h, washed again and incubated with streptavidin conjugated to horseradish peroxidase at room temperature for 30 min. Finally, the plate was incubated with tetramethylbenzidine at room temperature for 15 min in the dark. The reaction was stopped with 250 mM sulfuric acid. Extinction was measured at 450 nm in a Synergy HT multidetection reader. For Western Blot analysis, total soluble protein was extracted from leaf and seed samples as described above. Samples 100 mg of total soluble protein were resuspended in 16 sample buffer containing 10% glycerin, 150 mM Tris, 3% SDS, 1% bmercaptoethanol and 2.5% bromphenol blue. The samples were heated to 95uC for 5 min and separated under denaturing conditions by 15% SDS-PAGE and then electrophoretically transferred to a 0.45-mm PVDF membrane. The proteins were transferred at a constant 2 mA/cm2 at room temperature for 2 h in a Bio-Rad Trans-Blot semi-dry transfer cell using 50 mM Tris, 40 mM glycine, 0.01% SDS and 20% methanol 23727046 as the transfer buffer. The membrane was blocked with PBS containing 0.05% Tween-20 and 5% nonfat milk powder at room temperature overnight and then 
probed at room temperature for 2 h with a mouse monoclonal anti-human IL6 antibody or a rabbit polyclonal anti-human-IL6 antibody, each at 1:1000 dilution. After three washes, the membrane was probed at room temperature for 2 h with a horseradish peroxidase conjugated secondary antibody, either goat anti-mouse or donkey antirabbit, each at 1:10000 dilution. The signal was detected by ECL chemilumniscence and the membrane was exposed to Kodak Biomax light X-ray film for 1 min before it was developed and fixed. concentration of active plant-derived IL6 compared to a commercial standard. Statistical methods Statistical comparisons were carried out using the F-test with p#0.05 considered significant. The variability of different events and siblings was characterized by the relative coefficient of 1 s variation Vr %~ pffiffiffi – -100. The 15976016 experimental design was calculated with SPSS. n y Results Construction of expression vectors for stable transformation and transient expression The IL6 expression vectors used for stable expression and transient expression were constructed by incorporating the codon-optimized human IL6 gene. Several different variants of each vector were created to achieve protein targeting to the apoplast, ER and vacuole. The sequences of all the transformation vectors were verified by DNA sequencing. Molecular analysis of transgenic tobacco plants One of the objectives of this study was to investigate the suitability of the two commercial tobacco cultivars for the production of human IL6. Transgenic plants were selected on medium containing kanamycin and grown to maturity in the greenhouse. Total genomic DNA was prepared from the crude leaf extracts of putative T0 transgenic plants and non-transgenic controls, and was screened for the presence of the IL6 sequence and the nptII marker gene resulting in the expected amplification products. Transgene expression was analyzed by multiplex RT-PCR using the endogenous actin housekeeping gene as an internal control. Products of the expected size were detected in transgenic leaf tissue and the presence of the expected actin cDNA amplification product but the absence of the corresponding genomic product confirmed the absence of genomic DNA contamination. There appeared to be no difference in mRNA expression levels between the three targeted variants of IL6. IL6 6-Carboxy-X-rhodamine web accumulati
