| Summary: | 碩士 === 國立海洋大學 === 水產食品科學研究所 === 83 === Infectious pancreatic necrosis virus (IPNV) is an unportant viral pathogen to cause infectious fish disease. In Taiwan, IPNV has caused great damages and loss in the aquaculture of tilapia, ell, milk fish and clam. Therefore, how to effectively control the IPNV infection is an important issue to the aquacultures, Since using the antisense method to inhibit the virus replication has advantages of high specificity and low biotoxicity, in this study we constructed several antisense expression vectors, CMV21 (A), SV21 (A) and RSV21 (A), with the cytomegalovirus (CMV) immediately promoter or the SV40 early promoter as a promoter and the IPNV - EOS cDNA fragment, PB21, complementary to the B fragment gene, VPI, as an insert. These vectors and other vectors constructed before by our laboratory colleagues were introduced into the CHSE - 214 cells and 70 stable transfecied cell lines were selected. The result of virus titration determined on 27 cell lines showed that the transfected cell line SA21 (A) -3 has the best virus inhibition effect, whose virus titer decreased 2 log values. According to the result of the Southern blotting hybridization, the transfected DNA can not be detected in only three lines. As we extracted cellular proteins before virus infection and at 2, 4, 6, 8, 12 and 24 hours post - infection (at M. O. I. 1), and used the IPNV - EIS polyclonal antibody for the Western blotting hybridization, we found that the peak of virus protein 1 (VPI) production showed at 6 hour post - infection in the control, while in the cell line SV21 (A) -3, the peak was found at 8 hours post - infection and its highest yield was 33.17% less than the control. These results suggest that the antisense RNA complementary to VPI, of colony SV21 (A) can slow down and reduce the VPI production.
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