Candida Albicans H3K4 Methyltransferase Enzyme Expression And Application Of N-terminal Peptides

The morbidity and mortality of invasive candidiasis(IC) has been increasing globally in recent years, particularly in immunocompromised patients who were treated with chemotherapy, or those patients receiving high-dose corticosteroids or antibiotics. But up to now the diagnosis of IC is still quite difficult due to the lack of typical symptoms. Besides, the conventional diagnostic methods such as microbial culture and tissue examination, may lead to delaying treatment because of the lack of sensitivity at the early stages of infections. Therefore it is needed to develop a new rapid sensitive method. At present, the clinical studies has focused on the detection of the specific antigens and antibodies, including Candida albicans H3K4methyltransferase, which may be candidate antigen for clinical diagnosis of IC.ObjectiveTo express N-terminal region of C. albicans histone3lysine4methyltransferase (Setl-208p) by prokaryotic expression system, establish an ELISA method for detecting IgG antibody to Set1-208p in human sera, and evaluate its value in diagnosis of IC.MethodsMultiple-alignment program was used for alignment of the amino acid sequence of C. albicans histone3lysine4methyltransferase with those of other species. And the N-terminus of C. albicans Setlp (Setl-208p) does not demonstrate significant homology to any other human proteins and was selected for prokaryotic expression. The coding sequence of Setl-208p was obtained by PCR from genomic DNA of C. albicans (C1strain), cloned into the prokaryotic expression vector pET-28a (+), and finally introduced into E. coli BL21(DE3). The His6-tagged Setl-208p was produced under the induction of IPTG. The protein was identified by Western blot using the monoclonal antibody against His6-tag, and the antigenicity of this new recombinant product was confirmed by the sera from patients with systemic candidiasis. The specific antisera of rabbits to Setl-208p were also prepared. The ELISA method for detecting anti-Setl-208p antibody was established with recombinant Setl-208p as coating antigen. Usefulness of this method was evaluated by detecting sera from rabbit IC model and patients with invasive candidiasis. ResultsThe recombinant His6-tagged Setl-208p, about29KDa, was successfully expressed and purified. Western blot showed that the recombinant protein was specifically reactive to the sera of proven invasive candidiasis patients. Polyclonal antiserum specific to Setl-208p was raised from rabbits. An increased level of antibody to Setl-208p was detected in the rabbit since the4th day after infection. The sensitivity, specificity were79.2%,90%for diagnosis of IC. And little crossreaction was found with patients with bacterial infection and other fungal infections such as Aspergillus.ConclusionsAn immunogenic recombinant Setl-208p of C. albicans has been generated. An ELISA-based method for detecting anti-setl-208p antibodies was developed and the potential value in the diagnosis of IC has been demonstrated.