title: Bacterial synthesis of herpes simplex virus types 1 and 2 glycoprotein D antigens.
abstract: We have used elements of the E. coli lactose (lac) operon to produce a collection of herpes simplex virus types 1 and 2 glycoprotein D (gD-1 and gD-2) antigens. Our approach employed recombinant DNA techniques to construct plasmids with various segments of the gD-1 and gD-2 coding sequences fused to the lacZ gene. Such hybrid genes were expressed in a regulated manner in E. coli by joining them to the lac promoter-operator region. Efficient translation of these hybrid genes was facilitated by incorporating a coding sequence specifying a short peptide leader (lambda cro) in the plasmid expression vectors resulting in synthesis of chimeric Cro-gD-beta-galactosidase proteins. In addition, insertion of synthetic translation terminators at the junction of gD and lacZ enabled us to produce specific truncated gD polypeptide sequences unfused to beta-galactosidase. The gD antigens produced in E. coli were not glycosylated and were generally recovered as dense insoluble aggregates. Proteins containing portions of gD-1 or gD-2 were analyzed by immunoprecipitation using anti-HSV rabbit serum and a number of monoclonal antibodies recognizing different epitopes of gD-1. Initial animal studies were done with antigens that reacted with neutralizing antisera or monoclonal antibodies. When these bacterially produced proteins were injected into rabbits, antibodies were produced that specifically immunoprecipitated authentic gD polypeptides and neutralized the infectivity of both virus types. These studies suggest that gene fusion techniques can be used to produce immunogenic proteins in large quantity. These polypeptides are not only useful in analyses of gene structure and function, but also can provide novel diagnostic reagents and well-defined pure antigens for vaccine development.
mesh_terms: D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000235:genetics*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005814:Genes, Viral; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics*; D007763:Lac Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D018139:Simplexvirus / Q000235:genetics*
chemical_list: D000956:Antigens, Viral; D004274:DNA, Recombinant; D006023:Glycoproteins