为检测细胞凋亡过程中bcl-2基因的结合蛋白,用PCR技术扩增小鼠bcl-2基因(mbcl-2)调控区,经亚克隆后的序列分析证实其准确性.用5-氟尿嘧啶(5-Fu)诱导小鼠成纤维细胞(C3H 10 T1/2 Cl 8)凋亡,以此PCR产物作探针,对上述细胞的核蛋白质粗提物进行迁移位移法(EMSA)与DNA-蛋白质印迹分析.结果表明,细胞核内一分子质量为53 ku的结合蛋白与mbcl-2调控区的结合信号在5-Fu刺激12 h后明显增强,而一种100 ku的DNA结合蛋白与该区的结合信号在5-Fu刺激12 h后明显减弱.因而,p53蛋白有可能是bcl-2的负转录因子,100 ku的DNA结合蛋白有可能是bcl-2的正转录因子.
To explore the DNA binding proteins of mouse mbcl-2 regulatory region in the apoptotic process of mouse fibroblast cell line (C3H 10 T1/2 Cl 8) and its transformed counter part, the mouse bcl-2 (mbcl-2) regulatory region was amplified by PCR. The PCR products were subcloned into pGEM-T vector system and then checked by sequencing. Using the product as probe, DNA binding proteins of mbcl-2′s regulatory region in nuclear protein extracts of both cell lines were studied. Southwestern blotting results showed that a 53 ku protein binds with the probe.Its binding signal strengthened after exposing both cells to 5-Fu for 12 h, as another DNA binding protein (100 ku) also binds with the probe though its binding signal weakened after 5-Fu treatment. The results suggested that p53 protein may be the negative regulatory factor of mbcl-2 gene. An unidentified 100 ku protein may be the positive regulatory factor of mbcl-2 gene.
王文恭,童坦君.小鼠成纤维细胞凋亡与bcl-2结合蛋白[J].生物化学与生物物理进展,1999,26(6):563-566
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