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.
Casais
R
R
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, Oviedo, 33006, Spain.
Martín Alonso
J M
JM
Boga
J A
JA
Parra
F
F
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004175:Diptera / Q000201:enzymology*; D004478:Ectoparasitic Infestations / Q000175:diagnosis* / Q000662:veterinary; D004926:Escherichia coli; D005982:Glutathione Transferase; D006801:Humans; D007700:Kinetics; D007814:Larva; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; C070019:hypodermin C