Engineering and expression of a full length cDNA encoding Schistosoma japonicum paramyosin. Purification of the recombinant protein and its recognition by infected patient sera.
A cDNA encoding the complete open reading frame of the Schistosoma japonicum paramyosin has been constructed and cloned. The 2600 bp cDNA was engineered by PCR using a N-terminally truncated paramyosin clone (pmy25) as template and a 57-mer primer that introduced the eight missing amino acids and matched the 5' sequence of pmy25 in conjunction with a pmy specific reverse primer. After cloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and was shown to have a molecular mass of 99 kDa which is equivalent to the expected size of the full length recombinant fusion protein, comprising the 97 kDa paramyosin plus an additional 2 kDa for the N-terminal fusion peptide incorporating the six histidine residues required for purification. In Western blot assays it reacted specifically with anti-paramyosin antibodies in sera from vaccinated animals and patients with Asian schistosomiasis. The engineering of the full-length cDNA encoding Schistosoma japonicum paramyosin, its bacterial expression and purification will facilitate future studies aimed at determining its efficacy as an anti-schistosomiasis vaccine.
Kalinna
B H
BH
Molecular Parasitology Unit, Queensland Institute of Medical Research, Bancroft Centre, Royal Brisbane Hospital, Australia. berndK@qimr.edu.au
Becker
M M
MM
McManus
D P
DP
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000235:genetics* / Q000276:immunology; D014335:Tropomyosin / Q000235:genetics* / Q000276:immunology
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014335:Tropomyosin