An Improved Method for Purification of Soybean Mosaic Virus

Abstract

The particles of soybean mosaic virus (SMV) was successfully purified without inactivation by the following improved method. The soybean leaves infected with SMV were homogenized with two volumes of 0.3M Na-phosphate buffer (pH 7.0) containing 0.01M Na-DIECA and chloroform-butanol (each 8% v/v). The crude sap was filtered through two layers of cheesecloth and centrifuged at low speed (8, 500×g, 10min). The supernatant was supplied with 6% PEG and 0.1M NaCl. After 30min of incubation at 4C, the suspension was centrifuged at low speed, the precipitate obtained was resuspended in 0.05M Na-phosphate buffer (pH 7.0) containing 0.005M Na-EDTA, and ultrasonicated for 10 to 30 seconds. The virus fraction was collected by two cycles of differential centrifugation combined with ultrasonication just before each low speed centrifugation. The virus fraction was then subjected to the sucrose density gradient centrifugation for final step of purification. The purified virus thus obtained was highly infective. It showed the maximum ultraviolet absorption at 260nm and minimum at 247nm with a slight shoulder at 290nm. The ratio of A₂₈₀/A₂₆₀ was 0.76, indicating the nucleotide content of about 5.5%. The yield was 4.0 to 13.5mg per 1kg of the infected leaves.