Abstract:

Objective To obtain Streptococcus pneumoniae pneumolysin（PLY） through prokaryotic expression system,and to investigate its serological diagnosis role for infective pneumonia with Streptococcus pneumoniae. Methods The specific primers with cutting 33 nucleotides of carboxyl end were designed according to ply gene sequence of Streptococcus pneumoniae in GenBank. The ply gene was amplified by using the genome DNA of Streptococcus pneumoniae as template. The recombinant plasmid PET32a-ply was constructed,and it was expressed after transformation in Escherichia coli DH5aBL21（DE3）. Overexpression of recombinant protein without hemolysis activity in gene labeled with 6 infused amino acids was induced by isopropyl phosphorothioate（IPTG）. The immunocompetence of recombinant protein PLY was determined by western blotting after it was purified with Ni-NTA resin and identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. Enzyme-linked immunosorbent assay（ELISA） reactive mode was established by setting PLY as IgM antigen so as to determine community acquired pneumonia（CAP）,and the role of PLY in the diagnosis of infective pneumonia with Streptococcus pneumoniae was evaluated. Results The prokaryotic expression vector PET32a-ply was successfully constructed. The recombinant protein PLY without hemolysis activity had soluble expression,with good immunogenicity. The positive rate of anti-PLY IgM antibody in CAP patients was higher than that in healthy subjects（P<0.05）,and the sensitivities were higher than those of conventional sputum culturing and blood culturing（P<0.05）. Conclusions ELISA reactive mode based on the recombinant protein PLY could be used for the serological diagnosis of infective pneumonia with Streptococcus pneumoniae.

Key words: Streptococcus pneumoniae, Pneumolysin, Prokaryotic expression, Immunogenicity