Journal of General Virology, Vol 80, 563-570, Copyright © 1999 by Society for General Microbiology

ARTICLES

Development of porcine adenovirus-3 as an expression vector

PS Reddy, N Idamakanti, BH Hyun, SK Tikoo and LA Babiuk
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada. reddys@sask.usask.ca

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV- neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV- 3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.

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