AsPac J. Mol.
2013
Mol. Biol.
Biol.Biotechnol.
Biotechnol.
Vol. 21 (2), 2013
Vol. 21 (2) : 50-58
1
Characterization of T2 Tobacco Plants expressing the Hepatitis B
gene for Molecular and Morphological Characters
Asha Velayudhan Nair1*, Yogendra Kalenahalli Narasimhamurthy2,
Paramanahally Hanumanthegowda Ramanjini Gowda3
School of Biochemistry, University of Bristol, United Kingdom
Plant Science Department, McGill University, Montreal, Quebec, Canada
3
Department of Biotechnology, University of Agricultural Sciences, Bangalore, India
1
2
Received 9 March 2013 / Accepted 6 June 2013
Abstract. Hepatitis B virus (HBV) infection remains a worldwide health problem and immunization is the only means to prevent the occurrence of this disease. As the available vaccines are very expensive, there is still need for a less expensive vaccine source
especially in most of the developing countries. Plants provide great advantages over other expression systems for the production
of recombinant proteins effectively at low cost. In this paper we have shown the stable expression of the hepatitis B surface antigen (HBsAg) in transgenic tobacco plants into the second generation; the current primary focus in plant transformation is the
stability of transgene expression. The presence of the HBsAg gene in putative transgenics of the T2 generation of tobacco plants
was confirmed by PCR analysis that produced a 900 bp amplification product. Transgenic and untransformed (control) tobacco
plants were evaluated for the presence and expression of the hepatitis B surface antigen gene by SDS-PAGE, protein dot blot and
ELISA. The T2 generation seeds obtained from the transgenic tobacco plants were tested for germination rate in the presence
of kanamycin and were observed to have a segregation ratio of 3:1, indicating Mendelian inheritance. Observations of growth
and yield parameters of T2 generation tobacco plants transformed with the HBsAg gene were recorded at regular intervals. It was
found that the presence of the foreign gene did not inhibit the growth of the transgenic plants; transgenics showed similar growth
rate compared to that of the control plants.
Keywords:
Hepatitis B surface antigen (HBsAg), Hepatitis B vaccine, Kanamycin selection, Mendelian inheritance,
T2 generation, Tobacco, Transformation.
Abbreviations: ELISA, enzyme linked immunosorbant assay; HBsAg, hepatitis B surface antigen; kDa,
kilo Dalton; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
INTRODUCTION
On a global scale, Hepatitis B Virus infection is probably
the single most prevalent cause of persistent viraemia in
humans. There are about 350 million chronic carriers of
HBV, which is about 5% of the total world population.
It is estimated that 75-100 million of them will die of liver
cirrhosis and/or hepatocellular carcinoma (Kumar et al.,
2008).
Hepatitis B virus is a 42-nm coated DNA virus with
a circular, partially double-stranded 3.2 kb genome that
preferentially infects hepatocytes and is classified in the
group hepadnaviridae (Roberts, 2004). Hepatitis B virus vaccine has been recommended as a routine infant
vaccination worldwide since 1991 and as a routine
adolescent vaccination since 1995. Although there are no
federal laws requiring the vaccine for day-care or school
attendance, the World Health Organization (WHO)
recommends that all countries with routine hepatitis B
vaccination programs continue that practice, and
countries not currently immunizing against Hepatitis B virus
infection adopt such programs immediately. The vaccine is
delivered in a series of three intramuscular injections over
a six-month period. It requires refrigeration, and injections
must be administered by a medical professional, with the
total cost ranging between $100 and $150 per person. These
factors, coupled with transportation and distribution issues, make mass immunization, especially in Third World
countries, difficult.
* Author for correspondence: Asha Velayudhan Nair, School of Biochemistry,
Medical Sciences Building, University of Bristol, BS8 1TD, United Kingdom.
Tel.: 004401173312167,
Email - [email protected] or [email protected].
