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.

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.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2017.07.014

https://www.xmsyxb.com/EN/Y2017/V48/I7/1300

References

[1] BENFIELD D A, NELSON E, COLLINS J E, et al. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332)[J]. J Vet Diagn Invest, 1992, 4(2):127-133.
[2] STADEJEK T, STANKEVICIUS A, MURTAUGH M P, et al. Molecular evolution of PRRSV in Europe:current state of play[J]. Vet Microbiol, 2013, 165(1):21-28.
[3] CHA S H, CHOI E J, PARK J H, et al. Molecular characterization of recent Korean porcine reproductive and respiratory syndrome (PRRS) viruses and comparison to other Asian PRRS viruses[J]. Vet Microbiol, 2006, 117(2):248-257.
[4] KIM H S, KWANG J, YOON I J, et al. Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line[J]. Arch Virol, 1993, 133(3-4):477-483.
[5] JOHNSTONE R M, ADAM M, HAMMOND J R, et al. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes)[J]. J Biol Chem, 1987, 262(19):9412-9420.
[6] RAPOSO G, NILMAN H W, STOORVOGEL W, et al. B lymphocytes secrete antigen-presenting vesicles[J]. J Exp Med, 1996, 183(3):1161-1172.
[7] CHOI D S, CHOI D Y, HONG B S, et al. Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells[J]. J Extracell Vesicles, 2012, doi:10.3402/jev.v1i0.18704.
[8] ZERINGER E, LI M, BARTA T, et al. Methods for the extraction and RNA profiling of exosomes[J]. World J Methodol, 2013, 3(1):11-18.
[9] RAPOSO G, STOORVOGEL W. Extracellular vesicles:exosomes, microvesicles, and friends[J]. J Cell Biol, 2013, 200(4):373-383.
[10] MECKES D G Jr, GUNAWARDENA H P, DEKROON R M, et al. Modulation of B-cell exosome proteins by gamma herpesvirus infection[J]. Proc Natl Acad Sci U S A, 2013, 110(31):E2925-E2933.
[11] CHUGH P E, SIN S H, OZGUR S, et al. Systemically circulating viral and tumor-derived microRNAs in KSHV-associated malignancies[J]. PLoS Pathog, 2013, 9(7):e1003484.
[12] GOULD S J, BOOTH A M, HILDRETH J E K. The Trojan exosome hypothesis[J]. Proc Natl Acad Sci U S A, 2003, 100(19):10592-10597.
[13] ADMYRE C, JOHANSSON S M, QAZI K R, et al. Exosomes with immune modulatory features are present in human breast milk[J]. J Immunol, 2007, 179(3):1969-1978.
[14] RAMAKRISHNAIAH V, THUMANN C, FOFANA I, et al. Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells[J]. Proc Natl Acad Sci U S A, 2013, 110(32):13109-13113.
[15] IZQUIERDO-USEROS N, NARANJO-GÓMEZ M, ARCHER J, et al. Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway[J]. Blood, 2009, 113(12):2732-2741.
[16] MONTANER-TARBES S, BORRÁS F E, MONTOYA M, et al. Serum-derived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins[J]. Vet Res, 2016, 47(1):59.
[17] 李贺侠. 猪高热病主要病原HP PRRSV的病原学研究[D]. 扬州:扬州大学, 2008.
LI H X. Research on etiology of highly-pathogenic porcine reproductive and respiratory syndrome virus about major pathogen of swine high fever[D]. Yangzhou:Yangzhou University, 2008. (in Chinese)
[18] THÉRY C, AMIGORENA S, RAPOSO G, et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids[M]//Current Protocols in Cell Biology. New York:John Wiley & Sons, Inc, 2006.
[19] LÄSSER C, ELDH M, LÖTVALL J. Isolation and characterization of RNA-containing exosomes[J]. J Vis Exp, 2012(59):3037.
[20] LI J H, LIU K C, LIU Y, et al. Exosomes mediate the cell-to-cell transmission of IFN-α-induced antiviral activity[J]. Nat Immunol, 2013, 14(8):793-803.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	SONG Wenyan, ZHANG Hanwen, WU Aodi, ZHANG Liyan, LIU Zhao, YE Tongtong, CHEN Chuangfu, SHENG Jinliang. Screening of Nanobodies against Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein and Exploration of Their Inhibitory Effect on Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 258-270.
