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International Journal of Virology

Year: 2005  |  Volume: 1  |  Issue: 1  |  Page No.: 2 - 2

Molecular Cloning and Expression of the Coat Protein Gene of Plum Pox Virus El-Amar Strain in E.coli

Abou El- Nasr, M. A, Dougdoug, K. A., Hayam S. Abdelkader and Rehab A. Dawoud

Abstract

Plum pox potyvirus (PPV), the causal agent of Sharka disease of Prunus, inflicts severe crop losses in affected Amar areas. The virus isolated from EL-Amar apricot trees, was propagated on apricot healthy seedlings. Degenerated oligonucleotide primers were designed to amplify the N-terminal portion of the capsid protein (Nib-CP) of PPV. The amplified products were cloned into pGEM-T-Easy vector and hybridized to PPV DNA specific probe labeled with Dig-11dUTP. DNA sequencing of the of the capsid protein gene using fluorescent dideoxy nucleotides showed that the coat protein region of PPV-EA strain had about 65% sequence homology with other strains of PPV and 45% similarity to the CP of PPV-D strain. A PCR fragment coding for the 43 C-terminal amino acids of the Nib and the N-teminal part of the CP (complete variable region plus 19 amino acids of the conserved core) was cloned into the pQE-100 expression vector. Upon induction, the viral protein gene was expressed as 6xHis-tagged PPV fusion protein in E.coli M15 cells. The fusion protein was confin-ned by western blot analysis.

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