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

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.