nghiên cứu chuyển gen mã hóa protein bề mặt của virus h5n1 vào cây đậu tương phục vụ sản xuất vaccine thực vậ bản tóm tắt tiếng anh

MINSTRY OF EUCATION AND TRANING THAI NGUYEN UNIVERSITY –––––––––––––––––––– NGUYEN THU HIEN SOY BEAN AGROBACTERIUM - MEDIATED TRASFORMATION USING GENE ENCODING SURFACE PROTEIN FROMH5N

MINSTRY OF EUCATION AND TRANING

THAI NGUYEN UNIVERSITY ––––––––––––––––––––

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

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

Objection 1: Objection 2:

Supervisor: 1 Assoc Prof Chu Hoang Ha (PhD)

2 Prof Dr Chu Hoang Mau (PhD)

INTRODUCTION

1 Background

Flu is the most common transmitted disease and can be fatal Annually, there are about half of billion people suffered from the disease Currently, A/H5N1 flu virus, the most dangerous avian virus, has been spread over forty countries in Asia, Middle East, Europe and Africa In Vietnam, H5N1 Avian pandemic has been outbreak late 2003 resulted in avian industries and also human health Therefore, it has been necessary for scientists to carry out research to learn about the pathogenic pathway, epidemic mechanism so the 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

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

regenerated the tobacco carrying HA gene, HA1 gen region

- Developed and optimized a protocol to generate the formation of multiple adventitious shoots using cotyledonary node explants from young seedlings Evaluated the efficiency transformation of gus gen in DT12 and DT84 soybean genotypes

- Transferred the HA1 gen vector to the soybean genotypes using A tumefacien-mediated transformation system, and

determined the transient expression and regeneration rate of HA1 gene in T0 generation

- Analyzed HA1 gene expression of T1 transformed soybean lines using PCR and Western blot

4 Results

- HA gen and HA1 gen region vectors have been successfully

synthesized and recombined into A tumefaciens

- Transient infection efficiencies and regeneration rates of gus gen in DT12 and Dt84 soybean genotypes were 7.8% and 4.3%, respectively

- Eight T0 transgenic plant lines have been archived and expressed specific vector SLHEP-HA1 in seeds

- T0 transgenic plants produced positive T1 seedlings with HA1op gen and HA1 protein expression in seeds This transformed soybean genotype was named H11

5 Relevance and future direction

Scientifics: gene HA and Ha1 gene region have been created and expressed in plants The protocol for gen transformation of these genes using A tumefaciens has been developed and applied

In practice: The successful in engineering a HA1 genetic transformed soybean H11 genotype opens an opportunity for producing a soybean product that contains HA1 antigen This product

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

new sub-types Gen structure of A/H5N1 includes 8 separate regions and there is no gen encoding RNA repairing enzyme The region 4 encodes surface protein which is specific for each A flu virus This protein (hemaglutinin - HA) is an antigen and plays an important role

in viral lethal It can stimulate the host body to generate specific cytokines for each viral type to neutralise virus and prevent them from infecting the body again This is the principle for manufacturing vaccines

For human flu, the research and development of vaccine for poultry will not only prevent their disease spread out but also control the risk of the flu transmitting into human

There are several A/H5N1 flu vaccines such as traditional vaccines and new generation of vaccines using gen technology, synthesis vaccines, and edible vaccines In Vietnam, HA gen expression in tobacco plant has been successfully reported, and there are works on progress in expression of this gene into other plants such as soybean Also there has been many researches carried out, there is no plant vaccine manufactured for clinical use so far Even though, developing plant vaccine models for both human and animal

is a very promising pathway to prevent and control infectious transmitted diseases in Vietnam

Chapter 2 MATERIALS AND METHODS 2.1 Materials

Plans:

Soybean genotypes DT12 and DT84 were supplied by Centre for legume research and development, Institute of cereal plants, National 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

- 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)

- Proteins were extracted from seeds with 12.5% gel SDS polyacrylamide (Laemmli 1970)

- Western blot: total protein from SDS gel were transferred to nitrocellulose Hybond membrane (Amershame) Hybrid membrane then washed by TBS and blocked using 3% BSA for 1 hour And then second wash and incubated with c-myc in TBS supplemented with 0.5% BSA and 0.05% tween for 1 hour The membrane was washed again and incubated with Ig 2 with ALP or rat IgG inconbination with horseradish peroxidise (HRP – Abcam) After the

last wash the membrane were incubated with NBT/BCIP or using EDL kit (Amersham) to determine the recombined proteins

Chapter 3 RESULTS AND DISCUSSION 3.1 Transformed vectors with HA gen and HA1 gen region of H5N1 virus

3.1.1 Gen HA in the transformed vector

The immune cells do not recognise the whole antigen molecules They only identify certain part of the antigens called epitope There have been reported that the epitopes B and T of the

HA gen of H3N1 virus are in the region 91-108 and 307-319, respectively The HA sequence of A/H5N1 (AJ8670074) indicates a similar amino acide sequences of the above epitope The vectors express in the plants containing SLHEP structure with a peptide signal (2S2), B and T epitopes from HA, which can stimulate an immune on the intestine, the sequence to recognise endo-proteins (KDEL) and enzymes

3.1.1.1 Generation for HA gen containing triple codes highly express in plants

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

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)

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

Selection of recombined plasmid using restricted enzyme

To ensure that HA gen has been inserted in the vector, the

plasmid has been cut by enzyme XhoI/HindIII In theory, when the

plasmid cut by 2 restricted enzymes it will be resulted in two DNAs: inserted gen (1.7kb) and vector (2.5kb) They were determined using 0.8% agarose gel (fig 3.7)

Figure 3 7 Electrophoresis image of selection of recombined

plasmid p201-SLHEP-HA

Our results from agarose gel indicated that all of the three types showed that all of the DNA bands have the same expected size It means HA gen region has been inserted successfully into the vector

2,5kb 1,7kb

This recombined vector was the material for Gateway transformed method

3.1.1.3 Generation of vector carrying HAop expressing in seed

HAop gen and gens encoding functioning peptides were transformed in to pPhaso-dest vector by LR reaction (Gateway system) Re-examination of recombined vector using specific primer

XhoI-HA/HindIII-HA showed gen with the size of 1.7kb While

using restricted enzyme EcoRI resulted in 3 DNAs size 2.2kb, 5kb, and 10lb They were similar with what we have calculated (Fig 3.9) the first one has been selected for induced into bacterium A tumefaciens

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

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

3.1.2.1 HAlop gen transformation

HA1 gen isolation, amplification, and encoding

We use 50ng DNA recombined plasmid carrying HA gen in PCR to amplify HA1 using the specific primers The results show in fig 3.13

Figure 3 13 electrophoresis of HA1

The length of this DNA was 1.25kb which is the same size as HA1 The amplification also generated materials for selection process The encoding process was carried out using ABI – 313- DNA capillary electrophoresis system

Inserting HA1 into p201-SLHEP vector

The process is similar to the process of inserting HA gen described above We were then induced the vector into E coli Then examined the results using restricted enzymes and then start selection process

Recombined plasmid selection using restricted enzyme

The results of this process were two DNA sized 1.25kb and 2.5kb (fig 3.15)

Figure 3.15: Electrophoresis of recombined plasmid

p201-SLHEP_HA1

Gateway method for transforming HA1 into transformed vector 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

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