上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (5): 580-.doi: 10.3969/j.issn.1674-8115.2012.05.010

• 论著（基础研究） • 上一篇    下一篇

GST-p43/AIMP1融合蛋白表达和纯化以及与NF-L的相互作用

张珍珍1, 尹延青2, 周嘉伟2, 乐卫东1   

  1. 1.上海交通大学 医学院附属瑞金医院神经科, 上海 200025； 2.中国科学院上海生命科学研究院 |神经科学研究所, 上海 200031
  • 出版日期:2012-05-28 发布日期:2012-06-01
  • 通讯作者: 乐卫东, 电子信箱: wdle@sibs.ac.cn。
  • 作者简介:张珍珍（1984—）, 女, 硕士生；电子信箱: zhen-0720@163.com。

Expression and purification of GST-p43/AIMP1 fusion protein and its interaction with NF-L

ZHANG Zhen-zhen1, YIN Yan-qing2, ZHOU Jia-wei2, LE Wei-dong1   

  1. 1.Department of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China；2.Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
  • Online:2012-05-28 Published:2012-06-01

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摘要：

目的 构建人p43/AIMP1蛋白与谷胱甘肽转移酶（GST）重组的融合蛋白原核表达载体并进行表达纯化，通过GST pull-down方法验证其与神经中间丝轻链（NF-L）的体外直接相互作用。方法 以重组质粒pcDNA3.1-p43为模板，扩增p43/AIMP1基因，产物经纯化回收后与原核表达载体pGEX4T-1连接构建成新载体GST-p43/AIMP1，经鉴定完全正确后转化大肠埃希菌BL21(DE3)，通过异丙基-β-D-硫代半乳糖苷（IPTG）进行诱导表达，并纯化获得目的蛋白；将myc-NF-L体外转染HEK293T细胞，利用GST pull-down原理和方法验证人p43蛋白与神经中间丝轻链蛋白NF-L之间的相互作用。结果 酶切鉴定和测序结果显示，成功构建了GST-p43/AIMP1融合蛋白原核表达载体；考马斯亮蓝染色和Western blotting结果显示，成功获得有生物活性的GST-p43/AIMP1融合蛋白；GST pull-down实验结果证实，p43/AIMP1与NF-L存在直接相互作用。结论 获得有生物活性的GST-p43/AIMP1蛋白，并成功应用GST pull-down方法证实p43/AIMP1与NF-L在体外存在直接的相互作用。

关键词: p43/AIMP1, 蛋白表达纯化, GST pull-down, 神经中间丝轻链蛋白

Abstract:

Objective To construct and purify the prokaryotic expression vector of fusion protein of human p43/AIMP1 protein and glutathione-S-transferase (GST), and verify its direct interaction with neurofilament light subunit (NF-L) in vitro through GST pull-down assay. Methods p43/AIMP1 gene was amplified from pcDNA3.1-p43, and was inserted into prokaryotic expression vector pGEX4T-1 to generate novel vector GST-p43/AIMP1. After identification of GST-p43/AIMP1, Escherichia coli BL21 (DE3) was transfected, which was induced by isopropyl-β-D-thiogalactoside (IPTG), and target protein was obtained after purification. HEK293T cells were transfected in vitro with myc-NF-L, and the interaction between GST-p43/AIMP1 and myc-NF-L was detected using GST pull-down assay. Results Restriction enzyme digestion and sequencing indicated that prokaryotic expression vector of GST-p43/AIMP1 fusion protein was successfully constructed. Coomassie brilliant blue staining and Western blotting revealed that GST-p43/AIMP1 fusion protein with bioactivity was successfully obtained. GST pull-down assay verified that there was direct interaction between p43/AIMP1 and NF-L. Conclusion The fusion protein of GST-p43/AIMP1 with bioactivity has been successfully obtained, and the direct interaction between p43/AIMP1 and NF-L has been verified in vitro through GST pull-down assay.

Key words: p43/AIMP1, protein expression and purification, GST pull-down, neurofilament light subunit