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| Establishment of MDBK Cell Lines Stably Expressing Peste des petits ruminants virus (PPRV) Receptor Nectin-4 |
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Abstract Peste des petits ruminants virus (PPRV) is the causative agent of Peste des petits ruminants (PPR). Nectin-4 is the receptor of PPRV-infected host epithelial cells. In this study, Nectin-4 gene was synthesized to construct the recombinant plasmid pLOV-eGFP-Nectin-4. To obtain retrovirus-like particles,the recombinant plasmid was co-transfected into 293T cells with the packaging plasmid pSPAX2 and the outer membrane plasmid pMD2.G. To construct the MDBK (Madin-Darby bovine kidney) cell line stably expressing Nectin-4 receptor, the MDBK cells were firstly infected with the supernatant of cell culture which containing the retrovirus-like particles, and then screened and purified using puromycin. The MDBK cell line stably expressing Nectin-4 protein was obtained, which was named as MDBK-Nectin-4. The fluorescence of 293T cells co-transfected with pLOV-eGFP-Nectin-4, pSPAX2 and pMD2.G, respectively. Plasmids showed that the lentivirus was successfully packaged. The results of the minimal lethal concentrations of puromycin for MDBK cells showed that the best screening concentration of puromycin was 1 μg/mL. RT-PCR analysis showed that the Nectin-4 gene could be transcribed into mRNA in MDBK-Nectin-4 cells. The results of Western blot assay showed that Nectin-4 protein could be expressed stably in the MDBK-Nectin-4 cells. The results of indirect immunofluorescence assay (IFA) demonstrated that the expressed Nectin-4 protein was mainly distributed on the cell membrane. After continuous passaged to the 20th generation, the MDBK-Nectin-4 cell line still stably expressed the Nectin-4 protein, which indicated a good genetic stability of this cell line. MDBK-Nectin-4 cell line infected with PPRV was more rapidly cytopathic than the MDBK cells. The established cell line provides experimental materials for further investigation of the interaction mechanism between PPRV and receptor Nectin-4, as well as clinical rapid isolation of PPRV.
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Received: 05 March 2018
Published: 26 September 2018
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