Cloning and molecular analysis of genes encoding two 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, sequenced, and expressed in E. coli. Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a ;10 kDa protein. Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues of E. coli GroEL and GroES proteins, respectively. There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains of E. risticii. The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively. 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 mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a ;2 kDa lower molecular weight region. In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified. The 55 and 51 kDa antigens were overexpressed in E. coli using a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S. A.). The purified recombinant proteins cross-reacted with antisera to E. canis and E. sennetsu.
Vemulapalli
R
R
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD, 20742, USA.
Biswas
B
B
Dutta
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001483:Base Sequence; D015153:Blotting, Western; D018835:Chaperonin 10 / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics; D003001:Cloning, Molecular; D004285:Dogs; D004536:Ehrlichia / Q000235:genetics* / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005798:Genes, Bacterial / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics; D006736:Horses; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D014462:Ultrafiltration
D000907:Antibodies, Bacterial; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D016056:Immunodominant Epitopes; D011994:Recombinant Proteins