Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice

7/30/2019 Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice

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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 ofplant vaccines. The present study aimed to

develop transgenic tomato expressing CTBprotein, especially in the ripening tomato fruit

under the control of the tomato fruit-specific E8

promoter by using Agrobacterium-mediated

transformation. Transgenic plants were selectedusing PCR and Southern blot analysis. Exoge-

nous protein extracted from leaf, stem, and fruittissues of transgenic plants was detected by

ELISA and Western blot analysis, showing spe-

cific expression in the ripening fruit, with thehighest amount of CTB protein being 0.081% of

total soluble protein. Gavage of mice with ripe

transgenic tomato fruits induced both serum and

mucosal CTB specific antibodies. These results

demonstrate the immunogenicity of the CTB

protein in transgenic tomato and provide aconsiderable basis for exploring the utilization of

CTB in the development of tomato-based edible

vaccine against cholera. The rCTB antigenresulted in much lower antibody titers than an

equal amount of exgenous CTB in trangenic

fruits, suggesting the protective effect of the fi-brous tissue of the fruit to the exogenous CTB

protein against the degradation of protease inthe 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 watersources and improved sanitation (Steinberg et al.

2001). However, it is not affordable for many

poor countries to build large-scale water treat-ment and sanitation systems. A promising alter-

native is the production of vaccines in plants that

could be grown locally, as edible, plant-based

Xiao-Ling Jiang and Zhu-Mei He contributed equally tothis 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. Chinae-mail: [email protected]

Z.-M. He (&) Y. QiSchool of Life Sciences, Sun Yat-Sen University,Guangzhou 510275, P.R. Chinae-mail: [email protected]

Transgenic Res (2007) 16:169175

DOI 10.1007/s11248-006-9023-5

123

O R IG IN A L PA PE R

Cholera toxin B protein in transgenic tomato fruit induces

systemic immune response in miceXiao-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


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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 CTBsubunit retained its native antigenicity, and

induced the generation of both mucosal and sys-

temic anti-CT antibodies at levels sufficient togenerate protective immunity by oral immuniza-

tion in experimental mice. But its antigenicity was

lost by more than 50% after the potatoes werecooked (Hein et al. 1996; Arakawa et al. 1997,

1998, 1999). The Tomato plant is one of the

selected plants for transformation as oral vaccinesince its fruits are edible fresh. Jani et al. suc-

cessfully transferred the ctb gene controlled by

the CaMV 35S promoter into tomato plants, andthis transgenic plant expressed the CTB subunit

in leaves and fruits, which could specifically bindto G(M1)-ganglioside receptor, a special receptorfor CTB subunit (Jani et al. 2002, 2004).

A transgene driven by the constitutive

CaMV 35S promoter could be expressed in alltissues of a transgenic plant, such as fruit, stem,

leaf, and even flowers (Sandhu et al. 2000). If

the transgene is expressed specifically in fruit,the amount of expression should be increased.

The E8 promoter is a tomato fruit-specific

promoter, which drives the expression of the

E8 gene (Deikman and Fischer 1988; Deikmanet al. 1992, 1998). Sandhu et al. made RSV-F

antigen express specifically in transgenic cherrytomato fruit by using the E8 promoter. The

amount of the RSV-F antigen seemed a little

higher than that expressed in fruit with CaMV35S promoter (Sandhu et al. 2000). In this

study, we aimed to express the CTB specificallyin fruit and to increase the mount of expres-

sion in tomato fruit using the E8 promoter.

The immunogenicity of the CTB protein

expressed in tomato fruit was also evaluatedthrough determination of the serum and

mucosal anti-CTB antibody levels in experi-mental mice.

