Molecular cloning and expression of the gene encoding a cysteine proteinase of Spirometra erinacei.
A cDNA library constructed from plerocercoid of Spirometra erinacei (SEP) was immunoscreened using rabbit anti-plerocercoid proteinase polyclonal antibody. A 1.0-kb cDNA clone encoding a cysteine proteinase composed of 336 amino acids was isolated. The amino acid sequence predicted from the cDNA showed significant homology with human and mouse cathepsin L. N-terminal amino acid sequence of the native cysteine proteinase extracted from SEP was the same as that of mature proteinase predicted from the cloned gene. The gene encoding the proteinase was characterized by Southern and Northern blot analysis using the cDNA as a probe. The proteinase with a molecular mass of 34 kDa was demonstrated in in vitro translation products using anti-proteinase polyclonal antibody. A fusion protein derived from the cDNA synthesized by Escherichia coli (TB1) using the expression vector, pMAL-c2 was identified as an immunodominant antigen by epitope-selection method and had no cross-reactivity with other parasite-infected sera. A genomic DNA library derived from SEP was screened by the colony hybridization technique using the cDNA probe. A gene with 4.5 kb encoding the proteinase was obtained, which comprised three exons and two introns.
Liu
D W
DW
Department of Parasitology, Shinshu University School of Medicine, Matsumoto City, Japan.
Kato
H
H
Nakamura
T
T
Sugane
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000276:immunology; D015342:DNA Probes; D015723:Gene Library; D017238:Genes, Helminth; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017385:Sequence Homology; D013146:Spirometra / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic
D015342:DNA Probes; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases