AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vec...AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.展开更多
AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG→AGT)in vitro.METHODS: Two different monomers of anti-mtp53maxizyme (maxizyme right MzR, ...AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG→AGT)in vitro.METHODS: Two different monomers of anti-mtp53maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G5→A5) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR,pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme).Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEN-T vector under the T7 promoter control.The 32p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with 32p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis.RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 ℃ and 25 mM MgCL2. The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either.CONCLUSION: pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good fundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.展开更多
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...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.展开更多
基金the grant from Chinese Academy of Sciences,No.KSCX2-2-204
文摘AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.
基金the National Natural Science Foundation of China,No.30171061
文摘AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG→AGT)in vitro.METHODS: Two different monomers of anti-mtp53maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G5→A5) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR,pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme).Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEN-T vector under the T7 promoter control.The 32p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with 32p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis.RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 ℃ and 25 mM MgCL2. The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either.CONCLUSION: pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good fundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.
基金Ministry of Health(No 98-1-140)Chinese Academy of Sciences(No KJ951-B1-610)
文摘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.
