Transgenic Res (2007) 16:169175 DOI 10.

1007/s11248-006-9023-5

ORIGINAL PAPER

Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice
Xiao-Ling Jiang Zhu-Mei He Zhi-Qiang Peng Yu Qi Qing Chen Shou-Yi Yu

Received: 16 January 2006 / Accepted: 17 June 2006 / Published online: 16 January 2007 Springer Science+Business Media B.V. 2007

Abstract Cholera toxin B (CTB) subunit is a well-characterized antigen against cholera. Transgenic plants can offer an inexpensive and safe source of edible CTB vaccine and may be one of the best candidates for the production of plant vaccines. The present study aimed to develop transgenic tomato expressing CTB protein, especially in the ripening tomato fruit under the control of the tomato fruit-specic E8 promoter by using Agrobacterium-mediated transformation. Transgenic plants were selected using PCR and Southern blot analysis. Exogenous protein extracted from leaf, stem, and fruit tissues of transgenic plants was detected by ELISA and Western blot analysis, showing specic expression in the ripening fruit, with the highest amount of CTB protein being 0.081% of total soluble protein. Gavage of mice with ripe transgenic tomato fruits induced both serum and
Xiao-Ling Jiang and Zhu-Mei He contributed equally to this work. X.-L. Jiang Z.-Q. Peng Q. Chen S.-Y. Yu (&) School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China e-mail: yw@mmu.com Z.-M. He (&) Y. Qi School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, P.R. China e-mail: lsshezm@mail.sysu.edu.cn

mucosal CTB specic antibodies. These results demonstrate the immunogenicity of the CTB protein in transgenic tomato and provide a considerable basis for exploring the utilization of CTB in the development of tomato-based edible vaccine against cholera. The rCTB antigen resulted in much lower antibody titers than an equal amount of exgenous CTB in trangenic fruits, suggesting the protective effect of the brous tissue of the fruit to the exogenous CTB protein against the degradation of protease in the digestive tracts of mice. Keywords Cholera toxin B E8 promoter Edible vaccine Immunoglobins G and A Transgenic tomato

Introduction Cholera remains one of the most feared epidemic diseases throughout much of the world. Effective prevention of cholera depends on safe water sources and improved sanitation (Steinberg et al. 2001). However, it is not affordable for many poor countries to build large-scale water treatment and sanitation systems. A promising alternative is the production of vaccines in plants that could be grown locally, as edible, plant-based

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recombinant vaccines they are inexpensive, safe, and easy to administer (Giddings et al. 2000). Some studies have shown that the ctb gene was expressed in potato tubers. The expressed CTB subunit retained its native antigenicity, and induced the generation of both mucosal and systemic anti-CT antibodies at levels sufcient to generate protective immunity by oral immunization in experimental mice. But its antigenicity was lost by more than 50% after the potatoes were cooked (Hein et al. 1996; Arakawa et al. 1997, 1998, 1999). The Tomato plant is one of the selected plants for transformation as oral vaccine since its fruits are edible fresh. Jani et al. successfully transferred the ctb gene controlled by the CaMV 35S promoter into tomato plants, and this transgenic plant expressed the CTB subunit in leaves and fruits, which could specically bind to G(M1)-ganglioside receptor, a special receptor for CTB subunit (Jani et al. 2002, 2004). A transgene driven by the constitutive CaMV 35S promoter could be expressed in all tissues of a transgenic plant, such as fruit, stem, leaf, and even owers (Sandhu et al. 2000). If the transgene is expressed specically in fruit, the amount of expression should be increased. The E8 promoter is a tomato fruit-specic promoter, which drives the expression of the E8 gene (Deikman and Fischer 1988; Deikman et al. 1992, 1998). Sandhu et al. made RSV-F antigen express specically in transgenic cherry tomato fruit by using the E8 promoter. The amount of the RSV-F antigen seemed a little higher than that expressed in fruit with CaMV 35S promoter (Sandhu et al. 2000). In this study, we aimed to express the CTB specically in fruit and to increase the mount of expression in tomato fruit using the E8 promoter. The immunogenicity of the CTB protein expressed in tomato fruit was also evaluated through determination of the serum and mucosal anti-CTB antibody levels in experimental mice.

