Date of Award

1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ronald J. Siebeling

Abstract

Polar flagellar core protein was purified from Vibrio parahaemolyticus by differential centrifugation and cesium chloride isopycnic centrifugation. Twelve hybridomas secreting monoclonal antibodies against flagellar core protein (MAb-flc) were obtained from fusions. By chance, one purified flagellar core preparation retained flagellar sheath and as a consequence, two hybridomas were detected which secreted anti-sheath antibodies (MAb-fls). MAb-flc and MAb-fls reacted specifically with their corresponding antigens as demonstrated by immunogold labelling. Coagglutination reagents prepared with MAb-flc and MAb-fls were tested against 34 strains of V. parahaemolyticus and 34 heterologous Vibrio species. The coagglutination results revealed that the flagellar core was species-specific while the flagellar sheath was not. Preliminary adhesion studies of V. parahaemolyticus using rabbit intestinal model were done. The experimental conditions were established to be the following; 10$\sp{10}$ V. parahaemolyticus cells propagated in HIB were exposed to the formalin-preserved small intestinal tissue for 10 minutes and the number of adherent cells were determined by electron microscopy. The quantity of Fab prepared from MAb-flc and MAb-fls required for the adhesion inhibition test were determined by coagglutination inhibition assay. MAb-fls inhibited the adherence of V. parahaemolyticus cells to intestinal tissue, whereas MAb-flc pretreatment did influence adherence. It is likely that the sheath participates in the adhesion event but its function is not known. A genomic library of V. parahaemolyticus was constructed in order to isolate the gene encoding the polar flagellar core antigen. DNA fragments were cloned into Lambda ZAPII vector at the EcoRI cloning site and the recombinant DNA was transformed into Escherichia coli XL1-Blue strain. Recombinant plaques were screened with rabbit polyclonal antiserum produced against polar flagella and with monoclonal antibody produced against polar flagellar core protein. The pBluescript phagemid which contained the DNA fragment encoding the flagellar gene was rescued and analyzed. Additional investigation was carried out by mini-Mu transposon mutagenesis to map the coding region. The flagellar core coding region was mapped within 1.1 kb which encompasses the NcoI site present in the 6.5 kb insert.

Pages

128

Share

COinS