| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 99 === Classical swine fever virus (CSFV) belongs to Pestivirus of Flaviviridae, and has a single-strand, positive-sense RNA genome. The disease it caused, classical swine fever, makes pigs get fever and serious whole-body and visceral bleeding. It could transmit by direct, mechanic, biological, or non-biological contact of excrement, lachrymal gland, and nasal secretion. Prevalence and mortality are as high as 95%. Previously, we have discovered that the 3'' untranslated region (UTR) of CSFV can form a four-stemloop structure and is involved in translational regulation. In one of the constructs, pA/Luc/A, could transcribe the RNA (5''-Luc-3'') containing the CSFV 5''UTR, 3''UTR, and fire-fly luciferase (LUC) and inducing apoptosis-like phenomenon in 24 hours. After DNA laddering assay, we confirmed that these cells transfected with 5''-Luc-3'' RNA went to apoptosis. I am interested in understanding how do the untranslated regions trigger the cell apoptosis. I have used T7 or SP6 RNA polymerase to produce CSFV RNAs, 5''UTR, 3''UTR, and 5''-Luc-3'', and the capped Luc RNA as a control. Next, PK-15 or RK-13 cells were transfected with these RNAs with different dose and different post transfection time. We find that apoptosis level could be enhanced with dose increased and with the post transfection time extended. Besides, the severity of the cell apoptosis is different when treated with different RNAs. In the other hand, we trying to use the non-viral protein expression level by co-transfect p2luc, a plasmid could express Renilla luciferase (Rluc), into cells to evaluate the correlation of the level of protein expression with the severity of apoptosis. Results of these experiments show that the amounts of Rluc expressed in cells could reflect apoptosis level. 5''-Luc-3'' is the most severe, then 3''UTR and 5''UTR, and the capped Luc is the least severe in cells. Accordingly, the severity of 5''-Luc-3'' induced apoptosis is much stronger than the 5''UTR plus 3''UTR when transfected together suggesting a synergistic effect of the 5'' and 3'' UTRs. Therefore, we produced four kinds of RNAs, 5''-Luc with 3''UTR stem-loop 1, 2, 3, or 4. After DNA laddering assay, we find the stem-loop 1 in 3''UTR could have a role in synergistic effect with the 5''UTR and triggering apoptosis.
|
|---|
