Inhibition Of Hepatitis B Virus Expression By Malti-target Ribozyme In Vivo

There are approximately 350 million chronic carriers of hepatitis B virus(HBV)throughout the world. The infection is endemic to several populous regions of the world that include south east and east Asia, the western Pacific islands and sub-Saharan Africa. Common and usually fatal complications of HBV persistence include cirrhosis and hepato cellular carcinoma(HCC). While immunization effectively prevents HIBV infection, it is of little use to individuals who already carry the virus. Current drug treatment of HBV infection is usually ineffective and approaches using ribozyme has been a focus of developing new therapeutic strategies. 11EV is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. The core protein, the most important component in formation of HIBV nucleocapsid, is closely related to the replication of H7BV DNA. Cleavage of the pregenomic RNA and/or mRNA of core -4- gene, therfore, may interrupt the life cycle of HBV. Hammerhead ribozymes are short catalytic RNA molecules possessing endounclease activity. In this report, we describe multitarget ribozyme that inhibited the HBeAg expression in cell and in the transplanted tumor cell and the ribozymes expression in the cells. By study the multitarget ribozyme and analysis the secondaiy structure, we designed two mutatied ribozyme. Then we constructed plasmids which contained the mute-ribozyme. The results demonstrated that three trans-ribozymes can specifically cleave different cleavage sites on the target ribozyme. But the muta-ribozyme can抰 cleave the target ribozyme. The triple ribozyme gene: two cis-ribozyme and two muta-ribozyme gene have been cloned into five kinds of eukaryotic expressing plasmid. The HepG 2 cells were cotransfec ted with the vector and a plasmid p1.2 II carring geneome of adv-subtype HBV. The results demonstrated that the ribozyme and muta-ribozyme can express in the transfected cell. The inhibition rate of the ribozyme which was cloned into a retroviral vector downstream of tRNA gene was 81 %. The ribozyme and muta-ribozyme were cloned into adenovinis vector pAdCMV . By using the adenovirus expression system, the two plasmids were cotransfected into 293 cell line with adenovirus gene recombinant plasmids pJM17. The recombinant adenovirus were produced by plaque assay. Then the recombinant adenovirus were infected the 2.2.15 cells. The results demonstrated that the ribozyme can decrease the expression of H7BcAg up to 87.1 %. ?- The recombinant adenovirus infected the tumor cells in vivo which is from the nude mice of transpLated tumor.This is proved that the ribozyme can inhibitate the HBV gene express in vivo. The triple ribozyine gene were cloned into green fluorescent protein(EGFP)upstream of gene. To investigate the therapeutic utility of this ribozyme. We evaluated the cell express and the tissue distribution with this labeled form of the ribozyme. By subcutaneous or intravenous the ribozyme can express in the muscle cells and hepatic cells. This experiment laid a foundation for gene therapy of HBV infection.