Protein-protein interaction between ribonuclease inhibitor and angiogenin in mammalian cells

李林; 舒静; 刘玉林; 陈俊霞

您当前的位置：

首页 >

文章列表页 >

Protein-protein interaction between ribonuclease inhibitor and angiogenin in mammalian cells

更新时间：2026-01-09

- Protein-protein interaction between ribonuclease inhibitor and angiogenin in mammalian cells
- Journal of Army Medical University Vol. 36, Issue 14, Pages: 1454-1459(2014)
- 作者机构：
- 作者简介：
- 基金信息：
- DOI：
  CLC： R341;R394-33;R394.3
- Published：2014
- 稿件说明：
移动端阅览
李林,舒静,刘玉林,陈俊霞.核糖核酸酶抑制因子与血管生成素的相互作用[J].陆军军医大学学报(原第三军医大学学报),2014,36(14):1454-1459.

　Li Lin,Shu Jing,Liu Yulin,Chen Junxia.Protein-protein interaction between ribonuclease inhibitor and angiogenin in mammalian cells[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(14):1454-1459.
李林,舒静,刘玉林,陈俊霞.核糖核酸酶抑制因子与血管生成素的相互作用[J].陆军军医大学学报(原第三军医大学学报),2014,36(14):1454-1459. DOI：

　Li Lin,Shu Jing,Liu Yulin,Chen Junxia.Protein-protein interaction between ribonuclease inhibitor and angiogenin in mammalian cells[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(14):1454-1459. DOI：

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 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。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。

Abstract

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.

关键词

Keywords

references

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Construction of eukaryotic expression vector pcDNA3.1-RI and its effect on human umbilical vein endothelial cells

Up-regulation of ribonuclease inhibitor suppresses growth and apoptosis in murine melanoma B16 cells

Effect of down-regulating human angiogenin on proliferation and apoptosis in human bladder cancer T24 cells

Confirming direct association between APE1 and p53 protein in A549 and HeLa cells

Over-expression of human ribonuclease inhibitor suppresses invasion and migration in B16 melanoma cells

Related Author

熊东梅

姚雪

潘湘阳

李红彦

陈俊霞

彭源

潘湘阳

陈俊霞

Related Institution

No data

⁰