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 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 Objection 1: Objection 2: Supervisor: Assoc Prof Chu Hoang Ha (PhD) Prof Dr Chu Hoang Mau (PhD) INTRODUCTION 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 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 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 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 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 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 NA sub-types (N – N9) In principle, the combination of HA and NA sub-types will generate many more new sub-types Gen structure of A/H5N1 includes separate regions and there is no gen encoding RNA repairing enzyme The region 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 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 2.2 Methods 2.2.1 Methods and techniques used to generate gen transformed vectors - PCR - Gel electrophoresis - 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 hour And then second wash and incubated with c-myc in TBS supplemented with 0.5% BSA and 0.05% tween for hour The membrane was washed again and incubated with Ig 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 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 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 11 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 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 12 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 13 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 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 – 313DNA 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 14 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 p201SLHEP_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 randomised colony lines presented DNA bands with the expected size of 1.25kb Meaning the HA1 was transformed into E coli HD5 alpha 15 Figure 3.16: electrophoresis of transformed vector with pPhasoSLHEP-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 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 16 Figure 3.17 Electrophoresis of the vector transformed into CV58 (M: standard DNA ladder kb; – 3: colony lines of A tumefaciens) 3.2 HA1 transformed soybean 3.2.1 Regeneration methods for DT12 and DT84 soybean transformation 3.2.1.1 Optimising the disinfection time This process has big impact on the germination and the rate of infected culture The trial experiments provided an optimal disinfection time of 16 hours 3.2.1.2 Regeneration of plant After wounded and cut the growth apical, the samples were incubated in five different cultures (SIM – SIM 5) containing BAP from 0.5 to 2.5mg/L and multiple cotyledonary nodes were formed For DT12 soybean genotype, culture medium supplemented with 2mg/l of BAP seemed to be the most sufficient culture with the average rate of nodes/group reached 82.3% and 6.2 nodes per group For DT84the supplement of 1.5mg/l was the most with the rate of 17 41.6% and 3.4 nodes / group, much lower than DT12 However, we observed that DT84 has better cotyledons.The effect of GA3 and IAA to the shooting The selected cotyledons were transferred to growth cultures supplemented with GA3 and IAA at variety concentrations The results showed that when GA3 added, the shooting rate and the number of cotyledon grew were increased in both soybean genotypes However, when the chemical increases further, these numbers decreased We have worked out the optimal concentration of GA3 was 0.5mg/L and IAA was 0.1mg/L This optimal concentration provides bigger cotyledons with larger leaf and typical green The effect of IBA to rooting We found that the concentration of IBA of 0.1mg/L was the 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, DT12 and DT84 were positive given the efficiency of gus gen transformation of 7.8% and 4.3% respectively 18 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 Soybean seed A tumefaciens Surface disinfection Culture in broth (Clo gas x hours) Germination (in GM) Centrifuge then suspense bacteria repellent with CCM (OD600 0.6 – 1) Wounded cotyledon infected with bacteria suspension (30 – 40 minutes) Co-cultivation in CMM in dark environment (5 days) Shooting in SIM suppl 2mg/L BAP and 50mg/L Kanamycin (2 weeks) Shooting in SIM suppl 2mg/L BAP and 50mg/L Kanamycin (2 weeks) Removal of cotyledon, elongating in SEM suppl sung 0,5 mg/l GA3 + 0,1 mg/l IAA + 50 mg/l kanamycin (2 weeks) Rooting in RM suppl 0,1 mg/l IBA (14 – 20 days) 10 Plant in the glass house ( 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 T0 soybean lines In two vector gene transfer was successful designs (P201-SLHEPHA 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- 20 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 T0 soybean lines Three micro-litres of DNA extracted from T0 plant has been reacted with specific primer and then PCR Results of PCR showed T0 plants positive with HA1 transformed gen (1.2%) (Fig 3.27) They were then harvested and T1 plants continued to be analysed to determine the transformed stability 21 Figure 3.27 Electrophoresis imagines of T0 HA1 gen transformed soybean (M: standard DNA 1kb; -: negative control; WT: wild type; - 8: T0 lines; +: positive control) 3.3.2 Analysing of T1 plants Among T0 plants, only seeded (named H11 and H4) Their seeds, stems and leaves have been harvested for analysing by using PCR with XhoI-HA/HIndIII-HA primers As shown in fig 3.28, the plant DNA has the same size as HA1op gen Therefore, it is quit ascertained to confirm that HA1 gen has been transferred from T0 to T1 plant However, HA1 gen in H4 plant was undetectable 22 Figure 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 M – 55kDa 35kDa 40kDa Figure 3.29: HAlop protein in transformed seed – Western blot (M: marker; (-): negative control; and 2: seed samples from H11 soybean (T1 plant)) 23 CONCLUSION AND SUGESTION Conclutions 1.1 Successfully construct vectors carrying HA gen and HA1 gen region of A/H5N1 virus and express in soybean seeds and transformed them to a tumefaciens 1.2 Obtained transformed tobacco with Ha and HA1 genes All plants were PCR positive with specific primers and has DNA molecular weight of 1.7 kb (pPhaso-SLHEP-HA line) and 1.25kb (pPhaso-SLHEP-HA1 line) 1.3 Regenerated multiple codyledonary nodes from mature seed of DT12 and DT84 soybean genotypes using our own optimised protocol 1.4 DT12 and DT84 have been transformed with gus gen transformed efficiencies were 7.85 and 4.3% respectively 1.5 Sucessfully transformed SLHEP-HA1 to soybean and obtaine T0 positive plants 1.6 T1 plant named H11 stablised with HAlop gen and successfully express HA1 protein in soybean seed in this H11 soybean genotype 24 Suggestion 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 HAS PUBLISHED ARTICLESH RELATING TO DISSERTATION "Design vector surface antigen expression in influenza A/H5N1 plant", Scientific Report Biotechnology Conference 2009 Nationwide, pp.122-126 "Research and ability to regenerate transformed monocot gene armpit of two soybean (Glycine max L.) by Agrobacterium DT12 and DT84", Journal of Biotechnology, (38): pp 1305 -1310 "The study generated transgenic soybean plants carrying the gene expression structure encoding surface antigens of H5N1 virus for vaccine production plant," Journal of Thai Nguyen University of Technology, 89 (1): pp 123-127 "Expression of antigens in plants H5N1 virus," the Vietnamese Journal of Medicine, vol 411: pp 219-224 ... 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... 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 Specific... 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