| Summary: | 碩士 === 國立中興大學 === 獸醫微生物學研究所 === 89 === Classical swine fever (CSF) causes one of the most severe diseases in pig worldwide. Infected pigs developed pyrexia and leukopenia. Mortality rates may reach 90%~100% when acute infect, and outbreak of classical swine fever can causes large economic consequences. Classical swine fever virus (CSFV), the causative agent of classical swine fever, is a member of Pestivirinae within the family Flaviviridae. We constructed prokaryotic plasmids that express CSFV core and N-terminal half of envelope protein E2 with enhanced green fluorescent protein (EGFP). The results of fluorescent microscope and luminescent spectrophotometer examinations of fusion proteins CoreD2-EGFP and E2N-EGFP, extracted from inclusion body, showed that these fusion proteins could absorb UV light and emit green fluorescence. Results from SDS-PAGE and western blotting revealed that both fusion products have molecular masses as expected and exhibit strong reactivity to CSFV antibodies. ELISA results of immunogenecity analysis indicated fusion with EGFP would not change epitopes of CSFV structure proteins. Extracted products could be recognized by the sera of CSFV-infected swine in Dot-ELISA. The best score is using both CoreD2-EGFP and E2N-EGFP as antigen, while only E2N-EGFP is the second. Only using CoreD2-EGFP as an antigen is the lowest score of which by telling apart from SPF swine sera, the negative control. In this study, a very rapid, sensitive and specific Dot-ELISA was developed for immunological diagnosis in CSFV-infected swine. In laboratory diagnosis, CSFV-infected sera can be coated in 96-well plate after incubation with fusion proteins and examined by luminescent spectrophotometer, and the antibodies can be quantified. This immunological diagnosis method could be applied for discriminating diagnosis, when the CSFV subunit marker vaccines are used in the field.
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