Isolation of enterococcal antigen-encoding genes from genomic libraries

We have devised a procedure using immune sera to identify antigen-encoding genes of strains of Enterococcus faecalis. First, genomic cosmid libraries containing large inserts were constructed and screened with sera from patients with enterococcal infectious endocarditis and with serum from a rabbit immunized with surface proteins of an enterococcal endocarditis isolate. Immunopositive cosmid clones were analyzed by restriction enzyme digestions and clones containing distinct inserts were chosen for subcloning. Sublibraries were screened with one of the five sera, and immunopositive subclones were subjected to DNA sequencing. BLASTX and BLASTN at NCBI were used to search for database similarities.