Purification of turkey coronavirus by Sephacryl size-exclusion chromatography.
Sephacryl S-1000 size-exclusion chromatography was used to purify turkey coronavirus (TCoV) from infected turkey embryo. TCoV was propagated in the 22-day-old turkey embryos. Intestines and intestinal contents of infected embryos were harvested and homogenized. After low speed centrifugation, the supernatant was concentrated by ultracentrifugation through a cushion of 30 or 60% sucrose solution, or by ammonium sulfate precipitation. The purification methods included sucrose gradient and Sephacryl S-1000 size-exclusion chromatography. Ultracentrifugation through a cushion of 60% sucrose solution was better than the other two methods for concentration of TCoV from intestinal homogenate. The most effective method for purifying TCoV and removing extraneous materials was size-exclusion chromatography as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More spike-rich particles were observed in the sample purified by chromatography than those purified by sucrose gradient as examined by electron microscopy. Differentiation of turkey anti-TCoV antiserum from normal turkey serum was better achieved by ELISA plates coated with TCoV preparation purified by size-exclusion chromatography than that purified by sucrose density gradient. The results indicated that Sephacryl S-1000 chromatography was useful for purification of TCoV.
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Department of Veterinary Pathobiology and Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 47907-1175, USA.
Lin
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Wu
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Bryan
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Thacker
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Hooper
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Schrader
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D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004752:Coronavirus, Turkey / Q000254:growth & development / Q000302:isolation & purification*; D004625:Embryo, Nonmammalian / Q000821:virology; D004753:Enteritis, Transmissible, of Turkeys / Q000821:virology; D006386:Hemagglutination Tests; D007422:Intestines / Q000821:virology; D014422:Turkeys; D014461:Ultracentrifugation / Q000379:methods
D000956:Antigens, Viral