Cloning and characterization of a cDNA fragment encoding a Schistosoma mansoni actin-binding protein (Smfilamin).
To identify vaccine candidates for Schistosoma mansoni, the IgG fraction of rabbit antiserum raised against immature female worms affinity purified over a NP-40 extract of 3-h schistosomula was used to immunoscreen a cercarial lambdagt11 cDNA library. One clone with a 1.5-kb cDNA insert revealed an encoded peptide of 479 amino acids, which bears homology to human actin-binding protein (ABP-280=filamin). Northern blot analysis revealed a transcript of about 8.6 kb, indicating that the complete gene was not cloned. Overlapping clones, which encode a composite sequence of 983 amino acids (45% identity with filamin), were subsequently isolated from the cDNA library. The 1.5-kb insert was cloned into pGEX, overexpressed, and the 479 amino acid peptide purified. Western blot analysis using polyclonal antisera specific to the peptide identified a 280-kDa molecule in adult worm extracts. RT-PCR demonstrated that Smfilaimin is expressed in various stages. Immunofluorescence studies with specific antisera revealed a tegument-associated fluorescence in adult worms. IgG specific to the Smfilamin fragment showed 36.6% killing of schistosomules in an in vitro killing assay.
Mohamed
M R
MR
Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt.
Shalaby
K A
KA
LoVerde
P T
PT
Abd Allah
N M
NM
Karim
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D003285:Contractile Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D064448:Filamins; D005455:Fluorescent Antibody Technique; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000235:genetics / Q000254:growth & development / Q000276:immunology / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D000909:Antibodies, Helminth; D003285:Contractile Proteins; D018076:DNA, Complementary; D064448:Filamins; D015801:Helminth Proteins; D007074:Immunoglobulin G; D008840:Microfilament Proteins