Cloning and Expression of the Coat Protein Gene of Barley Yellow Dwarf Virus-PAV in Escherichia coli

Document Type: Research Paper


1 Department of Plant Pathology, Tarbiat Modarres University, P.O. Box: 14115-143, Tehran, I.R. Iran.

2 Department of Plant Science, Waite Institute, University of Adelaide, Glen Osmond, SA 5064, Australia.


Due to the restriction of Barley yellow dwarf virus (BYDV)-PAV particles to the phloem tissue and very low virus titers, purification of the virus is difficult. The aim of this study was to prepare antibody against viral
coat protein without purifying the virus. To produce recombinant coat protein, the coding sequence was
first amplified from a PAV full-length cDNA clone by polymerase chain reaction (PCR), ligated into a vector
(pBluescript SK+) to check the sequence, and subcloned into an expression vector (pGEX-2T). It was
then transformed into Escherichia coli DH5α by electroporation. The open reading frame 3 (ORF3) was
linked in-frame to the gene encoding glutathione-Stransferase (GST; 26 kDa) and expression induced by
IPTG. The expressed coat protein was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) for use as an immunogen. The antisera to BYDV-PAV recombinant coat protein reacted in
Western blot analysis with partially purified BYDV-PAV. These antisera were also used to detect BYDV-PAV by
immunogold electron microscopy of thin section of barley tissues. The results indicated that BYDV-PAV coat
protein can be produced in high yields by E. coli, which provides the ability of simple purification, and because
of proper antigencity, can be exploited for diagnostic applications.