Molecular cloning and characterization of a serine proteinase gene of Trichinella spiralis.
A serine proteinase gene was isolated from a cDNA library of Trichinella spiralis muscle larvae at 30 days post-infection (PI). The library was immunoscreened with T. spiralis-infected sera. A clone, designated Ts23-2, contained a cDNA transcript of 1,445 bp that encoded a putative signal peptide of 27 amino acids, a proregion of 20 amino acids, and a predicted mature enzyme of 374 amino acids. The predicted molecular mass of the Ts23-2 mature protein was 42.3 kDa. The enzyme comprised 2 regions, a catalytic domain of 234 residues and a C-terminal domain. The closest homologues of the Ts23-2 mature protein were serine proteinases from a wide range of organisms. The catalytic domain of the Ts23-2 clone was expressed as a proform in Escherichia coli. The recombinant protein cleaved serine proteinase-specific synthetic peptide substrates, and class-specific inhibitors of serine proteinases inhibited the enzymatic activity. Antibody against the Ts23-2 recombinant protein stained proteins migrating at about 51 and 33 kDa in crude extracts from 30-day PI muscle larvae and 18-day PI muscle larvae, but it failed to stain any proteins in crude extracts from newborn larvae and adult worms or in excretory-secretory products from 30-day PI muscle larvae. Production of the mRNA transcript for the Ts23-2 gene was mainly restricted to the 30-day PI muscle larvae, suggesting stage-specific expression. Intense staining with the anti-Ts23-2 serum was found within the parasites at the muscle stage of 30 days PI.
Nagano
Isao
I
Department of Parasitology, Gifu University School of Medicine, Tsukasa 40, Gifu 500-8705, Japan.
Wu
Zhillang
Z
Nakada
Takumi
T
Boonmars
Thidarut
T
Takahashi
Yuzo
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology / Q000469:parasitology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D017160:Trichinella spiralis / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D018509:DNA, Helminth; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases