Paper Information

Journal:   IRANIAN JOURNAL OF BIOTECHNOLOGY   April 2004 , Volume 2 , Number 2; Page(s) 84 To 89.
 
Paper: 

CLONING AND EXPRESSION OF THE COAT PROTEIN GENE OF BARLEY YELLOW DWARF VIRUS-PAV IN ESCHERICHIA COLI

 
 
Author(s):  SHAMSBAKHSH M.*, HENRY SYMONS R.
 
* TARBIAT MODARRES UNIVERSITY, TEHRAN, I.R. IRAN
 
Abstract: 
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 sub-cloned into an expression vector (pGEX-2T). It was then transformed into Escherichia coli DH5a by electroporation. The open reading frame 3 (ORF3) was linked in-frame to the gene encoding glutathione-S-transferase (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.
 
Keyword(s): BARLEY YELLOW DWARF VIRUS, EXPRESSION VECTOR, POLYCLONAL ANTIBODIES, E. COLI, COAT PROTEIN, PAV
 
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