Cloning and sequencing of yajC and secD homologs of Brucella abortus and demonstration of immune responses to YajC in mice vaccinated with B-abortus RB51

To identify Brucella antigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library of Brucella abor tus 2308 was screened for the expression of antigens reacting with immunogl obulin G2a antibodies from BALB/c mice vaccinated with B. abortus RB51. One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucle otide sequence analysis of this insert revealed two open reading frames (OR Fs). The deduced amino acid sequences of the first and second ORFs had sign ificant similarities with the YajC and SecD proteins, respectively, of seve ral bacterial species. Both the YajC and SecD proteins were expressed in Es cherichia coli as fusion proteins with maltose binding protein (MBP). In We stern blots, sera from mice vaccinated with B. abortus RB51 recognized YajC but not SecD. Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated with B. abortus 19 or 2308 or B. me litensis RM1 also produced antibodies to YajC. In response to in vitro stim ulation with recombinant MBP-YajC fusion protein, splenocytes from mice vac cinated with B. abortus RB51 were able to proliferate and produce gamma int erferon but not interleukin4. This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agen t.