Guohui Bai 1
Construction of a fusion anti-caries DNA
vaccine in transgenic tomato plants for
PAcA gene and cholera toxin B subunit
1 Special Key Laboratory of Oral Diseases Research, Higher Education
Institution in Guizhou Province, Zunyi Medical University, Zunyi, People’s
Republic of China
2 Hospital of Stomatology, Zunyi Medical University, Zunyi, People’s
Republic of China
3 Guiyang Hospital of Stomatology, Guiyang, People’s Republic of China
Abstract
Chronic bacterial infections in the oral cavity influence the
development of dental caries. Mutans streptococci are the
major pathogenic cause of dental caries. The World Health
Organization (WHO) ranks dental caries, cancer, and
cardiovascular diseases as the three major global diseases
that need urgent preventative and curative measures.
However, substantial evidence suggests that traditional
prevention and treatment strategies are inefficient in reducing
the prevalence of dental caries. For protection against caries, it
is important to develop effective vaccines that induce
anticolonizing immunity against Streptococcus mutans
infections. In the present investigation, we constructed a
fusion anti-caries DNA vaccine (PAcA-ctxB) through fusing A
Keywords: caries, Mutans streptococci, cholera toxin B subunit, transgenic
tomatoes
1. Introduction
Dental caries is a chronic, infectious disease that results in
dental calcified tissue localized dissolution and destruction
mainly caused by Streptococcus mutans. It remains one of the
most common global chronic diseases and is considered to be a
Abbreviations: CaMV, cauliflower virus; CTB, Cholera toxin B subunit
coding gene; GUS, β-glucuronidase gene; MCS, mamultiple cloning sites;
NOS, NOS terminator; PAcA, A region of cell surface protein PAc; SIgA,
secretary IgA..
∗ Address for correspondence: Jianguo Liu, PhD, Special Key Laboratory of
Oral Diseases Research, Higher Education Institution in Guizhou Province,
Zunyi Medical University, Zunyi, 563000. Tel.: 13087891001;
e-mail: 13087891001@163.com.
Received 7 June 2019; accepted 20 August 2019
DOI: 10.1002/bab.1806
Published online 5 December 2019 in Wiley Online Library
(wileyonlinelibrary.com)
924
Yuan Tian1,2
Jiayuan Wu1,2
Yu Gu1
Zhu Chen3
Fengjiao Zeng1,2
∗
Jianguo Liu1
region of cell surface protein PAc (PAcA) coding gene of
mutans streptococci with cholera toxin B subunit coding gene
(CTB). Afterward, the plasmids were integrated into tomato
genomes through agrobacterium-mediated plant
transformation technology. The presence of transgenes in the
tomato genome was confirmed by PCR, β-glucuronidase gene
(GUS), and western blot. The expression of genes was
confirmed at transcription and protein level. Altogether, the
results presented herein showed that transgenic tomatoes
may provide a useful system for the production of human
caries antigen. C 2019 International Union of Biochemistry and
Molecular Biology, Inc. Volume 66, Number 6, Pages 924–929, 2019
public health problem [1]. It is important to craft preventative
measures against dental caries. Since the molecular biology
technology has advanced, genetically modified transgenic
plants have become a new type of bioreactors [2]. Transgenic
plant expressed antigens are always considered natural
immunogens when compared with other vaccine production
methods [3]. The transgenic plant expressed antigens are
more cost effective and do not induce injection-related pain
in comparison to other bioreactors. The vaccine requires
low production cost, is biologically safe, and easy to store,
making it ideal for a large population. Thus, the application of
transgenic tomatoes in vaccine research has sparked interest
in many of scholars. Salazar-Gonzalez et al. [4] used transgenic
tomatoes expressing human beta-amyloid as a vaccine against
Alzheimer’s disease. Zhang et al. [5] used a carrot-derived vac-
cine candidate expressing UreB subunit against Helicobacter
pylori. This current study is the first to use transgenic tomatoes
to produce an oral vaccine against Streptococcus mutans.
 When the Streptococci mutans are present on the tooth
surface, they cause adhesion and aggregation of tissues that
causes caries [6]. The cell surface protein called PAc is consid-
ered to be a vital preventative target of the virulent factors from
Streptococcus mutans. The immune anti-caries protection uses
antibodies from the protective level. Alternatively, the mucosal
immunity that only uses soluble proteins or polypeptide
antigens cannot produce persistent secretory IgA (SIgA). Thus,
immune reagents and release systems must be used to enhance
the immune response of the mucous membranes and improve
the dental caries vaccine positive effects [7]. CTB is a strong
immune adjuvant, resembling high immunity levels alone or
in combination with other unrelated antigens. Particularly,
the activity of immunoadjuvant becomes stronger when it is
chemically/genetically fused to form a conjugated/fused protein
with an irrelevant antigen than when mixed with an irrelevant
antigen [8, 9]. To validate this hypothesis, we synthesized the
fusion DNA segment of the structural gene that is the important
Streptococcus mutans virulent factor, containing A region of
PAc protein-encoding gene and cholera toxin B subunit. Both
genes were properly expressed in Escherichia coli and we
used them as the target genes linked to the plant expression
vector via the intermediate vector. An anti-caries DNA vaccine
was constructed in tomatoes after all the procedures were
successfully completed.

