In order to study the pathogenesis of human papilloma virus type 11 (HPV11) and seek for a therapeutic approach of the disease caused by HPV11,we amplified the HPV11/E2 of 644bp length by PCR with HPV11 plasmid DNA,pG...In order to study the pathogenesis of human papilloma virus type 11 (HPV11) and seek for a therapeutic approach of the disease caused by HPV11,we amplified the HPV11/E2 of 644bp length by PCR with HPV11 plasmid DNA,pGEM T Easy was used as vector and a clone pTV 644 was obtained.The sequencing of inserted DNA was carried out after selecting and identification,according to the hammerhead structure described by Symon’s. We used computer to analyze the possible secondary cleavage sites on HPV11/E2 mRNA and to predict the secondary structure of substrate and ribozyme to exclude the analogous sequence of substrate combined with ribozyme found in the mRNA.The hammerhead ribozyme of RZ3281 against HPV11/E2 mRNA were selected to carry out cleavage reaction in vitro. Results of the experiment showed that 644bp substrate derived from HPV11/E2 can be cleaved site specifically by ribozyme in vitro,cleavage activity showed over 85% under the optimum reaction condition.Self cleavage hammerhead ribozyme can remove effects of gene joint transcript from the vector, that promise the consistent efficiency of ribozyme in vitro and in vivo as far as possible.Results of the experiment demonstrated that the ribozyme will become a highly effective and specific therapy against HPV11 infection.展开更多
文摘In order to study the pathogenesis of human papilloma virus type 11 (HPV11) and seek for a therapeutic approach of the disease caused by HPV11,we amplified the HPV11/E2 of 644bp length by PCR with HPV11 plasmid DNA,pGEM T Easy was used as vector and a clone pTV 644 was obtained.The sequencing of inserted DNA was carried out after selecting and identification,according to the hammerhead structure described by Symon’s. We used computer to analyze the possible secondary cleavage sites on HPV11/E2 mRNA and to predict the secondary structure of substrate and ribozyme to exclude the analogous sequence of substrate combined with ribozyme found in the mRNA.The hammerhead ribozyme of RZ3281 against HPV11/E2 mRNA were selected to carry out cleavage reaction in vitro. Results of the experiment showed that 644bp substrate derived from HPV11/E2 can be cleaved site specifically by ribozyme in vitro,cleavage activity showed over 85% under the optimum reaction condition.Self cleavage hammerhead ribozyme can remove effects of gene joint transcript from the vector, that promise the consistent efficiency of ribozyme in vitro and in vivo as far as possible.Results of the experiment demonstrated that the ribozyme will become a highly effective and specific therapy against HPV11 infection.
