Study On The Expression And Biological Functions Of Protein P10 Of Muscovy Duck Reovirus

Muscovy duck reovirus(MDRV)usually infected 4?45d old Muscovy ducks,especially the young one.Soft feet is the main clinical features.Lots of white piebald necrosis points on liver and spleen were the main pathological changes.Other pathogens infection often aggravated the disease because MDRV suppressed the immune function of sick ducklings.Now,MDRV infection has become one of the major infectious diseases in the large-scale poultry breeding industries,and has caused great economic losses.The non-structural protein p10 of ARV has the function of membrane fusion,which promote the cell formation to accelerate the cracking of ARV infection,but few studies on the non-structural protein p10 of MDRV have been reported.So it is very improtant to explore the biological function of protein p10.According to the p10 gene of MDRV-YB strain,a pair of specific primers were designed to amplify by RT-PCR.Then the PCR production was inserted into prokaryotic expression plasmid pET-32a(+)and expressed in E.coli BL21(DE3)with IPTG induction.SDS-PAGE analysis showed the molecular weight of recombinant protein(32a-p10)was 28.8ku.The results showed that the optimal induction conditions of protein(32a-p10)were 24h,37? and lmmol/L IPTG.MDRV-p10 had been correct expression that was detected by Werstern blot with MDRV positive serum.It was purified(32a-p 10)by Ni-NTA column and then was as antigen to prepare the rabbit anti-p10 polyclonal antibody.The analysis with ELISA and Western blot showed that the rabbit anti-MDRV-p 10 antibody was not only a good specificity but was high titer reach to 1:64 000 and 1:20 000.In order to further explore the biological functions of protein MDRV-p10,the recombinant plasmid pCI-neo-p10 was successfully constructed and transfected into DF-1 cells.Then,the transcription and expression levels of gene p10 were measured by RT-PCR and Western-blot,respectively.The results showed that recombinant plasmid pCI-neo-p10 was successfully expressed in the DF-1 cells.The results of Western-blot analysis also confirmed that the expressed protein p10 could react specifically with both anti-Flag mouse monoclonal antibody and anti-p10 rabbit polyclonal antibody.The analysis of Flow cytometry showed that the protein MDRV-p10 can induce apoptosis and cell cycle arrest at G1 phase in DF-1 cells.In addition,The analysis of Western blot showed that MDRV-p10 can activate and up-regulated the expression of Fas,Caspase-8,and Caspase-3 to induce apoptosis and the key proteins of cell cycle control,such as CDK2 and CDK4 were inhibited.So MDRV-p10 induce apotosis was related to the phosphorylation levels of p53.Above all,it was confirmed that MDRV-p10 was successfully expressed in E.coli BL21(DE3)and prepared the rabbit anti-MDRV-p10 polyclonal antibody.Besides,the recombinant eukaryotic plasmid of MDRV-p10 was successfully constructed to prove that the protein MDRV-p10 has the function of inducing apoptosis and regulating cell cycle.It suggest that MDRV-p10 may be related to Fas-mediated signaling pathway and p53 mediated cell cycle signaling pathway.