HBV颗粒的纯化及生物学活性鉴定

Purification of HBV virions and validation of its biological activity

目的优化纯化HBV患者血清中HBV颗粒的技术方法。方法在蔗糖介质中,分别以100 000 g和70 000 g进行不连续蔗糖梯度(60%,45%,35%,25%,15%)离心5 h,离心后,分段吸取6份收集液,PCR荧光定量检测各组分HBV滴度;收集前5份收集液进行超滤,去蔗糖,PCR荧光定量检测HBV超滤液;电镜观察70 000g纯化后的HBV超微形态;70000 g纯化后的HBV颗粒感染树鼩肝细胞,检验70 000 g纯化后的HBV感染活性。结果100 000 g离心造成大量HBV在离心过程中损失,收获率只有21%,70 000g离心不仅降低了离心中的病毒损失,而且可有效对HBV和血清进行分离,收获率达到78%以上,较100 000g还提高3.7倍多。电镜可观察到纯化后病毒液中有大量的病毒颗粒;纯化后的HBV颗粒感染活性良好,可有效感染原代树鼩肝细胞。结论70 000 g不连续蔗糖梯度离心可有效分离纯化HBV病毒,较100 000 g收获率明显提高,纯化后的HBV不但具有典型的HBV特征,还具有良好的生物学感染活性。

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 obsen＇ed 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.