| With the increasing of patients who caught immunologic deficiency disease,diabetes and organ transplantation, Candida albicans had already become the numberone in fungal infection. However, with the general useage of antifungal drugsincluding azole, resistance to antifungal drugs had become a tough problem of clinicalmedicine.The molecular mechanisms of how C.albicans resisted to antifungal agentswere quite complicated, it was generally thought that the main mechanisms were thechanges of azole target enzymes and overpression of two types of efflux pumps.Moreover, the changes in azole target enzymes played a very important role in theprocess of resistance to antifungal drugs. The lanosteral 14α-demethylase was thetarget enzymes of azole drugs. The changes of amino acids and three-dimensionalstructure in enzyme reacting region caused by the mutation in coding region of thetarget enzymes resulted in the decreasing affinity and adhesion between the targetenzymes and antifungal drugs, which made the drugs concentration in fungal cellimcompletly suppressing enzymatic activity, thus it became resistant. The changes ofthe gene regulation and corresponding regulatory gene made it overexpress the targetenzymes. Finally it produced a lot of target enzymes and it became resistant to thedrugs.For this reason, the study gave a primary reseach on the relationship betweenmRNA and protein expression of ERG11 and the resistance of the same C. albicansbefore and after drug resistance induction. These results could provide some scientificbasis for deeply studying the resistance to azole drugs in C.albicans.Diffent strains of Candida albicans (S,S1,L18,L18-1,L20,L20-1,T2 and T7) indifferent drug resistance were studyed. The MIC of these strains were 2μg/ml,128μg/ml, 8μg/ml, 64μg/ml, 4μg/ml, 128μg/ml, 128μg/ml and 256μg/ml respectively.According to the sequence of ERG11 gene in GeneBank and the introduction of thefluorescence gomphosis mean RT-PCR, a pair of primers was designed. With theextracted the total RNA of the tested strains, the real time fluorescent quantitationRT-PCR was carried out ,and the standard curve was drawn with the plasmid DNA.The mRNA expression of the artificially induced resistant strains and the clinicallyisolated resistant strains was higher than those of the standard strains and the clinicalsensitive strains. The results indicated that the mRNA expression was related to theirresistance.Refering to the relevant documents and the published sequence in GeneBank, theepitope domain of ERG11 gene was analysised, and the primers was designed withenzyme sites to amplified the fragment, which was 570 bp. ERG11 gene fragments ofthe resistant strains were amplified by PCR and cloned into pMD18-T vectors. Thepositive clones were identified by PCR and enzyme digestion. The target fragmentswere ligated with pET28a (+) vector and were inverted into the acceptor strain BL21(DE3). After induced by IPTG, the expression of the exogenous gene ERG11 weredetected. the rabbits were immunized by the purified expression product, the proteinswere detected by ELISA and Western-Blotting.The results showed that the proteinexpression of the artificial induced resistant strains and the clinical isolated strains washigher than that of the standard C.albicans and the clinical sensitive strains. Itindicated that the expression of Erg11p in different resistant strains was related to theirdrug resistance.The results indicated that the expression of Erg11p in different resistant C.albicans had positive correlation to their drug resistance. This study could providesome scientific basis for the investigation of the resistant mechanisms in C.albicans,clinical instruction of rational administration and invention of the new anti-fungalagents. |
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