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.
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.
Schleiss
Mark R
MR
Division of Infectious Diseases, Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. schlm0@chmcc.org
Jensen
Nancy J
NJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology; D003586:Cytomegalovirus Infections / Q000276:immunology / Q000517:prevention & control; D017931:DNA Primers / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006168:Guinea Pigs; D007313:Insecta; D008297:Male; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D014614:Vaccines, Synthetic / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology; D014765:Viral Vaccines / Q000235:genetics*; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C040495:glycoprotein B, Simplexvirus