Recombinant HyC antigen
The recombinant HyC protein was derived from P. silenus expressed in Escherichia coli (strain BL21 DE3 pLysS) transformed with the pET-32a(+) expression vector as described in Yadav et al.18 The rHyC was obtained as fusion protein with thioredoxin tag and used as antigen in the present study. Briefly, a confirmed positive recombinant clone was cultured in LB broth containing ampicillin and chloramphenicol, incubated at 37oC till it attains an OD of 0.6. Subsequently it was induced by adding IPTG to a final concentration of 1 mM. The culture was allowed to grow at 37oC with shaking at 200 rpm for 6 h and purified under denaturing condition using 8 M urea dialyzed against gradient urea concentration (8 M to 0 M) and finally in Tris buffer pH 8.0. The homogeneity of purified protein was analyzed by 12% SDS-PAGE and quantified by Bradford reagent.