| Hepatitis B virus (HBV) is a noncytopathic, enveloped, double-stranded DNA virus that causes acute hepatitis, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Human hepatitis B virus is the prototype for a family of viruses, referred to as Hepadnaviridae. It has at least 4 subtypes, ayw, adr, ayr, and adw, among which subtype adr is the most prevailing in China. The complete genomic DNA of subtype adr has been cloned in China. It is only 3.2kbp in length and different from the other 3 subtypes in DNA and protein sequence. Human hepatitis B virus encodes 4 overlapping reading frames. Among them HBx gene, which spans nucleotide positions 1374 to 1838 (465bp in length) in the viral genome, is essential for HBV infection and replication. It encodes a 17-kDa (154 amino acid) protein that has been attributed to a number of functions including transcriptional transactivation of viral and cellular genes, binding to p53, stimulation of signal transduction pathways, inhibition of nucleotide excision DNA repair, and involving in cell cycle and apoptosis. However, the physiological role of X protein during the course of HBV infection remains a major unresolved issue in hepadnavirus biology.In the present study, we constructed HBx gene expression vector pcDNA3-HBx, containing CMV promoter and HBx gene open reading frame by recombination DNA technique. It was identified by restriction endonucleases digestion and confined by DNA direct sequencing. The sequence of HBx gene was coincident with the reported HBx gene sequence. After restriction enzyme Salâ… digestion, the coding elements were microinjected into male pronuclei of mice zygotes. Following microinjection, the embyros were transferred to oviducts of psedopregnant females. 11 pups were born and survived. 5 of them were verified to integrate the HBx gene in their genomic DNA by multiplex PCR assay, named C57-TgN(HBx)SMMU. They expressed 17kDa X protein in liver tissue by Western blotting assay. With immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of C57-TgN(HBx)SMMU transgenic mice. Then the transgenic mice were mated to normal mice to establish transgenic mice strains. Now one of them had F4 offspring. We also analyzed the phenotype of the transgenic offspring. Histopathological analysis found the liver and lung of the transgenic mice developed gently degeneration. And X protein was expressed in the liver, spleen, lung and renal. In liver it was expressed in cytoplasm and/or nucleus of hepatocytes.In the present study, we also studied the relationship between X protein andapoptosis in vitro and in vivo. In vitro, the Hela cells, transfected with pcDNA3-HBx plasmids, resulted in typical apoptosis phenomena. Apoptosis peak appeared in these cells by Flow cytometer and the apoptotic percent in these cells was higher then control cells. In vivo, the C57-TgN(HBx)SMMU transgenic mice hepatocytes also appeared higher apoptotic percent than normal mice hepatocytes. These indicated that X protein could induce apoptosis and may be used to study the mechanism of X protein-induced apoptosis.Finally, we studied the pathogenicity of X protein by DNA-medicated immunization. The mice, immunized with pcDNA3-HBx plasmids, had abnormal renal. And the immunocomplex was deposited on glomerular basement membrane of immunized mice.
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