The schematic map of (A) pBinβΔC1/p24 and (B) pBinβΔC1/gag constructs.

<p>In (A) the CLCuMBΔC1 DNA with a direct repeat of 282 bp flanked the 696 bp DNA fragment of HIV-1 p24 and in (B) flanked the 1501 bp DNA fragment of HIV-1 gag. The direct repeat of the CLCuMB DNA is represented by the dotted rods. The restriction enzymes used in the construction of the cassettes and their nucleotide positions in CLCuMB DNA (Acc. No. AJ298903) are shown by the vertical bars. The positions of the polyadenylation signal and the TATA box in βC1 promoter, upstream of the initiation codon of the βC1 ORF are shown by the A and TATA signals on the diagram, respectively. The one sided arrows are used to show the position of the primers used in the construction of the constructs and further analysis of the resulted DNAs in inoculated plants. The two sided arrows are used to show the expected size of the amplified fragments. These constructs cloned separately as <i>Kpn</i>I/<i>Sna</i>BI fragments into the pBin20 binary vector were used in agroinoculation experiments.</p

Optical density values at 405 nm of the ELISA plate readings of protein samples from inoculated plants.

<p>Plants, as indicated at the top of the columns, inoculated with the constructs as indicated at the bottom of the columns. The value represented for each treatment is the mean of three replicates. Bars with different letter are significantly (<i>p</i> ≤ 0.05) different. Quantitation of HIV-1 p24 protein in plant extracts was estimated by ELISA using the positive antigen (Ag) control of the ELISA kit with a definite (100 pgml-1) concentration. The p24 protein concentration was estimated to be 407.1 ngml^-1 in a <i>N</i>. <i>bethamiana</i> plant extract (corresponding to a yield of 814.2 ng per g fresh plant tissue) whereas the concentration of the same protein in four <i>N</i>. <i>glutinosa</i> plant extracts ranged from 39.4 to 231.3 ngml^-1 (corresponding to a yield of 78.8 to 462.6 ng per g fresh plant tissue). This showed that <i>N</i>. <i>bethamiana</i> was a more suitable host for production of HIV-1 <i>p24</i> protein using the CLCuMB-based expression vector.</p

Transreplicon of recombinant βΔC1/p24 DNA (1735 bp) in <i>N</i>. <i>glutinosa</i> and <i>N</i>. <i>benthamiana</i> plants.

<p>The DNA fragments were obtained from plants, as indicated at the bottom of the lanes, co-agroinoculated with the pBinβΔC1/p24 construct and the helper virus TYLCV-[Ab] (lanes 1–8) by PCR using specific adjacent beta1285^V/1290^C primer pair (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190403#pone.0190403.t001" target="_blank">Table 1</a>). No DNA was amplified from the extracts of plants inoculated with the helper virus alone (lanes 9–10) and plants co-inoculated with the pBinβΔC1/gag construct and the helper virus (lanes 11–15) using the same primer pair. M, marker DNA ladder (Fermentas).</p

Amplification of the 142 bp p24 DNA fragments (A) and 112 bp tobacco elongation factor (Ef) 1-alpha DNA fragments (B).

<p>The DNA fragments were obtained by RT-PCR using the total RNA extracted from <i>N</i>. <i>glutinosa</i> plants inoculated with the constructs as shown at the top of the lanes using a pair of either p24 (A) or tobacco EF1 (B) specific primer pairs as shown at the bottom of each panel. The p24 142 bp DNA fragment and 112 bp tobacco EF1 DNA fragment were absent (lane 7 of each panel) in PCR control reactions without RT from the extracted RNA of <i>N</i>. <i>glutinosa</i> plants co-inculated with βΔC1/p24 construct in the presence of TYLCV-[Ab].</p

List, sequences and other details of oligonucleotide primers used in this study.

<p>List, sequences and other details of oligonucleotide primers used in this study.</p