摘要
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 T7 RNApolymerase and purified by denaturing PAGE. Cold HpRztranscript was incubated with 32p-labeled 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 MgCL2. Km = 26.31nmol/L, Kcat = 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 mayintracellularly 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 HpRz directed against the transcript of HBV core gene in vitro. METHODS:HpRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz.32p-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme in vitro.32p-labeled pKC transcript containing HBV core region as target-RNA was transcribed using T;RNA polymerase and purified by denaturing PAGE.Cold HpRz transcript 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℃ and 12 mM MgCL_2·K_m=26.31nmol/L,K_(cat)=0. 18/min.These results revealed that the design of HpRz was correct. CONCLUSION:HpRz prepared in this study possesses specific catalytic activity from the identification of cleavage activity.These results indicate that hairpin ribozyme may intracallularly inhibit the replication of HBV,therefore it may become a novel potent weapon for the treatment of hepatitis B.
基金
Ministry of Health(No 98-1-140)
Chinese Academy of Sciences(No KJ951-B1-610)
