GALNT14与MT2A相互作用验证

  • 左涛 ,
  • 刘学敏 ,
  • 单金帅 ,
  • 汤静珍 ,
  • 吴琛
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左涛(1989—),男,河南鲁山人,河北大学在读硕士研究生. E-mail:zuotao1123@163.com

收稿日期: 2015-06-12

  网络出版日期: 2016-01-25

基金资助

河北省自然科学基金资助项目(C2013201112);河北省教育厅优秀青年基金资助项目(Y2012024)

Validation of the interaction of GALNT14 and MT2A

  • ZUO Tao ,
  • LIU Xuemin ,
  • SHAN Jinshuai ,
  • TANG Jingzhen ,
  • WU Chen
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  • College of Life Sciences, Hebei University, Baoding 071002, China

Received date: 2015-06-12

  Online published: 2016-01-25

摘要

利用免疫共沉淀(Co-IP)和蛋白沉降实验(GST pull-down),分别从体内外对N-乙酰氨基半乳糖转移酶(GALNT14)与金属硫蛋白2A(MT2A)之间的相互作用进行了验证.将MT2A全长cDNA片段克隆到pET-DsbA和pCMV-Myc载体上.表达并纯化了His-Dsba-MT2A和GST-GALNT14蛋白,在体外通过GST pull-down技术分析,显示MT2A能与GALNT14结合,证实了二者在体外的直接相互作用.继而,共转染pCMV-Myc-MT2A和pCDNA3.1-Flag-GALNT14到人乳腺癌细胞株MCF-7中,通过免疫共沉淀实验发现抗Myc抗体可以将GALNT14从细胞裂解液中沉淀下来,证实了它们在胞内的相互作用.结果显示了GALNT14和MT2A在体内外条件下能够发生直接相互作用,这为进一步明确GALNT14的功能及作用机制奠定了基础.

本文引用格式

左涛 , 刘学敏 , 单金帅 , 汤静珍 , 吴琛 . GALNT14与MT2A相互作用验证[J]. 河北大学学报(自然科学版), 2016 , 36(1) : 65 -71 . DOI: 10.3969/j.issn.1000-1565.2016.01.011

Abstract

Co-immunoprecitipation assay and GST pull-down assay were applied to validate the interaction of GALNT14 and MT2A in vivo and in vitro.The cDNA fragment of MT2A was cloned into pET-DsbA and pCMV-Myc.His-Dsba-MT2A and GST-GALNT14 were expressed and purified,and the results of GST pull-down assay showed that MT2A could bound with GALNT14,which confirmed the directly interaction between them in vitro.Then pCMV-Myc-MT2A and pCDNA3.1-Flag-GALNT14 were co-transfected into human breast cancer cell MCF-7.The results of Co-IP illustrated that anti-Myc antibody can precipitated GALNT14 from cell lysates,which revealed MT2A interacted with GALNT14 in vivo.The results showed that GALNT14 could bind MT2A and interact with each other directly in vivo and in vitro,which provided some promising clue for the further exploration into the function of GALNT14.

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