Currently we are engaged in researches on increasing expression level of foreign proteins, and the animal trials of oral immunization of mice with transgenic tomatoes expressing HEV-E2 are also in progress. We have successfully introduced gene into tomatoes and identified the expression protein. and Southern dot blotting. The immunoactivity of recombinant protein extracted from transformed plants was examined by enzyme-linked immunosorbant assay (ELISA) using a monoclonal antibody specifically against HEV. ELISA was also used to estimate the recombinant protein content in leaves and fruits of the transformants. RESULTS: Seven positive lines of HEV-E2-transgenic tomato plants confirmed by PCR and Southern blotting were obtained and the immunoactivity of recombinant protein could be detected in extracts of transformants. The expression levels of recombinant protein were 61.22 ng/g fresh weight in fruits and 6.37-47.9 ng/g fresh weight in leaves of the transformants. CONCLUSION: gene was correctly expressed in transgenic tomatoes and the HCV-IN-3 recombinant antigen derived from them has normal immunoactivity. Transgenic tomatoes may hold a good promise for producing a new type of low-cost oral vaccine for hepatitis E virus. INTRODUCTION Research on using plants ARHGEF11 for expression and delivery of oral vaccine has attracted much academic attention and has become a hot spot of study since 1990 when Curtiss et al first reported the expression of surface protein antigen A (SpaA) in tobacco, and great progress has been made since then[1]. So far, more than 10 viral epitopes and subunits of bacterial toxins have been successfully expressed in plants, mainly including hepatitis B surface antigen (HBsAg)[2-9], heat-labile enterotoxin B subunit (LT-B)[10-15], cholera toxin B subunit (CT-B)[16], Norwalk virus capsid protein (NVCP)[17,18], rabies virus glycoprotein[19], CV. XiuNu) seeds were purchased from Xiamen Nong-You Seed Co., Ltd. Reagents, bacteria and plasmids Restriction endonucleases and T4 DNA ligase were obtained from Promega Co. Hygromycin and X-gluc staining solution from Calbiochem-novabiochem Co. and Amres Co., respectively. Double antibody sandwich-ELISA kit was provided by Beijing Wantai Biological Pharmaceutical Co. strain EHA105 was kindly presented by Professor Zhang Qi-fa, Huazhong Agricultural University. Plasmid pBPF7 containing CaMV35s promoter and nos terminator, and plant binary plasmid pCAMBIA1301 containing hygromycin-resistant gene, kanamycin-resistant gene and gene, were constructed and preserved in our laboratory. Construction of plant binary expression vector An 810 bp DNA fragment (named E2) of HEV ORF2 region, located between amino acid residue 394 and 604[23], was obtained by a PCR-based assembly from the patients serum and inserted into pBPF7 between CaMV35S promoter and nos terminator at strain EHA105 by freeze-thaw method. Open in a separate window Figure 1 Structure of plasmid p1301E2. Plant transformation and regeneration Tomato was transformed through leaf discs mediated by EHA105 with p1301E2. Shoots were generated from transformed callus after 3-4 weeks selected on medium containing 20 mg of hygromycin (Hyg) and 300 mg of cefotaxime per liter. The rooting was obtained in medium containing 20 mg of Hyg per liter, and the plantlets was transplanted to soil, and watered with 1/2 MS medium. Analysis of Gus HCV-IN-3 gene expression Both transformed and untransformed tissues were cut from tomato plants, immerged into Gus reaction buffer (X-gluc staining solution) for 12 to 24 hours at 37 C, then bleached with absolute alcohol, observed and photographed under dissecting microscope. Analysis of HEV-E2 gene integration PCR amplification Genomic DNAs extracted from leaves of tomato plants by CTAB[29] were used as PCR templates. The forward primer HEFP and reverse primer HERP were: 5-GGATCCATATGCAGCTGTTCTACTCTCGTC-3 and 5-CTCGAGAAATAAACTATAACTCCCGA-3, respectively (synthesized by BioAsia Co., Shanghai). PCR reaction was performed using 50 ng of template DNA, 0.5 HCV-IN-3 M of each primer in a total volume of 30 L. Cycling parameters were at 94 C for 10 min, followed by 35 cycles at 94 C for 50 s, at 57 C for 50 s, and at 72 C for 50 s, and a final extension at 72 C for 7 min. Southern dot blotting It was performed as reported previously[29]. Analysis of HEV-E2 gene expression ELISA Total soluble proteins were extracted from leaf and fruit tissues as described[29], and HEV-E2 recombinant protein was detected by HEV enzyme-linked immunosorbant assay (ELISA) kit, the protocol and positive determination were performed according to the instructions supplied with the kit. The expression levels of.