Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system

Nagy, Éva; Derbyshire, J.B.; Dobos, Peter; Krell, Peter J.

doi:10.1016/0042-6822(90)90012-g

Cited by 29 publications

(20 citation statements)

References 41 publications

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“…Virions are then formed by budding from the cell membrane, but the roles of the virion components in the initiation and control of the budding process are poorly understood, although the matrix protein is believed to be of major importance in this regard (Peeples, 1988). In our studies the matrix protein was lacking in the recombinant as shown by Western blot analysis with polyclonal antisera to NDV, and by the failure of antisera against extracts of cells infected with the recombinant to react with N D V proteins other than H N (Nagy et al, 1990). It is possible that a baculovirus protein such a s the 64K major envelope glycoprotein may substitute for the M protein by participating in the generation of the NDV-like envelopes, but there was no evidence of incorporation of baculovirus nucleocapsids within these envelopes.…”

Section: ~ Department Of Veterinary Microbiology and Immunology And Zmentioning

confidence: 73%

“…In previous studies in this laboratory (Nagy et al, 1990) the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV), an avian paramyxovirus, was cloned as a cDNA and inserted into a baculovirus expression vector. The baculovirus-HN recombinant expressed HN which was antigenically and electrophoretically similar to authentic HN, and had both haemagglutinating and neuraminidase biological activities.…”

Section: ~ Department Of Veterinary Microbiology and Immunology And Zmentioning

confidence: 99%

“…frugiperda cells as described (Nagy et al, 1990). The ECF was harvested after incubation of the cultures for 72 h, and virus was concentrated from the ECF by centrifugation at 100000 g for 30 min in a Beckman SW28 rotor.…”

Section: ~ Department Of Veterinary Microbiology and Immunology And Zmentioning

confidence: 99%

See 2 more Smart Citations

Synthesis of Newcastle disease virus (NDV)-like envelopes in insect cells infected with a recombinant baculovirus expressing the haemagglutinin-neuraminidase of NDV

Nagy¹,

Huber²,

Krell

et al. 1991

Journal of General Virology

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Section: ~ Department Of Veterinary Microbiology and Immunology And Zmentioning

confidence: 73%

Section: ~ Department Of Veterinary Microbiology and Immunology And Zmentioning

confidence: 99%

See 1 more Smart Citation

Synthesis of Newcastle disease virus (NDV)-like envelopes in insect cells infected with a recombinant baculovirus expressing the haemagglutinin-neuraminidase of NDV

Nagy¹,

Huber²,

Krell

et al. 1991

Journal of General Virology

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“…Glycosylation patterns (Martin et al, 1988) and processing of mitochondrial precursor proteins (Lithgow et al, 1991) appear to be species-specific for the insect cells. Most importantly, proteins are accurately targeted to the appropriate cellular compartment (Rice et al, 1987;Luchnow and Summers, 1988;Kang, 1988;Kurodaera/., 1990;Lithgow et al, 1991) in an enzymatically active form (Pennock et al, 1984;Nagy et al, 1990).…”

mentioning

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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“…The 263 bp HinfI fragment containing the promoter sequence was cut from a cloned 1.40 kb XbaI-BglIl fragment, blunt-ended with Klenow fragment and cloned into the Smal site of pGEM-7Z(+). The resultant 170 bp DraI-HindIII fragment was removed by digestion with HindIII (from the multiple cloning site in the plasmid) and DraL The 170 bp fragment was then cloned into pl8RHN (Nagy et aL, 1990) which was first digested with BamHI, blunt-ended with Klenow and then digested with ttindIII. The junction region between the L2R promoter and the HN gene in pL2RHN was verified by sequencing.…”

Section: Mapping and Northern Blot Analysis Of Highly Transcribed Framentioning

confidence: 99%

Partial transcriptional mapping of the fowlpox virus genome and analysis of the EcoRI L fragment

Jl¹,

Jb³

et al. 1996

Journal of General Virology

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Several fowlpox virus (FPV) DNA fragments were selected by differential hybridization using cDNA of transcripts that were strongly transcribed early and/or late after infection of QT-35 cells. The EcoRI L fragment contained three strongly transcribed FPV genes: L1L, a late 1452 bp partial (amino end) ORF; L2R, an early/late 522 bp ORF; and L3R, a late 948 bp ORF. The protein products of L1L, L2R and L3R shared homology with the products ofvaccinia virus ( W ) genes H4L (RAP94), H5R (Ag35) and H6R (topoisomerase), respectively, suggesting a conservation of gene structure and order between VV and FPV. The 5' upstream non-coding sequences of L1L and L3R were A + T rich and the sequence 5' TAAATG 3' overlapped the predicted translation start codon. Primer extension analysis of the L2R transcript mapped the transcriptional start sites of early and late mRNAs 14 nt downstream of a VV early promoter-like critical region sequence, AAAATTGAA-AAAAAAA. A VV-like TAAAT late transcriptional element was present 20 nt upstream of the L2R ATG translational start codon. A plasmid with the putative early L2R promoter cloned upstream of the Newcastle disease virus haemagglutinin-neuraminidase (HN) cDNA as a reporter gene was at least 6-fold more effective in generating HN mRNA than plasmids containing the P7.5 or P11 VV promoters in transient expression assays in FPV-infected CEF cells treated with cytosine arabinoside. The L2R promoter was also able to express an amount of HN mRNA equal to that expressed by the W promoters late in infection.

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Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system

Cited by 29 publications

References 41 publications

Synthesis of Newcastle disease virus (NDV)-like envelopes in insect cells infected with a recombinant baculovirus expressing the haemagglutinin-neuraminidase of NDV

Synthesis of Newcastle disease virus (NDV)-like envelopes in insect cells infected with a recombinant baculovirus expressing the haemagglutinin-neuraminidase of NDV

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

Partial transcriptional mapping of the fowlpox virus genome and analysis of the EcoRI L fragment

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