[2]	WANG Ying, LI Jiakang, ZENG Yue, SHI Kaituo, WEI Liting, PENG Jiajia, LI Qiuyan, CAO Longlong, ZHOU Dengyuan. Development of Monoclonal Antibodies against Feline Calicivirus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 401-405.
[3]	LI Huihui, DONG Keer, LIU Xinbo, ZHANG Chunxiao, MA Chao, CHEN Liping, ZHONG Qi, YAO Gang, MA Xuelian. Establishment and Application of Rapid Detection Method for Bovine Norovirus and Bovine Rotavirus Dual RAA-LFD [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 406-412.
[4]	ZHOU Jianhao, WANG Dongfang, LIU Ying, WANG Shujuan, MA Zhenyuan, XIE Caihua, ZHAO Xueli, YANG Haibo, FENG Guidan, KANG Taisheng, HU Yufeng, LI Bowen, YAN Ruoqian. Establishment and Preliminary Application of a Quantitative Droplet Digital PCR Assay for Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 413-418.
[5]	ZHANG Jiaqi, JIA Xining, ZHOU Qun, SONG Xin, ZHU Chenxi, LI Gege, ZHANG Bin. Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5143-5153.
[6]	ZHOU Likun, DONG Xinyuan, LI Jiahui, CUI Zhiying, ZHAO Shijie, XU Jie, CHEN Jing, ZHANG Yina, XIA Ping’an. Regulatory Role of MID2 on Porcine Reproductive and Respiratory Syndrome Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4735-4744.
[7]	ZHENG Ruwen, HUANG Tao, WU Daoyi, YU Guangmei, LI Fangfang, SUI Xin, MIN Tingyu. Establishment and Preliminary Application of LAMP-LFD Detection Method for Bovine Viral Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4745-4753.
[8]	JIA Yiquan, YU Mengting, LIU Weirong, FANG Shouguo, ZHANG Songbai. Baicalin Inhibits Infectious Bronchitis Virus Replication via Promoting the Expression of Type Ⅰ Interferon [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4839-4850.
[9]	CHEN Lu, CHANG Xinyu, SHEN Jiachen, SHEN Ruiting, ZHAO Zhenhua, HOU Xiaolin. Transcriptome Sequencing and Analysis of HD11 Cells Infected by Avian Infectious Bronchitis Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4860-4865.
[10]	HE Yeqing, ZUO Baijiao, LI Qinghao, SUN Juan, JIN Xin, XIA Lu, WEI Zhanyong. Histopathological Observation of Immune Organs of Piglets Infected with Transmissible Gastroenteritis Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4872-4879.
[11]	CHEN Wenzhe, ZHANG Xiangle, GU Fengxing, ZHAO Zhenxiang, LI Kangli, XUE Zhaoning, ZHENG Haixue, ZHANG Xiaoli, ZHU Zixiang. Evaluation of the Antiviral Effect of Porcine GRK2 against Foot-and-mouth Disease Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4350-4361.
[12]	ZOU Hong, XIA Yingju, LI Ling, XU Lu, ZHAO Junjie, WANG Tuanjie, ZHANG Qianyi, SONG Zhenhui. Establishment and Preliminary Application of a Dual Real-time RT-PCR Assay for CSFV and BVDV [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4422-4427.
[13]	YUAN Li, SUN Yangyang, ZHANG Lujie, ZHANG Jie, SUN Haifeng, BAI Juan, JIANG Ping. Production and Epitope Characterization of Monoclonal Antibodies against GP3 Protein of Porcine Reproductive and Respiratory Syndrome Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3424-3434.
[14]	CAO Liyan, KONG Xiangyu, LI Xiangtong, SUO Xuepeng, DUAN Yueyue, YUAN Cong, SHI Lei, ZHANG Yu, MA Guoxiang, ZHENG Haixue, WANG Qi. Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2487-2497.
[15]	ZHANG Senhao, WANG Xueying, CAI Limeng, XIE Weichun, KUANG Hongdi, WANG Xiaona, LI Jiaxuan, CUI Wen, JIANG Yanping, ZHOU Han, SHAN Zhifu, WANG Li, QIAO Xinyuan, LI Yijing, TANG Lijie. Establishment and Application of Rapid Diagnosis Method for RPA of Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2498-2508.