Generation of High Titre Antibodies to Pokeweed Antiviral Protein (PAP) in Rabbits Using Synthetic Peptides and their Use in Detecting PAP in Transgenic Petunia and Yeast

Abstract

Antibodies are important for the study of pokeweed antiviral protein (PAP), an important antiviral agent against many plant, animal and human viruses. As PAP is expressed only at a low level in pokeweed plants (Phytolacca americana L.), it is complex and time‐consuming to extract PAP from pokeweed plants for antibody preparation. Here, we describe an antigen‐designed method according to the amino acid sequence that translated from PAP gene cleaving the C‐terminus toxic region and N‐terminus signal peptide (Genbank No. ); the two peptides, DC15: DISGTERQDVETTLC and CR15: CRYPTLESKAGVKSR, were synthesized for generation of antibodies. The design strategy enabled straightforward antigen production and antibody generation. The antibodies can be used to detect PAP in transgenic petunia plants (Petunia hybrida Vilm.), pokeweed plants (P. americana) and transformed yeast (Pachia pastoris), which can express the PAP gene by western blotting. These antibodies generated against synthetic peptides will be useful for various assays such as for PAP detection, immunoprecipitation, protein purification and western blot analysis.

Improved Resistance to Cucumber mosaic virus in Petunia Transformed with Non‐Cytotoxic Pokeweed Antiviral Protein Gene

“…Subsequently, the expression product of mutant PAP in ten RT-PCR-positive transgenic lines was confirmed by Western blot assay. One band of 26 kDa protein from the six transgenic petunia lines (WR2①, WR10①, WR37①, WR39②, WR45② and WR48①) and expression product of mutant PAP in yeast have a strong reaction with the anti-PAP antibody, and a band of 29 kDa protein, which was natural PAP from pokeweed leaves collected in spring of pokeweed plants, had been detected with the anti-PAP antibody (Li et al 2012). In contrast, no signal was observed in the non-transgenic petunia (negative control).…”