The expression of recombinant varicella-zoster virus glycoproteins gpII and gpIII
The expression of recombinant varicella-zoster virus glycoproteins gpII and gpIII
Recombinant varicella-zoster virus (VZV) glycoproteins gpII and gpIII were expressed with the aim of evaluating their potential as components of a subunit vaccine or as diagnostic antigens. The glycoprotein genes were amplified by the polymerase chain reaction from the SGH strain fo VZV and the fidelity of the PCR products was confirmed by DNA sequencing. The genes were cloned into an SV40-based expression vector, pMAMneo, for transfection of COS-1 and Chinese hamster ovary (CHO-K1) cells and into an EBV-based vector, pREP9, for transfection of human embryonal kidney (293) cells. A subpopulation of 293 cells stably transfected with gpII developed an altered morphology, becoming fragile in appearance and seeming to fuse to form syncytia, which resulted in cell death. When examined by indirect immunofluorescence using anti-gpII MAbs as the primary probes, fluorescence was detected in association with the membranes of these cells. The implication is that VZV gpII is a cytotoxic glycoprotein. Indirect immunofluorescence and immunoblotting studies employing two anti-gpIII neutralizing antibodies (MAbs) failed to detect expression of VZV gpIII in any of the transfected mamalian cell lines, although the gpIII gene was shown by PCR to be present in stably transfected CHO-K1 cells.
An antigentic region of gpII was expressed in two fusion protein expression systems in E. coli and evaluated as a diagnostic antigen for determination of VZV immune status. This non-glycosylated product was inadequate for differentiating between VZV-positive and -negative sera in an ELISA, presumably because the tertiary structure of gpII recognized by antibodies in natural infection is dependent upon glycosylation for its formation.
The gpII and gpIII genes were cloned into a baculovirus transfer vector and co-transfected with linear baculovirus DNA into insect cells. Low level expression of recombinant VZV gpII was achieved.
University of Southampton
Douglas, Joanne Tracy
6c27ab1d-885b-4f39-a569-447d266fe3c6
1994
Douglas, Joanne Tracy
6c27ab1d-885b-4f39-a569-447d266fe3c6
Douglas, Joanne Tracy
(1994)
The expression of recombinant varicella-zoster virus glycoproteins gpII and gpIII.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Recombinant varicella-zoster virus (VZV) glycoproteins gpII and gpIII were expressed with the aim of evaluating their potential as components of a subunit vaccine or as diagnostic antigens. The glycoprotein genes were amplified by the polymerase chain reaction from the SGH strain fo VZV and the fidelity of the PCR products was confirmed by DNA sequencing. The genes were cloned into an SV40-based expression vector, pMAMneo, for transfection of COS-1 and Chinese hamster ovary (CHO-K1) cells and into an EBV-based vector, pREP9, for transfection of human embryonal kidney (293) cells. A subpopulation of 293 cells stably transfected with gpII developed an altered morphology, becoming fragile in appearance and seeming to fuse to form syncytia, which resulted in cell death. When examined by indirect immunofluorescence using anti-gpII MAbs as the primary probes, fluorescence was detected in association with the membranes of these cells. The implication is that VZV gpII is a cytotoxic glycoprotein. Indirect immunofluorescence and immunoblotting studies employing two anti-gpIII neutralizing antibodies (MAbs) failed to detect expression of VZV gpIII in any of the transfected mamalian cell lines, although the gpIII gene was shown by PCR to be present in stably transfected CHO-K1 cells.
An antigentic region of gpII was expressed in two fusion protein expression systems in E. coli and evaluated as a diagnostic antigen for determination of VZV immune status. This non-glycosylated product was inadequate for differentiating between VZV-positive and -negative sera in an ELISA, presumably because the tertiary structure of gpII recognized by antibodies in natural infection is dependent upon glycosylation for its formation.
The gpII and gpIII genes were cloned into a baculovirus transfer vector and co-transfected with linear baculovirus DNA into insect cells. Low level expression of recombinant VZV gpII was achieved.
This record has no associated files available for download.
More information
Published date: 1994
Identifiers
Local EPrints ID: 458552
URI: http://eprints.soton.ac.uk/id/eprint/458552
PURE UUID: 01854d7d-a1c2-4d2b-97f6-429a919d1fd1
Catalogue record
Date deposited: 04 Jul 2022 16:51
Last modified: 22 Feb 2023 18:54
Export record
Contributors
Author:
Joanne Tracy Douglas
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics