Cloning and expression in Escherichia coli of cDNAs encoding a 31-kilodalton surface antigen of Sarcocystis muris.
Using polyadenylated RNA isolated from Sarcocystis muris cyst merozoites, we have constructed a cDNA library in the expression vector lambda ZAP. Immunoscreening with monoclonal and polyclonal antibodies directed against a 31-kDa surface antigen of S. muris [1] yielded a number of clones with insert sizes ranging between 1.1 kb and 1.3 kb. An additional clone with an insert length of 1.55 kb was isolated by screening with a labeled DNA probe derived from one of the cDNA clones. The cDNA sequence was found to contain an open reading frame specifying a polypeptide of 280 amino acids with a predicted size of 29.7 kDa. The deduced amino acid sequence is rich in serine and threonine (22%) and harbors a hypothetical N-terminal signal peptide sequence as well as a C-terminal glycosyl phosphatidylinositol anchor attachment site. The predicted amino acid sequence has been confirmed by peptide sequencing and an analysis of the overall amino acid composition of the 31-kDa protein. A recombinant protein was obtained which was recognized by the polyclonal antibodies directed against the 31-kDa antigen. Antiserum raised against the purified fusion protein specifically reacted with a 31-kDa protein from S. muris cystozoites. Southern blot analysis indicated that the corresponding gene exists as a single copy within the S. muris genome.
Eschenbacher
K H
KH
Arbeitsgruppe Molekulare Genetik, Bochum, Germany.
Sommer
I
I
Meyer
H E
HE
Mehlhorn
H
H
Rüger
W
W
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D000954:Antigens, Surface / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012522:Sarcocystis / Q000235:genetics* / Q000276:immunology*
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins