切割HPV-6bE1和HPV-11E1通用核酶Rz 1282的体外活性鉴定

Identification of in vitro Activity of The Universal Ribozyme——Rz1282 Against HPV-6b/11 E1

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 .

A hammerhead ribozyme——Rz1282 had been designed to cleave the mRNAs of HPV 6b/11 at codon 1282 of HPV 6b gene(1276 of HPV 11)after analysis of the secondary structures of HPV 6b/11 mRNAs by computer.By means of in vitro transcription and precipitation with ethanol,anti HPV 6b/11 E1 ribozyme was prepared in vitro in large amounts.Cleavage reaction demonstrated that the ribozyme prepared in vitro could site specifically cleave the target E1 mRNA fragments of human papillomavirus type 6b and 11(HPV 6b/11)to yield two products (268 nt/52 nt and 231 nt/52 nt respectively).For HPV 6b, K m =13.8 nmol/L and k cat =0.07 min -1 ;for HPV 11, K m=23.0 nmol/L and k cat =0.24 min -1 .The results showed that Rz1282 prepared in vitro possessed a good specific catalytic cleavage activity and was universal for HPV 6b and HPV 11.It is hopeful that Rz1282 would be developed to be a nucleic acid drug that could inhibit the replication of HPV 6b/11 DNAs in vivo.