MINSTRY OF EUCATION AND TRANING
THAI NGUYEN UNIVERSITY
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NGUYEN THU HIEN SOY BEAN AGROBACTERIUM - MEDIATED
TRASFORMATION USING GENE ENCODING
SURFACE PROTEIN FROMH5N1 VIRUS FOR
PLANT VACCINE PRODUCTION

Specialty/Major: Genetics
Code: 62 42 01 21 PHD THESIS SUMMARY OF BIOLOGICAL
Thai Nguyen - 2013

The work was completed at:

Technology Division plant cells, Institute of Biotechnology
Department of Biology-Genetics modern, Department of Biology - Audit
College of education - Thai Nguyen University.

pandemic can be prevented and controlled.
Edible vaccine is made from plant and can stimulate both
humoral and cell-medicated immune systems. It is similar to other
common vaccines, but produced by plants and contained in plant’s
part such as leaves, roots, seeds, and fruits. There are several
advantages of plant vaccine such as low cost, easy to manufacture,
highly efficiency, and safe for use. Therefore, plant vaccine is being
considered suitable for public health strategies in developing
countries. In Vietnam, soybean is one of the main sources in food
supply for both human and animals. From that points of view, the
project titled “Soy Bean Agrobacterium-Mediated Transformation
Using Gene Encoding Surface Proteins from H5N1 Virus for Plant
Vaccine Production” has been carried out.

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2. General aims
The general purpose of this thesis project is to engineer a
genetic transformed soy bean genotype, expressing surface protein
from A/H5N1 virus, which can be used for producing edible vaccine.
In details, two specific aims of the project were
- To design and recombine genetic carrier vectors containing
HA gen and HA1 gen region from virus A/H5N1;
- To regenerate a HA1 genetic transformed soy bean cultivar
with recombined HA1 protein expression in seeds.
3. Specific aims:
- Design and synthesis transgenic vectors carrying HA gen and
HA1 region.
- Induced these genes into Agrobacterium tumefaciens and to
confirm that genes were delivered a tobacco genotype was
infected with recombined A. tumefaciens and then

transformed soybean H11 genotype opens an opportunity for
producing a soybean product that contains HA1 antigen. This product

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can be used for avian breeding and its protein may act as plant
vaccine to prevent the avian flu pandemic in the future.
We suggest to continue screening for the stable transformation
efficiencies of the transformed soybean genotypes in the next
generation and to examine the immune respond of the animals that
bred with the transgenic plant.
6. Thesis format
There are 110 pages in this thesis that consists of Introduction (4
pages); Chapter 1: literature review (43 pages); Chapter 2: materials
and methods ((14 pages); Chapter 3: results and discussion ( 43
pages); Conclusion (2 pages). The thesis consists of 36 figures and 16
tables and referred 156 references.
Chapter 1
LITERATURE REVIEW
The thesis referred 23 Vietnamese and 133 English references
which cover the related topics such as (1) Varian flu and A/H5N1
virus, (2) A/H5N1 flu vaccine, (3) the application of gen
transformation in plant vaccine manufacturing.
Avian flu is an acute transmitted disease caused by flu virus type
A Orthomyxoviridae. This viral type is classified into sub-types
based on their capsid surface antigen HA and NA. there are 16 HA
sub-types (H1 – H16), and 9 NA sub-types (N 1 – N9). In principle,
the combination of HA and NA sub-types will generate many more

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new sub-types. Gen structure of A/H5N1 includes 8 separate regions

Academy of Agriculture.
Bacteria and vectors
- pPTN289 vector and pDest-phaso were supplied by VUB.
- HA gene from A/H5N1 virus was provided by Institute for
biotechnology.
- E.coli and A.tumefaciens CV58 and EHA101 was from plant
cell technology laboratory –Institute for Biotechnology.
Chemicals and instruments used in this project provided by plant
cell technology laboratory and central gen technology laboratory,
institute for biotechnology.
2.2. Methods
2.2.1. Methods and techniques used to generate gen transformed
vectors
- PCR
- Gel electrophoresis

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- Inducing reaction: inducing into E. coli following published
heat shock method (Cohen 1972).
- Selection using PCR and selected enzyme method according to
methods of Sambroook el al. (2001).
- Gen transformed vector generated using Gateway technique.
- Inducing recombined vectors into A. tumefaciens
2.2.2. In vitro regeneration and transformation models
- Method for regeneration of tobacco
- Method for regeneration of soybean based on the method of
Olhoft et al (2001) using cotyledonary nodes from the mature seeds.
2.2.3. Analysis of transformed plants
- PCR: DNA from transformed plant genotypes were extracted
according to Edwards et al. (1991)

Recombined protein expression is the foundation of the
modern biotechnology. However, it is great challenge to express
proteins of one to another species. Some can be greatly expressed in
one plant but not in the other. The aim of this project was to express

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the HA gen from H5N1 virus into plants. Therefore, some triple
coded gens have been modified to amplify the HA gen expression in
the host cells. Based on the HA gen sequence and HA protein, we
have been able to recombine a new HA gen (HAop) which has high
level of expression in the plant. This recombined gen constructed of
1695 nucleotides and although the triple helix codes have been
modified, there was no change in acid amino sequence.
3.1.1.2 Designing recombined vector carrying HAop gen
HAop gen sequence was used to recombine a primmer. It has
been checked on 0.8% agarose gel with 1kb standard DNA ladder
(fig. 3.3) and found that the DNA of 1.7kb. This size was similar to
our calculation. Figure 3.3: PCR results of HAop gen using XhoI-HA/Hind III-HA
(M: standard DNA ladder 1kb)

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Insert HA gen region into p201-SLHEP vector
The gen product was cut by enzymes XhoI/HindIII and inserted
into p201-SLHEP vector between attL1 and attL2 region. The vector
also carries antibiotic resistant gen Kanamycin so we can select the
right plasmid during selection process. At the end of the process
recombined plasmid p201-SLHEP-HA generated.

tumefaciens.
Figure 3.9: recombined vector pPhaso-HAop A. A: PCR method,
B: restricted enzyme; M: standard ladder 1kb; 1, 2, 3: vector.

