In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus. (December 2020)

• Record Type: Journal Article
• Authors: Kokane, Sunil B.; Kokane, Amol D.; Misra, Pragati; Warghane, Ashish J.; Kumar, Pranav; Gubyad, Mrugendra G.; Sharma, Ashwani Kumar; Biswas, Kajal Kumar; Ghosh, Dilip Kumar
• Is Part Of: Molecular and cellular probes. Volume 54(2020)
• Publication Date: 2020-12
• Subjects: Citrus tristeza virus -- RNA binding Protein (CTV-p23) -- Western blot -- ELISA; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2020.101654
• Languages: English
• ISSNs: 0890-8508

Abstract

Abstract: Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein ( CTV-p25 ) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus. Highlights: CTV p23 gene was cloned and polyclonal antibodies raised against bacterially expressed recombinant CTV p23 protein. Produced polyclonal antibodies were used to develop a sensitive and economically affordable CTV detection tool. RT-PCR assay using CTV p23 gene specific primers was 1000 times more sensitive compared to coat protein gene based primers. In silico characterizations of CTV p23 protein has been accomplished.