Characterisation and expression of a cDNA encoding the 80-kDa large subunit of Schistosoma japonicum calpain.
We describe the cloning of a full length calpain-encoding cDNA constructed from two truncated cDNAs isolated from a cDNA library prepared with mRNA isolated from adult worms of the Philippine strain of Schistosoma japonicum. The cDNA sequence is 2.456 kb in length and predicts a protein of 758 residues with a molecular mass of 86.61 kDa and an isoelectric point of 5.34. Probes spanning the entire calpain cDNA hybridised to multiple bands in genomic DNAs of Philippine (SjP) and Chinese (SjC) S. japonicum, with some restriction fragment length polymorphisms evident between the two strains. Northern hybridisation analysis indicated that the cDNA codes for a single RNA transcript between 2.6 and 3.6 kb in size in the SjP and SjC genomes. After subcloning in the QIA express vectors pQE-31 and pQE-40 and subsequent expression, the recombinant protein was purified and shown to bind calcium. The availability of recombinant S. japonicum calpain will allow its future evaluation as a vaccine candidate, especially in light of recent work with the S. mansoni homologue which has provided evidence that this protein may be a target of protective immunity.
Scott
J C
JC
Molecular Parasitology Unit, Australian Centre for International and Tropical Health and Nutrition, Cooperative Research Centre for Vaccine Technology, University of Queensland, Australia.
McManus
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002154:Calpain / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D012549:Schistosoma japonicum / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D017931:DNA Primers; D018509:DNA, Helminth; D011994:Recombinant Proteins; D002154:Calpain