AsPac J. Mol. Biol. Biotechnol. Vol. 21 (2), 2013
Plant based production of vaccines for
Hepatitis B may be an economically feasible alternative to
conventional vaccines (Sharma et al., 1999). Initial
studies of plant-made vaccines utilized easily
transformed and manipulated model plants such as tobacco
(Mason et al., 1992). Subsequent work has focused on
crop plants, including carrot (Imani et al., 2002), potato
(Richter et al., 2000), banana (Kumar et al., 2005;
Elkholy et al., 2009), tomato (Salyaev et al., 2007;
Srinivas et al., 2008), cherry tomato (Guan et al., 2012), maize
(Hayden et al., 2012; Sala et al., 2005), lupin and lettuce
(Kapusta et al., 1999).
The hepatitis B surface antigen (HBsAg) produced in
transgenic tobacco plant is physically, biochemically and
immunologically similar to yeast-derived rHBsAg
(Mason et al., 1992; Thanavala et al., 1995). Despite
several studies on the expression and characterization of
Hepatitis B in several plants including tobacco, there is
a lack of studies on changes in morphological and yield
characteristics of successive generations due to the
introduction of the foreign gene (HBsAg). So, in the present
study we show the stable expression of HBsAg in the
second generation of transgenic tobacco plant. We have also
investigated the accompanying morphological and yield
characteristics in comparison with control plants in greenhouse conditions.
MATERIALS AND METHODS
Bacterial strain and plasmid vector.
The bacterial
strain used for transformation studies was Agrobacterium
tumefaciens strain LB4404 (received from the National
Centre for Biological Sciences, Bangalore, India) into which
the plant binary vector pHB118 (Nagesha et al., 2009;
Sojikul et al., 2002) was integrated using heat shock method
as described in Nagesha et al. (2009). The construct was
provided by Dr. Hugh S. Mason, Arizona Biodesign
Institute, USA and the tobacco plant variety used for the
experiments was kanchan (N. tabacum cv. ‘Kanchun’)
obtained from the central tobacco research institute,
Rajamundry, India.
PCR analysis. Total genomic DNA was isolated from
leaves of T2 generation transgenic tobacco plants and
control plants by the Cetyltrimethyl Ammonium Bromide
(CTAB) method, using 2% CTAB, 1.4 M NaCl, 20mM
EDTA, 0.2% b-Mercaptaethanol, 100mM Tris-Hcl and 1%
PVP. Genomic DNA was subjected to PCR analysis. A 25µl
PCR mix contained 10 pMol concentration of each primer,
0.3 units of Taq DNA polymerase, 2.5 µM of each dNTP,
1X Taq buffer (Bangalore Genei) and 200ng of genomic
DNA as template.
The PCR conditions were 94°C initial melting for 2
51
minute followed by 30 cycles of amplification with each
cycle consisting of following steps: 94°C for 30 seconds,
54°C for 30 seconds, and 72°C for 30 seconds with a final
extension of 5 minutes and hold at 4°C after all cycles were
completed (Nagaraju et al., 1998). The amplified products
were separated on 1.2% Agarose gel. The primer sequences
used to amplify the 900 bp fragment of the HBsAg gene were:
i.Forward primer - (5’- GCATTCTACTTCTATT
GCAGC-3’) and
ii.Reverse primer - (5’-ACGTGGTAACTTAGATGTA
CACCCAAAG-3’)
Protein extraction and analysis.
Total protein was
extracted
from
leaves
by
using
Protein
extraction buffer (pH 8.0, 0.1M Tris-Hcl, 1mM PMSF,
2- b-Mercaptaethanol, Sodium sulphate, 2% PVP)
from transgenic and control tobacco plants (Nagesha et al.,
2006). These crude proteins isolations were subjected to
ammonium sulphate precipitation and concentrated by
dialysis, electrophoresed through 12% SDS gel and
visualized through CBB staining.
