Purification of turkey coronavirus by Sephacryl size-exclusion chromatography

Abstract

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.

Fig. 1

Elution profile of size-exclusion chromatography with a Sephacryl S-1000 column (2.5×95 cm²) of turkey coronavirus Indiana isolate. The column was eluted at a flow rate of 0.5 ml/min with 0.02 M phosphate buffer (pH 7.2) containing 0.15 M NaCl. The fraction size was 5 ml. The absorbance of OD 280 of each fraction was determined. Turkey coronavirus particles were seen in only fractions of the first small peak.

Fig. 2

SDS-PAGE analysis of protein contents of purified materials. Purified preparations were electrophoresed in 10% gel under reducing conditions. Lanes 1, Mr markers; lane 2, supernatants from centrifugation of infected turkey embryo intestinal homogenates at 3000×g for 10 min; lane 3, opalescent band from ultracentrifugation of 100 000×g through a 5 ml cushion of 60% (w/v) sucrose solution for 1 h; lane 4, opalescent band from ultracentrifugation of 100 000×g through a continuous 40–60% (w/v) sucrose gradient for 16 h; lane 5, opalescent band from two consecutive ultracentrifugation of 100 000×g through a continuous 40–60% (w/v) sucrose gradient for 16 h; lane 6, the first small peak of the Sephacryl S-1000 size-exclusion chromatography; lane 7, the second large peak of the Sephacryl S-1000 size-exclusion chromatography.

Fig. 3

Immunoblotting analysis of protein contents of purified materials with turkey anti-TCoV antiserum. Purified preparations were electrophoresed, transferred onto nitrocellulose membrane, and reacted with anti-TCV antiserum. Lanes 1, Mr markers; lane 2, supernatants from centrifugation of infected turkey embryo intestinal homogenates at 3000×g for 10 min; lane 3, opalescent band from ultracentrifugation of 100 000×g through a 5 ml cushion of 60% (w/v) sucrose solution for 1 h; lane 4, opalescent band from ultracentrifugation of 100 000×g through a continuous 40–60% (w/v) sucrose gradient for 16 h; lane 5, opalescent band from two consecutive ultracentrifugation of 100 000×g through a continuous 40–60% (w/v) sucrose gradient for 16 h; lane 6, the first small peak of the Sephacryl S-1000 size-exclusion chromatography; lane 7, the second large peak of the Sephacryl S-1000 size-exclusion chromatography.

Fig. 4

Comparison of ELISA plates coated with turkey coronavirus (TCoV) preparations purified by sucrose density gradient or Sephacryl S-1000 size-exclusion chromatography for differentiation between turkey anti-TCoV antiserum (PC) and normal turkey serum (NC). The PC/NC is the ratio of anti-TCoV antiserum absorbance value to normal serum absorbance value.