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目的确定BLAP75蛋白与BLM蛋白及TopoⅢα蛋白的相互作用及作用位点。方法构建一系列携带MBP标记的不同BLAP75载体,通过体外纯化蛋白结合实验检测BLAP75与BLM、TopoⅢα的相互作用位点,并在真核细胞中通过免疫共沉淀方法进行验证;运用RNA干扰(RNAi)技术特异性抑制BLAP75的表达,并利用West-ern印迹法检测其对BLM和TopoⅢα蛋白表达的影响。结果 BLAP75与BLM和TopoⅢα蛋白的结合位点位于BLAP75的N端区域;BLAP75与BLM的直接相互作用需要DNA的介导;当BLAP75蛋白表达量减少时,BLM和TopoⅢα蛋白量也随之减少。结论细胞内BLM和TopoⅢα蛋白均结合在BLAP75的氨基端部分;BLAP75对TopoⅢα和BLM的蛋白水平具有重要影响。
Objective To determine the interaction and interaction between BLAP75 protein and BLM protein and Topo Ⅲ α protein. Methods A series of different BLAP75 vectors carrying MBP markers were constructed. The interaction sites between BLAP75 and BLM and Topo Ⅲ α were detected by in vitro purification of protein binding assay and verified by coimmunoprecipitation in eukaryotic cells. RNA interference (RNAi) Technically specific inhibition of BLAP75 expression, and West-ern blot assay BLM and Topo Ⅲ α protein expression. Results The binding sites of BLAP75 and BLM and TopoⅢα protein were located in the N-terminal region of BLAP75. The direct interaction between BLAP75 and BLM required DNA mediation. When BLAP75 protein expression decreased, the amount of BLM and TopoⅢα protein also decreased. Conclusion Both intracellular BLM and Topo Ⅲ α proteins bind to the amino terminal part of BLAP75. BLAP75 has an important effect on the protein levels of Topo Ⅲ α and BLM.