Study on Cleavage in vitro of HPVll/E2 Gene by Specific Ribozyme / 中国病毒学

ABSTRACT

In order to study the pathogenesis of HPVl1 and seek for a therapeutic approach of the disease caused by HPV1l. The HPVl1/E2 644bp was amplified by PCR with HPVll plasmid DNA. pGEM-T Easy was used as vector and a clone pTV-644 was obtained.The inserted DNA sequencing was carried out after selecting and identification. According to the hammerhead structure described by Symon's, the possible secondery cleavage sites on HPVll/E2 mRNA were analyzed and the secondery structure of substrate was predicted by computer and ribozyme, excluding analogous sequence of substrate combined with ribozyme were found in the mRNA.The hammerhead ribozyme of RZ2777 against HPV1l/E2 mRNA was seleted to carry out cleavage reaction in vitro. Results of the experiment showed that 644bp substrate derived from HPVll/E2 can be cleavaed site-specifically by ribozyme in vitro. The cleavage activity showed over 85% by choosing the optimun reaction condition, which was not affected by two cis-ribozymes on both 3′-and 5′-ends released by self cleavage,but two flan-king sequences of target RNA influenced the cleavage activity. Results demonstrated that the ribozyme will become a highly effective and specific therapy against HPVll infection.