Materials and methods

Construction of the plant expression vectors

Plant expression vectors containing the CTBcoding sequence controlled by the preferentially

core E8 promoter were constructed as following.The ctb gene fragment was cleaved from pRTL-CTB (gift from Military medical scientific insti-

tute, Beijing) with Kpn I and BamH I and ligated

into pBluescript II KS, and the resulting plasmidpCTB was confirmed by sequencing. The 1.1-kb

E8 promoter was amplified by PCR using the

forward primer (5-AAG CTT CTA GAA ATTTCA 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. Jinf-eng #1. The amplified fragment was cloned into

pMD18-T vector, generating pTe8. The ctb genecleaved by Kpn I and Sac I from pCTB was

ligated into pTe8 plasmid generating pE8-CTB,

in which the CTB gene was downstream to the1.1-kb E8 promoter. Plant expression vector

pJES1 (Fig. 1) was produced by insertion of the

E8-CTB gene fragment from pTe8 into pBI121 byHind III and Sac I sites, to replace the fragment

of CaMV 35S promoter plus gusA gene.

Tomato transformation and screeningof putative transgenic plants

Plasmid pJES1 was introduced into Agrobacte-rium tumefaciens strain LBA4404 and used totransform tomato (Lycopersicon esculentum cv.

Suifeng, purchased from the Vegetable Institute

of Guangzhou), following a modified protocoldescribed 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 overnight-

grown 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 of3-indoleacetic acid (IAA). Cotyledon explants

were then rinsed with sterilized deionized water,

blotted dry on a sterilized paper towel and placedonto a selection medium consisting of the pre-

scription of the former solid MS medium sup-

plemented with 100 mg/l of kanamycin and300 mg/l of carbenicillin. After 46 weeks, kana-

mycin-resistant shoot regenerants were removed

from the callus and transferred to an elongatingmedium 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. Elongatedshoots 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 thenwere acclimatized and transferred to a green-

house 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) tech-nique (Doyle and Doyle 1990) was used to extract

genomic DNA from the leaves of kanamycin-

resistant plants. In the PCR reaction, the forwardprimer (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 cyclingconditions were as follows: 94C for 30 s, 55C for

45 s, and 72C for 45 s for a total of 30 cycles. ForSouthern blot analysis, 10 lg of genomic DNA

from wild-type control plants and each of the

transformed plants was digested with Hind IIIand Sac I. The digested DNA samples were

seperated on a 2.0% agarose gel, transferred ontonylon membranes and hybridized using DIG-la-

beled 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, flower and fruit tissues, were deter-

mined by quantitative ELISA, as described by

Sandu et al. (2000) and Kang et al. (2004). Tis-sues (0.5 g fresh weight) from transgenic and

wild-type plants were homogenized by grinding inliquid 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 phenylmethyl-

sulfonyl fluoride, 0.05% Tween-20). The tissue

homogenates were centrifuged twice at 17,000 gfor 15 min at 4C and the insoluble cell debris was

removed. Total soluble protein was determined

by the Bradford protein assay (Sigma). StandardrCTB (gift from Military medical scientific insti-

tute, 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 quantitativerCTB, were coated at 100 ll per well into a 96-well

microtiter plate and incubated overnight. The platewas washed three times with PBST (PBS plus0.05% 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 timeswith 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 in0.01 M PBS containing 0.5% BSA for 2 h at 37C,

followed by three washes with PBST buffer. Theplates 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 readat 492 nm using an ELISA reader (BioRad), and

the amount of plant-expressed CTB was estimated

based on the known amount of purified CTB. Todetermine the expression levels of different tissues

in each transgenic line, three tissue explants were

analyzed for CTB expression and an averageexpression level was obtained.

