Identification And Verification Of The Potential Targets For Anti-HBV Drugs

Objective: Hepatitis B Virus (HBV) infection is a global problem, and our country is recognized as one of the Hepatitis B Virus infection highly popular area. Along with the development of research for Hepatitis B Virus structure and function, the research of targeting to the complete virus gradually attracted people's attention. The crux of the matter, limited the development of anti-HBV drugs, is due to the smaller viral genomes and the higher mutation. So the researchers suggest a idea that inhibiting host-cell proteins can prevent viral infection. The human genomes might contain more genes related to HBV infection and replication in comparing with the viral genome. Some research has shown that inhibiting these host of gene expression related to HBV replication can block HBV virus infection. Therefore, the aim of this study is to identify and validate the new potential cellular targets of viruses and host for anti-HBV drugs by using antisense oligonucleotide microarray technology.Methods:We design several antisense oligodeoxynucleotide targeting to the virus Through bioinformatics analysis. Meanwhile we construct a gene network related to HBV replication,basing on the prediction of bioinformatics and combining with the virus and host interaction network. According to the various genes nucleic acid sequence of mRNA in GeneBank,we develop computer aided design based on the secondary structure prediction and get 5 of antisense oligodeoxynucleotides targeting the above all genes. We screening and validation of the influence of HBV replication by semi-quantitative RT-PCR and western blot technology in the HepG2.2.15 cell model.Results: We have found 3 Virus targeted antisense oligodeoxynucleotides owning the activity of anti-HBVand two potential HBV drug host targets associated with HBV replication.The results of activity screening for Viruses targeting of antisense oligodeoxyn- ucleotides show that the sequence of self1820, self1852 and self2057 can obviously inhibit the levels of HBeAg and HBsAg in HepG2.2.15. The cell toxicity testing experiments show that antisense sequence self1820, self1852 and self2057 do not affect the HepG2.2.15 cell proliferation while the drug concentrationis in the range of 0.2 to 6.4μmol/L.The results of activity screening for host targeting of antisense oligodeoxynu- cleotides show that the sequence of COX6A1-2 and PPARα-2 can obviously inhibit the levels of HBeAg and HBsAg in HepG2.2.15 and present good dose dependent relationship. Meanwhile COX6A1-2 and PPARα-2 can also specificity and dose- dependent inhibit the expression of mRNA and protein in HepG2.2.15. The above results preliminarily determine the COX6A1 and PPARαmay become resistant HBV new targets.Conclusions:Collectively,these results show that antisense sequence self1820, self1852 and self2057 targeted HBV have good activity for anti-HBV and while preliminarily determine the COX6A1 and PPARαmay become resistant HBV new targets.