Baculovirus-directed high level expression of the hepatitis delta antigen in Spodoptera frugiperda cells

Kos, Ton; Molijn, Anco; Blauw, Bep; Schellekens, Huub

doi:10.1099/0022-1317-72-4-833

Cited by 13 publications

(6 citation statements)

References 20 publications

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“…However, during the course of natural infection with HDV, high titres of anti-HD of both IgM and IgG classes are regularly observed, and apart from possible differences in antigenic presentation to B cells, the lack of a humoral immune response observed in this study is not understood. Kos et al [1991], using recombinant HDAg expressed in a baculovirus system, induced a n anti-HD response in chimpanzees, which was, however, short lived and with antibody titres 105-fold lower than those following experimental infection with HDV.…”

Section: Discussionmentioning

confidence: 97%

Partial control of hepatitis delta virus superinfection by immunisation of woodchucks (Marmota monax) with hepatitis delta antigen expressed by a recombinant vaccinia or baculovirus

Karayiannis

Saldanha

Jackson

et al. 1993

Journal of Medical Virology

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We have successfully limited the level of hepatitis delta viraemia occurring after superinfection of hepadna-virus infected woodchucks by prior immunisation with the short form of the hepatitis delta virus antigen expressed by a recombinant baculovirus or vaccinia virus. This phenomenon occurred in the absence of detectable circulating antibody to hepatitis delta virus antigen and in the absence of evidence of priming of the humoral immune response and may reflect the induction of a cytotoxic T-cell response. The latter would control viraemia by rapid lysis of delta antigen expressing hepatocytes. It is suggested that the T-cell epitopes involved may be located on the carboxyl end of the delta protein (amino acids 77-195).

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Section: Discussionmentioning

confidence: 97%

Partial control of hepatitis delta virus superinfection by immunisation of woodchucks (Marmota monax) with hepatitis delta antigen expressed by a recombinant vaccinia or baculovirus

Karayiannis

Saldanha

Jackson

et al. 1993

Journal of Medical Virology

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“…It is possible that the original 3' untranslated region of the isoenzyme El cDNA did not have appropriate transcription stop or polyadenylation signals for the insect cell system (cf. Kos et al, 1991).…”

Section: Discussionmentioning

confidence: 95%

“…It was considered possible, therefore, that the transcription of the isoenzyme EI cDNA in the insect cells had resulted in a large and unstable mRNA transcript, consisting of nucleotide sequences of both the (1-3,1-4)-/3-glucanase isoenzyme EI and the viral polyhedrin (cf. Busto et al, 1988;Kos et al, 1991). Therefore, the 3' untranslated sequence of the isoenzyme El cDNA (378 bp) was replaced by the shorter sequence of the isoenzyme EII cDNA (175 bp), which included a poly(A) tail of 20 residues (Fig.…”

Section: Expression Of (1-3 L~4)-ß-glucanasesmentioning

confidence: 99%

Post-Translational Processing of Barley β-Glucan Endohydrolases in the Baculovirus–Insect Cell Expression System

Doan¹,

Høj²,

Collins³

et al. 1993

DNA and Cell Biology

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Two cDNAs encoding barley (1-->3,1-->4)-beta-glucanase (EC 3.2.1.73) isoenzymes EI and EII have been expressed in Spodoptera frugiperda (Sf9) cell cultures using the baculovirus AcNPV vector. Modifications to both the 5' and 3' ends of the cDNAs were required before satisfactory levels of expression were obtained. The modified cDNAs directed high levels of (1-->3,1-->4)-beta-glucanase expression in the Sf9 insect cell cultures, with yields of approximately 10 mg/liter of isoenzyme EI (expEI) and 15 mg/liter of isoenzyme EII (expEII). Amino acid sequence analyses showed that the expressed enzymes were processed correctly at their amino termini. However, affinity chromatography of the isoenzyme expEII on concanavalin-A (conA)-Sepharose indicated that, although the enzyme is glycosylated, the structures of the carbohydrate chains differ from those of the native enzyme. When a cDNA encoding the homologous barley (1-->3)-beta-glucanase (EC 3.2.1.39) isoenzyme GII was expressed in insect cells, aberrant amino-terminal processing of the nascent polypeptide was sometimes observed. The forms with incompletely removed signal peptides retained their substrate specificity, but exhibited slightly reduced catalytic efficiency, altered chromatographic behavior, and reduced stability at elevated temperatures. The results show that high levels of expression of recombinant plant proteins can be obtained in insect cells, but they emphasize the need to characterize thoroughly the products that are expressed in the heterologous insect cell system before comparisons are made with the native enzyme or with engineered enzyme mutants.

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“…In some cases, this heterogeneity has been attributed to the translation of two different HDV sequence variants corresponding to species of 195 and 214 amino acids, which often infect patients at the same moment [6,7]. The presence of more than a single-size product was also described for recombinant hepatitis 6 antigen products encoded by the 195-amino-acid sequence and was attributed either to proteolysis of a single precursor or to partial tRNA suppression of the termination code [4,8]. It is possible that both mechanisms occur, i.e.…”

Section: I I ~ K K D K D G E G a P P A K R A R T~h E~s G P R K R P mentioning

confidence: 99%

Characterization by mass spectrometry of a recombinant hepatitis δ virus antigen and its proteolytic products

Tecce¹,

Petracca²,

Giuliani³

et al. 1992

European Journal of Biochemistry

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The recombinant hepatitis 6 virus antigen was obtained as a chimaeric protein fused to the Cterminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i. e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of 6 antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis 6 antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35 -40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis 6 virus) antisera.Hepatitis 6 virus (HDV) is a defective single-stranded RNA virus which can only infect patients with acute or chronic infection by hepatitis B virus [l, 21. The hepatitis 6 antigen is essentially the only specific protein to be expressed as a consequence of HDV infection and which is immunogenic in infected patients [3]. It turned out that this protein is encoded by open reading frame (ORF) 5 in the viral genome and is specifically recognized by immune sera [4]. The diagnosis of hepatitis 6 virus infection is based both on the detection of the viral antigen and on the presence of the relative antibodies [l, 21. The availability of reproducible and safe preparations of the antigen is then necessary for the assembling of a clinical immunoassay for the detection of this viral disease. Viral preparations extracted from serum or liver tissue Now. The novel nucleotide sequence data published here have been submitted to the EMBL sequence data bank and are available under accession number X63373. The novel amino acid sequence data published here have been submitted to the EMBL sequence data bank. might be infective and yield a limited amount of material. To overcome these limitations, and to obtain from an unlimited and reproducible source a recombinant protein capable of substituting extractive antigens, the cloning and expression of the DNA sequence of hepatitis 6 antigen was undertaken.The recombinant protein was obtained together with several degradation products, similar to extracted hepatitis 6 antige...

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Baculovirus-directed high level expression of the hepatitis delta antigen in Spodoptera frugiperda cells

Cited by 13 publications

References 20 publications

Partial control of hepatitis delta virus superinfection by immunisation of woodchucks (Marmota monax) with hepatitis delta antigen expressed by a recombinant vaccinia or baculovirus

Partial control of hepatitis delta virus superinfection by immunisation of woodchucks (Marmota monax) with hepatitis delta antigen expressed by a recombinant vaccinia or baculovirus

Post-Translational Processing of Barley β-Glucan Endohydrolases in the Baculovirus–Insect Cell Expression System

Characterization by mass spectrometry of a recombinant hepatitis δ virus antigen and its proteolytic products

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