However, there is a long way to go before practical use of oral vaccine. p-Synephrine more and more researchers. The aim of this study that chose tomatillo as the plant acceptor was to investigate the feasibility of producing HBV oral vaccine with transgenic cherry tomatillo. MATERIALS AND METHODS Materials Plant materials Cherry tomatillo (Brot.). The seeds were obtained from Xiamen ITG Seed IMP&EXP CO., LTD. Bacteria and plasmids strain EHA105, was kindly provided by Prof. Zhang Qi-fa, Huazhong Agricultural University. p1301HBs, recombinant mini Ti plasmid of the binary expression vector (containing hygromycin-resistant gene, kanamycin-resistant gene, Gus gene and HBsAg-S gene), was constructed in our lab[20,21]. Animals in immune tests BALB/c mice aged 4-6 weeks were p-Synephrine purchased from the Center of Cancer Research, Xiamen University. Methods Construction of plant expression vector A 0.8 kb DNA fragment containing HBsAg-S gene was obtained by a PCR-based assembly from patients serum and inserted into pBPF7 between the constitutively-expressed cauliflower mosaic virus (CaMV) 35S promoter and Nos terminator at the I/I site by substituting the gene to form pBHBs, then the 2.4 kb DNA fragment containing the HBsAg-S gene, the CaMV35S promoter and the nos terimator was cleaved with PstI from pBHBs and ligated into the binary plant vector Cdh15 pCAMBIA1301 (containing hygromycin-resistant gene, kanamycin-resistant gene, Gus gene) digested by Pst I to give the resulting plasmid, p1301HBs (Figure ?(Figure11). Open in a separate p-Synephrine window Figure 1 Plasmid p1301HBs. Plant transformation and regeneration p1301HBs was introduced into strain EHA105 directly by the freeze-thaw method with slight modifications. Subsequently, carrying p1301HBs was used to transform cherry tomatillo cotyledons disks. Ten-days post germination cotyledons were excised from carrying p1301HBs on a shooting medium containing 2 mg/l 6-benzyladenine (6-BA) and 0.2 mg/l indole-3-acetic acid (IAA). Cotyledons were then rinsed with sterilized water added 500 mg/l cefotaxime to kill the on the surface p-Synephrine of explants, blotted dry on a sterilized paper towel, and placed onto a shooting medium added 500 mg/l cefotaxime for recovery. After seven days recovery period, the explants were transferred to a shooting medium added 300 mg/l cefotaxime and 25 mg/l hygromycin to select transgenic progeny. About eight weeks later, hygromycin-resistant shoot regenerants were removed to a rooting medium containing 0.2 mg/l IAA and 25 mg/l hygromycin. Rooted plantlets were acclimatized and transferred to a greenhouse for fruiting. Analysis of transgenic plants Detection of expression activity of reporter gene-Gus gene Histochemical quantification of Gus activity was performed. Different tissues were cut from transgenic plantlets and immerged into Gus reaction buffer (889 mg/l X-Gluc, 100 mg/l chloramphenicol, 50 mM NaH2PO4, 1 Triton-X100, 20% methanol, pH7.0-8.0). After incubation at 37 C for 24 h, the tissues were bleached with absolute alcohol, then observed and photographed under the dissecting microscope. Tissues from non-transformed cherry tomatillo were used as negative control. PCR amplification of plant genomic DNA Transgenic plants were checked for the presence of target gene in leaf tissue using PCR and dot-blot analysis. Genomic DNA samples extracted from leaves of regenerated cherry tomatillos plants by the CTAB method[26] were used as templates in PCR reactions using the following primers: forward primer 5-ATGGAGAACACAACATC-3; reverse primer 5-GGATCCTTTTGCGGAAGCCCA-3, such that amplification would yield a PCR product of 480 bps. The DNA was denatured at 94 C for 10 min. Forty cycles of PCR were performed at: 94 C for 50 sec, 45 C for 45 sec, 72 C for 50 sec; followed by a 72 C incubation for 7 min. A positive control (p1301HBs) and a negative control (DNA from non-transformed tomatillo leaves) were included in this.