<p><b>OBJECTIVE</b>To prepare pneumolysin as a new protein carrier of vaccine against otitis media with genetic engineeringtechnology and establish the base of the study on pneumococcal conjugative vaccines.</p><p><b>METHODS</b>Genomic DNA was isolated from streptococcus pneumoniae. A pair of primers which included two restriction sites was designed based on the published pneumolysin gene sequence. The pneumolysin gene was amplified from pneumococcal DNA with PCRtechnology. The restriction enzyme digested fragment was linked into the cloning vectorPET-28a and the recombinant plasmidDNA containing pneumolysin was then transfected into host cell E. coli JM109 (DE3).</p><p><b>RESULTS</b>DNA fragments were subcloned to construct the complete pneumolysin gene by a conventional coning and PCR. The inserted pneumolysin gene sequence was confirmed by DNA sequencing and the pneumolysin protein was successfully expressed. The relative molecular mass of the expressed product was 52 000. The expressed product amounted to 8% of the total host cellprotein.</p><p><b>CONCLUSIONS</b>The pneumolysin gene was successfully cloned into host cell using genetic engineeringtechnology. The recombinant pneumolysin was expressed and purified for preparation. This work laid a foundation of the preparation of pneumococcal conjugative vaccines.</p>