Materials and methods Construction of the plant expression vectors Plant expression vectors containing the CTB coding sequence controlled by the preferentially core E8 promoter were constructed as following. The ctb gene fragment was cleaved from pRTLCTB (gift from Military medical scientic institute, Beijing) with Kpn I and BamH I and ligated into pBluescript II KS, and the resulting plasmid pCTB was conrmed by sequencing. The 1.1-kb E8 promoter was amplied by PCR using the forward primer (5-AAG CTT CTA GAA ATT TCA CGA AAT-3) with BamH I site and the reverse primer (5-CTT CTT TTG CAC TGT GAA TGA T-3) and using the template of the total DNA of Lycopersicon esculentum cv. Jinfeng #1. The amplied fragment was cloned into pMD18-T vector, generating pTe8. The ctb gene cleaved by Kpn I and Sac I from pCTB was ligated into pTe8 plasmid generating pE8-CTB, in which the CTB gene was downstream to the 1.1-kb E8 promoter. Plant expression vector pJES1 (Fig. 1) was produced by insertion of the E8-CTB gene fragment from pTe8 into pBI121 by Hind III and Sac I sites, to replace the fragment of CaMV 35S promoter plus gusA gene. Tomato transformation and screening of putative transgenic plants Plasmid pJES1 was introduced into Agrobacterium tumefaciens strain LBA4404 and used to transform tomato (Lycopersicon esculentum cv. Suifeng, purchased from the Vegetable Institute of Guangzhou), following a modied protocol described by Hofgen et al. (1988). Cotyledons were excised from in vitro-germinated seedlings on MS (Murashige and Skoog) medium for 812 days, and co-cultivated for 48 h with an overnightgrown culture of A. tumefaciens (diluted 1:30) on the solid MS medium supplemented with 3.0 mg/l

Fig. 1 Schematic representation of plasmid pJES1 used for tomato transformation NOS-p: nopaline synthase promoter; NOS-t: nopaline synthase terminator; NPT II: neomycin phosphotransferase II gene

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of 6-benzyladenine (6-BA) and 0.2 mg/l of 3-indoleacetic acid (IAA). Cotyledon explants were then rinsed with sterilized deionized water, blotted dry on a sterilized paper towel and placed onto a selection medium consisting of the prescription of the former solid MS medium supplemented with 100 mg/l of kanamycin and 300 mg/l of carbenicillin. After 46 weeks, kanamycin-resistant shoot regenerants were removed from the callus and transferred to an elongating medium consisting of MS supplemented with 2.0 mg/l of BAP, 100 mg/l of kananmycin and 300 mg/l of carbenicillin for 2 days. Elongated shoots were then transferred to a rooting medium consisting of MS and supplemented with 0.2 mg/l of IAA for about 45 days. Rooted plantlets then were acclimatized and transferred to a greenhouse for fruiting. In order to determine the presence of the ctb gene in transgenic plants, PCR analysis and Southern blot analysis were performed. The cetyltrimethylammonium bromide (CTAB) technique (Doyle and Doyle 1990) was used to extract genomic DNA from the leaves of kanamycinresistant plants. In the PCR reaction, the forward primer (5-TTA AAT TAA AAT TTG-3) and the reverse primer (5-TTA ATT TGC CAT ACT AAT TG-3) were used, and 100 ng of genomic DNA was used as the template. The PCR cycling conditions were as follows: 94C for 30 s, 55C for 45 s, and 72C for 45 s for a total of 30 cycles. For Southern blot analysis, 10 lg of genomic DNA from wild-type control plants and each of the transformed plants was digested with Hind III and Sac I. The digested DNA samples were seperated on a 2.0% agarose gel, transferred onto nylon membranes and hybridized using DIG-labeled ctb as a probe (Roche), following standard procedures (Sambrook et al. 1989). Analysis of CTB protein in transgenic tomato tissues The presence and expression level of CTB protein in transgenic tomato plant tissues, including leaf, stem, root, ower and fruit tissues, were determined by quantitative ELISA, as described by Sandu et al. (2000) and Kang et al. (2004). Tissues (0.5 g fresh weight) from transgenic and

