HYPODERMA LINEATUM - EXPRESSION OF ENZYMATICALLY ACTIVE HYPODERMIN-C IN ESCHERICHIA-COLI AND ITS USE FOR THE IMMUNODIAGNOSIS OF HYPODERMOSIS

The cDNA coding for the mature hypodermin C from first instars of Hypo derma lineatum was cloned by reverse transcription and PCR amplificati on of total larval RNA using specific oligonucleotide primers. This cD NA was expressed in Escherichia coli as a glutathione S-transferase fu sion protein. Mature hypodermin C was released from the GST-fusion aft er glutathione-Sepharose 4B affinity chromatography and proteolytic cl eavage using factor Xa. The purified recombinant protein showed enzyma tic activity in gelatin-polyacrylamide gels and when azocoll was used as substrate. An enzyme-linked immunosorbent assay was developed using the recombinant antigen. Positive/negative cutoff values were calcula ted using the mean OD percentage (1.74%) of 113 negative sera plus thr ee standard deviations. Sensitivity and specificity according to the r esulting cutoff (10.74 %) were 85 and 98.2% respectively, (C) 1998 Aca demic Press.