Characterization of DNA binding protein of porcine adenovirus type 3.
To identify and characterize the protein encoded by the E2A region of porcine adenovirus (PAV)-3, DNA sequence coding for a portion (amino acids 102-457) of the DNA binding protein (DBP) open reaching frame was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase protein of Schistosoma japonica. The affinity-purified fusion protein was used to immunize rabbits. Immunoprecipitation/Western blot analysis demonstrated that the antisera specifically recognized a protein of 50 kD in PAV-3-infected cells. Immunoperoxidase staining detected the DBP protein predominantly in the nucleus of the cells. Western blot analysis demonstrated that DBP was detected as early as 6 h after infection and remained detectable throughout the infection. Based on these results, a novel assay for quantitation of PAV-3 was established. The assay is less time consuming and can be performed in different porcine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.
Zhou
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Y
Virology group, Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Pyne
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C
Tikoo
S K
SK
D017502:Adenovirus E2 Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D025662:Adenoviruses, Porcine / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D002461:Cell Line, Transformed; D005982:Glutathione Transferase / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013552:Swine; D010948:Viral Plaque Assay
D017502:Adenovirus E2 Proteins; D000914:Antibodies, Viral; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase