摘要
构建重组载体质粒pMCEfrt-Bcl-2,利用FIp-InTM定点重组系统,在CHO-dhfr-细胞内定点整合人Bcl-2基因,通过Western印迹检测重组细胞Bcl-2蛋白的表达。通过流式细胞仪和DNALadder检测在高NH4Cl条件下细胞的凋亡情况;用台盼蓝染色检测在无血清IMDM培养基中细胞的活细胞数目和活细胞比例。结果获得了稳定表达Bcl-2基因的细胞株CHO-Bcl-2,该细胞株能高水平表达Bcl-2蛋白。在无血清培养过程中,CHO-Bcl-2细胞比对照细胞保持高约15%的活细胞比例,细胞总数高25%。CHO-Bcl-2在高NH4+(50mmol/L)培养条件下具有较低的凋亡水平。建立了能够高表达Bcl-2基因并具有一定的抗凋亡能力的重组CHO/dhfr-细胞株。
To construct a new anti-apoptosis CHO/dhfr^- cell line by overexpessing integrated human Bcl-2 gene in the genome. Human Bcl-2 gene was integrated into the genome of CHO/dhfr^- cell by FIp-In^TM recombination system. The expession of Bcl-2 protein was detected by western-blot. The level of cell apoptosis was detected by FACS and DNA ladder. Cell viability and viable cell counts were preformed with trypan blue exclusion method. Recombination cell line CHO-bcl-2 that could overespress Bcl-2 protein was constructed, the apoptosis of CHO-Bcl-2 was considerably supressed and viability of cells were improved under conditions of serum withdrawal or treatment with ammonium chloride.
出处
《生物技术通讯》
CAS
2005年第6期605-608,共4页
Letters in Biotechnology
