Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis.
A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases. The 5' and 3' regions of the gene were amplified using rapid amplification of cDNA ends. The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the 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. Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin. The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites. The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis.
Na
Byoung-Kuk
BK
Department of Biology, College of Natural Science, Chung-Ang University, Seoul, South Korea.
Lee
Hye-Jeong
HJ
Cho
Shin-Hyeong
SH
Lee
Hyeong-Woo
HW
Cho
Jung-Hwa
JH
Kho
Weon-Gyu
WG
Lee
Joon-Sang
JS
Lee
Jong-Soo
JS
Song
Kyoung-Ju
KJ
Park
Po-Hyun
PH
Song
Chul-Yong
CY
Kim
Tong-Soo
TS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003003:Clonorchiasis / Q000175:diagnosis / Q000201:enzymology*; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018510:RNA, Helminth / Q000737:chemistry / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D015853:Cysteine Proteinase Inhibitors; D018509:DNA, Helminth; D018510:RNA, Helminth; D003546:Cysteine Endopeptidases