摘要
哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中 ,哺乳动物细胞生长活力差 ,且容易发生细胞凋亡 ,因而难以大规模培养。为解决此问题 ,应用双顺反子表达载体在CHO dhfr- 细胞中同时表达Igf 1 Bcl 2或Bcl 2 CyclinE基因组合 ,通过Bcl 2使细胞获得抗凋亡能力 ;通过Igf 1或CyclinE促进细胞生长分裂 ,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO dhfr- 细胞 ,应用Westernblot从G4 18抗性克隆中分别筛选到Bcl 2高表达克隆若干个 ,对其中表达Bcl 2最高的CHO IB3和CHO BC1做进一步Westernblot和流式细胞分析 ,确认此两个细胞株分别高表达Igf 1 Bcl 2和Bcl 2 CyclinE基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡 ,并以流式细胞术和DNALadder法检测细胞凋亡 ,证明CHO IB3和CHO BC1均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO dhfr- 对照细胞。在细胞培养瓶中的连续培养实验表明 ,CHO IB3和CHO BC1在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHO dhfr- 对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长 ,适于作为生物工程宿主细胞。
Serum used widely in mammalian cell culture is also a potential source of bacterial, mycoplasmal and viral contaminations. In addition, the complex biological components in serum make harder the subsequent product recovery process. High cost, high batch variation and potential source limitation are among the other shortcomings. So serum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO-dhfr- cells were genetically engineered to make them adapted to IMEM, a protein-free medium, and resistant to apoptosis. The genes in choice are insulin-like factor (Igf-1), Bcl-2 and cyclin E. Bcl-2 is a mitochondrial membrane-integrated protein. It can block the release of cytochrome c by maintaining the integrity of mitochondrial membrane, and thus inhibit apoptosis. Igf-1 is similar both in structure and function to insulin, a growth factor added to serum-free medium to promote cell growth and is the only protein component in many currently used serum-free media. cyclin E is a cell cycle protein expressed continuously in G1 phase. When cyclin E accumulates to certain amount, cell cycle was driven to S phase. So cyclin E is a proliferation-promoting protein. By co-express Igf-1/Bcl-2 or Bcl-2/cyclin E in CHO-dhfr- cells with a dicistronic expression vector, we constructed two cell lines: CHO-IB and CHO-BC. The high expression of each protein was confirmed by Western blot and flow cytometry. Apoptosis was analyzed by flow cytometry and DNA ladder detection, and the two cell lines were both found much more resistant to apoptosis induced by withdrawal of serum or addition of actinomycin D than the CHO-dhfr- parent cell. Cell proliferation assay by MTT method showed that the two cell lines proliferated much faster than CHO-dhfr- in IMDM medium without serum. Continuously culture assay proved that the two cell lines grow very well in IMEM protein-free medium supplemented with fibronectin and vitronectin to ease adherence. When compared to CHO-dhfr-, the two cell lines exhibited much more viable cell numbers and faster growth rate.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第1期66-72,共7页
Chinese Journal of Biotechnology
