Synthesis, Selection Of EP0 Gene Specific SiRNAs And The Effects On The Replication Of Pseudorabies Virus

Pseudorabies virus (PRV), a member of neurotrophic alphaherpesviruses, causes lethal infection in many domestic and wild animals except for pigs, in which leads reproductive failure and great economic loss in pig industry worldwide. PRV is easy to set up latency after infection of pigs. During latency no infectious viron and no virus shed could be detected in and from the pigs. However the latency could be reactivated under stress, resulting in a new outbreak of pseudorabies. Vaccination could largely reduce the morbidity risk of pigs, but could not block the virus infection, latency establishment and reactivation. So latency is one of the most important obstacles in the Pseudorabies Eradication Programs which are performing in many countries.The mechanism of herpesvirus latency is largely unknown. Several studies revealed that a latency-associated transcript (LAT) is produced during the latency, and its role in the latency is unclear. In the PRV genome, LAT gene is located in the same locus with the immediate early protein gene IE180 and early protein gene EP0 with a reverses transcription direction. Accordingly, traditional mutagenesis technology is not suitable to analyze the function of these genes and the relationship with the latency. RNA interference (RNAi) is a powerful approach to study gene functions at the RNA level without disruption of the genomic structure of the target genes. EP0, an early protein gene of PRV, plays an important role in the regulation of viral replication and might also in PRV latency. So, unraveling the spatio-temporal expression of EP0 gene may be an important step to explain the mechanism of PRV latency.In this study EP0 gene was highly expressed in E .coli. Anti-EP0 polyclonal antibodies were generated by immunizing rabbits with the purified recombinant EP0 protein, and a Western-blot method was established using the antibodies. This is very important to study the EP0 expression during PRV replication and latency.According to the design principles recommend by Ambion (www.ambion.com) and the EP0 gene sequence of PRV Ea strain, three siRNA templates, EP04, EP08 and EP12, were designed, synthesized, and then cloned into the downstream of the CMV promoter of the siRNA expression vector pSilencer 4.1-CMV neo, resulting in three recombinant plasmids p4.1-EP04, p4.1-EP08 and p4.1-EP12. Another recombinant plasmid pEP0-EGFP was also constructed by in-frame fusing the EP0 coding sequence to the 5' end of the EGFP gene in the expression vector pEGFP-N3. The recombinant plasmids p4.1-EP04,...