核糖核酸酶抑制因子与血管生成素相互作用的试验-第三军医大学学报.docVIP

_________________________________________ [基金项目] 国家自然科学基金81172424） [通信作者] 陈俊霞，电话:（023E-mail: chjunxia@126.com 核糖核酸酶抑制因子与血管生成素的相互作用研究 李林，舒静，刘玉林，陈俊霞 ( 重庆医科大学细胞生物学与遗传学教研室 重庆医科大学分子医学与肿瘤研究中心，重庆 400016) ［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（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(DE3)表达，并经谷胱甘肽琼脂糖凝胶4B（glutathione sepharose 4B）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。同时，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。免疫共沉淀实验结果也表明，RI与ANG蛋白在真核细胞反应体系内存在相互作用。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。 ［关键词］ RI蛋白；ANG蛋白；相互作用； ［中图法分类号］R392. 11; R394 - 33; Q78 ［文献标志码］ A Protein-Protein Interaction of Ribonuclease Inhibitor with Angiogenin in Mammalian Cells Li Lin, Shu Jing, Liu Yulin, Chen Junxia ( Department of Cell Biology and Genetics，Molecular Medicine ＆ Cancer Research Center，Chongqing Medical University，Chongqing， 400016，China) [Abstract] Objective To investigate the protein-protein interaction between ribonuclease inhibitor(RI) and angiogenin(ANG). Methods Immunofluorescence assay and laser scanning confocal microscopy analysis were used to observe the co-location between RI and ANG in 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 transformed into E. coli BL21(DE3) to express GST-RI fusion protein. The fusion protein GST-RI was purified by using glutathione- sepharose 4B. The interaction between RI and ANG in 293 cells was identified by glutathione-S-transferase (GST) pull-down assay and co-immunoprecipitation assay. Results We found that RI was co-localized with ANG in T24 cell. FRET study showed that RI inter