wild-type plants were homogenized by grinding in liquid nitrogen and resuspended in 1.0 ml of extraction buffer (200 mM TrisCl (pH 8.0), 100 mM NaCl, 400 mM sucrose, 10 mM EDTA, 14 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl uoride, 0.05% Tween-20). The tissue homogenates were centrifuged twice at 17,000 g for 15 min at 4C and the insoluble cell debris was removed. Total soluble protein was determined by the Bradford protein assay (Sigma). Standard rCTB (gift from Military medical scientic institute, Beijing) was diluted into the concentration grads of 1 lg/ml, 0.5 lg/ml, 0.25 lg/ml, 0.125 lg/ml, 0.063 lg/ml, and 0.032 lg/ml by coating buffer. Total soluble protein samples from transgenic and wild-type plants, together with the quantitative rCTB, were coated at 100 ll per well into a 96-well microtiter plate and incubated overnight. The plate was washed three times with PBST (PBS plus 0.05% Tween-20). The background was blocked with a 1% (w/v) BSA solution in PBS for 2 h at 37C, and the plate was again washed three times with PBST. The plates were then incubated with monoclonal antibody of rabbit anti-CTB IgG that was diluted 1:1000 in 0.01 M PBS containing 0.5% BSA for 2 h at 37C, followed by three washes with PBST buffer. Secondary labeling was done using horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Takara) with 1:3,000 dilution in 0.01 M PBS containing 0.5% BSA for 2 h at 37C, followed by three washes with PBST buffer. The plates were developed by the addition of 100 ll per well of TMB substrates (TakaRa) for 30 min at room temperature in the dark. The plate was read at 492 nm using an ELISA reader (BioRad), and the amount of plant-expressed CTB was estimated based on the known amount of puried CTB. To determine the expression levels of different tissues in each transgenic line, three tissue explants were analyzed for CTB expression and an average expression level was obtained. Western analysis was carried out following ELISA detection. Total soluble protein extracted from ripened fruit tissue of transgenic tomato lines 9 and 16, which showed CTB expression in ELISA, was separated on a 15% SDS-PAGE gel at 100 V in Trisglycine buffer (25 mM Tris (pH 8.5), 200 mM glycine). Samples of the plant homogenates, along with samples of 300 ng

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rCTB, were loaded directly onto the gel for 10 min prior to electrophoresis. The separated protein bands were transferred from the gel to a Hybond membrane (Roche), using the Mini transBlot electrophoretic transfer cell (BioRad) for 2 h at 140 mA in transfer buffer (50 mM Tris (pH 8.3), 40 mM glycine, 0.04% SDS, 20% methanol). Non-specic antibody reactions were blocked by incubation of the membrane in 25 ml of 5% non-fat dry milk in TBST buffer (TBS with 0.05% Tween-20) with gentle agitation at room temperature for 12 h. The membrane was incubated for 2 h at room temperature with gentle agitation in 1:3,000 dilution of monoclonal antibody of rabbit anti-CTB IgG in TBST buffer that contained 2.5% non-fat dry milk, followed by three washes with TBST buffer. The membrane was then incubated for 2 h at room temperature with gentle agitation in a 1:5,000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (TakaRa) in TBST buffer, and washed three times with TBST buffer and once with TMN buffer. Then the color was developed using TMB substrates (Roche) in TMN buffer (100 mM Tris (pH 9.5), 5 mM MgCl2, 100 mM NaCl) (Jani et al. 2004). Oral immunization with transgenic tomato fruits Twelve four-week-old BALB/c mice were fed ripe tomato fruit tissue from plant line 9 which showed the highest CTB expression with 10 g/ day/mouse. Each 10 g tomato fruit was divided into three pieces and consumed by each mouse in the morning, noon, and evening, respectively. In the morning, 3.3 g ripe tomato fruits were ground into homogenate, which were put into syringe and introduced directly into the digestive tract. After about a 4-h intermission, the operation was repeated at noon. Then the last gavage was done 4 h later. With the same method, six negative control mice were fed untransformed wild-type ripe tomato using the same quantity as the experimental group, and six positive control mice were fed rCTB 5 lg/day/mouse equal to the exogenous CTB protein of the transgenic tomato fruits. Feedings were done on days 0, 4, 14, 21, and 28 (Arakawa et al. 1999; Sandhu et al. 2000). Feces

were collected on day 32, and serum was collected from the vena orbitalis posterior on day 33. Antibody titers were analyzed by ELISA for production of antibodies to CTB. The serum and mucosal samples drawn from the immunized mice were diluted 1:100 by coating buffer, and 50 ll was added to each well of an ELISA microtiter plate coated with 10 lg/ml rCTB antigen in coating buffer. Secondary labeling was done using HRP-conjugated rabbit anti-mouse IgG or IgA, and ELISA readings were recorded at 492 nm. Data analysis was performed by MannWhitney test. The difference was considered statistically signicant if a two-tail P-value was less than 0.05. Statistical methods All data analysis was performed using SPSS for Windows 12.0 version (Chicago, IL, USA).

