Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen

Yadav, Vinita; Balamurugan, V.; Bhanuprakash, V.; Sen, Arnab; Bhanot, Vandna; Venkatesan, Gnanavel; Riyesh, Thachamvally; Singh, Rohit Kumar

doi:10.1016/j.jviromet.2009.07.014

Cited by 33 publications

(25 citation statements)

References 34 publications

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“…The expressed PPRV F protein in bacterial [51] and eukaryotic [80] system showed antigenicity and immunogenicity [159]. Further, the expressed truncated and full-length N protein of PPRV in E. coli showed reactivity in s-ELISA and tested as a coating antigen in c-ELISA for serological diagnosis of PPR infection [161]. Recently, Liu et al [84] produced polyclonal antibodies against the recombinant truncated PPRV M protein expressed in E. coli and checked its specificity in western blot and immunofluorescence.…”

Section: Recombinant Antigen Based Assaysmentioning

confidence: 99%

Diagnosis and control of peste des petits ruminants: a comprehensive review

Balamurugan¹,

Hemadri²,

Gajendragad³

et al. 2013

VirusDis.

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135

118

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Peste des petits ruminants (PPR) is an acute, highly contagious, world organization for animal health (OIE) notifiable and economically important transboundary viral disease of sheep and goats associated with high morbidity and mortality and caused by PPR virus. PPR is considered as one of the main constraints in augmenting the productivity of small ruminants in developing countries and particularly severely affects poor farmer's economy. The disease is clinically manifested by pyrexia, oculo-nasal discharges, necrotizing and erosive stomatitis, gastroenteritis, diarrhoea and bronchopneumonia. The disease can be diagnosed from its clinical signs, pathological lesions, and specific detection of virus antigen/antibodies/genome in the clinical samples by various serological tests and molecular assays. PPR is the one of the priority animal diseases whose control is considered important for poverty alleviation in enzootic countries. Availability of effective and safe live attenuated cell culture PPR vaccines and diagnostics have boosted the recently launched centrally sponsored control programme in India and also in other countries. This review article primarily focus on the current scenario of PPR diagnosis and its control programme with advancement of research areas that have taken place in the recent years with future perspectives.

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Section: Recombinant Antigen Based Assaysmentioning

confidence: 99%

Diagnosis and control of peste des petits ruminants: a comprehensive review

Balamurugan¹,

Hemadri²,

Gajendragad³

et al. 2013

VirusDis.

Self Cite

135

118

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“…The extracted RNA was used as a template for reverse-transcription (RT) by using random hexamer primer (MBI, Fermentas, MD, USA) and 200U of MMuLV reverse transcriptase (Promega, Madison, USA). The amino terminal portion of N Protein coding sequence was amplified from the synthesized cDNA, by PCR using Pfu DNA polymerase (MBI, Fermentas, MD, USA) and PPRV N gene-specific primers PPRNF or (EcoRI): 5'-ATCT-GAATTCATGGCTACTCTCCTTAAAAGC-3' and PPRNRev (NotI) (His): 5'-ATG GCGGCCGCATG-GTGATGGTGATGGTGGAGTCCGGCTTCGA-CAATATA-3' modified from earlier reported one (Choi et al, 2005a;Yadav et al, 2009), which were designed based on published sequence (Accession #AY560591). The applied cycling conditions were: preheating at 95°C for 3min, 35 cycles of 94°C for 45sec, 50-60°C (incremental step up annealing +1°C/cycle) for 1 min and 72°C for 2 min with a final extension of 72°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…Earlier recombinant antigen based diagnostic methods have been developed for the detection of several morbilliviruses such as rinderpest virus (RPV) (Kamata et al, 1993), PPRV (Ismail et al, 1995;Libeau et al, 1995;Choi et al, 2005b;Dechamma et al, 2006;Balamurugan et al, 2006;Yadav et al, 2009), canine distemper virus (CDV) (Latha et al, 2007) and Nipah virus (Yu et al, 2006). Among the structural proteins of morbilliviruses, N protein is the highly conserved immunogenic core protein (Lefebvre et al, 1991) and is expressed at a high level in infected cells (Diallo et al, 1994).…”

mentioning

confidence: 99%

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Development of Recombinant Nucleocapsid Protein based indirect ELISA for Serodiagnosis of Peste des Petits Ruminants in Sheep and Goats

Balamurugan¹,

Roy²,

SowjanyaKumari³

et al. 2016

Adv. Anim. Vet. Sci.

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| 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.

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“…Like c-ELI-SA, in this assay also live attenuated PPRV used as positive antigen. Attempts were made to develop a recombinant N protein based ELISA (Yadav et al, 2009). …”

Section: Sandwich Elisamentioning

confidence: 99%

Peste-Des-Petits-Ruminants: An Indian Perspective

Muthuchelvan¹

2015

Adv. Anim. Vet. Sci.

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Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen

Cited by 33 publications

References 34 publications

Diagnosis and control of peste des petits ruminants: a comprehensive review

Diagnosis and control of peste des petits ruminants: a comprehensive review

Development of Recombinant Nucleocapsid Protein based indirect ELISA for Serodiagnosis of Peste des Petits Ruminants in Sheep and Goats

Peste-Des-Petits-Ruminants: An Indian Perspective

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