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Ying
Ma, Yi Gao, Wen-Xin Luo, Jun Zhang, Ning-Shao Xia, The Key
Laboratory of the Ministry of Education for Cell Biology and Tumor
Cell Engineering, Xiamen University, Xiamen 361005, Fujian Province,
China
Ying Ma, Ministry of Education Key Laboratory for Marine
Environmental Science, Center for Marine Environmental Science,
Xiamen University, Xiamen 361005, Fujian Province, China
Shun-Quan Lin, College of Horticulture, South China
Agricultural University, Guangzhou, 510642, Guangdong Province,
China
Mei Li, Xiamen Overseas Chinese Subtropical Plant
Introduction Garden, Xiamen 361002, Fujian Province, China
Supported by a grant from the Natural Science Foundation of
Fujian Province, No. C9910004 and Xiamen Kaili Biologic Product
Limited Company
Correspondence to: Ning-Shao Xia, The Key Laboratory of the
Ministry of Education for Cell Biology and Tumor Cell Engineering,
Xiamen University, Xiamen 361005, Fujian Province, China. nsxia@jingxian.xmu.edu.cn
Telephone: +86-592-2184110
Fax: +86-592-2184110
Received: 2003-03-12
Accepted: 2003-07-15
Abstract
AIM: To transfer hepatitis E virus (HEV) ORF2 partial gene to
tomato plants, to investigate its expression in transformants and
the immunoactivity of expression products, and to explore the
feasibility of developing a new type of plant-derived HEV oral
vaccine.
METHODS:
Plant binary expression vector p1301E2, carrying a fragment of HEV
open reading frame-2 (named HEV-E2), was constructed by linking the
fragment to a constitutive CaMV35s promoter and nos terminator, then
directly introduced into Agrobacterium tumefaciens EHA105.
With leaf-disc method, tomato plants medicated by EHA105 were
transformed and hygromycin-resistant plantlets were obtained in
selective medium containing hygromycin. The presence and integration
of foreign DNA in transgenic tomato genome were confirmed by Gus
gene expression, PCR amplification 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:
HEV-E2 gene was correctly expressed in transgenic tomatoes and the
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.
Ma
Y, Lin SQ, Gao Y, Li M, Luo WX, Zhang J, Xia NS. Expression of ORF2
partial gene of hepatitis E virus in tomatoes and immunoactivity of
expression products. World J Gastroenterol
2003; 9(10): 2211-2215
http://www.wjgnet.com/1007-9327/9/2211.asp
INTRODUCTION
Research on using plants 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 Streptococcus mutans 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],
E.coli 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],
etc. The involved plant receptors are mostly mode species: tobacco,
potato, etc., some fruits and vegetables that can be consumed raw,
tomato, banana, lettuce and lupine have also been attempted.
Significant breakthrough has been achieved in increasing the
expression of vaccine components in plants. At present, the
plant-derived oral vaccines from potato for HBsAg and LT-B are
undergoing clinical trials[13,14], but there has been no
report about plant-derived oral vaccine for hepatitis E virus (HEV).
HEV is the major cause of
acute, enteric non-A, non-B (NANB) hepatitis in the world, and large
outbreaks occur primarily in underdeveloped countries. China is one
of the high epidemic areas. The highest infection rate is in young
adults (15-40 years). Although only 1 % to 3 % of non-pregnant
patients with HEV infection progress to fatal fulminant hepatitis,
the mortality can be as high as 20 % in pregnant patients[20].
There is neither effective antiviral drugs nor vaccine against HEV
available for commercial use at present. Since HEV pathogen is
difficult to culture, it is not easy to develop the live attenuated
strains for vaccine. A promising approach is to develop recombinant
subunit vaccines. Compared with gene engineering vaccine for
hepatitis B, the study of hepatitis E recombinant vaccine was only a
recent endeavor, but some progress has been made.
ORF2-encoded protein of
HEV is the most promising subunit vaccine candidate because it
possesses good antigenicity. So far, HEV ORF2 gene or its fragments
have been expressed in prokaryote cells[21-24], insect
cells[25], animal cells[26], and Pichia
pastoris[27], etc., and the expression products possessed
immunogenicity. HEV vaccine is about to undergo clinical trials[28].
