鸽毛滴虫外泌体的分离鉴定及蛋白质谱分析  

Extraction and Protein Profiling of Exosomes Derived from Trichomonas gallinae in Pigeon

作　　者：倪爱心 李云雷[1] 葛平壮 王攀林 Adamu Mani Isa 石雷[1] 范静 孙研研[1] 孙鸿 麻慧 陈继兰[1] NI Aixin;LI Yunlei;GE Pingzhuang;WANG Panlin;Adamu Mani Isa;SHI Lei;FAN Jing;SUN Yanyan;SUN Hong;MA Hui;CHEN Jilan(Key Laboratory of Animal (Poultry) Genetics Breeding and Reproduction of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;Beijing Youdi Pigeon Company, Beijing 101300, China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,农业部动物遗传育种与繁殖(家禽)重点实验室,北京100193 [2]北京优帝鸽业有限公司,北京101300

出　　处：《畜牧兽医学报》2020年第7期1737-1747,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：北京市家禽产业创新团队(BAIC04-2019);中央级公益性科研院所基本科研业务费专项资金(2019-YWF-YB-12);国家自然科学基金青年基金项目(31802058);中国农业科学院科技创新工程(ASTIP-IAS04)。

摘　　要：旨在研究鸽毛滴虫是否分泌外泌体及虫源外泌体的蛋白组成和功能,为探索外泌体在鸽毛滴虫寄生过程中的作用奠定基础。采集鸽毛滴虫感染个体口腔及咽部病灶处样本,进行分离培养及纯化,使用显微镜鉴定虫体形态,提取DNA并比对其序列,确定培养物是否为鸽毛滴虫虫株;通过对培养液上清进行超速离心,透射电子显微镜、Western blot技术和纳米颗粒追踪分析(nanoparticle tracking analysis,NTA)鉴定培养液上清中是否含有虫源外泌体;最后通过Label-free技术分析虫源外泌体的蛋白质谱表达。结果显示,虫体具有明显的毛滴虫形态及特征,与鸽毛滴虫ITS1/5.8S/ITS2基因比对率达98%。从虫体培养液中提取的囊泡经透射电子显微镜观察具有明显的茶托样结构;NTA结果显示,粒径集中于125.1 nm,峰值粒径为132.3 nm,占比99.3%;CD63和TSG101等外泌体标记蛋白的条带明显,表明提取物为外泌体。质谱结果显示,烯醇酶、3-磷酸甘油醛-脱氢酶、磷酸甘油酸变位酶和延伸因子为表达量较高的蛋白质。GO功能注释显示外泌体蛋白富集到细胞内和胞浆等细胞组分,发挥GTP连接和GTP酶活性功能,参与小GTPase介导的信号转导与糖酵解等过程。KEGG通路富集表明,虫源外泌体蛋白主要富集于代谢途径、糖酵解/糖异生、抗生素的生物合成和次级代谢产物的生物合成等通路。本研究发现鸽毛滴虫可以分泌外泌体,并对虫源外泌体进行蛋白质谱分析,提示虫源外泌体中的蛋白质在寄生虫的能量代谢、信号转导及生物合成等过程中发挥重要作用。The aim of this study was to determine whether Trichomonas gallinae(T.gallinae)secrete exosomes and investigate the protein composition of exosomes derived from T.gallinae.T.gallinae was isolated from upper digestive tract of infected pigeons and identified by microscopy and sequencing.Exosomes were extracted from serum absent culture medium,and further verified by transmission electron microscopy,Western blot and nanoparticle tracking analysis(NTA).Label-free was used to identify the proteins of exosomes.Results showed that the parasite had T.gallinae morphology,and sequence alignment of ITS1/5.8S/ITS2 gene showed an identity of 98%.The isolated vesicles presented a typical cup-shaped morphology;NTA results showed that particle size concentrated on 125.1 nm,percentage of peak diameter at 132.3 nm was 99.3%;proteins such as CD63 and TSG101 were conspicuously detected,suggesting that the extracts were exosomes.Mass spectrometry results showed that enolase,glyceraldehyde-3-phosphate dehydrogenase,phosphoglycerate mutase and elongation factor were the highly expressed proteins.GO pathways showed that proteins of exosomes mainly enriched into cellular components such as intracellular and cytoplasmic,played GTP binding and GTPase activity function,and participated in biological process of small GTPase mediated signal transduction and glycolytic process.KEGG enrichment analysis showed that proteins of exosomes were enriched in pathways as metabolic pathways,glycolysis/gluconeogenesis,biosynthesis of antibiotics and secondary metabolites.These results indicated that T.gallinae can secrete exosomes and proteins of exosomes might play roles in energy metabolism,signal transduction and biosynthesis.

关 键 词：鸽 毛滴虫 外泌体 Label-free技术 蛋白质谱分析 

分 类 号：S852.722[农业科学—基础兽医学] Q959.113.9[农业科学—兽医学]