外源PDI基因导入CHO细胞及其对HBsAg分泌的影响

在前期的研究工作中发现,当培养基中添加1.5%DMSO(二甲基亚砜)会使CHO细胞的HBsAg(乙肝表面抗原)产量提高80%以上。与此同时,DMSO引起了HBsAg在细胞胞内的大量积累,其含量提高了8倍。同时,DMSO引起了CHO细胞中PDI(二硫键异构酶)含量降低。由此怀疑是由于PDI不足造成了HBsAg的胞内积累。根据NCBI上查阅到的中华仓鼠小肠中PDI基因序列,通过RT-PCR的方法,从CHO细胞中获得了PDI序列,并构建重组质粒pGFP-PDI。通过阳离子脂质体转染技术,导入外源PDI,使各单克隆CHO细胞的PDI酶活提高30%～90%。但分析这些克隆在1.5%DMSO下HBsAg的胞内积累情况,发现没有改善,从而排除了PDI不足而引起外源蛋白积累的可能。

Study on Exogenous PDI Gene Transferred into CHO Cells to Assist in the Secretion of HBsAg

The productivity of hepatitis B surface antigen (HBsAg) in recombinant Chinese hamster ovary(CHO) cells was improved by over 80% at culture medium with 1.5% dimethyl sulfoxide (DMSO) compared to the control culture. At the same time,DMSO caused the decrease of protein disulfide isomerase (PDI) in CHO,which may be the reason for HbsAg intracellular accumulation. According to the determined gene sequence provided in NCBI,the PDI gene of CHO cells was amplified by RT-PCR,and then inserted into the vector. The recombinant plasmid pGFP-PDI was then transferred into cultured CHO cells with cationic polymer transfection reagent. The PDI activity of each monoclone was measured. The result showed PDI activity was increased by 30% to 90% compared to the control. But the overexpression of PDI did not result in the increase of HBsAg secretion. In conclusion, intracellular HBsAg accumulation of CHO cells by DMSO was unconcerned with the down-regulation of PDI.