Development of polyclonal antibodies against the recombinant protein of Barley yellow dwarf virus
Muhammad Khalid1, Najam us Sahar Sadaf Zaidi1, Naeem Rashid2, Muhammad Tahir1*
1Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan
2School of Biological Sciences (SBS), University of the Punjab, Lahore, Pakistan
Abstract
Barley yellow dwarf virus (BYDV) particle purification is challenging because of the limited phloem tissue and extremely low viral titers. The current study aimed to generate antibodies against the viral coat protein, without purification of the viral particles. To produce the recombinant coat protein, the genomic region of BYDV encoding the coat protein (CP), was cloned, and expressed in Escherichia coli (E. coli.) BL21 (DE3) strain. Physicochemical characteristics, subcellular localization, and immunogenicity of the BYDV CP (coat protein) were identified using an in-silico approach. The BYDV CP was synthesized by Synbio Technology, USA and cloned into the pET28a (+) expression vector, to produce recombinant fusion coat protein (rFCP-BYDV) in Escherichia coli (E. coli). The recombinant protein produced in inclusion bodies was denatured and purified with on-column refolding by affinity chromatography. Purified protein (rFCPBYDV) was used, as an antigen followed by four weekly intraperitoneal booster doses in mice to develop pAB antisera, which was collected by cardiac puncture, to raise polyclonal antibodies (pAB) in mice. The raised anti-BYDV CP immunoglobulins (IgGs) detected the recombinant BYDV CP even at 100 pg/mL and 1000-time diluted crude extract of proteins from BYDV-infected wheat plant leaves. Results from indirect ELISA titration showed that the anti-BYDVCP antiserum produced in mice had a titer of around 1:10,000. The findings offer a quick and simple immunodiagnostic technique for rapid detection of BYDV. To the best of our knowledge, this is the first report on the production of anti-BYDV CP pAB and their application for the diagnosis of BYDV disease in Pakistan.
