摘要
目的探讨核糖核酸酶抑制因子(ribonuclease inhibitor,RI)与血管生成素(angiogenin,ANG)的相互作用。方法利用免疫荧光标记结合共聚焦显微镜技术来确立RI与ANG蛋白在细胞内的共定位。利用激光共聚焦显微镜的荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术验证RI和ANG蛋白的相互作用。构建原核表达质粒pGEX-4T-RI,转化E.coli BL21大肠杆菌(DE3)表达,并经谷胱甘肽琼脂糖凝胶4B(glutathione sepharose4B)纯化GST-RI蛋白,考马斯亮蓝染色和免疫印迹鉴定表达情况,利用GST蛋白沉降技术检测RI与ANG蛋白在体外的结合作用。应用免疫共沉淀(co-immunoprecipitation,Co-IP)实验检测两蛋白在真核细胞内的结合作用。结果细胞内免疫荧光标记的RI和ANG蛋白在激光共聚焦显微镜观察下存在共定位现象。FRET结果显示RI与ANG在细胞内存在相互作用。另外酶切和测序结果证明原核表达质粒pGEX-4T-RI构建成功,GST-RI蛋白诱导表达正确,GST蛋白沉降结果显示RI和ANG蛋白在原核细胞反应体系中存在相互作用。结论 RI和ANG蛋白在原核细胞反应体系和真核细胞中均存在相互结合作用。
Objective To investigate the protein-protein interaction between ribonuclease inhibitor（RI） and angiogenin（ANG）.Methods Immunofluorescence assay and laser scanning confocal microscopy were used to observe the co-location between RI and ANG in human bladder cancer T24 cells.The molecular interaction between RI and ANG was detected by fluorescence resonance energy transfer（FRET）.The prokaryotic expressing plasmid pGEX-4T-RI was constructed and then transferred into E.coli BL21（DE3） to express glutathione-S-transferase-ribonuclease inhibitor（GST-RI） fusion protein.The fusion protein GST-RI was purified using glutathione Sepharose 4B.The interaction between RI and ANG in 293 cells was identified by GST pull down assay and co-immunoprecipitation.Results We found that RI was co-localized with ANG in the T24 cells.FRET study showed that RI interacted with ANG in the 293 cells.The prokaryotic expressing plasmid pGEX-4T-RI was verified by restriction enzyme digestion and DNA sequencing.Prokaryotically expressed products were purified and their sequences were confirmed.GST pull down and co-immunoprecipitation results showed that the interaction between RI and ANG occurred in both prokaryotic cells and mammalian cells.Conclusion The interaction between RI and ANG occurs in prokaryotic cells and eukaryotic cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第14期1454-1459,共6页
Journal of Third Military Medical University
基金
国家自然科学基金(81372203,81172424)~~
