Cloning, Expression And Identification Of Porcine Mx1 CDNA

Mx proteins are members of the dynamin superfamily of large GTPases, which are key components of the antiviral state induced by interferons and play an important role in antiviral infections by a wide range of viruses including negative-sense single-stranded RNA viruses, double-stranded RNA viruses, positive-sense single-stranded RNA viruses and the reverse-transcribing DNA virus at early stage of the replication cycle. Mx proteins include nuclear and cytoplasmic forms, among which murine Mx1 accumulates in the nucleus, while human MxA, murine Mx2 and porcine Mx1 in the cytoplasm. The nuclear form confers resistance to orthomyxoviruses such as FLUAV, while cytoplasmic form inhibits viral replication such as vesicular stomatitis virus and Bunyaviruses.Porcine Mx proteins include Mx1 (76KD) and Mx2 (73KD). The porcine Mx1 gene is mapped on chromosome 13 and contains an open reading frame encoding a polypeptide of 663 amino acids with anti-virus activity, while the similar activity of the Mx2 protein has not been reported. In this study, total RNA was extracted from porcine peripheral blood mononuclear cells after treatment with IFN-α. Mx1 cDNA was amplified by RT-PCR and subcloned into T vector for sequence analysis, which showed a 99.5% or 98.9% identity to the published mRNA sequence at nucleotide or amino acid sequence level. The cDNA was then subcloned into eukaryotic expression vector pcDNA3.1/Zeo(+) and the resultant recombinant vector pcDNA-Mx1 was used for transfection study. The cDNA was also subcloned into the pFastBac1 vector in Bac-to-Bac? Baculovirus expression system. The recombinant vector pFastBac1-Mx1 was transposited in transposition engineering bacteria DH10Bac containing a baculovirus shuttle plasmid-Bacmid and a helper plasmid. Sf9 cells were transfectd with recombinant shuttle plasmid rBacmid-Mx1 to obtain recombinant virus. MDCK cells were transfected with recombinant vector pcDNA-Mx1 or treated with recombinant porcine Mx1 protein expressed in Sf9 cells, followed by infection with vesicular stomatitis virus. Plaque reduction assay showed that both recombinant vector pcDNA-Mx1 and recombinant baculovirus expressed recombinant proteins with anti-viral activity, which lay the foundation for further studies of biological functions and application of the porcine Mx1 protein.