Development of recombinant Nucleocapsid protein based indirect ELISA for serodiagnosis of Peste des Petits ruminants in sheep and goats

Abstract

In this study, expression of immunogenic portion of Peste des Petits ruminants virus (PPRV) nucleocapsid (N) protein in Escherichia coli (BL21) was envisaged to evaluate the potential use of recombinant protein as a diagnostic antigen in polyclonal antibodies based indirect ELISA for serodiagnosis. The immunogenic region of N gene coding sequences from PPR vaccine virus (Sungri 96 strain) was amplified, cloned in pET32a vector and expressed in E. coli as fusion protein for bulk production and easy Ni-NTA His-tag purification. The recombinant PPRV N protein (rPPRVNP) was expressed in E. coli at an optimal temperature of 37 °C with 1 mM IPTG for 5 h post induction and characterized by SDS-PAGE and western blot using PPRV-specific monoclonal and polyclonal antibodies or serum or anti-His-tag conjugate that confirmed PPRV specific protein with a size of ~50kDa. The expressed rPPRVNP was in insoluble form and was purified under denaturation condition by Ni-NTA purification method followed by refolding, renaturation methods and further concentrated by protein cut-off concentrators for obtaining single protein band. The rPPRVNP was assessed for its immunoreactivity as diagnostic antigen by immunoblotting and ELISA using standard PPRV specific antibodies. The immunogenic reactivity of expressed rPPRVNP was optimized in indirect ELISA using known true positive and negative sera with respect to PPRV antibodies. On standardization of rPPRVNP based indirect ELISA using 661serum samples, the relative diagnostic sensitivity and specificity of the assay 83.76% and 83.13% respectively was observed at cut off level of 25 Per cent Positivity (PP) with an agreement of Cohen’s kappa value of 0.648 with good agreement. This indirect ELISA as additional diagnostic tool for diagnosis of PPR in sheep and goats and rPPRVNP could be a sustainable source of safe antigen in countries of non-endemicity without the need to handle infectious virus for sero-diagnosis