The pneumolysin (ply) gene, isolated from pneumococcal types 19F, 19A, 19B and 19C, has been cloned into a high-expression vector. The ply gene was prepared from a ClaI 5 kb fragment isolated from genomic DNA of group 19 strains, as well as 1.5 kb fragment synthesized by polymerase chain reaction from each strain. DNA samples were purified by gel electrophoresis, phenol extraction and ethanol purification. The DNA fragment was treated with a restriction endonuclease, such as pstI or EcoRI, dephosphorylated and ligated into a ClaI site of a vector pks(~), which contains a lac Z gene for fusion and ampicillin resistant gene for selection. The ply gene inserted vector was cloned into E. coli DH5a or XL1-Blue. Expression of the ply gene was studied in E. coli grown in Luria-Bertani medium supplemented with ampicillin. Synthesis of ply was induced by addition of isopropyl-b-D-thiogalactopyranoside (IPTG). Cells were lysed and the ply was purified by serial chromatography through DEAE-cellulose and Sephacryl S-200 columns. The presence of pneumolysin was monitored by hemolytic assay, using sheep erythrocytes. Initial results showed the transformed DH5a cells containing the ply gene expressed pneumolysin activity. Expression of the ply gene was studied under different experimental conditions and characterization of ply gene DNA fragments is continuing.
