Purification of HBV virions and validation of its biological activity

Abstract：In this study, we aimed to optimize the method for purifying HBV virions from serum of HBV patients. HBV virions were purified by using discontinuous sucrose density gradient (60%, 45%, 35%, 25%, 15% sucrose solutions) centrifugation at 100 000 g and 70 000 g for 5 h. After the centrifugation, six fractions were collected and real time fluorescent quantitative polymerase chain reaction was used to characterize the fractions containing HBV virions. The first 5 fractions were mixed and ultrafiltrated to remove sucrose, and then electron microscope was used to observe HBV virions. The infection of HBV virions was detected after incubating with tupaia hepatocyte. HBV was significant loss in the process of centrifugation at 100 000 g, so the harvest rate was only 19%. However, HBV virions were not significantly loss during the centrifugation at 70 000 g , which was effectively purified HBV virions. Furthermore, the harvest rate was 81%, four times higher than that in the former. A large number of HBV virions were observed in the liquid of purification at 70 000 g by electron microscope. Primary Tupaia hepatocyte cultures can be effectively infected with HBV virions, which indicated that the infectious activity of the purified HBV virions was good. The result mean that HBV virions could be effectively purified by using discontinuous sucrose density gradient centrifugation at 70 000 g, and purified HBV virions are not only displayed biological characteristics, but also have good biological infection.