The Effect Of DDx5 On Classical Swine Fever Virus NS5A Protein

Classical swine fever(CSV), as a violent and contagious disease in pig, is caused by classical swine fever virus(CSFV) also known as hog cholera, characterised by a generalized bleeding, high feverand immunosuppression. Swine fever is highly contagious disease, contact transmission is fast, high fatality rate, different ages and breeds of pigs can be infected, the world organization for animal health(OIE) will swine fever as a kind of animal infectious diseases that must be reported, will it as a kind of animal epidemics in China. CSFV is a enveloped virus, with a single-strand, positive-sense RNA genome, which is belong to Flaviviridae Pestiviruses member. NS5 A protein is a swine fever virus nonstructural protein, for swine fever virus replication play an important role, is also a part of the swine fever virus replication complexes. This experiment mainly around CSFV NS5 A protein and host cell intracellular proteins DDX5(RNA helicase DDX5, RNA- helicase p68), study the interaction between the two and the influence of DDX5 protein to swine fever virus genome replication, this experiment to get the following conclusion:1. Clone DDX5 gene from swine umbilical vein endothelial cell line and then we construct a recombinant plasmid pCDNA3.1-DDX5-Myc, CMV-DDX5 and pDsREDN1-DDX5. After identification of plasmid PCR, double enzyme digestion and sequencing, this plasmid used for following experiments.2. Tansfect the recombinant plasmid into 293 T cell and western-blot show that protein DDX5 can be espressed in 293 T cell nomal.3. CO-IP technology in intracellular and extracellular verify NS5 A and DDX5 protein were capable of binding to each other respectively.4. Use RT-qPCR method and show that, DDX5 protein can promote CSFV gene replication.Got these recombinant plasmid pCDNA3.1-DDX5-Myc 、CMV-DDX5andpDsREDN1-DDX5; CSFV NS5 A protein interact with the host DDX5 protein.; DDX5 protein can promote CSFV gene replication.