Study On The Inhibition Of Hepatitis B Virus (HBV) By Hepatitis Delta Virus (HDV) Ribozyme In Cells

Ribozymes are some catalytic RNA molecules which can specifically recognize their RNA substrates by the rule of Watson-Crick base pairs, and subsequently catalyze their cleavage. This feature makes ribozymes attractive as therapeutic tools for the inactivation of both viral RNAs and the mRNAs associated with various diseases. By now there are seven kinds of ribozymes have been discovered in nature including: hammerhead, hairpin, hepatitis delta virus (HDV) and Varkud Satellite (VS) ribozymes; group I and group II introns; and the RNA subunit of RNase P. Because of shorter segments, simpler structure and higher activity of catalysis, hammerhead and hairpin ribozymes have been studied more in the gene therapeutic researches of using ribozymes. But up to now they have not been applied to clinical treatments due to some obstacles including how to deliver them into the target cells specially and make them expressing efficiently under control, etc. It is important to find other ways, so that some advances about ribozymes might be done.Hepatitis delta virus (HDV) is the only virus found in human cells that containing ribozyme in it. It is a single-stranded circular RNA defective virion and its genome consists of about 1700 nucleotides. The replication of HDV in cells is RNA-directed transcriptional event via rolling circle like some viroids and plant viroid-like satellite RNAs, and uses its ribozyme, a self-catalytic RNA sequence, to cleave the multimeric strands into monomers. It must be packaged by HBsAg from HBV before it becoming a mature infectious virion, so HDV is named the satellite virus of HBV and always companied with HBV infection. In contrast to other catalytic RNAs, HDV ribozyme possesses several unique features, such as it might be more activity for its suitable human cellular enviroment, and more complex structure than hammerhead and hairpin ribozymes might make it more stable in cells. The more attractive is the unique relationship between HBV and HDV. The object in this study is to investigate how the inhibition of HBV by HDV ribozyme in HepG2.2.15 cells and prove whether or not HDV ribozyme is worthy of being used as a gene theraputic tool of HBV infection.Method: (1) Extracted the genome DNA of HepG2.2.15 cells and cloned HBV genomic full sequence which integrated in the genome of HepG2.2.15 cells via PCR. Purified products of PCR were inserted into pGEM-T clone vector and sequenced. (2) This sequence was aligned with 202 HBV full sequences of various genotypes which were retrieved from NCBI database to identify potential target sites conserved in most of the genotypes (homology > 90% and more than 15 nucleotides). (3 )Transcripted HBV full genome RNA in vitro and some sites located in C region of HBV gene were examined by RNase H hydrolysis with anti-oligonucleotides (AODNs) to find out the sites which are accessible for the ribozyme. (4) The cDNA of HDV trans-acting ribozyme was designed and synthetized according to the reports of others and selected site of ourself, and recombinated with pGEM-4Z vector that confirmed by sequencing. The HDV ribozyme was synthetized by transcription under TV-promoter in vitro and its target RNA segment was made in the same way. Then the activity of the HDV ribozyme was tested in vitro. (5) The cDNA of tRNAVal promoter was dezigned and synthetized, and recombinated with pUC19 vector to construct a vector named ptV. Then the eukaryote expression vector ptVHRz was constructed by recombination of HDV ribozyme and ptV using PCR. (6) HDV ribozyme gene was cloned by PCR using ptVHRz as the template and recombinated with pcDNA3.0 to construct the eukaryote expression vector pcDHRz. (7 ) In the same way as ( 6 ) constructed the other eukaryote expression vector pSURz using pSilencer 1.0-U6 vector. ( 8 ) Cells transfection: HepG2.2.15 cells were planted into 24-well plate the day before transfection until the cells was 90-95% confluent and transfected as following groups: Lipofectamine, pSURz, ptVHRz, pcDHRz, pSURz + ptVHRz, pSURz + pcDHRz, pSilencer 1.0-U6 and pcDN...