Objective      To investigate the interaction between heat shock protein DnaJ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

a key enzyme of glycolysis

in Streptococcus pneumoniae (S. pn).       Methods      The full-length gene of gap was amplified by PCR with specific primers from S.pn D39 and the

n cloned into the expression vector pET-28a(+). Recombinant pET-28a(+)-gap was transformed into E.coli BL21 (DE3) and induced with IPTG to express 6 His-tagged GAPDH. The affinity chromatography column was used to purify recombinant GAPDH

followed by SDS-PAGE to test its purity. The recombinant GAPDH was used to immunize kunming mice to prepare polyclonal antibodies. The antibody titer was tested by ELISA and the conservation of GAPDH was confirmed by Western blotting. The interaction between GAPDH and DnaJ was validated by BacterioMatch II two-hybrid system

direct binding assay and bio-layer interferometry (BLI).       Results      Recombinant GAPDH was successfully expressed with a purity of 90%

and its polyclonal antibodies (a titer of 10

7

) were obtained. Western blotting showed a specific band recognized by GAPDH antibody in the whole cell and culture medium from 7 serotypes of S.pn. BacterioMatch II two-hybrid system suggested that GAPDH interacted with DnaJ in vivo. Both direct binding assay and BLI demonstrated that GAPDH directly interacted with DnaJ in a concentration-dependent manner. BLI also showed that their affinity constant reach to 1.12×10

-7

mol/L.       Conclusion      GAPDH is a highly conserved secretion protein and directly interacts with DnaJ

which is important to determine the effects of DnaJ to GAPDH on its secretion and violence in S.pn.