利用免疫共沉淀联合质谱技术筛选盐藻MAPK的互作蛋白  

Detection Proteins Interacting With Dunaliella salina MAPK by Co-immunoprecipitation and Mass Spectrometry

作　　者：岳金荣 丛玉婷[1] 邢震宇 高相楠 王明芳 柴晓杰[1] YUE Jinrong;CONG Yuting;XING Zhenyu;GAO Xiangnan;WANG Mingfang;CHAI Xiaojie(Dalian Ocean University/Key Laboratory of Hydrobiology in Liaoning Province,Dalian,Liaoning 116023)

机构地区：[1]大连海洋大学/辽宁省省级高校水生生物学重点实验室,辽宁大连116023

出　　处：《核农学报》2020年第6期1187-1195,共9页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金(31472260、30972240)。

摘　　要：为进一步探究杜氏盐藻促有丝分裂原活化蛋白激酶(DsMAPK)的功能,采用免疫共沉淀联合质谱技术筛选盐藻MAPK的互作蛋白。将pGS21a-MAPK质粒转入大肠杆菌BL21中表达MAPK蛋白并制备多克隆抗体;将培养至对数生长期的盐藻细胞进行盐胁迫处理,然后提取盐藻总蛋白,并进行SDS-PAGE和Western blotting检测;以内源性靶蛋白为诱饵,将细胞总蛋白与MAPK抗体进行共孵育,将经蛋白A/G琼脂糖珠纯化的免疫共沉淀复合物进行质谱检测。结果表明,制备的多克隆抗体特异性良好;筛选出165种特有的差异蛋白。通过GO和KEGG分析发现,这些差异蛋白主要参与了新陈代谢、遗传信息的传递以及信号转导等生物学调控过程。蛋白质互作网络分析发现,直接与MAPK相互作用的蛋白有4种。本研究结果为深入研究杜氏盐藻响应盐胁迫的分子机制提供了参考。In order to explore the function of mitogen-activated protein kinase(DsMAPK) in Dunaliella salina, the interacting proteins with MAPK in D.salina were screened by immunoprecipitation-mass spectrometry. The pGS-21 a-MAPK plasmid was transfected into E. coli BL21 to express MAPK and prepare polyclonal antibodies. D. salina cells at logarithmic growth stage were treated with salt stress, then the total protein of D. salina was extracted and tested by SDS-PAGE and Western blotting. Using endogenous target protein as bait, total proteins of cell and MAPK’s antibody were co-incubated. The immunoprecipitation compound purified by protein A/G agarose beads were detected by mass spectrometry. The results showed that the prepared polyclonal antibody has good specificity and 165 specific differential proteins were screened out. Through GO and KEGG analysis, we found that these differentially expressed proteins are mainly involved in metabolism, genetic information transmission, signal transduction and other biological processes. Protein interaction network analysis revealed that 4 different proteins interact directly with MAPK. The results provide new information for further study on the molecular mechanism of D.salina responding to salt stress.

关 键 词：杜氏盐藻 MAPK 免疫共沉淀技术 质谱技术 生物信息学 

分 类 号：Q945[生物学—植物学]