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K. Sestak, L. Kim, K.-O. Chang and L. J. Saif, Food Animal Health
Research Program, Ohio Agricultural Research and Development Program,
The Ohio State University, Wooster, OH 44691
Summary
Three major structural proteins of the porcine coronavirus, transmissible
gastroenteritis virus (TGEV), membrane (M), nucleocapsid (N), and spike
(S), were expressed in a baculovirus expression system. A method for rapid
purification of the virus-specific proteins from cellular components was
developed by using continuous-elution polyacrylamide gel electrophoresis
(CE-PAGE). This approach might be a valuable preparative tool prior to
immunization studies, monoclonal antibody production, subunit vaccine
preparation, quantification of protein expression and for other experimental
scenarios where purified viral proteins are required.
Introduction
TGEV causes diarrhea and vomiting in pigs of all ages and produces 90100%
mortality in 12 week old sero-negative pigs (Bohl et al., 1972).
The economic impact of TGEV on the U.S. pork industry was estimated as
$200 million per year (Saif and Wesley, 1992). A possible vaccine includes
the three purified recombinant TGEV structural proteins embedded in immuno-stimulating
complexes (ISCOMs), microcapsules or other carriers.
Materials and Methods
The Sf9 insect cells containing the TGEV proteins were harvested from
150 cm2 stationary cell culture flasks (1.8 x 107 cells) and resuspended
in 1 ml of phosphate buffer saline (PBS), pH 7.3, mixed with 4 parts of
sample buffer (0.06 M Tris, pH 6.8; 10% glycerol; 2% (w/v) SDS; 5% mercaptoethanol
and 0.05% (w/v) bromophenol
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