RESUMO
OBJECTIVE: To detect the cleavage activity of Giardia canis virus (GCV) transfer vector-mediated hammerhead ribozyme for KRR1 in vitro transcript. METHODS: Giardia, a most primitive eukaryote, has KRR1 protein responsible for ribosome biosynthesis. cDNA encoding hammerhead ribozyme flanked with various lengths of antisense RNA was cloned into a viral vector pGCV634/GFP/GCV2174 derived from the genome of GCV, KRzS flanked with 21 nt KRR1 antisense RNA on each arm, or KRzL flanked with 288 nt and 507 nt KRR1 antisense RNA. At the same time, two control groups were established: PKR without the inserted ribozyme, and TRzL flanked with 324 nt and 380 nt triosephosphate isomerase (Tim) antisense RNA. The cleavage activity of GCV transfer vector-mediated hammerhead ribozyme for KRR1 in vitro transcript was then analyzed by absolute real-time quantitative RT-PCR. RESULTS: The in vitro cleavage activities on KRR1 mRNA of the two ribozyme KRzS or KRzL were 74.0% and 81.1% respectively by the absolute real-time quantitative RT-PCR. The two control groups, PKR or TRzL, showed no effect on KRR1 mRNA in vitro. CONCLUSION: The GCV transfer vector-mediated hammerhead ribozyme shows a high cleavage activity for KRR1 in vitro transcript, which demonstrates the feasibility of using a viral vector to express a ribozyme targeted at a specific mRNA in Giardia to reduce the expression of a specific gene.
