| Summary: | 碩士 === 國立中興大學 === 獸醫微生物學研究所 === 96 === Foot-and-mouth disease virus (FMDV), the only member of the genus Aphthovirus in the family Picornaviridae, composed of seven serotypes and more than 67 subtypes. The disease affects domestic cloven-hoofed animals, including cattle, swine, sheep, and goats, as well as more than 70 species of wild animals. The virus particle consists of a single-stranded RNA genome, and its capsid is made up by four different proteins, VP1 to VP4. The major immunodominant site on the virion surface resides on the G-H loop of C-terminal of VP1 which includes a highly conserved Arg-Gly-Asp (RGD) sequence. In Taiwan, a national vaccination program was carried out in 1997, and the FMD antigens used to formulate vaccines usually purified to reduce the non-structural proteins content. In order to demonstrate the optimal regions on structural proteins reacting with antibodies, we cloned the genes encoding structural proteins of VP1 and VP2 of O/Taiwan/97 full length, N-terminal deletion, internal deletion, and C-terminal deletion respectively. Proteins are expressed in E. coli system with subsequent purification using Ni2+ affinity column, and then analyses by ELISA and western blotting. Our results reveal that the reaction with serum is significantly reduced in that with C-terminal deletion, suggesting the existence of epitope within the C-terminal of VP1. In ELISA analysis, VP1 protein is more sensitive to antibodies than that of VP2 protein. In western blot analysis, VP2 protein is more sensitive to antibodies than that of VP1 protein, suggesting that native VP1 full-length and N-terminal deletion proteins are considered to be more responsive to antibodies, and the denatured VP2 protein is more sensitive to antibodies than the denatured VP1 protein.
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