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Biotecnología Aplicada

On-line version ISSN 1027-2852

Abstract

MAHFOUZE, Heba A; EL-DOUGDOUG, Khalid A; OTHMAN, Badawi A  and  GOMAA, Mostafa A. Isolation and cloning of the Phytolacca americana anti-viral protein PAP-I geneAislamiento y clonaje del gen de la proteína antiviral PAP-I de Phytolacca americana. Biotecnol Apl [online]. 2014, vol.31, n.1, pp.57-61. ISSN 1027-2852.

The pokeweed antiviral protein (PAP) isolated from Phytolacca americana and Phytolacca acinosa plants, inhibits protein translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. In this study, the P. americana PAP-I gene was isolated and sequenced, and further compared to other ribosome-inactivating proteins (RIPs) genes previously reported in GenBank(r). Total DNA was extracted from the late summer leaves of P. americana. A polymerase chain reaction (PCR) 868 bp-long DNA product was obtained, using gene specific primers, based on the expected gene size. The eluted product was purified and cloned into the pTZ57R/T vector, and mobilized into the Escherichia coli strain DH5a. After sequencing, the analysis of the PAP-I PCR product showed 98 to 82 % nucleotide and amino acid 94 to 26 % homologies, respectively, compared to previously reported RIPs. A phylogenetic analysis confirmed that the amplified PAP-I gene corresponds to the single chain Type-I RIP (PAP-I).

Keywords : pokeweed antiviral protein; cloning; PCR; DNA sequencing; phylogenetic alignment.

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