GALNT14与MT2A相互作用验证  

Validation of the interaction of GALNT14 and MT2A

作　　者：左涛[1] 刘学敏[1] 单金帅 汤静珍 吴琛[1] 

机构地区：[1]河北大学生命科学学院,河北保定071002

出　　处：《河北大学学报（自然科学版）》2016年第1期65-71,共7页Journal of Hebei University(Natural Science Edition)

基　　金：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)

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c DNA片段克隆到p ET-Dsb A和p CMV-Myc载体上.表达并纯化了His-Dsba-MT2A和GST-GALNT14蛋白,在体外通过GST pulldown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p CMVMyc-MT2A和p CDNA3.1-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.Co-immunoprecitipation assay and GST pull-down assay were applied to validate the interac- tion of GALNT14 and MT2A in vivo and in vitro . The cDNA fragment of MT2A was cloned into pET-Ds- bA and pCMV-Myc. His-Dsba-MT2A and GST-GALNT14 were expressed and purified,and the results of GST pull-clown assay showed that MT2A could bound with GALNT14 ,which confirmed the directly inter- action between them in vitro . Then pCMV-Myc-MT2A and pCDNA3.1-Flag-GALNT14 were co-transfect- ed into human breast cancer cell MCF-7. The results of Co-IP illustrated that anti-Myc antibody can precip- itated 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.

关 键 词：N-乙酰氨基半乳糖转移酶14(GALNT14) 金属硫蛋白2A(MT2A) 蛋白沉降实验(GSTpull-down) 免疫共沉淀(Co-IP) 

分 类 号：Q78[生物学—分子生物学]