GST pwn技术验证CIPK7蛋白激酶与CBL1蛋白的相互作用.doc

GST pull-down技术验证CIPK7蛋白激酶与CBL1蛋白的相互作用 第49卷第6期 兰州大学学报(自然科学版) Vol.49 No.6 2013年12月 Journal of Lanzhou University Natural Sciences Dec.2013 文章编号:0455-2059201306-0828-05 GST pull-down技术验证CIPK7蛋白激酶与 CBL1蛋白的相互作用 1,2 3 1,2 3 黄聪琳 , 丁 硕 , 姜 华 , 安黎哲 1.甘肃省中医院,兰州 730050 2.甘肃省中医药研究院,兰州 730050 3.兰州大学生命科学学院,兰州 730000 摘 要: 采用DNA重组技术,构建pGEX-4T-3-CIPK7和pET28-CBL1蛋白表达载体,转化大肠杆菌并诱导 表达目的蛋白,利用亲和层析纯化法纯化GST标签、His标签融合蛋白,通过GST pull-down试验验证CIPK7 与CBL1之间的相互作用. 成功构建了CBL1和CIPK7的重组质粒,经诱导表达及纯化获得了可溶性GST- CIPK7和CBL1-His融合蛋白, GST pull-down试验证实了CBL1能够与CIPK7结合. CIPK7蛋白与CBL1蛋 白之间存在直接的相互作用,为进一步研究蛋白激酶CIPK7的功能奠定了基础. 关键词: CBL1蛋白; GST pull-down; CIPK7蛋白;蛋白质相互作用 中图分类号: Q71 文献标识码: A Identi?cation of the interaction between CIPK7 kinase and CBL1 protein by GST pull-down assay 1,2 3 1,2 3 HUANG Cong-lin , DING Shuo , JIANG Hua , AN Li-zhe 1. Gansu Provincial Hospital of Traditional Chinese Medicine, Lanzhou 730050, China 2. Gansu Provincial Academy of Chinese Medicine, Lanzhou 730050, China 3. School of Life Sciences, Lanzhou University, Lanzhou 730000, China Abstract: With the DNA recombinant technique, the plasmids pGEX-4T-3-CIPK7 and pET28-CBL1 were constructed. Then the plasmids were transformed into E. coli and induced to express the target proteins. GST and His tagged fusion proteins were puri?ed by a?nity chromatography. The binding of CIPK7 and CBL1 was improved via GST pull-down assay. The expression vectors of CIPK7 and CBL1 were successfully constructed, and the soluble GST-CIPK7 fusion protein and CBL1-His tagged protein were obtained via expression and puri?cation. Thebindingofthetwoproteinswascon?rmedbyGSTpull-downassay. Thisworkprovedthatthere is a direct interaction between CIPK7 and CBL1, and provides some promising clue for the further exploration into the function of CIPK7 kinase. Key words: CBL1; GST pull-down; CIPK7; protein-protein interaction 次生代谢产物通常是药用植物的主要活性成 中药次生代谢产物积累的影响引起国内外学者的 [1] 分,是药材品质的物质基础植物的次生代谢产 不断关注和研究.干旱胁迫对提高银杏叶中槲皮 [5] 物是植物在长期进化过程中与环境相互作用的结 素含量有一定促进