Enhanced production of hepatitis B surface antigen in NIH 3T3 mouse fibroblasts by using extrachromosomally replicating bovine papillomavirus vector.

Abstract

We have constructed a recombinant pBR322 plasmid composed of a subgenomic transforming fragment of bovine papillomavirus DNA and the hepatitis B surface antigen gene from cloned hepatitis B virus DNA and used it for transfection of NIH 3T3 mouse fibroblasts. The transformed cells retain the plasmids in extrachromosomal form with a copy number of about 50 to 100 per cell. Expression of the hepatitis B surface antigen gene linked to bovine papillomavirus DNA is independent of its orientation relative to the bovine papillomavirus vector. Cell lines continuously secreting high amounts of hepatitis B surface antigen into the medium could be established. The antigen is released into the culture medium as 22-nm particles, having the same physical properties and constituent polypeptides as those found in the serum of hepatitis B virusinfected patients.

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

“…The production rate of HBsAg particles from MS 128 cells was 20 to 30 times higher than that of the well-known human hepatoma cell line PLC/PRF/5 (Macnab et al, 1976). Efficient production of HBsAg in mammalian cells (6 to 12 p.g/10 v cells/day) has been reported previously (Hsiung et al, 1984;Michel et al, 1984;Wang et al, 1983); the level of HBsAg production for MS128 cells was equivalent to, or higher than, those reported. The stable expression of HBsAg particles was maintained in MS128 cells passaged for at least 1 year (more than 400 generations).…”

“…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 Effect of Bovine Herpesvirus Type 1 Glycoproteins gI and gIII on Herpesvirus Infections

“…Although BPV vectors have been used to express the HSV-1 thymidine kinase gene (Lusky et al, 1983) and other surface proteins such as the envelope glycoprotein of human T cell leukaemia virus (Eiden et al, 1985), hepatitis B surface protein (Wang et al, 1983), and the influenza virus haemagglutinin (Sambrook et al, 1985), this represents the first expression of a herpesvirus glycoprotein gene using a BPV vector. The gI and glII proteins expressed by our transfected bovine cells were indistinguishable from native BHV-1 gI and glII by comparison of Mr and antigenic areas defined by all monoclonal antibodies used to date.…”

Comparative Expression of the Hepatitis B Surface Antigen Gene in Biochemically Transformed Human, Simian and Murine Cells

“…This expression in Veto cells was termed 'late transient' (Colbbre-Garapin et al, 1983) because its maximum occurs 3 to 14 days after transfection and then levels off. A similar kind of expression has been described in NIH 3T3 cells after transfection with a bovine papillomavirus vector carrying the HBs gene (Wang et al, 1983). Late transient expression is I Formerly known as Horodniceanu.…”

“…The expression of the human hepatitis B surface antigen (HBs Ag) has, however, been studied in vitro in some human hepatoma cell lines which spontaneously produce the HBs Ag (Alexander et al, 1976;Koike et al, 1983) and by gene transfer. Transient and/or stable expression have been developed using virus vectors (Moriarty et al, 1981;Liu et al, 1982;Stratowa et al, 1982;Crowley et al, 1983 ;Smith et al, 1983 ;Stenlund et al, 1983;Wang et al, 1983) or recircularized HBV DNA (Hirschman et al, 1980;Wang et al, 1982). Stable expression of the HBs Ag has been obtained in rodent cells following transfection with a selective marker (Dubois et al, 1980;Gough & Murray, 1982;Christman et al, 1982).…”