Secretion of the hepatitis B virus surface antigen from mouse cells using an extra-chromosomal eucaryotic vector.

Abstract

Recombinant DNA molecules which contained a subgenomic fragment of the hepatitis B virus (HBV) genome, the pML2 vector and the bovine papillomavirus type 1 (BPV) genome were constructed. The HBV fragment includes the entire transcription unit for the hepatitis B surface antigen (HBsAg). After propagation in Escherichia coli, the recombinant plasmids were cleaved with endonucleases SalI and PvuI to eliminate most of the bacterial sequences before transfection of mouse C127 cells. Foci were observed 10–14 days after transfection. Cells from selected foci were cloned and the supernatants were assayed for the presence of HBsAg. Most of the clones tested were found to secrete HBsAg particles into the growth medium. These particles appear to be similar to the 22 nm particles present in the serum of HBV chronic carriers. SDS‐polyacrylamide gel electrophoresis revealed that the particles contain two polypeptides, probably representing the glycosylated and unglycosylated forms of the HBsAg major polypeptide. An analysis of DNA from the transformed clones revealed that they contain multiple extra‐chromosomal copies of the recombinant, which, however, had suffered rearrangement.

The gene S promoter of hepatitis B virus confers constitutive gene expression

“…The similar sizes of the transcripts encoded by the plasmids pHI and pHDI suggest the existence of a splice in the mRNA, of about 300 bp and encompassing the BglII-EcoRI fragment in the pre-S region. This is in agreement with recent observations of Stenlund et al (21). Using the Si mapping technique these authors have observed that a splicing event takes place during the HBsAg mRNA maturation and have localized an acceptor site at position 3160.…”

Stable Expression of the Hepatitis B Virus Surface Antigen Containing Pre-S2 Protein in Mouse Cells Using a Bovine Papillomavirus Vector

“…Taking advantage of the episomal nature of the viral genome, bovine papillomavirus (BPV) DNA has been used as a vector to express the HBsAg gene in mouse cells (Denniston et al, 1984;Hsiung et al, 1984;Stenlund et al, 1983;Wang et al, 1983). Expression is, however, sometimes unstable in the cell cultures after a long passage.…”

The bovine papillomavirus genome and its uses as a eukaryotic vector

“…Coding information required for transformation must therefore be contained in this region and ORFs from this region are referred to as early (E) ORFs [11]. Five RNA species all mapping to this region were found in transformed mouse cells in culture [12]. RNA transcripts which hybridized with the non-transforming 31 % region were only found in tumours and fibropapillomas [8,13].…”