The study of anti HPV16E_6 ribozyme′s expression and cleavage activity in vitro
Objective To acquire a ribozyme against the E 6 gene of human papillomaviruses type 16 as a potential gene therapy tool on HPV related tumors. Methods According to Symon′s hammerhead structure, anti HPV16E 6 ribozyme (anti 16HRz) was designed by using computer programs. The DNA sequence encoding the ribozyme was synthesized, and incorporated into a pRG523 expressing vector to construct plasmid pRG16HRz. Ribozyme and HPV16E 6 mRNA were acquired by transcription in vitro , and ribozyme′s activity was identified by cleavage experiment in vitro. Results In vitro transcription of the pRG16HRz which had a cis ribozyme gene on both sides of the anti 16HRz gene showed that anti 16HRz could be trimmed to definite length by the 3′ cis Rz and 5′ cis Rz. The HPV16E 6 gene (+94-+296) was got by PCR amplification, then incorporated into pTZ19 to construct pTZ16E 6, which could generate HPV16E 6 mRNA fragment by transcription in vitro. In vitro cleavage reaction demonstrated that anti 16HRz could site specifically cleave the target HPV16E 6 mRNA fragment of 214nt to yield two products (82nt and 132nt). The temperature and time of reaction affected cleavage activity, which was maximum when reacting at 40℃ for 100 minutes. Conclusion Anti 16HRz may become a gene therapy tool on HPV16 related tumors as it can site specifically cleave the target HPV16E 6 mRNA.