title: Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri.
abstract: We previously cloned an antigenic gene (named nfa1) from a cDNA library of Naegleria fowleri by immunoscreening. The nfa1 gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of N. fowleri against Chinese hamster ovary (CHO) cells. Trophozoites of N. fowleri in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with N. fowleri, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. When examined using a light microscope, CHO cells grown in cocultures with N. fowleri trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with N. fowleri trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.
authors.affiliation: Department of Microbiology, School of Medicine, Ajou University, Suwon 442-749, Korea.
authors.forename: Myoung-Soo
authors.initials: MS
authors.lastname: Cho
authors.forename: Suk-Yul
authors.initials: SY
authors.lastname: Jung
authors.forename: Sun
authors.initials: S
authors.lastname: Park
authors.forename: Kyongmin Hwang
authors.initials: KH
authors.lastname: Kim
authors.forename: Hyung-Il
authors.initials: HI
authors.lastname: Kim
authors.forename: Seonghyang
authors.initials: S
authors.lastname: Sohn
authors.forename: Han-Jip
authors.initials: HJ
authors.lastname: Kim
authors.forename: Kyung-Il
authors.initials: KI
authors.lastname: Im
authors.forename: Ho-Joon
authors.initials: HJ
authors.lastname: Shin
mesh_terms: D000562:Amebiasis / Q000276:immunology; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D001921:Brain / Q000382:microbiology; D016466:CHO Cells; D003001:Cloning, Molecular; D018920:Coculture Techniques; D006224:Cricetinae; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007150:Immunohistochemistry; D051379:Mice; D008854:Microscopy, Electron; D016848:Naegleria fowleri / Q000276:immunology* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C460560:Nfa1 protein, Naegleria fowleri; D015800:Protozoan Proteins; D011994:Recombinant Proteins