Analysis Of The Interaction Between V Protein Of PPRV And STAT1of Host

Peste des Petits Ruminants virus, belongs to the genus Morbillivirus in the familyParamyxoviridae, mainly infected with small ruminants like goat and sheep. As PPRcauses a huge economic losses to animal breeding with it’s fast spread and high mortality,Food and Agriculture Organization&Office of International Epizootic all regard it as Akind of deadly infectious diseases. In China, PPR was first described in Tibet in2007,and was break out again in2010. So the study on PPRV must be strengthen for it’sserious threat to public health and sheep breeding industry.The V protein of PPRV expressed abundantly during the host infected with virus isnonstructural protein. It is reported that the nonstructural protein of genus Morbillivirusand even family Paramyxoviridae can interact with some key proteins of IFN signalpathway, such as V protein can interact with RIG-I or STATs to inhibit activation ofNF-κB or cell signaling of JAK/STAT, blocking the IFN induction or IFN signaling,thereby the antiviral defense of host, and then antagonizing the host antiviral defense ofIFN response.For further studying the function of V protein correlated to PPRV replication andpathogenesis, we expressed the V protein of PPRV China/Tib/07strain in Escherichiacoli and mammalian cells respectively, and purified it from prokaryotic expressionsystem. In addition, the STAT1gene of host was cloned and expressed. Finally, theinteraction between V protein and STAT1was identified through confocal assay, Co-IPassay and GST-pulldown assay, the mainly results are as follows:(1) Prokaryotic expression and purification of V protein of PPRV: we constructedprokaryotic expression plasmid of V gene of PPRV China/Tib/07strain, and then inducedit in different temperature, with different concentration of IPTG and at different timepoints. Moreover, we have the expressed protein purified and gained more fusion proteinof higher purity. Finally, we obtained the best inducible expression condition andpurification strategies of the fusion protein.(2) Eukaryotic expression of V protein of PPRV: Eukaryotic expression plasmid ofV gene was constructed and V protein was expressed abundantly in cos-7cell lines. (3) Clone and express of STAT1gene of the host: STAT1gene segment wasamplified from Vero cells by RT-PCR and cloned into pCAGGs vector, and thenexpressed abundantly in cos-7cell lines.(4) The interaction between V protein of PPRV and STAT1of host was confirmed:we confirmed the interaction between V protein and STAT1on three levels by confocalassay, Co-IP assay and GST-pulldown assay. The results show that they can interact witheach other both in vitro and in vivo.The V protein of PPRV China/Tib/07strain was expressed in Escherichia coli andmammalian cells respectively for the first time in this project. Furthermore, the STAT1gene of host was cloned into pCAGGs vector to construct a eukaryotic expressionplasmid, and was expressed abundantly in cos-7cell lines. At last, the interactionbetween V protein and STAT1was confirmed from different perspective. These worksprovided us a good foundation for further studying the biological function of V proteininteracting with the host and the replication of PPRV in the host cells.