摘要
研究了高效表达HBsAg的CHO-B43细胞系经转瓶培养所获液体的纯化流程,具体步骤是:细胞培养液经7 500r/min 4℃离心30分钟,除去脱落细胞及碎片,加固体硫酸铵达50％饱和度进行沉淀,离心后弃上清,再用45％的饱和硫酸铵重新悬浮,离心,保留沉淀物;正压超滤透析后,分别选用1.24g/cm^3和1.20g/cm^3的KBr浮力密度,相继进行两次32 000r/min 17℃ 46小时的平衡密度超速离心,收集HBsAg活性部分,过Sepharose 4B分子筛层析柱,再收集HBsAg活性峰,进行最后一次1.22g/cm^2 CsC1平衡密度梯度超速离心。由此流程HBsAg最终回收率为50％～65％,所获纯品纯度达95％以上,经电镜检查,牛血清残余量(<8.430ng/Dose)和细胞DNA残余量(<32pg/Dose),均符合WHO规定的基因工程HBsAg人体接种的标准。
Pooled supernatants containing HBsAg secreted by CHO-B43 cell line from rolling bottle culture were clarified and solid ( NH4 ) 2S04 was added to a final concentration of 0.5 saturation. The precipitate was collected by centrif ugation ( 30min at 7500r/min, room temperature ) and washed once with 45% ( NH4 ) 2SO4. The pellets were dissolved in a minimum volume of normal saline, dialysed and concentrated by ultrafiltration. KBr was added to a density of 1.24 and 1.20g/cm3 respectively and two successive runs of isopynic centrifugation were performed at 32,000rpm for 48hr in a SW40 rotor ( Beckman) at 17℃. Fractions were collected from the bottom and were assayed for HBsAg. Peak fractions were pooled and dialyzed, then passed through a Sepharose 4B column ( 2.6× 100cm) . The peak fractions of HBsAg were combined and dialyzed, CsCl was added to a density of 1.20g/cm3 and a third run of isopcynic centrifugatioa were performed in CsCl. The final recovery rate is 50%-65%. HBsAg purified from cell culture supernatant of B43 consisted of homogeneous 22-nm particles under the electron microscope. The amounts of residual calf serum and residual cellular DNA were less than 30ng/dose and 31.25pg/dose respectively. The quality of pure HBsAg is over the standards set by WHO.
出处
《病毒学报》
CAS
CSCD
北大核心
1990年第4期305-311,共7页
Chinese Journal of Virology
关键词
基因工程
HBSAG
纯化
rHBsAg CHO-B43 cell line Purication Determination of residual cellular(RC) DNA
