[Cloning of a protease gene family in Fasciola hepatica using the polymerase chain reaction (PCR)].
RT-PCR and degenerative Oligonucleotide primers derived from conserved cysteine protease sequences were used to amplify seven different Fasciola hepatica cysteine protease cDNA clones (Fcp1-7). Five of the clones showed homology to proteases of the cathepsin L type, whereas two appeared to represent the cathepsin B family. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA Ends PCR (RACE-PCR) protocol. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in Western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from immature and adult parasites proteins of 30 and 38 kDa were detected. In other stages, proteins of different molecular weight were recognized by anti-GST:Fcp1 antiserum, indicating stage-specific gene expression.
Heussler
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Institut für Tierpathologie der Universität Bern.
Dobbelaere
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D005210:Fasciola hepatica / Q000201:enzymology* / Q000235:genetics*; D017238:Genes, Helminth; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases
Die Klonierung einer Protease-Genfamilie von Fasciola hepatica mit Hilfe der Polymerase-Kettenreaktion (PCR).