Investigation On Biological Functions Of MDRV ?C And P10.8 Proteins

Muscovy duck reovirus(MDRV)mainly infects 10-day-old Muscovy ducklings and persists in infected ducklings flock to 6-week age,which has a mortality rate from 10%to 50%.The genome of MDRV consists of 10 segments of double-stranded RNA(dsRNA),each of which is monocistronic gene.The S4 gene includes two overlapping open reading frames(ORFs)and encodes p10.8 protein and ?C protein,respectively.As for Avian reovirus(ARV)and new-type MDRV(N-MDRV)or new-type goose reovirus(N-GRV),the only tricistronic S1 segment encodes p10,p17(p18)and ?C,respectively.?C protein is a component of the outer capsid of themature virions,and evidences has revealed that ARV ?C can induce neutralizing antibody and cell apoptosis.Although MDRV p10.8 protein also induces cellular apoptosis in cultured cells,the molecular mechanism of apoptosis is not clear and remains to be further studied.Nucleotide homology between p10.8 and ?C gene in MDRV and corresponding p10 and ?C gene in ARV is not high.And currently the functions of MDRV-?C protein is unclear,and the mechanism of p10.8 protein caused apoptosis needs further exploration.Therefore,in the present study,MDRV-?C protein was expressed in Escherichia coli(E.coli),and then rabbit polyclonal antibodies against the expressed MDRV-?C proteins were prepared.Next,MDRV-?C proteins were expressed in eukaryotic cells for further biologically functional studies.Additionally,we also explored the molecular mechanism about how p10.8 protein affected the endoplasmic reticulum stress and apoptosis in cultured cells,which would laid foundations for further functional explanation of MDRV S-class proteins.The main contents and results are as follows:1.Prokaryotic expression of MDRV-?C proteins and preparation of polyclonal antibody against MDRV-?C proteins.The MDRV ?C gene was amplified by PCR amplification with specific primers and inserted into prokaryotic expression plasmid pET-32a(+).The recombinant plasmid was identified via PCR identification,digestion identification,and DNA sequencing.Then the correct plasmid was transformed into BL21(DE3)for protein expression,and western blot analysis was performed to identify the expressed ?C proteins.After that,the expression conditions for recombinant ?C protein were optimized.The best induction optimization is 37?,1 mM IPTG for 4 h.Then ?C proteins were expressed with the optimized conditions for further purification using Ni-NTA Resin method,results showed that purification effect was better at 400 mM imidazole concentration.The purified ?C proteins were immunized into New Zealand rabbits according to the immunization program,after four times of injections,the rabbit polyclonal antibodies were obtained with a ELISA titer of 1:640000.2.Study on the biological functions of MDRV-?C proteins.Here we successfully constructed the eucaryotic expression recombinant plasmid of MDRV-?C gene as previously described,and transfected into DF-1 cells for protein expression,then identified the expressed ?C proteins in DF-1 cells with the prepared polyclonal antibodies.Next,the subcellular localization of the expressed ?C protein was detected by Indirect immunofluorescence assay(IIFA).The effect of ?C proteins on apoptosis,cell necrosis and cell cycle were detected with flow cytometry,simultaneously,the mRNA expression levels of CDK2,cyclin E,cIAP and MLKL gene were detected by fluorescence quantitative PCR method respectively.The results of IIFA showed that ?C protein mainly located in the cytoplasm but not in the nucleus.The results of flow cytometry showed that ?C transfection significantly increased the numbers of cells in G1 phase.The results of fluorescence quantitative PCR showed that mRNA levels of CDK2 and cyclin E were remarkably down-regulated in?C transfected DF-1 cells,indicating that MDRV ?C could induce DF-1 cell cycle arrested at G0/G1 phase.The results of flow cytometry showed that ?C transfection did not induced apoptosis,but probably induced cellular necrosis.And the results of fluorescence quantitative PCR showed that the transcriptional levels of cIAP and MLKL gene were obviously up-regulated.MLKL protein is an executive protein in the process of cell programmed necrosis,of which the increasing expression suggested that?C transfection induced cell programmed necrosis in DF-1 cells.3.Study on MDRV-p10.8 protein-induced endoplasmic reticulum stress(ERS)and apoptosis in DF-1 cells.In this study,to explore the effect of MDRV p10.8 protein on ERS and apoptosis in DF-1 cells,recombinant plasmid of MDRV p10.8 gene was transfected into DF-1 cells,followed by treatment of ERS inhibitors and activator,and cellular samples were collected for mRNA expression of ERS-and apoptosis-associated key genes(xbp-1,ATF6,CHOP and caspase 3)by fluorescence quantitative PCR method.Results showed that p10.8 transfection caused a significant increase in mRNA levels of the above genes in comparison with the blank control group,while pre-treatment with ERS inhibitor obviously reduced the mRNA expression of above genes when compared with p10.8 transfection group.Additionally,pre-treatment with ERS activator significantly enhanced the mRNA expression of above genes in comparison with the p10.8 transfection group.Collectively,the obtained results suggested that p10.8 transfection could induce ERS and apoptosis.In summary,here we successfully accomplish the prokaryotic and eukaryotic expression of MDRV-?C protein,and successfully prepare rabbit polyclonal antibody against the bacterially expressed ?C protein.The biologically functional studies indicate that MDRV-?C proteins can induce cell cycle arrest at G0/G1 phase and also cause cell programmed necrosis in DF-1 cells.In addition,MDRV-p10.8 proteins induce the endoplasmic reticulum stress-mediated apoptosis in DF-1 cells.