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Activity analysis of <i>N. benthamiana</i>-expressed rAH.

By Nobuyuki Matoba (211462), Adam S. Husk (364094), Brian W. Barnett (171386), Michelle M. Pickel (364095), Charles J. Arntzen (364096), David C. Montefiori (108747), Atsushi Takahashi (128295), Kazunobu Tanno (364097), Satoshi Omura (44367), Huyen Cao (318069), Jason P. Mooney (364098), Carl V. Hanson (364099) and Haruo Tanaka (364100)

Abstract

<p>(A) The gp120-binding capacity of rAH expressed in <i>N. benthamiana</i> was evaluated by gp120-ELISA using AH-expressing and control (uninfiltrated) leaf extracts prepared as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011143#s2" target="_blank">Methods</a>. Results of a representative sample obtained from a ∼2 g batch of leaf materials are shown. Data points are the means ± SD of triplicate analysis. This analysis was also used to determine the quantity of rAH in the extract based on a standard curve created by actinomycete-derived purified AH. (B) X-gal staining of syncytia formation by HeLa/Env/tat and HeLa/CD4/LacZ. The two cell lines were incubated for 18 h at 37°C with: <b>a</b>, media alone; <b>b</b>, 1/20-diluted non-infiltrated control leaf sample; <b>c</b>, 1 µM actinomycete-derived AH; and <b>d</b>, 1/20-diluted rAH-expressing leaf sample (containing approximately 0.32 µM, or 4.0 µg/ml, of rAH, as determined by gp120-ELISA). (C) Quantitative analysis of syncytia formation by HeLa/Env/tat and HeLa/CD4/LacZ. The two cell lines were incubated for 18 h at 37°C with serially diluted control or rAH-expressing leaf samples. See Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011143#s2" target="_blank">Methods</a> for the assay detail. Differences between the samples at each dilution point were evaluated by two-way analysis of variance followed by Bonferroni posttests (*** <i>p</i><0.001).</p

Topics: Medicine, Biotechnology, Virology, biotechnology/plant biotechnology, virology/antivirals, including modes of action and resistance, virology/immunodeficiency viruses, women's health/sexually transmitted diseases
Year: 2013
DOI identifier: 10.1371/journal.pone.0011143.g006
OAI identifier: oai:figshare.com:article/516275
Provided by: FigShare
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