Protein dot blot and western blotting. Total protein was
extracted from the leaves of both control and putatively
transgenic tobacco plants and analyzed for the levels of
HBsAg specific antibody. Mouse anti-Hepatitis B surface
antigen was used as the primary antibody and goat
anti-mouse IgG horse radish peroxidase conjugate as
the secondary antibody.
ELISA. The total protein isolated from the control and
second generation transgenic (T2) tobacco leaves were
assayed for levels of HBsAg expression. Both control and
transgenic proteins were incubated with the primary
antibody (Mouse anti-Hepatitis B surface antigen) and
secondary antibody (goat anti-mouse IgG horse radish
peroxidase), both from Sigma-Aldrich, for one hour each.
The detection was done using 0.4 mg/ml chromogen
orthophenylene diamine (Sigma-Aldrich) with 1 μl/ml
hydrogen peroxide as substrate. The protein extracted from
non-transgenic (control) tobacco plants was used as
negative control and a commercially-available vaccine was
used as positive control, with absorbance being recorded at
492 nm for all the samples.
Inheritance of the Hepatitis B surface antigen by
Kanamycin assay.
The selectable marker used for
transformation of tobacco plants with HBsAg gene
was npt II that shows resistance to kanamycin, which
allows the selection of putative transformants, enabling
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AsPac J. Mol. Biol. Biotechnol. Vol. 21 (2), 2013
the study of stable gene expression in transgenics over
generations. Seeds were collected from control plants and
putative transgenic tobacco plants and were tested for their
germination using kanamycin selection. The germination
studies were conducted by germinating seeds on 100ppm
of kanamycin. After 10 days of incubation observations
were taken by counting the germinated seeds and recording
the ratios between germinated and non-germinated seeds
Study of growth and yield parameters of transgenic and
control Tobacco plants. Control and transgenic (T2) plants
grown in the greenhouse were used for the study of growth
and yield parameters, under which conditions the following
phenotypes were recorded:
1.
2.
3.
4.
5.
Height of the plant
Number of leaves
Leaf area
Number of flowers
Number of pods per plant
Statistical analysis was carried out using single factor
ANOVA for the plant height, number of leaves, leaf area,
number of flowers and number of pods per plant. All the
characters were recorded at intervals of 30, 60, 90 and 120
days after sowing
RESULTS
PCR.
PCR analysis of the leaves of putative
transformed T2 generation of Tobacco plants was
conducted using genomic DNA from control plants
and plasmid DNA isolated from Agrobacterium. Both
putative transformed plants and plasmids showed the
presence of a 900 bp amplification product corresponding to the HBsAg gene, but it was absent in control plants
when primers specific to HBsAg gene were used (Figure 1).
SDS PAGE. SDS-PAGE was carried out by extracting total protein from the leaves of T2 transformed and control
Tobacco plants. The transgenic plants containing the
recombinant protein produced shown a thick band at around
24 KDa on a 12% acrylamide gel (Figure 2), revealing the
over-expression of the hepatitis B surface antigen gene.
Western blot analysis.
The crude protein samples
extracted from the T2 generation transgenic and control
tobacco plants were used as antigens for western blot analysis.
Following immunostaining, transgenic tobacco plant samples
indicated positive results of the 24 kDa antigenic Hepatitis B
surface protein, which was not detected in the control samples
(Figure 3). The results indicated that the transgenic tobacco
plants contain the immunogenic hepatitis B surface antigen.
Figure 1. PCR amplification of Hepatitis B surface antigen in T2 generation transgenic tobacco plants with HBsAg with
gene specific primers. M : 1 Kb DNA Marker. C+ : Positive Control (Plasmid DNA). C- : Negative Control (Non transgenic
tobacco plant). P1- P14: Putative transformants.
AsPac J. Mol. Biol. Biotechnol. Vol. 21 (2), 2013
53
Figure 2. Confirmation of expressed HBsAg protein in T2 generation tobacco plants by
SDS-PAGE. M – Protein molecular weight marker. C : Crude protein from control tobacco.
