Obtaining recombinant nucleocapsid protein of PPR virus for disease serodiagnostic

Abstract

Peste des petits ruminants is a highly contagious, acute or subacute viral disease of sheep and goats, characterized by fever, sores in the mouth, haemorrhagic gastroenteritis, lesions in lymphatic system and pneumonia. (E.P.J. Gibbs et al., 1979; A. Diallo et al., 1989; T.M. Ismail et al., 1995). Because of high morbidity of 50-100 % and mortality of 50-90 %, Peste des petits ruminants belongs to a number of emerging diseases, having a significant threat to livestock production in countries where the disease is notified (R.A. Kock et al., 2015; E.M.E. Abu-Elzein et al., 1990). The etiological agent of PPR is a Morbillivirus (PPRV) of Paramyxoviridae family (M.H.V. Van Regenmortel et al., 2000). The PPRV antigens are similar to antigens of other Morbilliviruses (G. Libeau et al., 2014). Severity of the clinical signs depends on different factors, e.g. PPRV line, animal species, breed, and immune status. Because of that, the final diagnosis must be confirmed by laboratory methods. In diagnostics and monitoring serological testing, the preference is given to sensitive and automated Enzyme-linked immunosorbent assay (ELISA). Modern methods of PPRV serodiagnostic are developed on the basis of virus-specific recombinant proteins and primary nucleocapsid N protein (A. Diallo et al., 1994; G. Libeau et al., 1995; M. Munir et al., 2013; N.V. Vavilova et al., 2006.), which is superior to the other Morbilliviruses’s proteins in antigenic and immunogenic characteristics (P.C. Lefevre et al., 1991; M. Yunus et al., 2012). The other protein N advantage is that, as an antigen, it is the most conservative of the PPR virus proteins (M. Muhammad, 2013). The purpose of this paper was to obtain recombinant nucleocapsid N protein of PPR virus as an antigen and virus-specific antiserum of pigs as a source of antibodies for serodiagnostic of disease by competitive ELISA. A gene construct was designed which contained a sequence of protein N gene fragment of 1530 kb in length in the express plasmid vector pET32a. After polypeptide screening by SDS-PAGE and immunoblotting we found clones of Escherichia coli pET32а/N/10 which express 70 kDa virus-specific major polypeptide. It was shown, that in competitive ELISA the optimal dose of recombinant protein N purified by Immobilized Metal Chelate Affinity Chromatography method is 0.25 μg per well. The ratio of OD450 values for negative and positive control goat sera was 11.52. So the electrophoretically purified and immunochemically pure recombinant protein N can be used in competitive ELISA for PPRV serodiagnostic. For obtaining specific antisera, pigs were inoculated with purified PPRV. The antibody titers in antisera samples from the pigs in a neutralization test with PPRV were 1:64-1:128. These values are comparable with antibodies titers in sera of sheep and goats vaccinated against PPR (A.V. Konstantinov et al., 2017). However pigs’ antisera were less effective in competitive ELISA than positive goat sera.