Cloning and expression of the guinea pig cytomegalovirus glycoprotein B (gB) in a recombinant baculovirus: Utility for vaccine studies for the prevention of experimental infection

Mark R. Schleiss; Nancy J. Jensen

doi:10.1016/S0166-0934(02)00258-6

Cloning and expression of the guinea pig cytomegalovirus glycoprotein B (gB) in a recombinant baculovirus: Utility for vaccine studies for the prevention of experimental infection

Mark R. Schleiss, Nancy J. Jensen

Research output: Contribution to journal › Article › peer-review

14 Scopus citations

Abstract

The guinea pig cytomegalovirus (GPCMV) is unique among the cytomegaloviruses of small mammals, insofar as during pregnancy it crosses the placenta, causing infection of the fetus. Although the guinea pig model is well suited to vaccine studies, the lack of cloned, recombinant forms of immunogenic GPCMV proteins, such as envelope glycoproteins, has hindered experimental evaluations of subunit immunization for prevention of fetal disease. Since the glycoprotein B (gB) is a major target of neutralizing antibody responses, the GPCMV gB was cloned and expressed in a recombinant baculovirus. A recombinant was generated which expressed gB, truncated at codon 692, upstream of the putative transmembrane domain. Processing and expression of the recombinant protein, designated Bac-gB, was assessed, and the protein was characterized immunologically. Anti-gB antibodies were immunoreactive with Bac-gB by enzyme linked immunosorbent assay (ELISA) and immunoblot assay. Immunoprecipitation with polyclonal anti-GPCMV antisera identified protein species of 120, 80 and 30 kDa by reducing SDS-PAGE, suggesting that authentic cleavage and processing of Bac-gB occurred in insect cells. Sera from guinea pigs immunized with lectin-column purified native glycoproteins had high ELISA titers to Bac-gB. Recombinant GPCMV gB expressed in insect cells should prove useful in defining correlates of protective immunity in the GPCMV congenital infection model.

Original language	English (US)
Pages (from-to)	59-65
Number of pages	7
Journal	Journal of Virological Methods
Volume	108
Issue number	1
DOIs	https://doi.org/10.1016/S0166-0934(02)00258-6
State	Published - Mar 2003
Externally published	Yes

Bibliographical note

Funding Information:
The authors acknowledge the technical assistance of Leah Hamel and the gift of reagents and useful discussions from Teresa Compton, University of Wisconsin/Madison. The authors also acknowledge the gift of sera from Nigel Bourne. This work was supported by National Institutes of Health HD38416-01 and AI65289 and March of Dimes 6-FY98-0416 and FY01-226.

Keywords

Baculovirus
Cytomegalovirus vaccine
Glycoprotein B
Guinea pig cytomegalovirus

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title = "Cloning and expression of the guinea pig cytomegalovirus glycoprotein B (gB) in a recombinant baculovirus: Utility for vaccine studies for the prevention of experimental infection",

abstract = "The guinea pig cytomegalovirus (GPCMV) is unique among the cytomegaloviruses of small mammals, insofar as during pregnancy it crosses the placenta, causing infection of the fetus. Although the guinea pig model is well suited to vaccine studies, the lack of cloned, recombinant forms of immunogenic GPCMV proteins, such as envelope glycoproteins, has hindered experimental evaluations of subunit immunization for prevention of fetal disease. Since the glycoprotein B (gB) is a major target of neutralizing antibody responses, the GPCMV gB was cloned and expressed in a recombinant baculovirus. A recombinant was generated which expressed gB, truncated at codon 692, upstream of the putative transmembrane domain. Processing and expression of the recombinant protein, designated Bac-gB, was assessed, and the protein was characterized immunologically. Anti-gB antibodies were immunoreactive with Bac-gB by enzyme linked immunosorbent assay (ELISA) and immunoblot assay. Immunoprecipitation with polyclonal anti-GPCMV antisera identified protein species of 120, 80 and 30 kDa by reducing SDS-PAGE, suggesting that authentic cleavage and processing of Bac-gB occurred in insect cells. Sera from guinea pigs immunized with lectin-column purified native glycoproteins had high ELISA titers to Bac-gB. Recombinant GPCMV gB expressed in insect cells should prove useful in defining correlates of protective immunity in the GPCMV congenital infection model.",

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Cloning and expression of the guinea pig cytomegalovirus glycoprotein B (gB) in a recombinant baculovirus: Utility for vaccine studies for the prevention of experimental infection

Abstract

Bibliographical note

Keywords

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