2. Materials and Methods

The following materials were used in this study: E. coli JM109,
Agrobactium tumefaciens EHA105, vector pCAMBIA2301 (gift
from Professor Ma Xinrong, Associate Institute of Biology,
Chinese Academy of Sciences), vector pBPC55 (gift from Dr.
Zhang Xiaodong, Beijing Academy of Agriculture and Forestry),
Intermediate vector pBPC55 (gift from Dr. Zhang Xiaodong, Bei-
jing Academy of Agriculture and Forestry Sciences), plasmids
pEAC10 and pEAC5 (provided by the dental caries laboratory Plasmid construction flowchart
of Zunyi Medical College). FIG. 1

2.1. Construction of plant expression vector

pCAMBIA–PAcA-CTB
The plasmid construction procedures are shown in Fig. 1.
Initially, we extracted a fragment containing 35 s promoter,
NOS terminator, and multiple cloning site (size 1.1 kb) from
the intermediate vector pBPC55 using EcoRI and HindIII
endonuclease to enable the efficient expression of the foreign
gene. The cloning site on the plant binary expression vector
pCAMBIA2301 was replaced with the obtained fragment to
initiate expression of the foreign gene, and then the recombi-
nant plant expression vector pCAMBIA-35s was constructed.
Second, to extract the fusion gene PAcA/CTB of surface globulin
PAc gene A region and cholera toxin B subunit, the following
procedures were done. A pair of PCR primers including both
ends sequences of chimeric genes PAcA-CTB and restriction
endonuclease sites were designed according to the sequencing
results of the recombinant plasmids pEAC (containing A region
of PAc protein-encoding gene and cholera toxin B subunit) =
Upstream: 5 -T GAT TCT AGA ATG GAT GAA ACG ACC ACT
ACT AG-3 , containing Xba I restriction site; Downstream: 5 -A
TCG GGT ACC TTA ATT TGC CAT ACT AAT TGC-3 , containing
Kpn I restriction sites (synthesized by Chengdu Fama genetic
company). Finally, the purified chimeric gene PAcA-ctxB
was used as an exogenously inserted gene fragment. The
recombinant vector pCAMBIA-35s was cleaved with Xba I
and Kpn I, and the exogenous inserted gene fragment was
ligated into the downstream segment of 35S promoter in
the recombinant plasmid pCAMBIA-35s to obtain the plant
expression vector pCAMBIA–PAcA-ctxB. The pCAMBIA–PAcA-
ctxB was transformed into E. coli JM109 to obtain many
transformants. The recombinant plasmid pCAMBIA–PAcA-CTB
was constructed successfully after confirmation using the re-
striction enzyme identification with XbaI and KpnI. The verified

