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| Establishment of a MARC-145 cell line expressing Porcine (Sus scrofa) SARM1 |
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1. Huazhong Agricultural University, Wuhan,China
2.
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Abstract Porcine (Sus scrofa) SARM1 (sterile-α and TIR motif containing protein 1) plays an important role in innate immune response during Porcine reproductive and respiratory syndrome virus (PRRSV) infection. Establishment of a MARC-145 cell line expressing Porcine (Sus scrofa) SARM1 is an important step to study function of porcine SARM1 gene. In the present study, the recombinant expression vector (pEGFP-N1-pSARM1) was successfully constructed, and transfected into MARC-145 cells using LipofectamineTM 2000. Fluorescence imaging using confocal microscope demonstrated localization of porcine SARM1-GFP protein in mitochondrion of MARC-145 cells. The stably transfected MARC-145 cell line was screened with 800 μg/mL of G418 for 6 d. The monoclonal cell was acquired by limiting dilution from the polyclonal mass of stably transfected cells. The stably transfected MARC-145 cell line had normal morphology and exhibited strong green fluorescence. After continuous passage culture, green fluorescence in stably transfected cell line could be observed. Porcine SARM1 and SARM1 protein stably expressed in stable transfected cell line by reverse transcription PCR and western blot, respectively. 0.1 multiplicity of infection (MOI) of PRRSV was used to infect stably transfected cell line to test whether this cell line could support PRRSV replication in vitro. Result of qRT-PCR showed that PRRSV ORF7 expressed in stably transfected cell line at 12 hour post infection (hpi), and the expression of ORF7 at 12, 24 and 48 hpi was decreased extremely significantly compared with that in normal MARC-145(P＜0.01). 50% tissue culture infective dose(TCID50) assay also showed viral load of PRRSV in stable transfected cell line was lower. The study provides foundation for investigating role of porcine SARM1 in immune regulation with PRRSV infection in future.
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Received: 17 June 2014
Published: 01 March 2015
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