”type”:”entrez-nucleotide”,”attrs”:”text”:”AY172957″,”term_id”:”27728676″,”term_text”:”AY172957″AY172957). untranslated leader sequence of tobacco etch virus; SP, endoplasmic reticulum signal peptide; KDEL, ER retention signal; 35S-T, terminator of Cauliflower mosaic virus 35S gene. (b) Structure of recombinant proteins. (c) strain LBA4404 by electroporation. Transgenic tobacco plants were generated by plants were grown in a greenhouse under controlled conditions. 2.3. PCR Amplification of Genomic DNA Gly-Phe-beta-naphthylamide Genomic DNA was isolated from the fresh leaf tissue of transgenic and non-transgenic plants using a DNA extraction kit (iNtRON Biotechnology, Seoul, Republic of Korea) according to the manufacturer’s recommendations. PCR amplification of genomic DNA was performed in order to confirm the presence of the recombinant genes using the following primer pairs: for GA733K, forward primer 5-GCG TCG ACA CGG CGA CTT TGC CGC TCA GGA A-3, reverse primer 5-GCT CTA CAT CAG AGT TCA TCT TTT TTT AGA CCC TCG ATT GAG-3; for GA733-FcK, forward primer 5-GCG TCG ACA CGG CGA CTT TTG CCG CAG CTC AGG AA-3, reverse primer 5-GCT CTA GAT CAG Gly-Phe-beta-naphthylamide AGT TCA TCT TTA CCC GGG GAC AGG G-3; for GA733-Fc, forward primer 5-GCG TCG ACA CGG CGA CTT TGC CGC AGC TCA GGA A-3, reverse primer 5-GCT CTA GAT CAA CCC GGG GAC AGG GAG AG-3. PCR was performed with 38 cycles of 94C for 60?s, 55C for 60?s, and 72C for 60?s. Non-transgenic plants were used as unfavorable control, while a T-easy vector (Promega, Madison, WI, USA) made up of the GA733-FcK gene was used as a positive control. The expected size of the DNA products for GA733K, GA733-FcK, and GA733-Fc was 768, 1483, and 1471?bp, respectively. 2.4. RNA Isolation and Semiquantitative RT-PCR The transcription levels of GA733K, GA733-FcK, and GA733-Fc mRNA were quantified by performing semi-quantitative RT-PCR. Total RNA was extracted from transgenic and non-transgenic plants using the RNeasy herb mini kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol. To remove the genomic DNA, 600?ng of total RNA was treated using a TURBO DNA-free kit (Ambion, Austin, TX, USA) in a reaction volume of 20?conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA) diluted in blocking buffer at 1?:?3,000 and then incubated for 1?h and 30?min at RT with secondary antibody goat anti-mouse IgG (Animal Genetics, Daejeon, Republic of Korea) diluted in Gly-Phe-beta-naphthylamide blocking buffer at 1?:?3,000. Anti-Fcrecognizes the Fc portion of GA733-Fc, while anti-human GA733 antibody detects GA733. Protein bands were visualized by exposing the membrane to an X-ray film (Fuji, Tokyo, Japan) using a chemiluminescence substrate (Pierce). Non-transgenic plants and the recombinant human EpCAM/TROP-1 Fc chimera (R&D systems), of which the human EpCAM/TROP-1 is usually fused with the Fc fragment of human IgG1, were used as negative and positive controls, respectively. 2.6. Confocal Microscopy Analysis To Gly-Phe-beta-naphthylamide Gly-Phe-beta-naphthylamide reconfirm the expression of recombinant proteins, confocal microscopy analysis was carried out. The leaf samples were collected from the 3 transgenic plants (GA733K, TGFBR2 GA733-FcK, and GA733-Fc) and a non-transgenic herb and were then fixed in formalin-acetic acid-alcohol solution for 24?h. The fixed leaves were dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks. The paraffin blocks were sectioned into 7-conjugated to horseradish peroxidase (Jackson ImmunoResearch) (1?:?3,000) diluted in PBS for 2?h at RT and was detected using soluble TMB (3.3, 5.5-tetramethylbenzidine) substrate (KPL, Gaithersburg, MD, USA). The antibody titers in 3 wells per tested sample were estimated by determining the optical density at.