A recombinant E1-deleted porcine adenovirus-3 as an expression vector.
Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B(large) coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B(large) of PAV-3 and also complemented PAV214 (E1A+E1B(small) deleted). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B(large) coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B(small) + E1B(large)) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B(large) was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines.
Zakhartchouk
Alexander
A
Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
Zhou
Yan
Y
Tikoo
Suresh Kumar
SK
D017498:Adenovirus E1 Proteins / Q000235:genetics*; D000260:Adenoviruses, Human / Q000235:genetics; D025662:Adenoviruses, Porcine / Q000235:genetics* / Q000254:growth & development; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D018388:Codon, Terminator; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D017353:Gene Deletion; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D016865:Reassortant Viruses; D011994:Recombinant Proteins / Q000096:biosynthesis; D013552:Swine; D014162:Transfection
D017498:Adenovirus E1 Proteins; D018388:Codon, Terminator; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins