抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究  

Improvement of IVCC and Increase of Productivity by Over-expression of bcl-xl Gene in Recombinant Cell Line

作　　者：何太平 唐菁燕 宋春雷 聂艳桃 雷韬 杨波 

机构地区：[1]成都蓉生药业有限责任公司抗体药物开发研究室,成都610041

出　　处：《中国细胞生物学学报》2012年第7期674-679,共6页Chinese Journal of Cell Biology

摘　　要：用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分(integral viable cell concentration,IVCC),提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl-xL的工程细胞的IVCC提高了50%,最终目的蛋白表达增加超过200%,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。Serum-free medium and chemical-defined medium are widely used for large-scale commercial production of therapeutic proteins. However, recombinant glycoproteins like coagulation factors and glycoprotein hormones are not expressed efficiently in these media. One of the most important reasons for this low productivity is due to apoptosis of cells during the cell culture process, which subsequently causes the low viable cell concentration and the short culture longevity. Over-expression of anti-apoptotic gene in the recombinant cells can improve IVCC （integral viable cell concentration）, and therefore, increase the productivity. In this study, bcl-xl gene was transferred into an established CHO-FSH cell line. CHO-FSH/Bcl-xL, a stable clone expressing Bcl-xL protein and the interested protein, was developed by screening high-producing clone and investigating its anti-apoptosis ability. Compared with the parental cell line, IVCC of the cell line over-expressing bcl-xl gene was 150%, and its productivity increased over 200%. These results demonstrated that expression of anti-apoptotic gene bcl-xl could alleviate apoptosis, enhance culture performance and increase productivity under serum-free suspension cell culture, which suggested that this strategy could be used for large-scale producing trace glycoproteins including coagulation factors and glycoprotein hormones in serum-free suspension conditions.

关 键 词：细胞凋亡 BCL-XL CHO细胞 无血清培养基 糖蛋白 表达 

分 类 号：R363[医药卫生—病理学]