Synthesis and Selection of siRNAs Specifically Inhibiting the Expression of the EP0 Gene of Pseudorabies Virus
Abstract: Abstract：EP0, an early protein gene of pseudorabies virus (PRV), plays important roles in viral replication and possibly in PRV latency. In order to select an siRNA that specifically inhibits the expression of EP0, three siRNA templates, EP04, EP08 and EP12, were designed and synthesized according to the EP0 sequence of PRV Ea strain and the siRNA design guidance of Ambion, and then cloned into the siRNA expression vector pSilencer 4.1-CMV neo containing a CMV promoter. This resulted in three recombinant plasmids p4.1-EP04, p4.1-EP08 and p4.1-EP12. Another recombinant plasmid pEP0-EGFP was also constructed by an in-frame fusing the EP0 coding sequence to the 5’-end of the EGFP gene in the expression vector pEGFP-N3. The p4.1-EP04, p4.1-EP08, p4.1-EP12 and a negative control p4.1-NK were separately co-transfected into IBRS-2 cells. Fluorescence microscopic observation, flow cytometric and RT-PCR analysis revealed that all the three siRNA could inhibit EP0 expression in the model as well as in PRV replication, with EP08 being the most efficient one, followed by EP12 and EP04. Our data are important to further study the function of EP0 in PRV replication and latency.