| dc.description.abstract | Saprolegnia parasitica is a pathogenic oomycete within the phylum of stramenopiles that mainly infects salmon and trout. It is found all over the world and causes a disease called saprolegniosis that often results in death of the fish. S. parasitica contributes to a huge economical loss for the aquaculture industry, and there are currently no successful treatments available. Ricin B is a protein that binds to galactose residues on the cell membrane, and it is found in many organisms, including S. parasitica. Its function is unknown, but it has been associated with various functions that are important for the infection process of the pathogen.
In this thesis, Ricin B was studied to determine if it functions as a translocation module in S. parasitica. Three Ricin B constructs were transformed and expressed in E. coli cells before the proteins were purified. A HisTrap HP column was used to separate the proteins from native bacterial proteins and a centrifugal filter was used to concentrate them. The purified proteins were added to RTGill W1 cells and the protein uptake was analysed with a flow cytometer and confocal microscope.
All three Ricin B proteins studied were translocated in a pathogen-independent manner into the RTGill W1 cells and seen in vesicle-like structures outside the nuclei of the cells. The cells with Ricin B_2 had the highest protein uptake. The experiments did not show that there was any significant advantage to Ricin B_DB with two Ricin B domains compared to Ricin B_1 and Ricin B_2 with only one domain, which indicated that two domains did not increase the translocation efficiency. | |