28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Tetrahymena thermophila As An Expression System For Recombinant Vaccine Antigens Yelena A. Bisharyan and Theodore G. Clark Department of Microbiology and Immunology, Cornell University, Ithaca, NY, 14853 Tetrahymena thermophila, offers a unique system for large-scale expression of eukaryotic membrane proteins.  We are using this system to develop a vaccine against Ichthyophthirius multifiliis, a protozoan pathogen of freshwater fish.   Ichthyophthirius multifiliis expresses abundant GPI-anchored membrane proteins, referred to as immobilization antigens (or i-antigens) that elicit protective immunity in fish.  Nevertheless, Ichthyophthirius cannot be readily cultured, and effective vaccines against this agent will require the use of recombinant DNA techniques to express the corresponding proteins on a commercial scale.  We had previously shown that i-antigen genes from Ichthyophthirius could be introduced into T. thermophila by homologous recombination, and expressed at high-level under the control of a cadmium-inducible, metallothionein gene promoter.  Nevertheless, a recently identified gene whose product is thought to play a role in protection against the most common I. multifiliis serotype (namely, D), had yet to be expressed.  We sought to optimize expression of the IAG52B[G5] gene by examining a variety of parameters including cadmium concentration, time of induction, and composition of the Tetrahymena growth medium.  Western blotting on whole cell lysates, indirect immunofluorescence microscopy of fixed cells, and immobilization of live cells using i-antigen specific antibodies monitored protein expression levels.  Results indicated that maximum levels of expression occurred at 12-16 hrs following induction with 2 mg/ml CdCl2.  Recombinant cells expressing the IAG52B[G5] gene are now being tested as vaccines. Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page