自我剪切锤头状核酶体外切割HPV11/E2的研究  

Studies on Cleavage of HPV11/E2 Gene by Self-cleaved Hammerhead Ribozyme

作　　者：侯化 杨光彩 黄杨中[2] 王曙[3] 

机构地区：[1]广州同和第一军医大学生化教研室,广州501515 [2]美国弗吉尼亚大学生物部,美国96102 [3]上海复旦大学生命科学院遗传学研究所,上海200092

出　　处：《病毒学报》2001年第3期261-264,共4页Chinese Journal of Virology

基　　金：国家自然科学基金 (3 970 0 187);广东省自然科学基金(970 3 3 2 )

摘　　要：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.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.

关 键 词：人乳头瘤病毒11型 E2基因 垂头状核酶 体外剪切 

分 类 号：R373.9[医药卫生—病原生物学] Q783.1[医药卫生—基础医学]