Plant materials Cherry tomatillo (Physalis ixocarpa Brot.). The seeds were obtained from Xiamen ITG Seed IMP&EXP CO., LTD.

Bacteria and plasmidsAgrobacterium tumefaciens 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 purchased from the Center of Cancer Research, Xiamen University.

Construction of plant expression vector A 0.8 kb DNA fragment containing HBsAg-S gene was obtained by a PCR-based assembly from patient’s serum and inserted into pBPFΩ7 between the constitutively-expressed cauliflower mosaic virus (CaMV) 35S promoter and Nos terminator at the Xho I/EcoR I site by substituting the Ω 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 pCAMBIA1301 (containing hygromycin-resistant gene, kanamycin-resistant gene, Gus gene) digested by Pst I to give the resulting plasmid, p1301HBs (Figure 1).

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Figure 1 Plasmid p1301HBs.

Plant transformation and regeneration p1301HBs was introduced into Agrobacterium tumefaciens strain EHA105 directly by the freeze-thaw method with slight modifications. Subsequently, Agrobacterium tumefaciens carrying p1301HBs was used to transform cherry tomatillo cotyledons disks. Ten-days post germination cotyledons were excised from in vitro-germinated seedlings, and co-cultivated for 48-72 h with an overnight-grown culture of Agrobacterium tumefaciens 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 A. tumefaciens on the surface 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.

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 experiment. Analysis of PCR products was carried out by loading samples onto a 1.2% agarose gel containing ethidium bromide (EB), followed by electrophoresis and visualization via ultraviolet transillumination. A molecular weight standard (DL-2000, Takara) was used.

Dot blotting The denatured genomic DNA samples were loaded onto a nitrocellulose membrane (Millipore). A positive control (p1301HBs) and a negative control (DNA from non-transformed tomatillo leaves) were included in this experiment. Pre-hybridization was followed by DNA hybridization using a DIG-labelled probe generated from the same 480 bps fragment that was amplified from p1301HBs. Pre-hybridization and hybridization conditions were both at 42 °C. After washing procedure, the membrane was incubated with alkaline phosphatase-conjugated anti-DIG antibody for 2 h at room temperature. The membrane was finally soaked in the color solution at room temperature for up to 4 h in the dark, and the reaction was stopped by rinsing the membrane in distilled water when satisfactory color was observed.

ELISA Different organs of Cherry tomatillo were harvested, frozen in liquid N₂, and ground to a fine power. The power was suspended in extraction buffer (50 mM Tris-HCl, 0.029% NaN₃, pH9.5) at room temperature overnight. The slurry was then centrifuged at 12000 rpm for 10 min at 4 °C, and supernatants were tested for HBsAg by ELISA (Double Antibody sandwich-ELISA Kit, provided by Beijing Wantai Biological Medicine Co.) following the corresponding protocol. Total soluble protein was quantified using Molecular Biology Assistant (MBA2000, Perkin-Elmer Co.).

Oral primary immunization Two four-week-old BALB/c mice were fed 20 g transgenic tomatillo tissues (containing the equivalent of approximate 1 µg HBsAg) every day. Two control mice were fed wild-type tomatillo tissues. After four weeks, two groups of mice were all boosted parenterally with a subimmunogenic dose (containing 0.5 µg HBsAg) of commercial vaccine (Chuangchun Institute of Biological Products).

Oral booster immunization Four four-week-old BALB/c mice were administered parenterally with commercial vaccine (containing 2 µg HBsAg) as primary immunization. Once the serum antibody levels of the mice decreased apparently, three mice were fed transgenic cherry tomato tissues (20 g/day, containing the equivalent of approximately 1 µg HBsAg) and negative control one was fed wild-type tomatillo tissues.

Antibody assays Mice sera were collected once every week and evaluated for anti-HBsAg-specific antibodies using a commercially available enzyme immunoassay (EIA) diagnostic kit (Double Antigen sandwich-ELISA Kit, Beijing Wantai Biological Medicine Co.).

The plasmid for expression of HBsAg in plants (p1301HBs, Figure 1) was introduced into Agrobacterium tumefaciens and used in transformation experiments. Hygromycin incorporated into tissue culture media allowed for selection of transformed callus tissue from which shoots and thereafter mature cherry tomatillo plants were regenerated over a period of about 2-3 months. Ten independent hygromycin-resistant lines of transgenic plants were generated finally. Some lines grew poorly in tissue culture. Such effects could result from insertional mutagenesis. Four lines appearing similar in morphology to wild-type plants were transferred to a greenhouse for fruiting (Figure 2). And F1 progeny of one line was obtained.

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Figure 2 Transgenic cherry tomatillo. A, Transgenic plant in tissue culture; B, Transgenic plant in greenhouse; C, Green transgenic fruits; D, Ripe transgenic fruits.

The transformed plants, containing Gus gene as the report gene, were tested for the Gus activity (Figure 3). The results demonstrated that Gus gene was stably integrated into nuclear chromosomal DNA of tomatillo and different organs all presented a remarkably high Gus activity.