Western analysis was carried out followingELISA detection. Total soluble protein extracted

from ripened fruit tissue of transgenic tomato

lines 9 and 16, which showed CTB expression inELISA, 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 planthomogenates, along with samples of 300 ng

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rCTB, were loaded directly onto the gel for10 min prior to electrophoresis. The separated

protein bands were transferred from the gel to a

Hybond membrane (Roche), using the MinitransBlot 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-specific antibody reactions were

blocked by incubation of the membrane in 25 ml

of 5% non-fat dry milk in TBST buffer (TBS with0.05% Tween-20) with gentle agitation at room

temperature for 12 h. The membrane was incu-

bated for 2 h at room temperature with gentleagitation in 1:3,000 dilution of monoclonal anti-

body of rabbit anti-CTB IgG in TBST buffer that

contained 2.5% non-fat dry milk, followed bythree washes with TBST buffer. The membrane

was then incubated for 2 h at room temperaturewith gentle agitation in a 1:5,000 dilution ofhorseradish peroxidase-conjugated anti-rabbit

IgG (TakaRa) in TBST buffer, and washed three

times with TBST buffer and once with TMNbuffer. Then the color was developed using TMB

substrates (Roche) in TMN buffer (100 mM Tris

(pH 9.5), 5 mM MgCl2, 100 mM NaCl) (Janiet al. 2004).

Oral immunization with transgenic tomato

fruits

Twelve four-week-old BALB/c mice were fedripe tomato fruit tissue from plant line 9 which

showed the highest CTB expression with 10 g/

day/mouse. Each 10 g tomato fruit was dividedinto three pieces and consumed by each mouse in

the morning, noon, and evening, respectively. In

the morning, 3.3 g ripe tomato fruits were groundinto homogenate, which were put into syringe and

introduced directly into the digestive tract. After

about a 4-h intermission, the operation wasrepeated at noon. Then the last gavage was done

4 h later. With the same method, six negative

control mice were fed untransformed wild-typeripe tomato using the same quantity as the exper-

imental group, and six positive control mice werefed 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 collectedfrom the vena orbitalis posterior on day 33.

Antibody titers were analyzed by ELISA for

production of antibodies to CTB. The serum andmucosal samples drawn from the immunized mice

were diluted 1:100 by coating buffer, and 50 ll

was added to each well of an ELISA microtiterplate 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 statisticallysignificant if a two-tail P-value was less than 0.05.

Statistical methods

All data analysis was performed using SPSS forWindows 12.0 version (Chicago, IL, USA).

Results

Molecular detection of exogenous CTB genein transgenic tomato plants

A total of 26 independent transgenic tomatoplants were regenerated after Agrobacterium-

mediated transformation, and the presence of thectb gene in the genomic DNA of leaf tissues wasdetermined using PCR analysis. Fourteen tomato

plants were confirmed to have the exogenous ctb

gene with the size of 404 bp as expected. Southernblot analysis was also performed and showed the

integration of the ctb gene into the genome of the

14 lines (Fig. 2).

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

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Identification of exogenous CTB protein

in tomato

To measure the presence of CTB protein in the 14

confirmed transgenic tomatoes, ELISA analysis

of the total protein from fruit, leaf, stem andflower tissues was performed along with known

amounts of purified rCTB. The transgenic plant

lines 9 and 16 showed higher OD value in thetotal soluble protein from the fruit tissues com-

pared with other transgenic lines. All tissues ex-

cept for fruit in these 2 lines, as well as theuntransformed control, showed no expression of

CTB protein. As shown in Fig. 3, the levels of

CTB protein varied among the different devel-oping 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 to0.081% and 0.075% of total soluble protein) in

3140 days fruit tissues of transgenic tomato lines,

9 and 16, respectively (Table 1). Immunoblotanalysis of protein extracted from transgenic

tomato fruits of lines 9 and 16 showed specific

binding with a 46-kDa protein, which is consistent

with the standard rCTB (Fig. 4). Thus, transgenictomato 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 12mice for five times during a 28-day period to test

the ability of the tomato-expressed CTB protein

to induce mucosal and serum responses. CTB-specific antibody induction in feces and serum

from orally rCTB-immunized, transgenic fruit-

immunized and the wild-type tomato-immunizedmice was determined by using ELISA. The six

rCTB-immunized mice showed a significant

response in serum and feces. The untransformedcontrol did not produce detectable anti-CTB

antibodies. Among the 12 mice fed transgenic

Table 1 Amount of CTB in fresh ripe tomato fruits

Days TSP (mg/ml) CTB (lg/ml) CTB/TSP (%) CTB/fresh fruit (lg/g)