Since hepatitis E occurs
primarily in developing countries and impoverished regions with poor
environmental sanitation, where administration of various medicinal
vaccines may be hampered by the relatively high cost. Plant-derived
hepatitis E vaccine is a promising approach that may solve the
problem because of its low cost for delivery and administration, and
its safety for humans.
Tomato is a
nutrition-rich fruit that can be consumed raw and easily
transformed, so it is an ideal plant carrier for oral vaccine. In
this paper we reported that a plant expression vector of HEV antigen
gene was constructed and transformed into tomato plants with Agrobacterium
tumefaciens, and its expression in plants and immunoactivity of
the expression product were examined. This study would lay the
foundation for further research on the development of a new type of
plant-derived hepatitis E oral vaccine or other oral vaccines, and
promote their practical application.
MATERIALS
AND METHODS
Plant material
Tomato (Lycopersium esculentum 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. Agrobacterium tumefaciens
strain EHA105 was kindly presented by Professor Zhang Qi-fa,
Huazhong Agricultural University. Plasmid pBPFW7
containing CaMV35s promoter and nos terminator, and plant binary
plasmid pCAMBIA1301 containing hygromycin-resistant gene, kanamycin-resistant
gene and Gus 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 patient's
serum and inserted into pBPFW7
between CaMV35S promoter and nos terminator at BamHI/EcoRI site to
form pBE2. The fragment containing "P35S+W+E2+Tnos"
was isolated by gel extraction from plasmid pBE2 after PstI
restrictive digestion and then subcloned into plasmid pCAMBIA1301
that had been digested by the same restriction endonuclease to yield
the reconstructed plant binary expression plasmid p1301E2 (Figure
1). Confirmed by restriction digestion, p1301E2 was directly
introduced into Agrobacterium tumefaciens strain EHA105 by
freeze-thaw method.
Figure
1(PDF) Structure of
plasmid p1301E2.
Plant
transformation and regeneration
Tomato was transformed through leaf discs mediated by Agrobacterium
tumefaciens 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 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'-GGA TCC ATA TGC AGC TGT TCT ACT CTC GTC-3' and
5'-CTC GAG AAA TAA ACT ATA ACT CCC GA-3', respectively (synthesized
by BioAsia Co., Shanghai). PCR reaction was performed using 50 ng of
template DNA, 0.5 mM
of each primer in a total volume of 30 ml.
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 HEV-E2
in transformants were quantified by ELISA. The extract of
transformant was diluted several fold until it could reach the same
OD value (measurement wavelength: 450 nm) as the standard HEV-E2
protein (1 ng/mg), then HEV-E2 expression levels in transformants
could be calculated according to the sampling quantity, and diluting
times.
RESULTS
Regeneration of transgenic tomato plants
The plasmids for expression of HEV-E2 in plants allowed
morphogenesis of transformants on selective medium containing
hygromycin. Through about 1 month of selection, 7 independent Hyg-resistant
plantlets were obtained. These transformants bore fruits and the
seeds were collected. The flowers and fruits of the transformants
resembled those of wild-type tomato plants and the seeds were plump.
Each fruit produced approximately 70 seeds. This demonstrated that
the introduction of foreign gene into tomato plants did not
influence the normal growth and development of the transformants
(Figure 2).
Gus
expression in transgenic plants
Untransformed tissues appeared colourless when stained with
Gus reaction solution and bleached with absolute alcohol, whereas
transformed tissues presented blue spots even after bleached with
absolute alcohol, the spots were generally large in size, with a
deep blue color (Figure 3). The results indicated that Gus gene was
stably integrated into the genomic DNA of transformed tomatoes.
Figure
2 Transgenic plants
in greenhouse. A:
Flowers of transformants, B:
Fruits of transformants.
Figure 3 Analysis
of Gus gene expression. A:
Leaf of untransformed tomato plant, B:
Leaf of transformed tomato plant, C:
Flesh of untransformed tomato, D:
Flesh of transformed tomato.
Figure 4 Analysis
of HEV-E2 gene integration. A:(PDF)
PCR
amplification of genomic DNA of tomato plants, B:
Southern dot blotting of genomic DNA of tomato plants. A1: DL-2000
marker, A2: Positive control (p1301E2), A3: Total DNA of wild-type
control plant, A4-A10: Total DNA of independent transformants. B1:
Total DNA of wild-type control plant, B2: Positive control
(p1301E2), B3-B8: Total DNA of independent transformants.
