The antigenicity of Varicella Zoster virus gpII studied using recombinant forms of the glycoprotein

Gittins, John Robert (1991) The antigenicity of Varicella Zoster virus gpII studied using recombinant forms of the glycoprotein. University of Southampton, Doctoral Thesis.

Abstract

The VZV gpII gene and a truncated form, gpIIt, were cloned into a baculovirus transfer vector, and recombinant viruses expressing these genes produced. Expression levels upon infection of insect cells were very low. The `anchor minus' form, gpIIt, was not secreted from the cells. The baculovirus system was apparently not suited to the production of recombinant gpII. It was concluded that the coding sequence for the gpII signal peptide had been omitted from the cloned gene due to an error in the published sequence. An initial attempt to express gpIIt in E. coli was unsuccessful, so fragments of the gpII gene encoding the external domain were amplified by PCR. A nested set of six gene fragments encoding polypeptides deleted from the N-terminus in 100 amino acid steps were produced. Three fragments were expressed at low level using a pTTQ vector. An antigenic region was identified within the polypeptide encoded by the second smallest fragment, PCR5. This reacted specifically upon Western immunoblotting with polyclonal rabbit and human antisera, and with six out of a panel of eight McAbs to gpII. High level expression of the gpII PCR fragments PCR5 and PCR6 was achieved using a modified pGEMEX^TM vector and the gpII antigenic region was mapped to within the 157 amino acid PCR6 polypeptide. Fusion protein inclusions were purified and the PCR6 polypeptide partially cleaved from the T7 gene 10 protein hybrid by acid hydrolysis of an Asp-Pro coupling bond. Unfortunately, the PCR6 polypeptide could only be retained in solution in conditions unsuited for antigenic analysis ie. below pH3 or in high concentrations of chaotropes. Subfragments of PCR6 for expression in E. coli were produced by deletion and PCR. Upon Western blotting of the gene 10 fusion proteins, a weak reaction was obtained, only with one half of the PCR6 polypeptide, by one McAb, 3,10, and rabbit anti-VZV serum. The other antisera failed to react with these smaller polypeptides. Attempts were made to precisely map the gpII antigenic site with synthetic solid-phase decapeptides, using an ELISA assay with different antisera. McAb clone 3,10 gave the only conclusively positive reaction, with peptides containing the sequence Threonine-Threonine-Arginine-Cysteine-Tyrosine. The gpII antigenic site identified in this study was destroyed when its coding sequence was expressed as separate halves, and epitopes comprising it could not be mapped using solid-phase decapeptides. It was concluded that either the site was conformation-dependent, or composed of large epitopes spanning the centre of the PCR6 polypeptide. This site is apparently highly antigenic in three mammalian species, and warrants further study.

This record has no associated files available for download.

Contributors

Download statistics