Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1983;2(5):669–673. doi: 10.1002/j.1460-2075.1983.tb01482.x

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

A Stenlund, D Lamy, J Moreno-Lopez, H Ahola, U Pettersson, P Tiollais
PMCID: PMC555167  PMID: 6641713

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.

Full text

PDF
669

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Binétruy B., Meneguzzi G., Breathnach R., Cuzin F. Recombinant DNA molecules comprising bovine papilloma virus type 1 DNA linked to plasmid DNA are maintained in a plasmidial state both in rodent fibroblasts and in bacterial cells. EMBO J. 1982;1(5):621–628. doi: 10.1002/j.1460-2075.1982.tb01218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brawerman G., Mendecki J., Lee S. Y. A procedure for the isolation of mammalian messenger ribonucleic acid. Biochemistry. 1972 Feb 15;11(4):637–641. doi: 10.1021/bi00754a027. [DOI] [PubMed] [Google Scholar]
  3. Crosnier J., Jungers P., Couroucé A. M., Laplanche A., Benhamou E., Degos F., Lacour B., Prunet P., Cerisier Y., Guesry P. Randomised placebo-controlled trial of hepatitis B surface antigen vaccine in French haemodialysis units: I, Medical staff. Lancet. 1981 Feb 28;1(8218):455–459. doi: 10.1016/s0140-6736(81)91847-x. [DOI] [PubMed] [Google Scholar]
  4. Dubois M. F., Pourcel C., Rousset S., Chany C., Tiollais P. Excretion of hepatitis B surface antigen particles from mouse cells transformed with cloned viral DNA. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4549–4553. doi: 10.1073/pnas.77.8.4549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  6. Hirschman S. Z., Price P., Garfinkel E., Christman J., Acs G. Expression of cloned hepatitis B virus DNA in human cell cultures. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5507–5511. doi: 10.1073/pnas.77.9.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lowy D. R., Dvoretzky I., Shober R., Law M. F., Engel L., Howley P. M. In vitro tumorigenic transformation by a defined sub-genomic fragment of bovine papilloma virus DNA. Nature. 1980 Sep 4;287(5777):72–74. doi: 10.1038/287072a0. [DOI] [PubMed] [Google Scholar]
  8. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  9. MACPHERSON I., MONTAGNIER L. AGAR SUSPENSION CULTURE FOR THE SELECTIVE ASSAY OF CELLS TRANSFORMED BY POLYOMA VIRUS. Virology. 1964 Jun;23:291–294. doi: 10.1016/0042-6822(64)90301-0. [DOI] [PubMed] [Google Scholar]
  10. Morein B., Helenius A., Simons K., Pettersson R., Käriäinen L., Schirrmacher V. Effective subunit vaccines against an enveloped animal virus. Nature. 1978 Dec 14;276(5689):715–718. doi: 10.1038/276715a0. [DOI] [PubMed] [Google Scholar]
  11. Moriarty A. M., Hoyer B. H., Shih J. W., Gerin J. L., Hamer D. H. Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2606–2610. doi: 10.1073/pnas.78.4.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Persson H., Jansson M., Philipson L. Synthesis and genomic site for an adenovirus type 2 early glycoprotein. J Mol Biol. 1980 Feb 5;136(4):375–394. doi: 10.1016/0022-2836(80)90396-4. [DOI] [PubMed] [Google Scholar]
  13. Pourcel C., Louise A., Gervais M., Chenciner N., Dubois M. F., Tiollais P. Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA. J Virol. 1982 Apr;42(1):100–105. doi: 10.1128/jvi.42.1.100-105.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  15. Sarver N., Gruss P., Law M. F., Khoury G., Howley P. M. Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector. Mol Cell Biol. 1981 Jun;1(6):486–496. doi: 10.1128/mcb.1.6.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  17. Steffen D., Weinberg R. A. The integrated genome of murine leukemia virus. Cell. 1978 Nov;15(3):1003–1010. doi: 10.1016/0092-8674(78)90284-2. [DOI] [PubMed] [Google Scholar]
  18. Szmuness W., Stevens C. E., Harley E. J., Zang E. A., Oleszko W. R., William D. C., Sadovsky R., Morrison J. M., Kellner A. Hepatitis B vaccine: demonstration of efficacy in a controlled clinical trial in a high-risk population in the United States. N Engl J Med. 1980 Oct 9;303(15):833–841. doi: 10.1056/NEJM198010093031501. [DOI] [PubMed] [Google Scholar]
  19. Tiollais P., Charnay P., Vyas G. N. Biology of hepatitis B virus. Science. 1981 Jul 24;213(4506):406–411. doi: 10.1126/science.6264599. [DOI] [PubMed] [Google Scholar]
  20. Valenzuela P., Medina A., Rutter W. J., Ammerer G., Hall B. D. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature. 1982 Jul 22;298(5872):347–350. doi: 10.1038/298347a0. [DOI] [PubMed] [Google Scholar]
  21. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES