Objective      To investigate the enzyme activity and protein expression localization of pyruvate oxidase encoded by

spxB

gene from Streptococcus pneumoniae (S.pn)

its conservation in different serotypes of S.pn，and its virulence mechanisms.       Methods      The full-length 

spxB

gene of S.pn D39 was amplified by PC

R with specific primers

and then inserted into pET-28a(+). Recombinant plasmid pET-28a(+)-spxB was transferred to E. coli BL21(DE3) and then the positive clones were induced with IPTG to express His-tagged rSpxB recombinant protein. The recombinant protein was purified by affinity chromatography column

and the protein purity was checked by SDS-PAGE. The enzyme activity assay was performed in a commercial pyruvate oxidase quality control method. The KM mice were immunized with the purified rSpxB protein to obtain polyclonal antibody. The antibody titers were determined by ELISA

and the antibody specificity and protein conservation were determined by Western blotting. The antibody was used for protein expression localization of SpxB in S.pn by flow cytometry. The virulence mechanism was studied preliminarily through the construction of spxB-deficient mutant strain and co-culture with Haemophilus influenzae.       Results      Soluble expression of SpxB with pyruvate oxidase activity was realized. Antiserum of high titer against SpxB protein was obtained from KM mice. Western blotting showed high antiserum specificity to SpxB and the conservation of spxB in the 1

2

4

6B

14

19F

and 23F serotypes of S.pn. Flow cytometry results showed that the fluorescence signal of SpxB shifted to the right compared to the negative control

but much weaker than that of the positive control. The wild D39 strain which produced hydrogen peroxide inhibited the growth of Haemophilus influenzae

while the SpxB-deficient strains showed much weaker activity.       Conclusion      Pyruvate oxidase encoded by SpxB is highly conserved in 7 different serotypes of S.pn and has mostly intracellular expression. SpxB helps S.pn hold advantages to Haemophilus influenza in co-culture.