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3.1.1.4. Generation of a. tumefaciens
The recombined vector has been induced into a. tumefaciens by
electrical pulse method. PCR examination as showed in fig. 3.10
confirmed that the pPhaso-HAop vector has been successfully
transferred into the bacterium.

Figure 3.10. Electrophoresis of pPhaso-HAop vetctor induced in
a. tumefaciens CV58.
3.1.2 Generation of transformed vector containing HA1 gen
region
HA gen region is relatively large (1.7kb), and it epitope B and T
located at 91 – 108 and 307 – 319 at the beginning end of the HA.
Therefore, we selected a HA gen region with the size about 0.5kb
smaller than whole HA gen. This region named HAlop. To insert
this region into the vector p201-SLEHP, we used XhoI-HA1 and
HindIII-HA1 primers to amplify HA1 gens. The end of HA1 contain
codes for restricted enzymes XhoI and HindIII.

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And to selct the desired types we have determined the products
using colony-PCr reaction and restricted enzymes.
The results of Colony – PCR in fig. 3.16 confirmed that all 5
randomised colony lines presented DNA bands with the expected
size of 1.25kb. Meaning the HA1 was transformed into E. coli HD5
alpha.

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Figure 3.16: electrophoresis of transformed vector with pPhaso-
SLHEP-HA1. Upper line: PRC using XhoI-HA1/HindIII-HA1;
lower line: cut using HindIII

Induction of pPhaso-SLHEP-HA1 into agrobacterium
50 – 100ng plasmid pPhaso-SLHEP – HA1 transformed into A.
tumefaciens. They were incubated at 280C in antibiotic selected
medium for 2 days. The desired colonies were selected and tested
using colony – PCR
The results showed in fig. 3.17 revealed that selected colonies
were positive on PCR gel with the only band sized 1.25kb. it means
the vector has been transformed into a. tumefaciens.

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Figure 3.17. Electrophoresis of the vector transformed into CV58
(M: standard DNA ladder 1 kb; 1 – 3: colony lines of A.
tumefaciens)

most optimal culture medium for rooting.
3.2.2. Transformation of gus gen into soybean using A.
tumefaciens
We used 395 DT12 and 480 DT84 soybean samples to
transform gus gen in co-cultivation with Agrobacterium and selection
cultivation with Kanamycine. There were 114 DT12 and 70 Dt84
germinations developed in the medium with 50mg/L antibiotic.
Among them, 9 DT12 and 3 DT84 were positive given the efficiency
of gus gen transformation of 7.8% and 4.3% respectively.

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As results, we have proposed a process model of regeneration
for soybean gen transformation using cotyledonary node (fig. 3.24). Figure 3.23. gus gen expression in transformed soybean. A:
cotyledon of non-transformed soybean; B: cotyledon of
transformed soybean; C: Leaf of non-transformed soybean; and
D: Leaf of transformed plant; E: gus gen transformed soybean 19
1
Soybean seed

( in smoked husk and golden sand, 1:1 ratio)
Figure 3.24: model of regeneration process of transformed
soybean DT12.
3.2.3 Confirmation the present of HA1 in T
0
soybean lines
In two vector gene transfer was successful designs (P201-SLHEP-
HA and vector-SLHEP-HA1) we chose P201-structured vector
carrying SLHEP-HA1 genes HA1 transferred into DT12 soybean.
Conduct a bacterial infection A.tumefaciens recombinant P201-

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bearing structures into armpits SLHEP-HA1 cotyledons were hurt
DT12 of soybean under the process has been optimized. HA1 gene
translocation experiments in DT12 soybean was conducted with 650
samples, collected over 65 shoots lasting SEM selective medium
containing 50 mg / l kanamycin, in which 45 shoots rooted and
capable of receiving the 32 T0 plants growing on the substrate
(Figure 3:26).

Figure 3.26. T0 transformed soybean in the glass house.
3.3. HA1 transformed soybean
3.3.1. Confirmation the present of HA1 in T
0
soybean lines
Three micro-litres of DNA extracted from T0 plant has been
reacted with specific primer and then PCR. Results of PCR showed 8
T
0
plants positive with HA1 transformed gen (1.2%) (Fig. 3.27).

Figure 3. 28: electrophoresis image of HA1 gen in t1 plants (M:
marker; (-): negative control; (wt): wild type; H4 and H11: T1
plants)
Protein expression in transformed seeds
Total protein in transformed seeds of T1 plant has been
extracted and analysed by using Western blot (Fig. 3.29). The protein
expressed here has its weight of around 40kDa equivalent to HA
protein. Primarily, we conclude that HA1 protein has been
transformed and expressed in soybean seeds. Further study will
determine its immune capacity to A/H5N1.

Figure 3.29: HAlop protein in transformed seed – Western blot
(M: marker; (-): negative control; 1 and 2: seed samples from
H11 soybean (T1 plant)).
M – 1 2
35kDa
55kDa
40kDa

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CONCLUSION AND SUGESTION
1. Conclutions
1.1. Successfully construct vectors carrying HA gen and HA1
gen region of A/H5N1 virus and express in soybean seeds