Biotechnology and Applied Biochemistry 925


pCAMBIA–PAcA-ctxB plasmid was transformed into competent
cells of Agrobacterium EHA105 using the electroporation and
was cultured at 28 ◦ C. After 48 H, the transformants were ran-
domly picked, and then plasmids were extracted and detected
by PCR. The transformants were amplified with specific primers
of chimeric gene PAcA/CTB and the original donor plasmid
pEAC was used as a positive control. Agrobacterium-mediated
transformation of the tomato was performed referring to the
guidelines from Frary and Hamilton [10].
2.2. Plants transformation
Many sterile tobacco tube seedlings were harvested, crushed
in a sterilized mortar and a liquid medium with a similar
composition as the co-culture medium was added to prepare a
tobacco extract. The verified Agrobacterium was expanded and
then centrifuged, and the obtained bacteria were suspended in
the tobacco extract. The seeds were sterilized with 75% alcohol
for 30 Sec, soaked in 20% sodium hypochloride solution for
10 Min and washed four times with sterile distilled water. The
sterilized seeds were cultivated on Murashige and Skoog tissue
culture medium at 25 ◦ C with 16 H of sunlight per day. Fifteen
days later, the fully sprouted cotyledons were used for the plant
transformation. The cotyledons were cut into 5 mm2 , soaked
in smoke tobacco extract for 5 Min, and then placed onto
Petri dishes for 3 days. The resistant seedlings that had grown
approximately 1 cm of height were cut and put into a strong
seedling growth medium (selection pressure is kanamycin
100 mg L−1 ). The well-grown resistant differentiated shoots
were transferred into a rooting medium for root selection,
with 25 mg/L selection pressure. Among them, the roots of
the resistant plants were identified and cut into segments to
continue breeding in the culture flasks.
2.3. Identification of tomato transgenic plants
After 6 weeks of transformation, the trans-
formed/untransformed plant roots were cut into small
pieces and soaked in GUS dye solution at 37 ◦ C overnight.
They were decolorized with 70% ethanol until the negative
control turned white, after observing the blue material under
the microscope. The uneven total DNA of the transgenic plant
was extracted according to the method given in Refs. 11–13
and used as a template for the positive and negative controls
simultaneously. Synthetic primers were designed to amplify a
portion of the NPT II gene based on the coding sequence of NPT
II as follows: Upstream: 5 -TCCGGCCGCTTGGGTGGAGAG-3
and Downstream: 5 -CTGGCGCGAGCCCCTGATGCT-3 . The
specific primers PAcA-CTB of the target gene were used as
primers for PCR amplification and derived from the total DNA
of the transformed plants as the template. The negative control
(total DNA of untransformed plants) and a positive control
(plasmid pCAMBIA–PAcA-CTB) were set up simultaneously to
identify the transgenic plants. The southern blot hybridization
of the transformed materials was performed to verify the
integration of foreign genes on the chromosomes of plants and
further identification of the transgenic plants. The DNA from
926
Biotechnology and
Applied Biochemistry
Validation map of recombinant plasmid
FIG. 2 pCAMBI-35s
the transformed and untransformed plants was extracted.
After treating with alkali, the DNA was spotted on a nylon
membrane and the probe was subjected to the following
processes: prehybridization, hybridization, membrane
washing, and autoradiography via random primer method.
3. Results
3.1. Construction and identification of recombinant
plasmid pCAMBIA–PAcA-ctxB
The plasmids, namely pCAMBI-35s and pBPC55, were digested
by EcoRI and HindIII to excise a 1.1 kb DNA fragment including
the CaMV35S promoter, NOS terminator, and MCS, while the
corresponding pCAMBIA2301 was absent. The plasmid namely
A2301 was digested by EcoRI and HindIII to obtain a multiple
cloning site of 50 bp size and pCAMBIA2301 vector. The 1.1 kb
fragment of lanes 3 and 7 were equivalent to the size of the
35S promoter of plasmid pBPC55, the multiple cloning site, and
the NOS terminator, indicating that the recombinant plasmid
pCAMBI-35s was successfully constructed (Fig. 2).The PCR
product of plasmid pEAC was confirmed by 1% agarose gel
electrophoresis to obtain a gene of interest of approximately
1,734 bp (Fig. 3).The XbaI and KpnI double digestion results
of the plant expression vector pCAMBIA–PAcA-ctxB must have
two 1.7 kb and 23 kb bands, and the identification process
indicated that the target gene had been integrated into the
vector pCAMBIA-35s. Therefore, the plant expression vector
pCAMBIA–PAcA-ctxB was successfully built as shown in Fig. 4.
3.2. Agrobacterium transformation and identification
of plant expression vector pCAMBIA–PAcA-ctxB
To demonstrate that pCAMBIA–PAcA-ctxB plasmid was suc-
cessfully transferred to A. tumefaciens EHA105, we used PCR
An Anti-caries Vaccine in Transgenic Tomato Plants
 Validation diagram of plasmid pEAC
FIG. 3