Figure 5 HEV-E2
expression levels in transgenic tomato plants. 1: Leaves of
wild-type control plant, 2-8: Leaves of seven independent
transformants, 9: The mixed flesh tissue of 3 independent
transformant fruits.
PCR amplification
The expected 810 bp fragments were amplified from 7
transformed lines of tomatoes, the length of the fragments was
identical with that amplified from p1301E2 (positive control),
whereas there was no PCR product in wild-type tomato plants
(negative control). The results primarily verified that the target
gene was integrated into the genomic DNA of transformed tomatoes
(Figure 4A).
Southern
dot blotting
To further verify the integration of foreign gene into
tomato plants, the total genomic DNA of transformed tomatoes was
hybridized by a DIG-labeled probe (encompassing the coding region of
HEV-E2 gene) generated by PCR amplification from p1301E2 with HEFP
and HERP primers, and all the transformants gave the same
hybridization spots as the positive control(p1301E2) did, whereas
the untransformed plant showed no detectable hybridization signal
(Figure 4B). Thus the results further confirmed that the target gene
was integrated into the tomato plants.
Expression
of HEV-E2 gene in transgenic tomato plants
The leaf extracts of 7 lines of transformants and the mixed
fruit tissue extracts from 3 transformants were tested by ELISA for
the presence of HEV-E2 expression products, and foreign protein
could be detected in all of the examined samples. The reaction was
specific because wild-type tomato showed no detectable expression
products. The result demonstrated that HEV-E2 gene was expressed in
transformed tissue. The expression level of HEV-E2 protein was lower
as compared to HBsAg in transgenic tomatoes[8], the
maximal expression level was only 47.9 ng/g fresh weight in leaves.
The expression levels were different between different transformants
and between different organs of the same plant, which indicated that
the inserting site of foreign DNA into the plant genome was random.
The expression in fruits (61.22 ng/g fresh weight) was higher than
that in leaves, which was similar to that of HBsAg in transgenic
tomatoes[8]. The higher expression of target protein in
edible tissue mightbe helpful for producing oral vaccine.
DISCUSSION
The most striking advantage of using tomato as oral vaccine
vector is that the plant-derived vaccine is cheap and tomato is a
freshly-eaten fruit, which allows the target population to acquire
immunity at the same time when they enjoy the delicious fruits. But
accumulation of protein in tomato itself is low, the expression
levels of foreign protein in it are much lower. This will make it
difficult when administering the plant-derived vaccine. However, the
expression of foreign protein in plants might be increased by
several modifications, including the use of stronger promoters, the
use of plant-derived leader sequences and signal peptide, and
targeting the protein for retention in edible tissue and so on[1].
For example, Mason et al[2] increased the level of
HBsAg 11 fold in transgenic tobacco by linking CaMV35S promoter to
the tobacco etch virus (TEV) 5 leader sequence (acts as a
translational enhancer). In 1998, Mason et al[12]
increased the expression of LT-B in transgenic potatoes 3-14 times
by designing and constructing a plant-optimized synthetic gene
encoding LT-B. Tackaberry et al[30] reported the
synthesis of recombinant glycoprotein B which expressed specifically
in tobacco seeds, with expression level reaching 70-146 ng/mg
extracted protein. Lauterslager et al[11] made a
synthetic gene coding for LT-B and optimized it for expression
specifically in potato tubers and accumulation in endoplasmic
reticulum, which resulted in a high expression level about 13 mg/g
fresh weight of LT-B in potato tubers. Besides, by using chloroplast
expression system, Cosa et al[31] introduced
foreign genes into the chromoplast genome, which enabled the foreign
protein to accumulate at 46 % of the total soluble protein in leaves
of transgenic plants. All these studies provided us successful
experiences in improving expression level of HEV-E2 in transgenic
tomatoes. In our study, we only inserted an enhancer W
in the promoter, there were many reconstruction possibilities to
achieve high expression. 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 HEV-E2 gene into tomatoes and identified the expression
protein. The expression product possessed HEV specific antigenicity
in transgenic plants. Previous studies also demonstrated that
plant-derived vaccines were safe and functional. It is cheap to
produce and store, easy to deliver and administer, it has many
advantages over other vaccines. Although this technology is not
likely to produce great commercial value in the short term, the
present advance in plant genetic engineering demonstrates that there
is a tremendous potential to develop various low-cost recombinant
vaccines by using plants.
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