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

doi:10.11843/j.issn.0366-6964.2017.07.014

Abstract

Abstract:

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 Optiprep^TM 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.

Key words: MARC-145 cells, exosomes, PRRSV particles, rate-zonal centrifugation, immune magnetic

CLC Number:

S852.659.6

ZHANG Liang, WANG Shuai-shuai, CHEN Ren-xia, TAO Jia-li, FAN Kuo-hai, DUAN Ma-kui, YI Yan-yan, JIANG Jun-bing. Separation and Identification of Exosomes from MARC-145 Cells Infected with Porcine Reproductive and Respiratory Syndrome Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(7): 1300-1305.

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Separation and Identification of Exosomes from MARC-145 Cells Infected with Porcine Reproductive and Respiratory Syndrome Virus

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