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Continuous-Elution Electrophoresis for Purification of the Baculovirus-Expressed Coronavirus Structural Proteins, Rev A

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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