title: Cloning and expression of Legionella pneumophila antigens in Escherichia coli.
abstract: To isolate and characterize Legionella pneumophila antigens, we constructed a genomic library of L. pneumophila serogroup 1 (strain 130b). L, pneumophila DNA fragments (2.5 to 7.5 megadaltons) obtained by partial digestion with Sau 3A endonuclease and size fractionation on a sucrose density gradient were inserted into the dephosphorylated BamHI site of vector pBR322; CaCl2-treated Escherichia coli cells of strain HB101 were transformed with hybrid plasmids. To detect expression of antigens, 2,559 ampicillin-resistant transformants were transferred to nitrocellulose paper, lysed in situ, and screened by enzyme immunoassay (EIA) with E. coli-absorbed rabbit anti-L. pneumophila sera. A total of 77 (3%) of the colonies were reactive by EIA; 31 (1.2%) were strongly reactive, and 6 were strongly reactive by EIA without colony lysis. Analysis of 29 stable, strongly reactive clones by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting showed antigenic bands in 18 clones by EIA with E. coli-absorbed antisera. Absorption of antisera with heat- and Formalin-killed L. pneumophila antigen eliminated or diminished the reactivity of the antigenic bands in representative clones. These studies confirm that several L. pneumophila antigens can be cloned and expressed in E. coli.
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D007875:Legionella / Q000235:genetics / Q000276:immunology*; D008970:Molecular Weight; D010957:Plasmids; D014158:Transcription, Genetic
chemical_list: D000942:Antigens, Bacterial; D007106:Immune Sera; D004262:DNA Restriction Enzymes