Preparation And Identification Of Polyclonal Antibodies Against Several Antigenic Determinants Of Candida Albicans CYP51

14α-demethylase (P45014DM, CYP51), the key enzyme in sterol biosynthesis, which results in depletion of ergosterol and accumulation of lanosterol and other 14-methyl sterols ultimately leading to the growth inhibitionon of fungal cells.Inhibition of the ergosterol biosynthetic pathway in fungi has thus attracted intense interests in the application of such antifungal compounds.According to the bioinformatics analysis and prediction of the CYP52A1,hydrophilicity and antigencity of CYP52A1 protein,a specific amino acid(position 307 to 518) was derived from full length.In this study,this antigenic determinant genes(starting from nucleotide 916 and ending with nucleotide 1549)have been amplified successfully from C.albicans genome and then were cloned into a cloning vector,and the DNA sequencing proved that the fractions amplified by PCR are totally corrent.The correct gene fragments were cloned into a prokaryotic expression vector to generate fusion protein named GST- CYP52A1.GST-LINK respectively.which were induced by IPTG and well expressed in E.coli DH5α.The purification of the GST- CYP52A1 protein for preparation of a polyclonal rabbit was achieved by glutathione-agarose affinity chromatography of E.coli cell extracts by standard procedure described by the manufacture(Amersham).