GST pull-down联合质谱分析技术筛选鸡NOS2的互作蛋白  

Proteins Interacting with Chicken NOS2 were Screened by GST pull-down Combined with Mass Spectrometry

作　　者：周鑫琦 王颖洁[1,2] 张文慧 张亚妮[1,2] ZHOU Xinqi;WANG Yingjie;ZHANG Wenhui;ZHANG Yani(College of Animal Science and Technology,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Key Laboratory of Animal Breeding Reproduction and Molecular Design,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学动物科学技术学院,江苏扬州225009 [2]江苏省动物遗传繁育与分子设计重点实验室,江苏扬州225009

出　　处：《中国家禽》2020年第9期19-25,共7页China Poultry

基　　金：江苏省大学生创新创业训练计划项目(201811117064Y);江苏高校优势学科建设工程资助项目。

摘　　要：为了筛选NOS2在鸡雄性生殖细胞形成过程中的互作蛋白,通过克隆NOS2序列,构建pGEX-6p-NOS2原核表达载体且诱导表达NOS2蛋白,采用GST pull-down联合质谱分析技术筛选与鸡NOS2相互作用的蛋白质。结果显示:经GST pull-down质谱分析鉴定到316个与鸡NOS2相互作用的候选蛋白质,其中12个高评分蛋白具有相同的功能注释,参与精子运动调控,且主要分布在细胞质、细胞核等细胞组分中,可能通过与其它蛋白质、核酸等分子结合而参与MAPK、Notch等信号通路;质谱分析还发现GOT1蛋白与NOS2互作最为明显,该蛋白参与Notch信号通路,后续将作为重点候选互作蛋白以探究其与NOS2蛋白的互作方式。该结果为进一步探究NOS2在鸡胚胎干细胞向雄性生殖细胞分化中的调控机制奠定了实验基础。In order to screen the interaction proteins of NOS2 in the formation of chicken male germ cells, the prokaryotic expression vector pGEX-6 p-NOS2 was constructed by cloning the NOS2 sequence and inducing the expression of NOS2 protein.The proteins interacting with chicken NOS2 were screened by GST pull-down combined mass spectrometry.The results showed that 316 candidate proteins interacting with chicken NOS2 were identified by GST pull-down mass spectrometry, 12 of the highly rated proteins had the same functional annotations;it participates in the regulation of sperm motility and is mainly distributed in cytoplasm, nucleus and other cellular components.It may be involved in MAPK,Notch and other signaling pathways by binding to other proteins, nucleic acids and other molecules. Mass spectrometry showed that GOT1 protein had the most obvious interaction with NOS2, which was involved in Notch signaling pathway.In the future, GOT1 protein will be used as a key candidate protein to explore its interaction with NOS2 protein. The results lay an experimental foundation for further exploring the regulation mechanism of NOS2 in chicken embryonic stem cell differentiation into male germ cells.

关 键 词：NOS2 GST pull-down 质谱分析技术 蛋白质相互作用 

分 类 号：S831.2[农业科学—畜牧学]