Preparation And Identification Of Universal Ribozymes—Rz1198&Rz1282against HPV-6b/11 E1,and Construction Of A Shotgun-type Ribozyme—pRE1

Condyloma Acuminatum (CA) is a benign hyperproliferative disease of the cutaneous and mucosal epithelia induced by Human Papillomavirus type 6 and/or 11 (HPV-6/1 1), and belongs to sexually transmitted disease (STD). Nowadays, there is not a therapy that could effect a radical cure, because of the replication of HPV DNA that affect the tissue latently. CA is one of the most troublesome virus diseases for clinicians. We thought, if the expression of El 梩he key protein for HPV DNA replication was disturbed, the replication of HPV DNA would be inhibited correspondingly. Thus, the proliferation of HPV in affected tissues would be controlled and the immunity of the body could eliminate the viruses and the cure of CA would be achieved bonsequently. In this study, we described a new method of inhibiting the replication of HPV DNA by a hammerhead ribozyme directed against HPV El niRNA. Having analyzed the homologous sequences between HPV-6bEl and HPV-l IE1 mRNAs by computer, we have designed two universal hammerhead ribozymes桼z1198 and Rz1282, and studied their cleavage activities in vitro. The synthesized genes for R.z1198 and Rz1282 were cloned into p1.5.1 that possesses 5?cis-Rz and 3?cis-Rz for self-trimming and the ribozymes were got by in vitro transcription, respectively. Parts of HPV-6bEl and HPV- 1 1E1 genes were cloned into T-vectors, and target RNAs were got by in vitro transcription labeled with 32P, and the in vitro cleavage reactions for ribozymes were processed. The kinetics showed that the two ribozymes prepared in vitro could cleave both target RNAs specifically and perfectly, and they possessed the enzyme catalytic activities. Rzl 198 and Rz1282 could cooperate with each other too. So, we cloned both of them together to get a shotgun-type ribozyme 梡RE1, and in vitro transcription demonstrated its feasibility. The present study showed that the ribozymes designed against HPV- 6bEl and HPV-l 1E1 universally possessed perfect cleavage activities in vitro, ?? and the shotgun-type ribozyme梡RE1 was hopeful to inhibit the replication of HPV-6b and HPV-1 1 DNAs in vivo. A new gene therapy would come true for the cure of CA.