Inhibition Of Gene Expression And Replication Of Foot-and-Mouth Disease Virus By SiRNA

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious disease among cloven-hoofed animals and was listed on the top of the List A of animal infectious diseases by the International Animal Health Organization (Office International des Epizooties, OIE).The FMDV genome contains a unique open reading frame. It is translated into a polyprotein which is subsequently cleaved into several mature structural proteins and nonstructural proteins by proteases. 3C gene encodes a kind of protease, which can cleave the P12A precursor into four structural proteins, VP4, VP2, VP3 and VP1, which form the capside of the viron. So, 3C gene expression is very important for FMDV replication. RNA interference (RNAi) is the process of sequence specific, posttranscriptional gene silencing (PTGS) in animals and plants. There was evidence that short than 30nt dsRNA molecules, termed small interfering RNA (siRNA), exhibit inhibitory effect on the expression of target gene. The 3C gene is highly conserved among the different serotypes of FMDV, so this study ought to investigate the inhibitory effects of 3C gene specific siRNAs on FMDV replication.In this study 3C and 3D genes were cloned from an infectious genomic clone of FMDV serotype O, and highly expressed in E. coli. Polyclonal antibodies were generated by immunizing rabbits with the purified recombinant 3C and 3D proteins respectively, and Western blot methods were established using the antibodies. This is very important to study the expression and function of nonstructural protein genes in FMDV replication.The coding sequences of 3C genes from the seven serotypes of FMDV were compared, which included serotypes O, A, C, SAT1, SAT2, SAT3 and Asia 1. According to the conserved sequence of 3C gene and the design principles recommend by Ambion (www.ambion.com), two siRNA templates, 3C07 and 2C12, 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 two recombinant plasmids pSilencer-3C07 and pSilencer-3C12. Another recombinant plasmid p3C-EGFP was also constructed by...