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

发布时间：2018-05-29 20:55

本文选题：MARC-细胞 + 外泌体　；参考：《畜牧兽医学报》2017年07期

【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30~100nm,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。
[Abstract]:The aim of this study was to find an effective method to isolate exocrine from MARC-145 cells infected with PRRSV and to lay a foundation for exploring the role of exocrine in PRRSV infected cells. The exocrine produced by MARC-145 cells was extracted by the kit, the exocrine produced by MARC-145 cells was identified by electron microscopy negative staining and Western blot technique, and the exocrine of MARC-145 cells was isolated by centrifugation with iodoxanol rate zone. The distribution of exocrine was detected by Western blot technique. After infecting MARC-145 cells with PRRSV, the exocrine produced by MARC-145 cells was isolated with kit, and further purified by immunomagnetic bead capture technique. Western blot showed that the exocrine produced by MARC-145 cells had specific marker proteins CD63Tsg101 and Alix, and there was no exocrine unlabeled protein EEA1GRP94 or Cytochrome C.The negative staining of electron microscope showed that exocrine size produced by MARC-145 cells was negative. The size of an inch is at 30 ~ 100 nm, which is in line with the characteristics of exocrine body. The samples collected in the rate zone centrifugation of iodoxanol were analyzed by Western blot. The results showed that the exosomes produced by MARC-145 cells were distributed in the density interval of 18% iodoxanol. The results of Western blot showed that there was no exososomal marker protein Tsg-101 in the control group obtained by the method of immunomagnetic bead capture. These results suggest that MARC-145 cells can produce exocrine bodies and can be isolated from MARC-145 cells infected with PRRSV by immunomagnetic bead capture method.
【作者单位】：山西农业大学动物科技学院;
【基金】：山西省科技攻关项目(20100312016)
【分类号】：S852.651

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