Identification of Agrobacterium expression vector

FIG. 5

Breeding of transgenic tomato plants

FIG. 6

for further identification. The results of agrobacterium expres-

sion vectors showed that the recombinants amplified the same
size specific bands as the positive control. They can be utilized
for the genetic transformation procedures of plants (Fig. 5).

3.3. Identification of transformed tomato plants

The tomato sprouts were observed on the bud differentiation
medium at 2 weeks (Fig. 6A). Then a lot of clustered differ-
entiated shoots on explant callus were observed after being
cultivated in a medium for approximately 3–4 weeks (Fig. 6B)
and the differentiated budded explants transitioned to strong
seedlings medium for growth (Fig. 6C).
Verification map of plant expression vector
FIG. 4 pCAMBIA–PAcA-ctxB 3.4. GUS activity assay results of transformed plants
Tissue materials were destained with anhydrous ethanol.
We noticed that the positively transformed plants showed a
clear blue color, while the pseudo-transformed plants and the

Biotechnology and Applied Biochemistry 927


Detection of GUS activity in transgenic plants
FIG. 7
Amplification of the marker gene NPT II
FIG. 8
untransformed plants did not show a clear blue color (Fig. 7),
which illustrates that GUS gene was expressed in tomato plants.
3.5. PCR amplification of NPT II
The marker gene NPT II was amplified by PCR. The transfor-
mants were amplified to the expected size fragments; they had
initially demonstrated the introduction of foreign genes that
had been transferred into the plants genome (Fig. 8).
3.6. PCR identification of transgenic plants
The positive control and transformed plants were able to
amplify a fragment of approximately 1,730 bp. Some of the
pseudo-transformants did not amplify the expected fragments,
while the untransformed plants did not show the corresponding
bands. This shows that the gene of interest had been introduced
into resistant plants (Fig. 9).
3.7. Southern blot analysis of transgenic plants
Five of the six transformed plants had positive spots in the
total DNA, but one lacked the hybrid spots. Although the
nontransformed plants that served as the negative controls
lacked the hybridized spots, the presence of positive spots in
the total DNA indicated that the total DNA of the transgenic
plants had homology with the probes and that the gene of
interest was evidently integrated into the tomato genome
(Fig. 10).
928
Biotechnology and
Applied Biochemistry
Verification of target genes in transgenic plants
FIG. 9
Southern blot analysis of transgenic plants
FIG. 10
4. Discussion
Over the years, research on active immunization of dental
caries has undergone various stages of development as follows:
inactivated, live attenuated, purified antigen subunit, recom-
binant antigen, genetic recombinant, and nucleic acid vaccine
stages [14]. The inactivated vaccine destroys and loses certain
antigenic structures during the inactivation process. It requires
repeated inoculations and it resembles poor immune effects.
The live attenuated vaccines have insufficiently weakened viru-
lence or repeated mutations. Since Curtiss proposed the use of
transgenic plant expression systems to produce edible vaccines
in 1990, several plants have been used as bioreactors for
large-scale production of recombinant vaccines or recombinant
antibodies [15]. The genetically engineered transgenic plants
are becoming important foreign protein expression systems that
are cost effective. Several studies have confirmed that trans-
