The Expression And Subcellular Localization Of Protein ?A Of Muscovy Duck Reovirus

Muscovy duck reovirus(MDRV)causes an acute infectious disease in Muscovy ducks.MDRV infection is an immunosuppressive disease,which is susceptible to secondary infection of other pathogens,thus increasing difficulties for control of MDRV and leading to huge economic losses in Muscovy duck industry.MDRV and chicken reovirus(CRV)are both members of the Avian reovirus of Orthoreovirus genus subgroup II,nevertheless,studies on biological functions of MDRV-associated proteins are fewer in comparison with CRV.? A protein is a component of inner capsid which MDRV and CRV shares,CRV ?A protein has been demonstrated to play an important role in the resistance of interferon-induced cellular response against CRV,as well as gene expression and regulation in host cells,while functions of MDRV ?A protein remain unknown.In the present study,MDRV aA protein was first expressed in Escherichia coli(E.coli),and then rabbit polyclonal antibodies against the expressed ?A proteins were prepared.In addition,expression of MDRV ?A proteins in eukaryotic cells and their subcellular localization were successfully accomplished,which would lay foundations for further ?A-associated studies.1.Prokaryotic expression of aA protein of MDRV and preparation of polyclonal antibody.The ?A gene of MDRV-YB strain was cloned into expression plasmid pET-32a(+),and then transformed into E.coli BL21(DE3)with IPTG induction.The expressed products were analyzed by SDS-PAGE and further confirmed by Western-blot assays.SDS-PAGE analysis showed that the fusion protein with molecular weight of 64.2 ku was expressed mainly in insoluble fractions.The IPTG induction time,concentration and temperature were optimized to be 6 h,1.2 mmol/L and 24?27 ?,respectively.Then the inclusion bodies were purified to homogeneity by Ni2+-affinity chromatography and could be recognized by the MDRV positive serum in Western-blot assays,which indicated that the purified His-?A proteins still had high reactogenicity.The purified His-aA proteins were then injected into New Zealand rabbit for preparation of polyclonal antibody,the titer of antiserum detected by indirect ELISA was approximately 1:32 000.Western-blot analysis further confirmed that the prepared polyclonal antibody could react specifically with MDRV-?A protein,which provided a basic and efficient tool for further functional studies on MDRV a A protein.2.Expression of MDRV ?A gene in different eukaryotic cells.The ?A gene was amplified and successfully inserted into eukaryotic expression plasmid pCI-neo-flag,the recombinant plasmid pCI-flag-?A was identified with PCR,restriction enzyme digestion and DNA sequencing analysis and then transiently transfected into DF-1 cells or Vero cells,and mRNA and protein levels of ?A gene were determined with semi-quantitative(sq)RT-PCR and Western-blot analysis,respectively.Sequencing results showed that the recombinant plasmid pCI-flag-?A was constructed correctly with no frameshift mutations.The results of both sqRT-PCR and Western-blot assays showed that MDRV ?A gene could be successfully expressed in both kinds of cell lines,with increasing levels of products at 0-60 h post-transfection.3.Subcellular localization of the expressed MDRV ?A proteins in different cell lines.To determine the exact distribution of the expressed MDRV ?A proteins in eukaryotic cells,bio-analysis software was used for primary prediction,followed by further confirmation through indirect immunofluorescence assay(IFA)and Western-blot analysis.The prediction based on software and relative websites showed that MDRV ?A protein might be located at nuclear lamina,while lacking nuclear localization sequence(NLS)and nuclear export sequence(NES).IFA and Western-blot analysis of cytoplasm-nucleus separated extracts suggested that ?A protein were present in both cytoplasm and nucleus.