Expression And Anti-Classical Swine Fever Virus Activity Of Porcine MX1Protein

The interferonl-induced Mx proteins of vertebrates are dynamin-like GTPases and have a very broad antiviral spectrum. It is important to discover the mechanisms underlying these various antiviral activities.As a number of Mx protein family porcine Mxl capable of blocking the influenza virus and vesicular stomatitis virus have been identified.However, anti-CSFV activity of porcine Mxl protein has not been reported to date.Classical swine fever is a serious contagious disease of pig caused by classical swine fever virus (CSFV). It is a notifiable (previously List A) disease by the World Organization for Animal Health (OIE). Porcine Mx1was cloned into prokaryotic eukaryotic and expression system. Antiserum was prepared and antiviral test of pocine Mx1was performed well.1.Prokaryotic expression and identification of porcine Mxl gene and preparation of polyclonal antibodiesBased on a pair of specific primers, the open reading frame of porcine Mx1(1992bp) gene was amplified from pT-poMxl. The coding region of Mx1gene was cloned into the prokaryotic expression vector of pGEX-6P-1and verified by enzyme digestion and DNA sequencing. Then this recombinant plasmid was transformed into E.coli BL21(DE3) for overexpression under opitimization conditions. The expression product had a molecular mass about99KDa as expected. The poMxl protein was separated in gel slices and used to immunize Balb/c mice. Antiserum was prepared and specificity analyzed by western blot. High titer of antiserum against porcine Mx1was obtained. The successful expression of poMxl protein in E.coli BL21and the preparation of poMxl specific mouse antiserum laid foundation for the further study on the antiviral mechanism poMxl protein.2.Prokaryotic expression,purification and antiviral activity of porcine Mxl proteinThe full coding sequence or poMxl was amplified by polymerase chain reaction (PCR) using specific primers with a PTD sequence and the purified PTD-poMxl fragment was inserted into pCold-I expression vector that provides six His residues at the N-terminus of the expressed protein. Positive clones were confirmed by sequencing and transformed into Rosetta2cells for expression induced by IPTG.SDS-PAGE analysis showed the fusion protein was essentially present in inclusion bodies.After purification,denaturation and refolding PTD-poMxl protein was transducted into Vero cells and detected in6h by wester blot.80ug/ml was selected as a working cencentrantion.Anti-VSV activity test of PTD-Mxl in Vero cells were detected by cytopathic effect inhibition,virus plaque assay and SYBR Green Real Time PCR. Analysis of result revealed significant changes in PTD-poMx1(+) group and PTD-poMx1(-) group. High concentration of fusion protein presented more protection than other groups. It was shown obviously PTD-poMxl confers resistance to VSV on Vero cells.TCID50test and SYBR Green Real Time PCR were performed to detect the anti-CSFV activity on PK-15cells.The results showed PTD-poMxl was able to inhibit the replication of SHIMEN strains and24h after infection the inhibition of fusion proteins were more.3.Establishment of PK-15cell line stably expressing porcine Mx1gene and its effect on replication of classical swine fever virusBased on a pair of specific primers, the open reading frame of porcine Mx1gene was amplified from pT-poMxl. The coding region of poMxl gene was cloned into the eukaryotic expression vector pEGFP-C1and verified by enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP-poMxl was transferred into PK-15cells by liposome. The positive PK-15/EGFP-poMxl cells were obtained by G418selection.Transcription of EGFP-poMxl was detected by RT-PCR and expression product was identified by Western blot. Fluorescence microscope showed that the granular green fluorescence was observed in cytoplasm of PK-15/EGFP-poMx1cell line.Antiviral activity of poMxl was determined by TCID50determination, indirect immunofluorescence assay and SYBR Green I real-time PCR assay.The expression of EGFP-poMxl fusion protein could restrain the replication of CSFV on PK-15cells and progeny virus production was decreased. PK-15/EGFP-poMxl cell line was infected with CSFV to investigate the fluorescent colocalization of E2of CSFV and EGFP-poMxl by confocal microscope observation.It was showed that both of them were aggregated to perinuclear region.These results would contribute to further antiviral mechanism studies of poMxl.