T Lymphocyte Activation Triggered By Dendritic Cells Transfected With FMDV VP1 Gene

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals,including cattles, pigs and sheep.This disease is widely endemic all over the world and often results in considerable economic losses. Current FMD vaccines are mainly based on inactivated viruses, although effective, but outbreaks of FMD have been directly associated with incomplete inactivation of virus, and even some time contribution to the escape of virus from vaccine manufacturing facilities. FMDV capsid contains 60 copies of each of four structural proteins, VP1, VP2, VP3, and VP4. VP1 is composed of 213 amino acids, also named ID protein, occupying 2977-3615 of FMDV RNA genome. VPl plays a major role in inducing FMDV neutralization antibody.In this study, the expression vector pcDNA3.1(+) and pMD18-VP1gene were digested with restriction endonucleases EcoR I and Hind III, respectively.The VP1 gene was inserted into the eukaryotic expression vector pcDNA3.1. Positive clone, a recombinant plasmid named as pcDNA3.1-VP1 with interest gene, was identified by restriction analysis and DNA sequencing. Then the recombinant plasmids were transformed into E.coli DH5αfor VP1 expression. The interest genes were identified by restriction analysis and DNA sequeneing. The homology of the sequences of VP1 gene among foot-and-mouth disease virus O isolate O/NYOO of FMDV registered in GenBank was compared (AY333431.1). It was found that the pMD18-VP1 gene shared highest homology with foot-and-mouth disease virus O isolate O/NYOO strain(100%). In order to identify whether the interest gene of the VP1 can be expressed in eukaryotic cells, the recombinant plasmids of pcDNA3.1-VP1 were used to transfect mouse monocyte-derived dendritic cells(MoDC) with pcDNA3.lA empty vector transfected MoDC as a negative control. Western blot results showed that there is an obvious protein of 60 kD, and empty vector had no hybrid zone in the corresponding position. In order to elucidate whether MoDC transfected with FMDV VP1 antigen gene are able to activate T lymphocytes, MoDC transfected with FMDV VP1 gene were cocultured with T cells, the MoDC which were pulsed with inactivated FMDV(FMDV+DC)were cocultured with T cells as a control. Negative control was performed by coculturing lymph node T cells with MoDC. MoDC transfected with FMDV VP1 gene have the potential to activate CD8+T lymphocytes and stimulate high levels of expression of IFN-γat the indicated timepoints. These data pave the smooth way for FMDV antigen presentation of DC and imply new strategy for novel vaccine development.