Results Molecular detection of exogenous CTB gene in transgenic tomato plants A total of 26 independent transgenic tomato plants were regenerated after Agrobacteriummediated transformation, and the presence of the ctb gene in the genomic DNA of leaf tissues was determined using PCR analysis. Fourteen tomato plants were conrmed to have the exogenous ctb gene with the size of 404 bp as expected. Southern blot analysis was also performed and showed the integration of the ctb gene into the genome of the 14 lines (Fig. 2).

Fig. 2 Identication of the ctb gene in transformed plants by Southern blot analysis (degested by Hind III and Sac I). Lane1: Plasmid pJES1; Lane 2: Total DNA extracted from the leaves of untransformed wild-grown plant; Lanes 35: Total DNA extracted from the leaves of transformed plant. Lane 4 and lane 5 showed that the ctb gene occurred in lines 9 and 16, respectively

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Transgenic Res (2007) 16:169175 Fig. 3 CTB-ELISA result of transgenic plant lines 9 and 16 and untransgenic plant (OD value) X-axis: Total proteins extracted from different part of tomato plant (S: Stem; L: Leaf; FL: Flower; FR1: 010 days fruit; FR2: 1120 days fruit; FR3: 2130 days fruits; FR4: 3140 days fruit)

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Identication of exogenous CTB protein in tomato To measure the presence of CTB protein in the 14 conrmed transgenic tomatoes, ELISA analysis of the total protein from fruit, leaf, stem and ower tissues was performed along with known amounts of puried rCTB. The transgenic plant lines 9 and 16 showed higher OD value in the total soluble protein from the fruit tissues compared with other transgenic lines. All tissues except for fruit in these 2 lines, as well as the untransformed control, showed no expression of CTB protein. As shown in Fig. 3, the levels of CTB protein varied among the different developing period fruits of the same plant and showed an increase over time. The highest level was up to 0.455 lg/g and 0.385 lg/g fruit fresh (equally to 0.081% and 0.075% of total soluble protein) in 3140 days fruit tissues of transgenic tomato lines, 9 and 16, respectively (Table 1). Immunoblot analysis of protein extracted from transgenic

tomato fruits of lines 9 and 16 showed specic binding with a 46-kDa protein, which is consistent with the standard rCTB (Fig. 4). Thus, transgenic tomato fruits expressing cholera toxin B (CTB) protein under the control of the E8 promoter were obtained. Induction of both serum and mucosal antibodies of mice Tomato fruits from line 9 were orally fed to 12 mice for ve times during a 28-day period to test the ability of the tomato-expressed CTB protein to induce mucosal and serum responses. CTBspecic antibody induction in feces and serum from orally rCTB-immunized, transgenic fruitimmunized and the wild-type tomato-immunized mice was determined by using ELISA. The six rCTB-immunized mice showed a signicant response in serum and feces. The untransformed control did not produce detectable anti-CTB antibodies. Among the 12 mice fed transgenic

Table 1 Amount of CTB in fresh ripe tomato fruits Days 9th fruit 1~ 11~ 21~ 31~ 1~ 11~ 21~ 31~ TSP (mg/ml) 1.59 1.03 0.88 1.09 1.35 1.15 0.89 1.03 CTB (lg/ml) 0 0.422 0.624 0.909 0 0.394 0.594 0.769 CTB/TSP (%) 0 0.041 0.071 0.081 0 0.034 0.067 0.075 CTB/fresh fruit (lg/g) 0.000 0.211 0.312 0.455 0.000 0.197 0.297 0.385

16th fruit

TSP: Total soluble protein; CTB: Exogenous CTB protein in tomato fruits

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Fig. 4 Western blot analysis of protein extracted from transgenic tomato fruits Line 1: protein from untransformed wild-type plant; Line 2: protein from fruit of transgenic tomato plant line 9; Line 3: protein from fruit of transgenic tomato plant line 16; Line 4: standard rCTB

fruit, 11 showed signicant serum and mucosal response and produced anti-CTB antibodies. The results showed that the titers of serum and mucosal antibody produced by transgenic fruits are signicantly higher than that of rCTB-immunized mice and wild-type tomato-immunized mice (P < 0.05), demonstrating successful oral immunization by the transgenic fruit and showing that fruit-derived CTB was active as an oral immunogen (Fig. 5).