genic plant vaccines can effectively stimulate specific mucosal
and humoral immune responses in animals and have no ad-
verse side effects on experimental animals [16–19]. The tomato
is the main host plant that produces oral vaccines. Currently, it
is the most widely used edible vaccine transgenic plant due to
its flavorful and nutritious traits. The oral vaccines are easier
to use for large populations compared to other vaccinations.
Currently, the research on transgenic tomato is extensive but
lacks in the area of enhancing the agronomic traits of the toma-
toes. Many antigenic proteins have been expressed in tomato
An Anti-caries Vaccine in Transgenic Tomato Plants
including cholera toxin, hepatitis B surface antigen HBsAg [20],
and rabies virus glycoprotein [21]. The tomato transformation
technology is relatively established when compared to its
formative stages in the early 1990s. However, its application
has some limitations such as perishability, which limits the
storage duration. From this limitation, the Hindfi fragment
of the PG gene was cloned between the 35S promoter of the
cauliflower virus (CaMV) promoter and the 3 -untranslated
region (NOS) terminator of the plant transformation vector
Bin19 through reverse orientation clone technology. The
transformed plants were obtained through the co-cultivation of
agrobacterium with the cotyledon explant of aseptic seedlings
of tomatoes. In the tomato fruit transformed with antisense
PG gene, the level of PG mRNA and PG enzyme activity were
significantly reduced during the fruit ripening stage. The
oral plant vaccines are new concepts for advancing modern
vaccines and this is a potential area for further research and
advancement in preventing dental caries.
In this experiment, the A region of pac gene of Strepto-
coccus mutans and mosaic plasmid of B subunit of cholera
toxin were transferred to the tomato and their integration
in the tomato genome was confirmed. In recent years, many
researchers have focused on the protein and gene vaccines
encoding multiple adhesion molecules [22]. Improvements in
the vaccines include identification of protective epitopes as-
sociated with functional thresholds, single or multicomponent
vaccines in combination to enhance potency, multiple routes
of coimmunization, and in-depth studies of immune adjuvants
[23]. The improvement and advancement of molecular biologi-
cal experimental techniques influence the synthesis of effective
anti-caries vaccines. This study therefore indicates that the use
of an oral plant vaccine is a novel concept for creating modern
vaccines, which provides a platform for further research and
development of preventative measures for caries.
5. Acknowledgments
This study was supported by funds from the NSFC (National
Natural Science Foundation of China) (Grant No. 81560186):
Research on the preparation of anti-porphyromonas gingivalis
FimA and HagA DNA vaccine and its mechanism of action; the
Construction Program for the Key Characteristic Laboratory
in High Institutions of Guizhou Province (Grant No. Qian Jiao
He KY[2013]109); the Science and Technology Innovation
Talents Training Project of Zunyi (Grant No. Zun Ke He Ren
[2014]8): Development of a new type of mouthwash and the
Biotechnology and Applied Biochemistry
therapeutic effect analysis; and Guizhou Province Science and
Technology Innovation Talent Team “Guizhou Province Oral
Disease Prevention Research and Technology Innovation Talent
Team” (Grant No. Yankehe Talent Team [2013] 4026).
The authors declare no conflict of interest.
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