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Abstract
Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA), and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV) [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus.
Highlights
Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV) for downstream use in virus characterization
We describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA), and for use in production of a rabbit polyclonal antibody
These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV) [1]
Summary
The entire RacPyV VP1 gene sequenced from tumor tissue (Rac 2) plus a terminal sequence encoding six histidines was cloned into the baculovirus expression vector pFastBac. Recombinant Baculovirus was generated using the Bac-to-Bac system (Life Technologies/Fisher Scientific, Illkirch, France). Tni (Trichoplusia ni) insect cells (Expression Systems, LLC, Davis, CA) were infected with recombinant Baculovirus at an MOI of 3. Insect cells were pelleted, lysed on ice in 1 Â cobalt buffer (0.3 M NaCl, 50 mM Na2HPO4 in milliQ water at pH 7.4) plus protease inhibitor (cOmpleteTM EDTA free protease inhibitor cocktail tablets, Roche). VP1 protein was purified by incubation overnight at 4 °C with HisPur cobalt resin (Thermo Scientific, Rockford, IL, USA) followed by washes with increasing concentrations of imidazole (10 mM, 20 mM, and 40 mM imidazole in PBS) and elution with 150 mM imidazole elution buffer. Purified virus like particles (VLPs) were coated onto 96 well Maxi-sorp plates for ELISA and serosurvey of collected raccoon sera [1]
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