Molecular Cloning and Expression of the Coat Protein Gene of Plum Pox virus El-Amar Strain in E. coli
Molecular Cloning and Expression of the Coat Protein Gene of Plum Pox virus El-Amar Strain in E. coli
M. A. Abou El- Nasr1, Kh. A. Dougdoug1, Hayam S. Abdelkader2, and Rehab A.
Dawoud2
ABSTRACT
Plum pox potyvirus (PPV), the causal agent of Sharka disease of Prunus inflicts severe crop losses in affected El-Amar area. 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 of PPV. The amplified products were cloned into pGEM-T-Easy vector and hybridized to PPV DNA specific probe labeled with Dig-I 1dUTP. DNA sequencing using fluorescent dideoxy nucleotides showed that the capsid protein region of PPV-EA strain had about 65% sequence homology with other strains of PPV and 45% similarity to the CP or PPV-D strain. A PCR fragment coding for the 43 C-terminal amino acids of the Nib and the N-terminal part of the CP (complete variable region plus 19 amino acids of the conserved core) was cloned and expressed into the pQE- 100 plasmid vector. Upon induction, the viral protein coat gene was expressed as 6xHis-tagged PPV/CP fusion protein in E. coli M15 cells. The fusion protein was confirmed by western blot analysis.
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