P1- P6 : Crude protein from putative transformants
Figure 3. Western blot analysis showing the expression of the 24 kDa Hepatitis B surface
antigen from the T2 generation transgenic tobacco. C - : Crude protein from control tobacco. C+ :
Positive control (commercial vaccine). P1-P6: Crude protein from putative transformants
ELISA. Crude protein samples were extracted from T2
generation transgenic tobacco leaves and coated at the
concentration of 1 mg per well as an antigen. The highest
absorbance at 492nm was 0.198, produced by one of the
transgenic samples, significantly higher than the negative
control which gave an absorbance of 0.012, whereas the
commercial vaccine (positive control) gave a value of 0.325
(Figure 4). This shows the immunogenic nature of the
sample confirming the presence of the recombinant HBsAg,
and also validates the earlier SDS-PAGE and Western blot
results.
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AsPac J. Mol. Biol. Biotechnol. Vol. 21 (2), 2013
Inheritance analysis of Hepatitis B surface antigen gene
in the seeds obtained from transgenic tobacco. To study
the inheritance of hepatitis B surface antigen gene, the
seeds obtained from the transformed tobacco plants were
tested for their germination in presence of kanamycin.
The hepatitis B surface antigen segregation ratio was 3:1
as expected from Mendelian inheritance, when they were
germinated in presence of 100 ppm of kanamycin (Figure 5).
Morphological characters and Statistical analysis of
transgenic and non transgenic Tobacco progenies.
To investigate the differences in phenotypic variations
between T2 transgenic progenies and control Tobacco
plants, 3 independent T1 lines were selected and seeds were
obtained from them separately to generate T2 plants. A
total of 135 plants were selected from the T2 generation
(45 from each independent T1 line) to study their
phenotypic characteristics. Transgenic plant height was
greater (42.77cm, 75.51cm, and 101.48 cm) than the
control (42.23 cm, 74.10 cm, and 98.59 cm) at 60,
90, and 120 days post-sowing, respectively, while the
control plant height (20.67 cm) was greater than that of
the transgenic plant (20.20 cm) in the 30 days observation
(Figure 6). However, these differences were not significant.
No significant difference was seen in the number of leaves
either, though the trend was for a greater number of leaves
in transgenic plants (6.15, 14.69, 19.61 and 25.84) than in
control plants (5.84, 14.53, 18.61 and 24.00) 30, 60, 90,
and 120 days post-sowing, respectively. In the case of leaf
area, transgenic plants showed a greater leaf area (183.73,
381.85, 777.13, and 887.01cm2) than the control plants
(177.53,
361.69,
748.20,
and
864.06
cm2)
in
all
the
growth
stages.
There was no significant difference between
Figure 4. ELISA absorbance values recorded at 492nm for T2 generation
of transgenic and control tobacco leaf protein (crude) sample.
Figure 5. Germination of seeds obtained from nontransgenic (control)
and transgenic T2 tobacco plants in presence of 100 ppm kanamycin.
AsPac J. Mol. Biol. Biotechnol. Vol. 21 (2), 2013
55
Figure 6. Comparison of T2 generations of Transgenic and Control Tobacco
plants grown in a green house. (a) Transgenic plans grown in the green house.
(b) Control plants grown in a green house.
transgenic and control plants in the number of
flowers or seed pods: the average number of flowers in
transgenic plants was 197.46 compared to 187.00 in
control plants; and the average number of pods per plant in
transgenics was 170.23 and in control plants was 170.69.
DISCUSSION
In recent years, the expression of subunit vaccine antigens
in plants has been developed as a “convenient, safe and
potentially economical platform technology, with the
potential to provide a novel biotechnological solution to
vaccine production and delivery” (Thanavala et al., 2006).
