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.

Equine Herpesvirus Type 1 Unique Short Fragment Encodes Glycoproteins with Homology to Herpes Simplex Virus Type 1 gD, gI and gE

“…A transmembrane segment from residues 356 to 372 was predicted as a membrane anchor element. Comparison of the amino acid sequence of the putative EHV-1 ORF 2-encoded protein with proteins in the Swissprot database revealed significant homology with HSV-1 gD (McGeoch et al, 1985;Watson et al, 1982) and pseudorabies virus (PRV) gp50 (Petrovskis et al, 1986a) glycoproteins (Table 2). The third ORF (ORF 3) extends from nucleotide positions 2287 to 3558.…”

Antigenic and Mutational Analyses of Herpes Simplex Virus Glycoprotein B Reveal Four Functional Regions

“…Cells were incubated at 37°C for an additional 24 h, fixed with methanol and acetone (2:1 [vol/vol]) for 30 min, and air dried. Plaques were stained by using a cocktail of polyclonal antibody (PAb) to gB, gC, gD, and gH/gL, followed by protein A-HRP and substrate, and then counted (47). The neutralization titer was measured as a 50% plaque reduction.…”

Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli

“…The firA200(Ts) protein was partially purified from a culture of ID18 containing pID4 by making an aggregate protein preparation (29) (23). The best results were obtained with plasmid DNA that had been purified by two equilibrium density gradient centrifugations, as previously described (28 [firA200(Ts) rpoB] and ID21 (rpoB).…”