Prepartion and identification of activity of anti-HPV-6b/11E1 universal ribozyme——Rz1198 in vitro

Aim: To study the preparation and cleavage activity of Rz1196 directed against HPV-6bE1 and HPV-11E1 (HPV-6b/11E1) transcripts in vitro. Methods: HPV-6b/11E1 gene fragments were cloned into T-vector under the control ofT7 promoter, ～(32)P-labeted HPV-6b/11E1 transcripts as target-RNAs were transcribed in vitro and purified by PAGE.Rz1198 gene designed as a universal ribozyme for both HPV-6b/11E1 transcripts was cloned into vector p1.5 between5'-cis-Rz and 3'-cis-Rz. a2P-labeled Rzl198 transcript was gel-purified, incubated with target-RNAs at different condi-tions and autoradiographed after denaturing gel-electrophoresis. Results: Rz1198 was active at 37℃. The optimaltemperature was 50℃. For HPV-6bE1, k_m = 12.2 nmol/L, k_cat = 0.18 min～(-1); For HPV-11E1, k_m= 14.7 nmol/L,k_cat =0.14 min～(-1). All these revealed that the design of Rz1198 was correct. It could be a universal ribozyme for thetwo substrates--HPV-6bE1 and HPV-11E1 transcripts. Conclusion: Rz1198 prepared in vitro possesses the perfectspecific catalytic cleavage activity. It leads to the expectation that, in the future, it will be possible to develop a newnucleic acid drug from Rz1198 which can efficiently inhibit the replication of HPV-6b/11 DNA in vivo. (Asian J An-drol 1999 Dec; 1: 195-201)

切割HPV-6bE1和HPV-11E1通用核酶Rz 1282的体外活性鉴定

利用计算机分析 HPV- 6b E1和 HPV- 1 1 E1 m RNA的同源序列 ,设计出通用于两者的锤头状核酶—— Rz1 2 82 (HPV- 6b基因 1 2 82位 ) ,通过体外转录建立了体外大量制备 Rz1 2 82的方法 .体外的切割实验表明 ,Rz1 2 82可在体外准确和有效地切割 HPV- 6b/1 1 E1靶 RNA,形成 2 68nt/52nt和 2 31 nt/52 nt大小的切割产物 .对于 HPV- 6b,Km和 kcat值分别为 1 3.8nmol/L和 0 .0 7min-1;对于 HPV- 1 1 ,Km 和 kcat值分别为 2 3.0 nmol/L和 0 .2 4 min-1.结果表明 ,体外制备的 Rz1 2 82具有较好的特异催化切割活性 ,并通用于 HPV- 6b及 HPV- 1 1 .它有望发展成为在细胞内有效抑制HPV- 6b/1 1 DNA复制的核酸药物 .