Development of a diagnostic reagent of foot-and-mouth disease using gene-expressed recombinant protein
| Autor: | chia-kai Lin, 林家凱 |
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| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 Foot-and-mouth disease (FMD) outbreaks usually result in huge economic losses. FMD is a severe, clinically acute, vesicular disease of cloven-hoofed animals including domesticated ruminants and swine, FMDV belongs to the Picornaviridae family, and is the only member of the genus Aphthovirus, Serologically, FMDV can be classified into 7 antigenically distinct serotypes, O, A, C, SAT1, SAT2, and SAT3, and Asia 1. The virus has a predilection to replicate in epithelial cells including those lining the distal oropharynx and the dorsal surface of the soft palate. In these sites, infectious virus has been found for several months or even years in a proportion of recovered ruminants but not pigs. Detection of FMDV is important for monitoring vaccination status and detection of the carries. Therefore, it is important to develop diagnostic methods for the detection of FMDV antibody. In this study, FMDV VP1 and VP2 gene of foot-and-mouth disease virus were amplified by RT-PCR. The DNA containing FMDV VP1 or VP2 gene sequences was further cloned into a prokaryotic expression vetor pET21d, respectively, named pET21d FMDV-VP1 or pET21d FMDV-VP2, respectively. The recombinant FMDV VP1 or VP2 protein was expressed by the host E. coli BL21(DE3). The protein with expected size about 25.8 kDa or 26.4 kDa was obviously observed in SDS-PAGE. The recombinant FMDV VP1 and VP2 proteins were identified by western blot assay with FMDV-infected swine serum. Expressed FMDV VP1 and VP2 proteins were purified by passing through the Ni-NTA Superflow column. Using the purified proteins as a coating antigen, FMDV-infected swine serum and vaccinated swine serum were used for ELISA test. Our results demonstrated that the recombinant FMDV VP1 protein has a better specificity than that of the VP2 recombinant protein. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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