To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core region in vitro, HRz gene designed by computer targeting the transcript of HBV core gene was cloned into the...To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core region in vitro, HRz 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. 32 P-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme. 32 P -labeled pKC transcript containing HBV core region as targets-RNA was transcribed by using T 7 RNA polymerase and purified by PAGE. Cold HpRz transcript was incubated with 32 P-labeled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis. The results showed that HpRz had the ability of cleavage at 37 ℃ and 12 mmol/L MgCl 2 and the design of ribozyme was correct. It is concluded that HpRz prepared in vitro possesses specific catalytic activity, indicating that it is possible for HpRz to intracellularly inhibit the replication of HBV. It may be developed into a nucleic acid drug in the treatment of hepatitis B in the future.展开更多
基金This project was supported by a grantfrom the Ministry ofHealth (No.98- 1- 140 ) and Chinese Academy of Science(No.KJ95 1- B1- 6 10 )
文摘To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core region in vitro, HRz 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. 32 P-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme. 32 P -labeled pKC transcript containing HBV core region as targets-RNA was transcribed by using T 7 RNA polymerase and purified by PAGE. Cold HpRz transcript was incubated with 32 P-labeled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis. The results showed that HpRz had the ability of cleavage at 37 ℃ and 12 mmol/L MgCl 2 and the design of ribozyme was correct. It is concluded that HpRz prepared in vitro possesses specific catalytic activity, indicating that it is possible for HpRz to intracellularly inhibit the replication of HBV. It may be developed into a nucleic acid drug in the treatment of hepatitis B in the future.
