Molecular intervention in Pierce's Disease

Doctoral thesis English OPEN
Azizi, Armaghan
  • Subject: UOW2

Pierce's Disease (PD) is one of the most devastating diseases threatening grapevines\ud in the USA caused by the Gram negative bacterium Xylella fastidiosa. There is no\ud effective control method for Pierce’s Disease and control is mainly achieved by\ud suppressing transmission of the glassy winged sharpshooter (GWSS) insect vector.\ud The study of the surface proteins of X. fastidiosa may provide targets that may be\ud attractive sites for intervention. MopB is an outer membrane protein, which has been\ud identified on the X. fastidiosa surface. Blocking MopB with antibodies may prevent\ud or reduce transmission of the pathogen.\ud The main goal of this research was to generate recombinant antibodies to be used to\ud investigate the interaction of MopB with the plant and GWSS. Towards this goal,\ud recombinant MopB has been produced and used with sera from five mice immunized\ud with whole heat killed X. fastidiosa in an Enzyme-Linked Immunosorbent assay\ud (ELISA). The ELISA results revealed the presence of antibodies against the\ud recombinant MopB protein. In vitro combinatorial antibody ribosome display\ud libraries were assembled from immunoglobulin transcripts rescued from the spleens\ud of mice immunized with heat-killed X. fastidiosa. The libraries were used in a single\ud round of selection against the outer-membrane protein MopB, resulting in the\ud isolation of a panel of recombinant antibodies. The potential use of selected anti-\ud MopB antibodies was demonstrated by the successful application of the 4XfMopB3\ud antibody in ELISA, Western blot and immunofluorescence assays.\ud Pantoea agglomerans, a candidate bacterium for PD paratransgenic approach was\ud engineered to express and secrete selected anti-MopB (4XfMopB3) single chain\ud antibody. The expression cassette that include the pelB secretion signal was created\ud and tested for its efficiency\ud in secreting the 4XfMopB3 antibody in E. coli and\ud P. agglomerans. The 4XfMopB3 antibody was successfully expressed and secreted in\ud E. coli while it was only expressed in P. agglomerans.
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    112 references, page 1 of 12

    Table 2.1 Primers (5' to 3' direction) used in PCR. ...............................................................................42  

    Table 3.1 Total RNA extraction concentrations and A260/280 and A260/230 ratios obtained from 5 mice spleens previously immunized with whole heat-killed X. fastidiosa. ...........................................83  

    Table 3.2 Junction analysis and alignment of the VL and VH CDR3 sequences of the selected antiMopB scFv's (4XfMopB1-4). ......................................................................................................88  

    Table 3.3 Pantoea agglomerans antibiogram zones of inhibition.........................................................99   HE, M. & TAUSSIG, M. J. 2002. Ribosome display: cell-free protein display technology. Briefings in Functional Genomics and Proteomics, 1, 204-212.

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