CLONING AND EXPRESSION OF A CYSTEINE PROTEINASE GENE FROM PARAGONIMUS WESTERMANI ADULT WORMS

摘要

A gene encoding a cysteine proteinase from Paragonimus westermani has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription–polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from the conserved active site of the cysteine proteinase. The 5′ and 3′ regions of the gene were amplified using a PCR technique for the rapid amplification of cDNA ends. The cloned gene has an open reading frame of 687 bp and deduced amino acid sequence of 229. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form a catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. The expressed protein reacted with the sera of patients with paragonimiasis but not with the sera of fascioliasis and clonorchiasis. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of paragonimiasis.