. This shuttle plasmid was used in cotransfections with
digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin
resistance gene to replace the deleted E3.
In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were
not isolated. It seems that other approaches should be applied to make a final
conclusion on BAV3 infectivity