Purification and characterization of a 7-kDa protein from Clonorchis sinensis adult worms.
A 7-kDa protein was purified from extracts of adult Clonorchis sinensis by a combination of ammonium sulfate precipitation, anion exchange chromatography, cation exchange chromatography, gel-filtration chromatography, and reversed-phase FPLC. The 7-kDa protein exists in the excretory-secretory products of adult C. sinensis, but not in extracts of adult Paragonimus westermani. Also, the 7-kDa protein reacted with the sera of patients with clonorchiasis but not with paragonimiasis or normal human sera. To observe the localization of the 7-kDa protein in the tissue of adult C. sinensis, an immunogold labeling method was followed using anti-7-kDa antibody. The gold particles were observed in the basal layer below the tegumental syncytium, in the interstitial matrix of the parenchyma, and in the content of the uterus. The 7-kDa cDNA was obtained through reverse transcription-polymerase chain reaction using a primer designed from N-terminal sequence analysis. Rapid amplification of cDNA ends (5'-RACE) was used to obtain the complete protein coding sequence. The sequence encodes a 90-amino acid polypeptide. The deduced amino acid sequence of the 7-kDa protein revealed no homology with proteins of different organisms reported so far. These results suggest that the 7-kDa protein is a fluid antigen and may be valuable as a tool for the immunodiagnosis of clonorchiasis.
Lee
Hye-Jeong
HJ
Graduate School of Biotechnology, Korea University, Seoul, South Korea.
Lee
Chang-Seok
CS
Kim
Beom-Su
BS
Joo
Kyoung-Hwan
KH
Lee
Joon-Sang
JS
Kim
Tong-Soo
TS
Kim
Hak R
HR
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003003:Clonorchiasis / Q000097:blood / Q000276:immunology; D003004:Clonorchis sinensis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008297:Male; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D018510:RNA, Helminth / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000947:Antigens, Helminth; D018076:DNA, Complementary; D018510:RNA, Helminth