国家重点基础研究发展规划项目(973)(2003CB515508).
This work was supported by a grant from The Special Funds for Major State Basic Research of China (2003 CB515508).
建立FKBP12/Fas双聚体可诱导细胞凋亡生物效应的细胞模型,为FK506类小分子化合药物的初步筛选提供一个可应用的技术平台.首先利用RT-PCR方法钓取人源性Fas胞浆区基因,测序鉴定正确后,插入到载体pC4M-FV2E中,构建pC4M-FV2E/Fas表达质粒.然后将Myr-FKBP12-Fas融合基因从pC4M-FV2E/Fas质粒克隆进pEGFP-N1中.最后将重组质粒pEGFP-N1/FKBP12-Fas转染进HEK293细胞,并利用G418筛选融合基因FKBP12-Fas稳定表达的细胞株.实验结果显示,阳性化合物AP20187作用4~6 h后,稳定转染的靶细胞发生凋亡,基因组呈现典型的“DNA Ladder”,而FKBP12的天然结合蛋白FK506可竞争性地阻断这种阳性药物诱导的细胞凋亡.研究提示:所构建的细胞模型,可用于以FKBP12为靶标、基于FK506空间构型设计合成的FK506类小分子化合药物的初步筛选.
The gene encoding the cytoplasmic domain of human Fas from 175aa to 304aa was amplified from U937,which was then determined by sequence analysis and cloned into pC4M-FV2E. The resulting Myr-FKBP12-Fas fusion gene was subcloned into pEGFP-N1.Stable cell clones as named 293/ FKBP12-Fas were obtained by transfecting the recombinant plasmid pEGFP-N1/FKBP12-Fas into HEK293,which was following selected by G418. The integration and expression of FKBP12-Fas fusion gene were identified by RT-PCR and Western-blotting respectively. Both the observation of the condensation and margination of the chomatin on nuclear membrane under electron microscopy and the detection of typical “DNA Ladder” proved the apoptosis induced by AP20187. However, FK506 could competitively block such cell apoptosis, indicating that the inducible apoptosis cell model established as above offered an applicable technical platform for the screening of FK506-like small molecular compounds.
肖鹤,于鸣,李松,沈倍奋,黎燕.用于FK506类小分子化合物筛选的细胞模型的建立[J].生物化学与生物物理进展,2004,31(10):895-901
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