Prokaryotic Expression And Application Of Fructose Diphosphate Aldolase In Candida Albicans

The incidence and prevalence of invasive candidiasis(IC) have increased in the large population of those hospitalized with serious underlying diseases and immunocompromised patients. Candida albicans, one of the major opportunistic pathogen, which has become the main cause of IC. However, it is still difficult for the diagnosis of IC due to non-specific clinical signs and lack of unequivocal diagnostic methods. Therefore, non-culture methods with high sensitivity and specificity are needed for improvement of etiologic diagnosis of IC. And now, the clinical studies have focused on the detection of the specific antibodies of C. albicans. Fbal (fructose-bisphosphate aldolase)-a glycolytic enzyme, is one immunodominant antigen of the C. albicans. Basing on the detection of anti-Fbal antibodies, the serological test could distinguish IC from colonizations,so it might be valuable for clinical diagnosis of IC.Objective:By using molecular cloning and prokaryotic expression technology, we obtained recombinant protein of Candida albicans Fbal; and evaluated rapid diagnosis of IC serological detection methods by using the recombinant Fbal.Methods:The Fbal’s full-length gene was obtained by PCR from genomic DNA of C.albicans. The amplified fragment was cloned into pMD18-T vector. After sequencing, the recombinant plasmid pMD18-T/Fbal was digested by the restriction enzymes, and the target fragment was inserted into pET-28a (+) vector. Then it was used to transform E. coli BL21(DE3) and induced expression by IPTG. The recombinant protein was analyzed by SDS-PAGE, and then it was purified by TALON metal affinity resins. Finally, the antigenicity of the protein was confirmed by Western blot with patients’ sera of IC.Using this recombinant protein as coating antigen, an ELISA was developed for detecting antibodies against Fbal. The specificity, repeatability was investigated and cut-off value was determined. Sera from healthy individuals and patients with candidemia, colonizations, or bacteremia were detected.Results:The gene of C. albicans Fbal was cloned, and it was expressed highly in E. coli BL21(DE3). This recombinant Fbal was specifically reactive to anti-Fbal antibodies in sera from IC patients by Western blot.The ELISA using recombinant Fbal as coating antigen has a good precision. Intra-assay variation was1.81%, inter-assay variation was14.85%. The specificity of ELISA was confirmed by blocking assay. According to ROC curve, the absorbance value (A) of0.61was detected as the cut-off value. The sensibility in candidemia was74.5%(82/110), and the specificity was92.8%(322/347). Overall the anti-Fbal antibodies detection is meaningful (p<0.001) in diagnosing candidemia.Conclusions:An immunogenic recombinant C. albicans Fbal was highly expressed and purified. The ELISA method for detecting anti-Fbal antibodies by using recombinant Fbal as coating antigen might be valuable in the diagnosis of IC.