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目的 从癫痫模型大鼠海马凋亡神经元中克隆caspase - 3的新底物。 方法 制作癫痫模型大鼠海马组织cDNA文库 ;PCR获得caspase- 3的P12和P17两亚基 ,然后分别定向插入pBridge质粒 ,构建三杂交诱饵载体 ;进行酵母三杂交筛库。结果 建立成功癫痫模型大鼠海马组织cDNA文库 ,构建了大鼠caspase- 3基因的酵母三杂交诱饵载体 ,并通过了caspase - 3与eIF2α之间的相互作用验证 ,筛库获得caspase- 3的新底物PIAS1。 结论 用酵母三杂交系统寻找caspase- 3下游底物具有可行性 ,从癫痫模型大鼠海马的cDNA文库中筛库获得caspase - 3的新底物PIAS1,为进一步研究caspase -3在癫痫发作引起的海马损伤中的作用奠定了基础
Objective To clone a new substrate for caspase - 3 from apoptotic neurons in hippocampus of epilepsy rats. Methods The hippocampal cDNA library of epilepsy model rats was made. The two subunits of P12 and P17 of caspase-3 were obtained by PCR, and then inserted into pBridge plasmid respectively to construct three-hybrid bait vector. Results The cDNA library of hippocampus of rats with epilepsy was successfully established, and the yeast three - hybrid bait vector containing rat caspase - 3 gene was constructed and verified by the interaction between caspase - 3 and eIF2α. The new screen of caspase - 3 Substrate PIAS1. Conclusion It is feasible to find the downstream substrate of caspase - 3 by the yeast three - hybrid system. The new substrate of caspase - 3, PIAS1, is obtained from the cDNA library of hippocampus of epileptic model rats. To further investigate the effect of caspase - 3 on seizure The role of hippocampal damage laid the foundation