9th fruit 1~ 1.59 0 0 0.00011~ 1.03 0.422 0.041 0.21121~ 0.88 0.624 0.071 0.31231~ 1.09 0.909 0.081 0.455

16th fruit 1~ 1.35 0 0 0.00011~ 1.15 0.394 0.034 0.19721~ 0.89 0.594 0.067 0.29731~ 1.03 0.769 0.075 0.385

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

Fig. 3 CTB-ELISAresult of transgenic plantlines 9 and 16 anduntransgenic plant (ODvalue) X-axis: Totalproteins extracted fromdifferent 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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fruit, 11 showed significant serum and mucosal

response and produced anti-CTB antibodies. The

results showed that the titers of serum andmucosal antibody produced by transgenic fruits

are significantly higher than that of rCTB-immu-

nized mice and wild-type tomato-immunized mice(P < 0.05), demonstrating successful oral immu-

nization by the transgenic fruit and showing that

fruit-derived CTB was active as an oral immu-nogen (Fig. 5).

Discussion

CTB is a non-toxic unit of CT and well charac-

terized as an antigen against cholera because of

its ability to bind to GM1-ganglioside on thesurface of mammlian intestinal epithelial cells.

Gavage with transgenic fruits could induce serumand mucosal responses in BALB/c mice, which

proved that CTB subunit retained its native

antigenicity. Our result is consistent with previousstudies in which mice were fed raw transgenic

potato plant expressing antigens, for example,

hepatitis B surface antigen (HBsAg) (Masonet al. 1996; Kong et al. 2001; Thanavala et al.

2005) or the CTB subunit (Haq et al. 1995; Wang

et al. 2001). This study confirmed that the oralchannel of rCTB and trangenic fruits produced

the antibodies in blood. rCTB antigen was set as

the control and resulted in much lower antibodytiters than an equal amount of exogenous CTB in

trangenic fruits, suggesting the protective effect of

fibrous tissue of fruit to the exogenous CTBprotein against the degradation of protease in the

digestive tract of mice.

Among the full-length 2.2-kb E8 promoter, the1.1-kb core part can regulate gene expression

specifically in fruits during ripening, and the otherpart of the full-length 2.2-kb E8 promoter is due tohigher levels of expression of the gene (Deikman

et al. 1998). Sandhu et al. had transformed RSV-F

antigen into cherry tomato plants with the full-length 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, whichseemed higher than that under CaMV 35S pro-

moter 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, theCTB subunit was especially well expressed intransgenic ripening tomato fruit, and the highest

amount of CTB subunit expressed in ripening

fruits was 0.075% and 0.081% of total soluble

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

Fig. 5 Amount of IgGand IgA in serum andfeces stimulated byfeeding fruits oftransgenic tomato line 9

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protein, which was a little higher than that of anearlier report with 0.04% using CaMV 35S pro-

moter (Jani et al. 2002). So it is reasonable to raise

a hypothesis that the expression may be increasedif a transferred gene can be expressed specifically

in fruit. However, more experiments are needed.

In conclusion, E8 core promoter provided us anew way to enhance the expression of the CTB

subunit, specifically in edible fruits of the tomato

plant. Transgenic tomato plants expressing CTBprotein specially in fruits will move us closer to

achieving a low-cost, convenient, effective and

safe strategy for the prevention of cholera in hu-mans, especially in poor regions where conven-

tional vaccines are unaffordable or unavailable.

Acknowledgements This research was supported by

Natural Science Foundation in Guangdong Province ofChina (Grant No. 013075). Thanks are due to Prof. Zhong-Ju Xiao at Southen Medical University, China, Terry W.Hill at Rhodes College, TN, USA, and Dr. Chun-Quan Ouat HongKong University, China for providing valuablesuggestions. The authors also wish to thank School of LifeSciences, Sun Yat-Sen University, where part of theexperiments were conducted.

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