定点整合人Bcl-2基因的CHO/dhfr-细胞株的建立

构建重组载体质粒pMCEfrt—Bcl-2，利用FIp—In^TM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DNA Ladder检测在高NH4C1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。

Construction of Anti-Apoptosis CHO/dhfr- Cell Line by Overexpressing Integrted Bcl-2 Gene

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.