Plant expression systems have a significant advantage
compared to other methods of recombinant protein
production since plants are much cheaper and easier to
cultivate than cell cultures. The potential of plant viral
vectors for production of pharmaceuticals is being realised
(Canizares et al., 2006), with various plant-based vaccines
having been developed, including the expression of the
binding subunit of Escherischia coli heat labile enterotoxin
(LT-B) in potato and tobacco (Tariq et al., 1995), plague
vaccine in tomato fruit (Alvarez et al., 2006), in planta
production of the recombinant vaccinia virus B5
antigenic domain (pB5), an attractive component of a subunit
vaccine against smallpox (Golovkin et al., 2007), and the
HBsAg production in tobacco plants (Mason et al., 1992).
An effective Hepatitis B vaccination strategy may eventually
consist of a combination of plant-derived parenteral and oral
vaccines, or a partial substitution of parenteral vaccines by an
oral formula for economical reasons (Pniewski, 2012; 2013).
The stable expression and inheritance of the
Hepatitis B surface antigen was demonstrated in T2
transgenic plants by PCR analysis, and expression
shown by western blotting and ELISA, confirming the
results of Zhong et al. (2005) and Nagesha et al. (2009).
Zhong et al. transformed Nicotiana benthamina plants with
pHB117 and pMHB plasmid constructs, and Nagesha et al.
(2009) transformed tobacco plants with pHB118 plasmid.
ELISA analysis indicated the immunogenic nature of the
transgenic plant sample, and the absorbance values showed
that the T2 transgenic plants expressed the HBsAg antigen
but at a lower level than a commercially-available vaccine.
The hepatitis B surface antigen segregated in a 3: 1
ratio when T1 generation transgenic tobacco seeds were
germinated in the presence of kanamycin, confirming
Mendelian inheritance. This result is in confirmation of that
of Vasil et al. (1993) who transferred the gene coding for
phosphinothricin acetyl transferase (PAT) into wheat plants;
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AsPac J. Mol. Biol. Biotechnol. Vol. 21 (2), 2013
Table 1. Kanamycin assay for study of transgene
expression in transgenic (T2) and non transgenic tobacco
seeds by germination on 100 ppm of kanamycin.
100 ppm of kanamycin.
Generations
No of
seeds
sown
Germinated
Nongerminated
Non Transgenic
40
0
40
T1
40
27
13
T2
40
30
10
T3
40
28
12
PAT activity was detected in a 3: 1 ratio in R1 generation
plants following cross- or self-pollination. Both male and
female transmission of the PAT gene and its segregation
as a dominant Mendelian ratio in R1 and R2 plants were
demonstrated. The growth and yield characteristics were
measured for all the transgenic plants at regular intervals
and found no significant difference in the transgenic plants
compared to the control plants. This shows that the
insertion and expression of the HBsAg has not affected the
growth of the plants.
Demonstration of the stable integration and
normal growth of T2 transgenic tobacco plants gave us the
opportunity to maintain the transgene expression in
the second generation produced from the T1 seeds thus
producing transgenic plants without performing a new
transformation each time.
CONCLUSION
In conclusion, our study has shown that the Hepatitis B
gene is inherited in the Mendelian ratio in transformed
tobacco plants in successive generations from T0 to T1
to T2. The integration of recombinant HBsAg gene into
transgenic tobacco plants and its expression was proven
through molecular analysis. The stable integration and
expression of the gene in T2 generation was confirmed,
and no inhibition of growth due to the integration of the
transgene was observed. Further studies in the subsequent
generations should be performed to know the integration
and expression pattern of the HBsAg gene in tobacco in the
longer term. Immunogenicity studies on the
HBsAg S-protein expressed in tobacco have to be conducted
in experimental animals, and if results are promising the
investigation should be extended to large scale clinical trails
to study the effectiveness of the plant-derived vaccine in
animals and humans.
ACKNOWLEDGEMENTS
We would like to thank Dr. Hugh S. Mason, Arizona
Biodesign Institute, USA for giving us the pHB 118 construct.
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