Cloning and characterization of a new cysteine proteinase secreted by Paragonimus westermani adult worms.
The cysteine proteinases of Paragonimus westermani are known to play important roles in invasion and pathogenesis to hosts and in immune modulation and nutrient uptake. In this study, we have cloned a new cysteine proteinase of P. westermani, PwCP2, from adult worms and tested its diagnostic usefulness. The PwCP2 gene had an open reading frame of 816 base pairs and a conserved catalytic triad of cysteine, histidine, and asparagine residues. The mature form of recombinant PwCP2 (rPwCP2) lacking a proregion was overexpressed in Escherichia coli and used to produce antiserum. Western blot and immunohistochemical analyses using this antiserum showed that PwCP2 was expressed as a mature form, 24-kD product in a crude extract and in the excretory-secretory product of P. westermani, and was localized mainly in the intestinal epithelium of the adult worm. Western blot analysis using the rPwCP2 showed not only high sensitivity (90%) and specificity (100%) to sera from patients with paragonimiasis westermani, but also no cross-reactivity with sera from patients with clonorchiasis, sparganosis, or cysticercosis. Furthermore, an enzyme-linked immunosorbent assay using rPwCP2 exhibited a sensitivity of 93% and a specificity of 93% with sera of rats infected with P. westermani metacercariae. These results suggest that the excretory-secretory PwCP2 can be used for the diagnosis of paragonimiasis.
Yang
See Hoon
SH
Departments of Internal Medicine, Pathology, and Parasitology, College of Medicine, Department of Pathology, College of Oriental Medicine, Wonkwang University, Iksan 570-749, Korea.
Park
Jung-Ok
JO
Lee
John Hwa
JH
Jeon
Byung Hun
BH
Kim
Won Sin
WS
Kim
Suk-Il
SI
Yun
Ki-Jung
KJ
Jeong
Eun Taik
ET
Lee
Kil-Whoan
KW
Kim
Yong Man
YM
Lee
Mi Hyang
MH
Park
Hyun
H
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D010237:Paragonimiasis / Q000175:diagnosis / Q000276:immunology; D010238:Paragonimus / Q000201:enzymology* / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases