Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells.
DNA sequence coding for a portion of DNA binding protein (amino acids 3-58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine 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, Sask., S7N 5E3, Saskatoon, Canada.
Pyne
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C
Tikoo
S K
SK
D017502:Adenovirus E2 Proteins / Q000032:analysis* / Q000235:genetics; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D007124:Immunoenzyme Techniques; D017921:Mastadenovirus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics; D010948:Viral Plaque Assay
D017502:Adenovirus E2 Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase