CLONING AND MOLECULAR ANALYSIS OF GENES ENCODING 2 IMMUNODOMINANT ANTIGENS OF EHRLICHIA-RISTICII

Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism. To understand the role of 55 and 51 kilodalton immunodominant antigens of E. risticii in strain variation, their genes from the 25-D and 90-12 strains were cloned, s equenced, and expressed in E. coli. Sequence analysis revealed that th e gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a similar to 10 kDa protein. Homology searches indi cated that the 55 kDa antigen and the 10 kDa protein were homologues o f E. coil GroEL and GroES proteins, respectively. There was no nucleot ide sequence difference between the genes of the 55 kDa antigen, nor b etween the entire operons, from both strains of E. risticii. The seque nce-based estimation of the sizes of the putative mature 51 kDa antige ns of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectiv ely. The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences. The difference in some of the amino acids may be responsible for variation in their mobiliti es on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a similar to 2 kDa lower molecular weight region. In Western blots, a 155 kDa protei n that appeared to be a trimer product of the 51 kDa antigen was ident ified. The 55 and 51 kDa antigens were overexpressed in E. coli using a commercial expression system, pRSET A,B,C (Invitrogen inc., San Dieg o, CA, U.S.A.). The purified recombinant proteins cross-reacted with a ntisera to E. canis and E. sennetsu. (C) 1998 Academic Press.