中文摘要：

目的研究血管生成素（ANG）突变体与核糖核酸酶抑制因子（RI）的相互作用。方法构建p CMV-3×flagANGH13R和p CMV-3×flag-ANGH114R突变体质粒;用免疫荧光标记结合共聚焦显微镜技术来确立RI与ANG突变体蛋白在BIU-87细胞内的定位;用GST融合蛋白沉降技术检测RI与ANG突变体蛋白在细胞外的相互作用;用免疫共沉淀（Co-IP）实验检测两蛋白在人膀胱癌BIU-87细胞内的相互作用。结果 p CMV-3×flag-ANGH13R和p CMV-3×flag-ANGH114R突变体质粒构建成功;RI和ANG突变体蛋白存在共定位现象;GST-RI融合蛋白诱导表达正确,RI和ANG突变体蛋白在原核反应体系中存在相互作用;RI与ANG突变体蛋白在真核细胞内存在相互作用。结论 RI和ANG突变体蛋白在原核反应体系和真核细胞中都存在相互作用,并且两突变型与RI相互作用存在一定的差异。

英文摘要：

Objective To research the protein-protein interaction between ribonuclease inhibitor（ RI） and angiogenin（ ANG） mutant. Methods To construct p CMV-3 × flag-ANGH13 Rand p CMV-3 × flag-ANGH114 Rplasmid. Immunofluorescence assay and laser scanning confocal microscopy analysis were used to observe the co-localization between RI and ANG mutant in BIU-87 cells. The interaction between RI and ANG mutant in BIU-87 cells was identified by glutathione-S-transferase（ GST） pull-down assay and co-immunoprecipitation assay. Results The expressing plasmid p CMV-3 × flag-ANGH13 Rand p CMV-3 × flag-ANGH114 Rwere verified by restriction enzyme digestion and DNA sequencing. We found that RI was coloc-alized with ANG mutant in BIU-87 cell by laser scanning confocal microscopy analysis. Prokaryotically expressed products were purified and their sequences were confirmed. GlutathioneS-transferase（ GST） pull down and co-immunoprecipitation results showed that the interaction between RI and ANG mutant occurred in BIU-87 cells. Conclusions The interaction between RI and ANG occurs in BIU-87 cells. The difference between RI and ANG mutant exists in BIU-87 cells.