Discussion CTB is a non-toxic unit of CT and well characterized as an antigen against cholera because of its ability to bind to GM1-ganglioside on the surface of mammlian intestinal epithelial cells. Gavage with transgenic fruits could induce serum and mucosal responses in BALB/c mice, which proved that CTB subunit retained its native

antigenicity. Our result is consistent with previous studies in which mice were fed raw transgenic potato plant expressing antigens, for example, hepatitis B surface antigen (HBsAg) (Mason et al. 1996; Kong et al. 2001; Thanavala et al. 2005) or the CTB subunit (Haq et al. 1995; Wang et al. 2001). This study conrmed that the oral channel of rCTB and trangenic fruits produced the antibodies in blood. rCTB antigen was set as the control and resulted in much lower antibody titers than an equal amount of exogenous CTB in trangenic fruits, suggesting the protective effect of brous tissue of fruit to the exogenous CTB protein against the degradation of protease in the digestive tract of mice. Among the full-length 2.2-kb E8 promoter, the 1.1-kb core part can regulate gene expression specically in fruits during ripening, and the other part of the full-length 2.2-kb E8 promoter is due to higher levels of expression of the gene (Deikman et al. 1998). Sandhu et al. had transformed RSV-F antigen into cherry tomato plants with the fulllength E8 and CaMV 35S. The average levels of RSV-F antigen in fruits was 12.68 2.55 lg/g fruit fresh weight under the E8 promoter, which seemed higher than that under CaMV 35S promoter at 9.01 1.87 lg/g fruit fresh weight of expression (Sandhu et al. 2000). In our study, under the control of the core E8 promoter, the CTB subunit was especially well expressed in transgenic ripening tomato fruit, and the highest amount of CTB subunit expressed in ripening fruits was 0.075% and 0.081% of total soluble

Fig. 5 Amount of IgG and IgA in serum and feces stimulated by feeding fruits of transgenic tomato line 9

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175 5-anking region of the ripening-related E8 gene. Plant Mol Biol 37:10011011 Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:11511155 Hofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877 Haq TA, Mason HS, Clements JD, Arntzen CJ (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268:714716 Hein MB, Yeo TC, Wang F, Sturtevant A (1996) Expression of cholera toxin subunits in plants. Ann NY Acad Sci 792:5056 Jani D, Meena LS, Rizwan-ul-Haq QM, Singh Y, Sharma AK, Tyagi AK (2002) Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Res 11:4754 Jani D, Singh NK, Bhattacharya S, Meena LS, Singh Y, Upadhyay SN, Sharma AK, Tyagi AK (2004) Studies on the immunogenic potential of plant-expressed cholera toxin B subunit. Plant Cell Rep 22:471477 Kang TJ, Loc NH, Jang MO, Yang MS (2004) Modication of the cholera toxin B subunit coding sequence to enhance expression in plants. Mol Breed 13:143153 Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS, Thanavala Y (2001) Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc Natl Acad Sci USA 98:1153911544 Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Pro Natl Acad Sci USA 93:5335 5340 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York, USA Sandhu JS, Krasnyanski SF, Domier LL, Korban SS, Osadjan MD, Buetow DE (2000) Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response. Trans Res 9:127135 Steinberg EB, Greene KD, Bopp CA, Cameron DN, Wells JG, Mintz ED (2001) Cholera in the United States, 19952000: trends at the end of the twentieth century. J Infect Dis 184:799802 Thanavala Y, Mahoney M, Pal S, Scott A, Richter L, Natarajan N, Goodwin P, Arntzen CJ, Mason HS (2005) Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci USA 102:33783382 Wang XG, Zhang GH, Liu CX, Zhang YH, Xiao CZ, Fang RX (2001) Puried cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity. Biotechnol Bioeng 72:490494

protein, which was a little higher than that of an earlier report with 0.04% using CaMV 35S promoter (Jani et al. 2002). So it is reasonable to raise a hypothesis that the expression may be increased if a transferred gene can be expressed specically in fruit. However, more experiments are needed. In conclusion, E8 core promoter provided us a new way to enhance the expression of the CTB subunit, specically in edible fruits of the tomato plant. Transgenic tomato plants expressing CTB protein specially in fruits will move us closer to achieving a low-cost, convenient, effective and safe strategy for the prevention of cholera in humans, especially in poor regions where conventional vaccines are unaffordable or unavailable.
Acknowledgements This research was supported by Natural Science Foundation in Guangdong Province of China (Grant No. 013075). Thanks are due to Prof. ZhongJu Xiao at Southen Medical University, China, Terry W. Hill at Rhodes College, TN, USA, and Dr. Chun-Quan Ou at HongKong University, China for providing valuable suggestions. The authors also wish to thank School of Life Sciences, Sun Yat-Sen University, where part of the experiments were conducted.

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