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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

1 Microbiology Department, Faculty of Agriculture, Ain Shams University. Shubhra EIkheima, Cairo, Egypt.
2 Plant Virus and Phytoplasma Research Department, Plant Pathology Research Institute, Agricultural Research Center. Giza. Egypt.

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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Journal of Virological Sciences

July

Vol. 3, Iss. 1

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