Expression of avian leukaemia virus env-gp85 in Spodoptera frugiperda cells by use of a baculovirus expression vector

Noteborn, Mathieu H. M.; Boer, G. F. de; Kant, A.; Koch, G.; Bos, Johannes L.; Zantema, A.; Eb, A. J. Van Der

doi:10.1099/0022-1317-71-11-2641

Cited by 14 publications

(12 citation statements)

References 33 publications

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“…Sf9 cells were transfected with mixtures of 1 µg purified linearized AcRP23-lacZ DNA and 5 µg pUW-VP1\VP2 by the calcium phosphate method of Graham & Van der Eb (1973) as modified by Smith et al (1983). The recombinant-VP1\VP2 baculoviruses were selected as described by Noteborn et al (1990).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope.

Noteborn

Verschueren

Koch

et al. 1998

Journal of General Virology

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Chicken anaemia virus (CAV) expresses three proteins, VP1, VP2 and VP3, but its capsid contains only the VP1 protein. In this paper, we report that for production of the neutralizing epitope, cosynthesis of (recombinant) VP1 and VP2 has to take place. We show via immunofluorescence that recombinant-baculovirus-infected Sf9 cells synthesizing VP1 (or VP2) alone react very poorly with CAVspecific neutralizing antibodies. In contrast, Sf9 cells co-infected with VP1-and VP2-recombinant baculo-

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Section: Methodsmentioning

confidence: 99%

Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope.

Noteborn

Verschueren

Koch

et al. 1998

Journal of General Virology

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“…2). All the proteins that migrated faster than native gp51 could represent incomplete and/or incorrectly glycosylated molecules, as is frequently observed in insect cells (16,19) and different mammalian cell lines (1,26).…”

Section: Discussionmentioning

confidence: 99%

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

Giuseppe

Feliziani

Rutili

et al. 2004

Clin Vaccine Immunol

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The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH 2 -terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.Bovine leukemia virus (BLV), the etiological agent of enzootic bovine leukosis (EBL), is a C-type retrovirus of cattle with a worldwide distribution (5). The virus induces a persistent lymphocytosis and in some cases lymphoid tumors in domestic cattle.The BLV envelope (Env) glycoprotein, which consists of the gp51 outer membrane glycoprotein and the gp30 transmembrane glycoprotein, is directly involved in infectivity events and, like the p24 major structural protein, can elicit a strong immune response in infected cattle (23). Serological diagnosis of EBL is mainly based on screening of field samples for gp51 antibodies. It follows that diagnostic procedures that use native gp51 constitute a prerequisite for the design of an efficient EBL eradication program. In recent decades different serological methods, such as the agar gel immunodiffusion test (AGID) and the enzyme-linked immunosorbent assay (ELISA), have been developed. ELISAs are based on the use of partially purified BLV gp51 and monoclonal antibodies (MAbs) against BLV gp51 epitopes (4, 17). These procedures are particularly useful for samples with low antibody titers, such as milk samples or pooled sera. A permanent fetal lamb kidney (FLK) cell line chronically infected with BLV (FLK-BLV) is used to produce gp51. Although highly productive BLV-infected cell lines are available, their production is rather laborious, expensive, and time-consuming.In recent decades different investigators have described the expression of the BLV Env glycoproteins in heterologous expression systems such as Escherichia coli (2, 21, 22), Saccharomyces cerevisiae (11), and recombinant vaccinia virus (10, 18) systems and, recently, a baculovirus system (9, 19). However, the use of recombinant gp51 (rgp51) in an ELISA method has not yet been described. The aim of this paper is therefore to describe the production of secreted rgp51 by recombinant baculovirus in insect cells and its use in an ELISA for the detection of BLV antibodies. MATERIALS AND METHODS Cells and viruses.BLV-infected and uninfected FLK cells (National Veterinary Laborat...

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“…No sialylation was observed Kuroda et al, 1990;Noteborn et al, 1990). Complex carbohydrate structures were only described by Castellino and his coworkers (Davidson et al, 1990(Davidson et al, , 1991Davidson and Castellino, 1991 a,b).…”

mentioning

confidence: 90%

Expression of human interferon‐α2 in Sf9 cells

Sugyiama

Ahorn²,

Maurer‐Fogy³

et al. 1993

European Journal of Biochemistry

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Human interferon a2 (IFN-a2) was expressed in Spodopteru frugiperdu Sf9 insect cells using the baculovirus expression system. The protein purified by immunoaffinity chromatography exhibited biological activity identical to that of leukocyte-derived 'natural' IFN-a2. However, the protein was found to be heterogeneously glycosylated, partially truncated by proteolysis and partially lacking a disulfide bridge. The major product was shown to be 0-glycosylated at the same position as natural human IFN-a2. Enzymatic cleavage, reverse-phase HPLC peptide mapping and plasma-desorption mass spectroscopy analysis revealed the presence of two types of 0-linked carbohydrates. The major 0-linked carbohydrate was found to be the disaccharide galactosyl(/31-3)-N-acetylgalactosamine, the minor component the monosaccharide N-acetylgalactosamine. No evidence for sialylation was found. The non-glycosylated species representing about 40% of the total material were shown to partially lack the C-terminal three amino acids. In addition an unglycosylated, reduction-sensitive dimer was observed. This was formed due to the lack of the N-terminal cysteine normally forming an intramolecular disulfide bridge. Furthermore, a minor species was identified which contains Cysl and Cys98 in a modified form, thereby hindering the formation of a disulfide bridge between these two residues.

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Expression of avian leukaemia virus env-gp85 in Spodoptera frugiperda cells by use of a baculovirus expression vector

Cited by 14 publications

References 33 publications

Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope.

Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope.

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

Expression of human interferon‐α2 in Sf9 cells

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