猪繁殖与呼吸综合征病毒感染的MARC-145细胞中外泌体的分离与鉴定

摘要

旨在寻找有效方法,能够从PRRSV感染的MARC-145细胞中分离细胞产生的外泌体,并为探索外泌体在PRRSV感染细胞时的作用奠定基础.使用试剂盒提取MARC-145细胞产生的外泌体,利用电镜负染色法和Western blot技术对MARC 145细胞产生的外泌体进行鉴定;采用碘克沙醇速率区带离心分离MARC-145细胞产生的外泌体,并用Western blot技术检测外分泌体的分布区间.用PRRSV感染MARC-145细胞后,使用试剂盒对MARC-145细胞产生的外泌体进行初步分离后,采用免疫磁珠捕获技术进行进一步纯化,并使用Western blot进行验证.Western blot结果显示MARC-145细胞产生的外泌体存在特异性标记蛋白CD63 、Tsg101和Alix,不存在外泌体非标记蛋白EEA1 、GRP94和Cytochrome C;电镜负染色结果显示MARC-145细胞产生的外泌体尺寸大小处于30～100 nm,符合外泌体特征.利用Western blot对碘克沙醇速率区带离心中收集到的样品进行分析,结果显示MARC-145细胞产生的外泌体分布于7.2％～18％碘克沙醇密度区间.Western blot结果显示免疫磁珠捕获法获得的对照组不存在外泌体标记蛋白Tsg-101.以上结果表明MARC-145细胞能够产生外泌体,并且通过免疫磁珠捕获法能够从PRRSV感染的MARC-145细胞中将其分离.%The objective of this study was to find an efficient method to isolate the exosomes from MARC-145 cells infected with PRRSV,and lay the foundations for explore the role of exosomes in PRRSV infection.Exosomes were isolated from MARC-145 cells using Total Exosomes Isolation.Exosomes were identified by using Western blot and electron microscopy.The rate-zonal centrifugation was designed to isolate the exosomes,its distribution range was identified by Western blot.Total Exosomes Isolation and immune magnetic bead capture technology were used to isolate the exosomes from MARC-145 cells infected with PRRSV,its characteristic were detected by Western blot.Results were as follows:CD63,Alix and Tsg101,three exosomal protein markers,were expressed in the purified exosomes from MARC-145 cells.EEA1,GRP94 and Cytochrome C did not expressed,which were not exosomal protein markers.The isolated exosomes were verified as small vesicles of approximately 30-100 nm in size by using the electron microscopy (TEM) assay.Using CD63 as an exosomes marker,we demonstrated that OptiprepTM velocity gradients were efficient in isolating exosomes,with exosomes distributed in 7.2％-18％ zones.The samples obtained by immunoaffinity capture were tested by Western blot,the results showed that isotype control magnetic beads did not express exosomal marker Tsg-101.These results indicated that exosomes are released into the extracellular space from MARC-145 cells,and suggested that the exosomes were successfully purified from MARC-145 cells infected with PRRSV by using immune magnetic bead capture technology.