Candida Albicans Enolase And Its N-terminal Peptides Of Prokaryotic Expression And Applied Research

Candiada albicans(C. albicans), one of opportunistic pathogen, has became the major pathogen of invasive candidiasis(IC). The morbidity and mortality of IC has been increasing global in recent years, particularly in immunocompromised patients which were treated with chemotherapy, or those patients receiving high-dose corticosteroids or antibiotics. But untill now, the diagnosis of IC is still difficult due to the lack of specific clinical features and a definitive diagnostic method. At present, the clinical studies has focused on detection of the specific antigens and antibodies of C. albicans. Enolase, a glycolytic enzyme, is an immunodominant antigen of the C. albicans. the serodiagnosis of IC which detect antibodies against C. albicans enolase can distinguish invasive Candida albicans infections from colonizations, and may useful for clinical diagnosis of IC.Objective:To obtain full-length protein and N-terminal fragment of Candida albicans enolase, prepare polyclonal antibody against recombinant enolase, and evaluate recombinant fragment usefulness in diagnosis of IC.Methods:The full-length coding sequence of enolase was obtained by PCR from genomic DNA of C. albicans and cloned into the prokaryotic expression vector pET-28a(+). A His6-tagged enolase was produced under the induction of IPTG in E. coli BL21(DE3), analyzed by SDS-PAGE and purified by TALON metal affinity resins. The antigenicity of the recombinant product was evaluated by Western blot with patient's sera of systemic candidiasis.Using this protein as antigen, the specific antisera of rabbits and goats to ENO1 were prepared. The specificity of those antisera was identified by Western blot. DNA encoding ENO1-319P, which has low homologous to a-ENOl of Homo sapiens, was cloned into pET-28a(+) and expressed in E.coli. BL21(DE3). Usefulness of this N-terminal fragment was evaluated by ELISA-based method for detecting human anti-ENO1-319P antibody.Results:The full-length gene of C. albicans enolase was cloned and recombinant enolase was expressed highly in E. coli. A 319 amino acids recombinant peptide (1-319aa) of ENO1 also has been expressed and purified. Western blot showed that both recombinant proteins were specifically reactive to antibody in sera from systemic candidiasis patients. Polyclonal antiserum specific to ENO1 was raised from rabbits and goat.Results of Western bot analysis demonstrate the specificity of the antibody. Results of ELISA using recombinant peptide ENO1-319P as coating antigen as follow:the positive rate of IC patients and healthy people were 84.8%(56/66) and 3.0%(6/200).Conclusions:A immunogenic recombinant enolase of C. albicans has been expressed and purified. The polyclonal antiserum specific to enolase of C. albicans was prepared. The N-terminal fragment of candida albicans enolase, called ENO1-319P, would be used to prepare more specific antibody for serodiagnosis of IC.