HBx-DNA probe preparation and its application in study of hepatocarcinogenesis

AIM:To study the role of HBV especially HBx Open Reading Frame (ORF) in the development of hepatocellular carcinoma (HCC).METHODS:HBV 3.2kb fragment was retrieved by digesting recombinant plasmid pBR(322)-2HBV with EcoR II, and HBx 0.59kb fragments by digesting HBV-DNA with BamH I and Bgl II. These fragments were labelled with digoxigenin to get HBV-DNA and HBx-DNA probes. HBV-DNA was detected in HCC by dot blot and Southern blot hybridization with HBV-DNA probe, so the positive specimens in which HBV-DNA were integrated were selected. HBx-DNA was subsequently detected in the selected specimens with HBx-DNA probe.RESULTS:HBV-DNA was detected in 75% HCC, among which integrated type, integrated + free type covered 63.6% and 36.4%. There was no free type. HBx-DNA was detected in 90.5% specimens of integrated type.CONCLUSION:Hepatocarcinogenesis was highly related to HBV-DNA integration, and HBV-DNA mainly integrated into chromosome with incomplete virus DNA fragments among which HBx fragment was the predominant one.