Objective   To detect the expression of in vitro amplified pneumolysin gene in E.coli and verify its biological activity.    Methods   DNA was isolated from cultured S.pneumoniae D39. Pneumolysin gene was amplified in vitro by PCR and cloned into pET-28(α) expression vector. Recombinant pneumolysin protein was transferred to E.coli Rosetta(DE3) by sequencing. Over-expression of recombinant pneumolysin gene labeled with 6 infused amino acids was induced by IPTG. Biological activity  of the recombinant pneumolysin gen

e were detected after it was purified with Ni-NTA resin and identified with SDS-PAGE.    Results    The sequence of cloned pneumolysin gene was consistent with that in the GenBank. The pneumolysin gene was expressed a soluble manner. The purity of recombinant pneumolysin protein was 95%. Hemolysis testing showed that the hemolytic activity of pneumolysin increased in human red blood cells in a dose-dependent manner more significantly in experimental group than in control group (P0.05) . Cytotoxicity testing showed that pneumolysin inhibited the human umbilical vein endothelial cells in a dose-and-time-dependent more significantly in experimental group than in control group (P0.05). The IC

50

(24 h) was 1.525 μg/ml.    Conclusion   The amplified pneumolysin protein can be identified by sequencing

expressed and purified with a hemolytic activity and a cytotoxicity.