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Peptide heterogeneity of the C-protein alpha antigen of group B Streptococcus

by Kling, David Eric

Abstract (Summary)
The C-protein alpha and beta antigens of group B Streptococcus (GBS) are protective, surface-associated proteins. These potential virulence factors are candidates for use in a GBS conjugate vaccine. The genes encoding the alpha (bca) and beta (bcb) antigens had previously been cloned and expressed in Escherichia coli. Both the native and the cloned bca gene products are expressed as laddering peptides. This dissertation describes the characterization of bca and bcb gene clones (pJMS23 and pJMS1), the localization of two protective epitopes within the alpha antigen, the elucidation of the alpha antigen laddering mechanism, and the identification and characterization of spontaneous alpha antigen mutants in GBS. The bca gene was localized in a subclone of pJMS23 and the bcb gene was localized in pJMS1, to facilitate molecular characterization of these genes. Previous studies of the recombinant bcb gene product from pJMS1, indicated that the entire gene was not present. To obtain the full-length bcb gene, another bcb gene clone (pDEK4) was isolated from a library of GBS DNA. The gene product from this clone was characterized biochemically and immunologically. Two regions of the alpha antigen were characterized that contain protective epitopes, the N-terminus and the repeat region. The epitope bound by alpha antigen monoclonal antibody, 4G8, was localized to the repeat region. Antibodies raised to the recombinant alpha antigen N-terminus are opsonic and protect mice against alpha antigen-bearing strains of GBS. The basis of alpha antigen laddering was studied in both GBS and E. coli. Although, laddering appears to be the result of proteolytic processing in GBS, it could not be determined if the recombinant alpha antigen is processed in E. coli. In GBS, a serine proteolytic activity that formed laddering peptides was localized to the intracellular fraction of GBS. Spontaneous mutants of the alpha antigen were identified and characterized at the phenotypic and genotypic levels. These mutants contain deletions localized to the tandem repeat region. However, these deletions did not affect resistance to opsonophagocytosis in the absence of alpha antigen-specific antibodies. Thus, the size of the alpha antigen does not affect susceptibility to opsonophagocytosis in the absence of antibodies.
Bibliographical Information:

Advisor:

School:University of Massachusetts Amherst

School Location:USA - Massachusetts

Source Type:Master's Thesis

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ISBN:

Date of Publication:01/01/1996

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