MOLECULAR ANALYSIS AND CHARACTERIZATION OF A PROTEIN INVOLVED IN THE REPLICATION OF INTRACELLULAR TOXOPLASMA-GONDII

Previous studies in our laboratory have identified a cytoplasmic prote in (p97) of T. gondii that is involved in the process of intracellular parasite replication. Monoclonal antibody inhibits parasite replicati on in vitro and recognizes a protein of approximate 97 kDa by Western blot analysis. Using biotinylation, we demonstrate that p97 is not exp ressed on the surface of the tachyzoite. Polyclonal sera raised agains t the purified native protein was used to isolate a cDNA of 3.3 kb fro m a library. The product of this gene expresses a protein of approxima te M-r 97 kDa that is reactive to the antibody (1B8) raised against th e native antigen. The protein sequence of this product suggests that i t is within the cytoplasm as suggested by the lack of a signal sequenc e or hydrophobic trans-membrane domain. This protein fails to dissocia te into a monomer in the presence of non-ionic detergents as shown by gel filtration and density gradient. Southern blot analysis demonstrat es a homologous gene sequence in two closely related Apicomplexa, Neos pora caninum and Besnoitia jellisoni suggesting this protein is conser ved among certain species of the Sarcocystidae. (C) 1997 Published by Elsevier Science B.V.