Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against schistosomiasis japonica.
We report the cloning, molecular characterization, and purification of functionally active recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the human bloodfluke Schistosoma japonicum. The GAPDH homolog from the related species Schistosoma mansoni has shown correlation of antibody titer to resistance to reinfection. A 1,164-bp cDNA (C1) was isolated from an S. japonicum lambda ZapII cDNA expression library immunoscreened with hyperimmune rabbit serum raised against soluble adult S. japonicum proteins. The open reading frame of C1 encodes a protein of 338 amino acids exhibiting 90% identity to the amino acid sequence of S. mansoni GAPDH. The inferred molecular mass of the protein is 36,589 daltons, and in vitro translation of the cDNA with [35S]methionine produced a radiolabelled band of the predicted size. Antibodies to C1 selected from hyperimmune rabbit serum by affinity purification recognized an S. japonicum protein doublet of 37 kDa but did not cross-react with a corresponding protein in S. mansoni extracts. The S. japonicum GADPH appears to be translated from a single mRNA encoded by a single-copy gene. After subcloning in the QIAexpress vector pQE-10 and subsequent expression, the recombinant protein was purified under nondenaturing conditions and shown to exhibit functional GAPDH enzymatic activity.
Waine
G J
GJ
Molecular Parasitology Unit, Queensland Institute of Medical Research, Bancroft Centre, Brisbane, Australia.
Becker
M
M
Yang
W
W
Kalinna
B
B
McManus
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D012554:Schistosomiasis japonica / Q000517:prevention & control; D014614:Vaccines, Synthetic / Q000276:immunology*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases