Expression in Escherichia coli of hepatitis B virus DNA sequences cloned in plasmid pBR322

Burrell, C. J.; Mackay, Patricia; Greenaway, Peter; Hofschneider, Peter Hans; Murray, Kenneth

doi:10.1038/279043a0

Cited by 232 publications

(96 citation statements)

References 24 publications

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“…After the 24-h chase period, the presence of this protein within the cells was diminished (Fig. 6A, lane d (7,42) and HBsAg (15,57), although the coding capacity of the genome is clearly sufficient to specify additional polypeptides (15,57,58). In addition, the mechanisms of synthesis, glycosylation, assembly, and secretion of HBsAg are poorly understood.…”

Section: Resultsmentioning

confidence: 99%

“…This ignorance is largely a consequence of the inability of HBV to propagate in any tissue culture system or to infect convenient experimental animals (for reviews, see references 47 and 55). Recently, the 3,200-base pair (bp) viral genome has been molecularly cloned from infectious virus isolated from serum (8,50), and the determination of the complete nucleotide sequence of the cloned DNA has provided definitive evidence that at least two viral antigens are encoded by the genome of the virus (7,15,42,57). These include the core antigen, a basic polypeptide of 19,000 daltons which represents the major viral nucleocapsid protein, and the surface antigen (HBsAg).…”

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confidence: 99%

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Plasmid-directed synthesis of hepatitis B surface antigen in monkey cells.

Crowley¹,

Liu²,

Levinson³

1983

Mol. Cell. Biol.

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We introduced the gene encoding the hepatitis B virus surface antigen (HBsAg) into simian virus 40 (SV40)-based plasmids capable of autonomously replicating in both Escherichia coli and permissive monkey cells. After introduction into monkey cells by transfection, these plasmids directed the synthesis of high levels of HBsAg, as determined by immunofluorescence, radioimmunoassays, and identification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides comprising the antigen. Expression was dependent upon the presence of an SV40 promoter, with both the early and late promoters able to effectively initiate transcription. Using expression of HBsAg to assay promoter function, we demonstrated that an intact copy of the SV40 72-base pair repeat, which constitutes an essential element of the SV40 early promoter during the lytic SV40 cycle and which can enhance the transcriptional activity of heterologous promoters, was not required for HBsAg expression, suggesting that the hepatitis genome contains an enhancer element capable of complementing that provided by the 72-base pair repeat element of SV40. The antigen appears to be glycosylated after synthesis in transfected cells and is apparently secreted, as evidenced by the localization of [35S]cysteine-labeled antigen to the medium of transfected cultures.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Plasmid-directed synthesis of hepatitis B surface antigen in monkey cells.

Crowley¹,

Liu²,

Levinson³

1983

Mol. Cell. Biol.

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“…Purified, formaldehyde-treated antigen, isolated from human plasma, is an effective vaccine for the prevention of hepatitis B infection (7). Recently, the HBsAg gene has been cloned in yeast (8)(9)(10)(11) and a vaccine has been made that is safe and immunogenic in humans (12,13) and is effective in preventing hepatitis B infection in chimpanzees (14).…”

Section: Introductionmentioning

confidence: 99%

Multiple chemical forms of hepatitis B surface antigen produced in yeast.

Wampler¹,

Lehman²,

Boger³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis B surface antigen (HBsAg) has been extracted from yeast cells that produce HBsAg. These cells contain the gene for surface antigen carried on a plasmid that replicates in the cells. Analysis of the yeast-derived HBsAg by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis shows that the antigen that is initially released from yeast cells is a high molecular weight aggregate of the fundamental Mr 25,000 subunit. Unlike HBsAg derived from human plasma, the yeast antigen is held together by noncovalent interactions and can be dissociated in 2% NaDodSO4 without the use of reducing agents. During in vitro purification of the yeast antigen, some disulfide bonds form spontaneously between the antigen subunits, resulting in a particle composed of a mixture of monomers and disulfide-bonded dimers. Treatment with 3 M thiocyanate converts the 20-nm particles into a fully disulfide-bonded form that is not disrupted in NaDodSO4 unless a reducing agent is added. This disulfidebonded particle resembles the naturally occurring, plasmaderived surface antigen particle, and the in vitro formed particle has been used to prepare a vaccine for humans against hepatitis B virus infection.

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“…The plasmids used in the study for either labelling (by nick translation) or cRNA synthesis in vitro were pBR 322 and its derivative, pHBV i38 which contains a I94O nucleotide-long HBV DNA fragment (Pasek et al I979). The isolation of the plasmid has been described earlier (Burrell et al 1979).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus (HBV)-specific Structures found in Cytoplasmic Extracts of Cells producing HBV Surface Antigen (HBsAg) in vitro

Zaslavsky

Marquardt

Wong

et al. 1980

Journal of General Virology

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SUMMARYTwo kinds of hepatitis B virus-specific particles are present in cytoplasmic extracts of hepatoma cells synthesizing hepatitis B virus (HBV) surface antigen, One class of particle contains the surface antigen of the virus, is 2oS in size and has a buoyant density (in CsCI) of 1.2 g/ml. The second class of particle is a deoxyribonucleoprotein (DNP) with t. 3 g/ml buoyant density (in CsC1) and is 3oS in size, the DNA of which contains HBV sequences thus proving virus specificity.

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Expression in Escherichia coli of hepatitis B virus DNA sequences cloned in plasmid pBR322

Abstract: Fragments of hepatitis B virus DNA isolated from Dane particles have been inserted into the Escherichia coli plasmid pBR322 and cloned. Cells carrying the hybrid plasmid synthesise antigenic material that reacts specifically with antisera to hepatitis B viral antigens.

Cited by 232 publications

References 24 publications

Plasmid-directed synthesis of hepatitis B surface antigen in monkey cells.

Plasmid-directed synthesis of hepatitis B surface antigen in monkey cells.

Multiple chemical forms of hepatitis B surface antigen produced in yeast.

Hepatitis B Virus (HBV)-specific Structures found in Cytoplasmic Extracts of Cells producing HBV Surface Antigen (HBsAg) in vitro

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