慢病毒载体介导的稳定表达eIF5A细胞系的建立及其对猪繁殖与呼吸综合征病毒增殖的影响

doi:10.11843/j.issn.0366-6964.2023.03.028

Abstract

Abstract: The purpose of this study was to construct a MARC-145 cell line with stable expression of eukaryotic translation initiation factor 5A (eIF5A) by means of a lentiviral vector system to provide a research tool. First, the target eIF5A sequence was synthesized and the lentivirus expression vector PLV-CMV-MCS-EF1A-PURO was constructed after double enzyme digestion. HEK293T cells were co-transfected with pLV-CMV-MCS-EF1a-Puro, the lentivirus packaging plasmid psPAX2, the envelope protein pMD2.G, and the lentivirus expressing eIF5A was packaged. The harvested lentivirus was transduced into MARC-145 cells. The MARC-145 cell line that could stable overexpress eIF5A was obtained and named as MARC-145-eIF5A cell line. MTS assay confirmed that the cell viability of the constructed cell line did not change significantly. Real-time PCR, Western blot and Indirect immune fluorescence assay were used to verify the gene and protein level of PRRSV N in the constructed cell lines and control cells, the results showed that both the gene and protein level of PRRSV N in MARC-145-eIF5A cell line were significantly increased. PRRSV propagation was significantly promoted in the MARC-145-eIF5A cell line compared with the control group. In this study, the MARC-145-eIF5A cell line which is stable over expression eIF5A protein was successfully constructed. PRRSV propagation experiment confirmed that stable expression of eIF5A could promote the PRRSV propagation in MARC-145 cells.

Key words: porcine reproductive and respiratory syndrome virus, lentiviral vector, eIF5A, MARC-145 cell, infection

CLC Number:

S852.659.6

LI Huawei, WANG Xuying, QIAO Hongxing, LI Xinfeng, JI Xiangbo, GUO Kewei, YANG Zhongyuan. Establishment of a Lentiviral Vector-mediated Stable Expression of eIF5A Cell Line and Its Effect on Porcine Reproductive and Respiratory Syndrome Virus Propagation[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1169-1176.

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Establishment of a Lentiviral Vector-mediated Stable Expression of eIF5A Cell Line and Its Effect on Porcine Reproductive and Respiratory Syndrome Virus Propagation

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