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Figure 3 Analysis of Gus activity in transformed cherry tomatillo tissues. A, Leaf of transformed cherry tomatillo; B, Leaf of non-transformed cherry tomatillo; C, Fruit of transformed cherry tomatillo; D, Fruit of non-transformed cherry tomatillo.

To verify that these plants contained the target gene, genomic DNA extracted from young leaves was subjected to PCR amplification and the PCR products were electrophoresed through agarose. Samples containing the target gene were identified by the presence of an amplified product of 480 bp, representing a fragment of the HBsAg-S coding sequence in p1301HBs. The results obtained demonstrated the presence of an amplified product of the expected size in all the hygromycin-resistant plants tested, which was absent in non-transformed plants (Figure 4 A).

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Figure 4 Identification of HBsAg gene in transgenic cherry tomatillo by PCR (A) and dot blot (B). A1, DNA marker; A2, Positive Control; A3, Non-transformed plants; A4-9, Trans-formed plants. B1-6, Transformed plants; B7, Non-transformed plants; B8, Positive control.

To further confirm that HBsAg coding gene was stably integrated into nuclear chromosomal DNA of transgenic cherry tomatillo, genomic DNA was analyzed by dot blotting, which showed that all the transgenic samples had the same hybridization signals as the positive control (p1301HBs) (Figure 4 B).

Plants positive in PCR and dot blotting were further tested by ELISA to detect and quantitatively analyze the target protein. The rHBsAg levels in different organs from a representative one of several tactile lines are presented in Table 1. The results indicated that the expression levels of rHBsAg varied among different organs of the same plant. Apparently, the amount of rHBsAg produced in leaf was much higher than any of the other organs. rHBsAg protein levels in fruit and stem were in a similar range. However, rHBsAg/TSP (total soluble protein) value of fruit was somewhat higher than that of leaf. No significant variation was observed among the rHBsAg/TSP values of the same kind of organs in different individuals reproduced from the same line, but striking difference in the absolute expression levels of rHBsAg was observed and the level of TSP in leaves of the plantlets in tissue culture was significantly lower than that in leaves of the plants in greenhouse. The difference might be due to growth condition of these plants.

The rHBsAg levels in leaves of tissue-cultured plantlets of four different lines were shown in Table 2. ELISA analysis demonstrated that rHBsAg was expressed in these different lines ranging from 21 to 90 ng/g fresh weight.

The immunogenicity of plant-derived rHBsAg was tested by oral immunizing BALB/c mice with transgenic tissues from plant line with highest production of rHBsAg. Then vaccinated mice were bled weekly, and the sera were assessed for the presence of specific antibodies by ELISA.

Oral immunization of two naive mice by feeding transgenic tissues did not evoke primary antibody within four weeks. Then mice in two groups were both injected with a subimmunogenic dose (0.5 μg HBsAg) of commercial HBV vaccine via an intramuscularly route as booster. However, none of the mice developed measurable specific serum antibodies in the following examination. There was no significant difference between serum antibody titers of the mice primed with transgenic tissues and those of the control mice in this experiment (Figure 5).

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Figure 5 Serum antibody response elicited by oral prime with transgenic cherry tomatillo. A.B: Mice were fed non-transformed tissues until week 4, then boosted with a subimmunogenic dose of commercial vaccine; C.D: Mice were fed transformed tissues until week 4, then boosted parenter-ally with a subimmunogenic dose of commercial vaccine.

After a single dose (2 μg HBsAg) of commercial vaccine as priming, titers of serum antibody in mice rose sharply in response to antigen initially and then declined. Three mice primed were orally immunized with tissues of the transgenic tomatillo tissues as booster, and elicited an immediate high-level recall antibody response within the following 3-5 weeks. Antibody titers in serum were augmented significantly. In comparison, one negative control mouse fed with wild type tomatillo tissues did not showed any booster response. Serum titers of antibody remained same (Figure 6).

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Figure 6 Serum antibody response elicited by oral boost with transgenic cherry tomatillo. A: Mouse was primed parenter-ally with 2 µg commercial rHBsAg, and when its serum anti-body level descends to OD < 1.0, mouse was fed with untransformed tissues; B.C.D: Mice were primed parenterally with 2 µg yeast-derived rHBsAg, and when their serum anti-body levels descend to OD < 1.0, mice were fed with transformed tissues as boost.

We concluded that immune memory cells were established in mice following parenteral immunization of commercial vaccine and a recall response was elicited by oral delivery of HBsAg in transgenic tissues.

Our results showed that tomatillo-derived rHBsAg had same immunogenicity as commercial vaccine in stimulating production of HBsAg-specific antibodies in animal. The present experiment was limited by the total amount of plant tissues that mice ingested in 24 h and the amount of rHBsAg produced in the transgenic tissues. Absence of expected anti-HBsAg response in the oral priming study might be due to inadequate immunization schedule, such as low doses of antigen, or low sensitivity of the detection system and interference of plant material with development of an immune response. In contrast, active immune response to orally administrated antigens in parenterally primed animals might be due to altered antigen processing in favoring of active antibody production rather than oral immunotolerance.