慢病毒载体介导稳定表达CD163 的PAM细胞系的建立及对PRRSV感染的研究

doi:10.11843/j.issn.0366-6964.2013.11.014

Abstract

Abstract:

The objectives of this study were to use the lentiviral vector pTrip-CMV-IRES-pur plasmid to deliver porcine CD163 into a porcine alveolar macrophage (PAM) cell line (CRL-2843), to generate a cell line stably expressing CD163 (designated PAM-CD163), and to evaluate its permissibility for PRRSV infection. The porcine CD163 coding sequences were amplified by PCR from pJET1.2-CD163 plasmid and inserted into downstream of CMV promoter in the lentiviral vector pTrip-CMV-IRES-pur plasmid. Monolayer of 293-T cells were cotransfected with three plasmids psPAX2, pMD2.G and pTrip-CMV-CD163-IRES-pur. The recombinant lentivirus expressing CD163 was harvested in the culture fluid. For transduction, the immortalized PAM cells (CRL-2843) were exposed to lentivirus in the presence of polybrene. The transduced cells were selected with puromycin and single cell colonies were isolated and expanded for PRRSV infection assay. The CD163 gene was transcribed by RT-PCR and the protein was expressed as identified by indirect immunofluorescence assay, Western blot and flow cytometry analyses. A PAM cell line (CRL-2843) stably expressing CD163 (designated PAM-CD163) was susceptible to PRRSV infection with the titer of 10^5.0 TCID₅₀·mL^-1. A PAM cell line stably expressing CD163 was constructed and permissive to PRRSV infection. This cell line could be a valuable tool for PRRSV propagation and PRRSV cellular receptors study.

CLC Number:

WANG Xiang-peng, WEI Rui-fang, XIAO Shu-qi, ZHOU En-min. Generation of a Porcine Alveolar Macrophage Cell Line Stably Expressing CD163 by Lentiviral Vector for the Production of Porcine Reproductive and RespiratorySyndrome Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1797-1804.

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Generation of a Porcine Alveolar Macrophage Cell Line Stably Expressing CD163 by Lentiviral Vector for the Production of Porcine Reproductive and RespiratorySyndrome Virus

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