Cloning and characterisation of a highly immunoreactive 37 kDa antigen with multi-immunoglobulin domains from the swine roundworm Ascaris suum.
Antigens from larval stages of Ascaris suum have been shown to induce protection against challenge infection with infective A. suum eggs. We previously identified several antigens that reacted strongly with serum from pigs inoculated with infective eggs containing L3. In this study, we isolated an antigen with a molecular mass of 37 kDa and a pI of 4.8 (As37) from A. suum infective eggs using two-dimensional electrophoresis, and obtained a full-length cDNA by reverse transcription-polymerase chain reaction using primers designed based on the internal amino acid sequence of As37. The cDNA sequence consisted of 1,540 bp coding for a protein of 321 amino acids with a complex domain organisation. Simple modular architecture research tool (SMART) analysis indicated that As37 contains three immunoglobulin domains, indicating that it is a member of immunoglobulin superfamily (IgSF). A homology search of GenBank showed that As37 has significant similarity to Caenorhabditis elegans DIM-1 protein and has low similarity to part of the multi-repeat Ig domain from nematode twitchin and mammalian skeleton muscle titin, and to members of the IgSF at the amino acid sequence level. Localisation analysis revealed that antibodies to Escherichia coli-expressed recombinant As37 (rAs37) bound to muscle cells and the hypodermis. The antibodies identified a 37 kDa native antigen in human and dog roundworms, suggesting that there are As37 homologues in ascarid nematodes. Sera from mice, rabbits and pigs immunised with A. suum infective eggs reacted with rAs37 in immunoblot analyses. The potential use of rAs37 for protection against A. suum infection is discussed.
Tsuji
Naotoshi
N
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organisation, Tsukuba, Ibaraki 305-0856, Japan. tsujin@affrc.go.jp
Kasuga-Aoki
Harue
H
Isobe
Takashi
T
Arakawa
Takeshi
T
Matsumoto
Yasunobu
Y
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000302:isolation & purification*; D001196:Ascariasis / Q000276:immunology*; D017165:Ascaris suum / Q000276:immunology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D013552:Swine; D013553:Swine Diseases / Q000276:immunology*
D000947:Antigens, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth