Separation And Identification Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) And Exosomes In Marc-145 Cells

The Exosome is a lipid double membranous capsule that is secreted by eukaryocytes. Its main function is mediating the communication between cells,the formation and properties with cell specificity. Its role in virus infection is called Exosome way for virus infection. Because of the Exosome and PRRSV particle have similar buoyant density and size, they cannot be separated by the traditional centrifugal methods.This study aims to: Establish the Marc-145 cell culture model to collect and isolate the Exosomes. The Exosomes were identified by using both negative staining TEM measurement and western blot. The rate-zonal centrifugation was designed to isolate the Exosomes, its distribution range was identified by western blot. The PRRSV infection model was established for Exosome study and the results were observed by IFA; After successful isolating the Exosomes, immune magnetic bead capture technology were used to isolate the Exosomes and PRRSV particles, its characteristic were detected by Western blot.Results: Morphology observation showed that the cells displayed normal shape and satisfactory refractivity, there was no significant difference compared with control group. It suggested that the model was suitable for follow-up study. The results of negative staining TEM measurement showed the sizes of Exosomes produced by Marc-145 cells ranged from 40nm to 100nm and their shapes were round or oval which are typical characteristics of the Exosomes. The results of Western blot showed the Exosomes produced by Marc-145 cells expressed CD63、Alix、Tsg-101 and β-actin, which were the markers of Exosome, also they didn’t expressed proteins such EEA1, GRP94 and Cytochrome C, which were not Exosome markers. The rate-zonal centrifugation indicated Exosomes distributed in 3-12 zones. The results of IFA showed the model of PRRSV infection was successful. The samples obtained by using immune magnetic bead capture technology were tested by western blot, results showed that isotype control magnetic beads didn’t express Exosome marker Tsg-101 and suggested that the Exosomes were successfully purified.