Cloning, expression, purification and serodiagnosis evaluation of three Francisella tularensis proteins

Serological methods are currently the most widely used approach for diagnosing tularemia. Identifying and evaluating immunoreactive antigens of Francisella tularensis could significantly enhance the development of an advanced diagnostic method for tularemia. This study aimed to assess three F. tularensis recombinant proteins of F. tularensis for their potential application in serodiagnosis. In the present study, three proteins were selected for evaluation. Outer membrane protein A (FopA, FTT0583), a conserved hypothetical protein (FTT0975), and GroEL protein (FTT1696). Genes from F. tularensis subsp. Holarctica LVS NCTC 10857 were cloned and expressed using the pET28a-E. coli BL21 system. The recombinant proteins were analyzed by SDS-PAGE and confirmed via western blot. After purification, immunoblot assays were performed on sera from tularemia patients and healthy controls to detect anti-F. tularensis IgG antibodies. Successful cloning of all three genes was confirmed by PCR, restriction enzyme digestion, and sequencing. The recombinant proteins were effectively expressed and purified. Immunoblotting with sera from tularemia patients showed seroreactivity rates of 36% for FopA, 24% for FTT0975, and 52% for GroEL. The diagnostic values for combination of these proteins were as follows: FopA-FTT0975 (44%), GroEL-FTT0975 (58%), FopA-GroEL (64%), and FopA-GroEL-FTT0975 (66%). No false positives were found in control sera. Although all three recombinant proteins exhibited moderate diagnostic value individually, they demonstrated enhanced performance when combined. Therefore, these proteins are recommended for use alongside other immunogenic proteins in the development of improved diagnostic tests for tularemia.