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

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Title Development of recombinant Nucleocapsid protein based indirect ELISA for serodiagnosis of Peste des Petits ruminants in sheep and goats
Not Available
 
Creator Balamurugan V
Roy M
Sowjanyakumari S
Abraham S
Apsana R
Nagalingam M
Hemadri D
Rahman H
 
Subject PPR virus
Recombinant
nucleocapsid
E. coli
indirect ELISA
serodiagnosis
 
Description Not Available
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
Not Available
 
Date 2018-11-05T09:17:15Z
2018-11-05T09:17:15Z
2016-06-16
 
Type Research Paper
 
Identifier Not Available
2307-8316
http://krishi.icar.gov.in/jspui/handle/123456789/9338
 
Language English
 
Relation Not Available;
 
Publisher Nexus Academic Publisher