旋毛虫肌幼虫期外泌体的分离和小RNA鉴定  

Isolation of exosomes from Trichinella spiralis muscle larvae and identification of small RNAs

作　　者：高欣 杨勇 刘蕾 刘晓雷 刘明远 白雪 GAO Xin;YANG Yong;LIU Lei;LIU Xiao-lei;LIU Ming-yuan;BAI Xue(Institute of Zoonosis,Key Laboratory of Zoonosis Research,Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学人兽共患病研究所/人兽共患病研究教育部重点实验室/动物医学学院,长春130062

出　　处：《中国人兽共患病学报》2020年第4期261-266,共6页Chinese Journal of Zoonoses

基　　金：国家重点研发项目(No.2017YFC1601206,No.2017YFD0501300);吉林省优秀青年人才基金项目(No.20180520042JH);中央高校基本科研业务费专项资金(No.45119031D082)。

摘　　要：目的分离纯化旋毛虫肌幼虫分泌的外泌体,并进行鉴定,为研究旋毛虫免疫逃逸机制提供新的线索。方法采用超速离心法提取旋毛虫肌幼虫期外泌体,通过透射电子显微镜、纳米颗粒跟踪分析、免疫印记和小RNA测序对其进行鉴定。结果旋毛虫肌幼虫期外泌体为有膜的泡状物,直径约80~200 nm,表达外泌体特异性标记蛋白CD63和Enolase,含1266个已知的miRNA。结论经形态、粒径及标记蛋白分析证实成功分离旋毛虫肌幼虫期外泌体,同时构建旋毛虫肌幼虫期外泌体的小RNA文库,鉴定出多种功能性小RNA,为深入研究旋毛虫肌幼虫期外泌体内小RNA分子的应用提供数据。Trichinella spiralis(T.spiralis)is an important food-born parasite that can regulate the host immune response to achieve long-term parasitic.Recently,it has been found that exosomes derived from parasites can not only transmit virulence factors between parasites,but also regulate the immune response of hosts.We purified and identified T.spiralis muscle larvae exosomes,providing a new direction for the research on the immune regulation ability of T.spiralis.In this experiment,exosomes of T.spiralis muscle larvae were extracted by ultracentrifugation and were identified by transmission electron microscope,nanoparticle tracking analysis,western blotting and small RNA sequencing.T.spiralis muscle larvae exosomes were membrane-structure vesicles with a diameter of 80-200 nm and expressed exosomes-specific markers CD63 and Enolase,containing 1266 known miRNAs.Our study indicated that the exosomes from Trichinella spiralis muscle larvae were successfully extracted and were comfirmed by the morphology,particle size and markers analyze.Meanwhile,small RNA libraries of exosome were constructure to indentify a variety of functional small RNAs.These findings provide data for further study the application of small RNAs in exosomes.

关 键 词：旋毛虫 肌幼虫期 外泌体 MIRNA 

分 类 号：R383.15[医药卫生—医学寄生虫学]