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 Hypoderma lineatum was cloned by reverse transcription and PCR amplification of total larval RNA using specific oligonucleotide primers. This cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Mature hypodermin C was released from the GST-fusion after glutathione-Sepharose 4B affinity chromatography and proteolytic cleavage using factor Xa. The purified recombinant protein showed enzymatic 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 calculated using the mean OD percentage (1.74%) of 113 negative sera plus three standard deviations. Sensitivity and specificity according to the resulting cutoff (10.74%) were 85 and 98.2% respectively.