& Objectives: Haemophilus influenzae is a Gram-negative bacterium that is part of the normal nasopharyngeal flora of most humans. H. influenzae strains were defined in two categories: encapsulated (typeable) and non-capsulated (nontypeable) strains. Outer membrane protein P6 is a highly conserved and stable protein in the outer membrane of both encapsulated and non-capsulated strains of H. influenzae. As an immunogen, P6 protein is a potential candidate vaccine against H. influenzae strains.

more »

... aim of this study was to produce recombinant protein P6 as a carrier protein for production of conjugate vaccines. Methods: The sequence (324 bp) coding P647-153 protein of H. influenzae was successfully cloned in pJET1.2 and subsequently in pET28a (+). Expression of the recombinant protein was induced with 1mM IPTG. Recombinant P647-153 was purified through dissolving inclusions in 8M urea buffer, absorbing to Ni-NTA resins, washing by buffers with decreasing urea concentration and finally eluted in Imidazole solution. Imidazole was removed by dialysis against PBS (pH 7.4). The recombinant p6 47-153 was confirmed by western blot analysis using rabbit anti H. influenzae polyclonal antiserum. Results: The recombinant P647-153 was successfully expressed in E. coli BL21 (DE3) and purified (4 mg /lit of broth culture). The immunoblotting showed that recombinant P647-153 conserved its native antigenic structure. Conclusion: Western blot results, along with that of sequencing, confirmed accurate production of recombinant P647-153 and partially retention of its conformational epitopes.