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AIM:To study the preparation and cleavage activity of HpRzdirected against the transcript of HBV core gene in vitro.METHODS:HpRz gene designed by computer targeting thetranscript of HBV core gene was cloned into the vector p1.5between 5’-cis-Rz and 3’-cis-Rz.32p-labeled HpRz transcriptproved whether the vector fit for the preparation of hairpinribozyme in vitro.32p-labeled pKC transcript containing HBVcore region as target-RNA was transcribed using T;RNApolymerase and purified by denaturing PAGE.Cold HpRztranscript was incubated with 32p-labaled target-RNAs underdifferent conditions and radioautographed after denaturingpolyacrylamide gel electrophoresis.RESULTS:HpRz has the specific ability of cleavage of target-RNA at 37℃ and 12 mM MgCL_2·K_m=26.31nmol/L,K_(cat)=0.18/min.These results revealed that the design of HpRz wascorrect.CONCLUSION:HpRz prepared in this study possessesspecific catalytic activity from the identification of cleavageactivity.These results indicate that hairpin ribozyme mayintracallularly inhibit the replication of HBV,therefore it maybecome a novel potent weapon for the treatment of hepatitisB.
AIM: To study the preparation and cleavage activity of HpRzdirected against the transcript of HBV core gene in vitro. METHODS: HpRz gene designed by computer targeting the transcription of HBV core gene was cloned into the vector p1.5between 5’-cis-Rz and 3 ’-cis-Rz.32p-labeled HpRz transcriptproved whether the vector fit for the preparation of hairpinbozyme in vitro.32p-labeled pKC transcript containing HBVcore region as target-RNA was transcribed using T; RNApolymerase and purified by denaturing PAGE. Cold HpRztranscript was incubated with 32p-labaled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis. RESULTS: HpRz has the specific ability of cleavage of target-RNA at 37 ° C and 12 mM MgCl_2 · K_m = 26.31 nmol / L, K_cat = 0.18 / min.These results revealed that the design of HpRz wascorrect. CONCLUSION: HpRz prepared in this study possessesspecific catalytic activity from the identification of cleavageactivity. The results of which that hairpin ribozyme mayintracallularly inhibit the replication of HBV, therefore it maybecome a novel potent weapon for the treatment of hepatitisB.