Luận văn thạc sĩ purification of dengue virus like particle from insect cell culture

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY BACH THI DUNG Topic title: PURIFICATION OF DENGUE VIRUS LIKE PARTICLE FROM INSECT CELL CULTURE BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2012 – 2016 Thai Nguyen, 15/08/2016 n THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY BACH THI DUNG Topic title: PURIFICATION OF DENGUE VIRUS LIKE PARTICLE FROM INSECT CELL CULTURE BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2012 – 2016 Supervisors : Asst Prof Dr Kanokwan Poomputsa Dr Nguyen Xuan Vu Thai Nguyen, 15/08/2016 n DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Biotechnology Student name Bach Thi Dung Student ID DTN1253150043 Thesis title Purification of dengue Virus-like-particle from insect cell culture Supervisors Asst Prof Dr Kanokwan Poomputsa Dr Nguyen Xuan Vu Abstract: Dengue infection, a disease caused by the Dengue virus infection, is one of a global health problem in the tropics and subtropical areas To prevent the spreading of this disease, vaccination using Dengue vaccine is needed One of the vaccine candidates against Dengue virus (DENV) is Dengue virus like particle (VLP) Dengue VLP which is a particle with structure is identical to the native Dengue virus with no genetic material is potential to highly and safely elicit the immune response In this study, Dengue VLP produced from stable transfected insect cells cultured in TNMFH medium supplemented with 10% feval bovine serum (FBS) was purified by three methods: affinity column chromatography, tangential cross-flow filtration, and aqueous two-phase system (ATPS) The result showed that ATPS was the best method that can separate Dengue VLP from albumin as well as the biggest protein impurities in the culture medium Key words Purification, DENV, Dengue virus like particle, Concentration, affinity column chromatography, tangential cross-flow filtration, ATPS Number of pages 37 i n ACKNOWLEDGEMENT Attain this thesis; I would like to express my deep gratitude to my thesis advisor Asst Prof Dr Kanokwan Poomputsa from the School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Thailand The door to her office was always opened whenever I ran into a trouble spot or had questions about my research or writing She consistently allowed this paper to be my own work, but steered me in the right direction whenever she though I need it I would also like to thank Dr Nguyen Xuan Vu from the Biotechnology and Food Department of Thai Nguyen University of Agriculture and Forestry (TUAF) who used to help, support and give me encouragements during this thesis implementation I would also like to extend my heartfelt thanks to my teachers of Biotechnology and Food Department, TUAF who imparted me a lot of knowledge through four years of university The knowledge not only helped me with my research but also created a basic and soul foundation for me to start the job in the future Further, I would also like to express my sincere gratitude to Dr Duong Van Cuong and Msc Trinh Thi Chung for providing me the opportunity to develop my skills by doing an internship abroad I sincerely thank to the teachers, the laboratory staffs and students at Animals Cell Culture laboratory for their regards and giving me an opportunity to research in the laboratory I would also especially thank Msc Marlita who always helped, cared and taught me as my sister during my practicing in Thailand I am especially grateful to my dear mother, Duong Thi Nga and all my family and to the many old and dear friends who have stood by my side trough the many ups and downs of this long campaign Many thank you and best regards, Student Bach Thi Dung ii n CONTENTS LIST OF TABLE vi LIST OF FIGURES iv LIST OF ABBREVIATION viii PART 1: INTRODUCTION 1.1 Background 1.1.1 Dengue infection/ disease 1.1.2 Dengue virus 1.2.2.1 Structure 1.2.2.2 Life cycle 1.2.2.3 Immune system interaction 1.1.3 Dengue vaccine 1.1.3.1 Dengue vaccine types 1.1.3.2 Dengue vaccine production 10 1.1.4 Production of dengue virus like particle (VLP) from insect cell culture 11 1.1.5 Dengue virus like particle (VLP) purification from cell culture 12 1.1.5.1 HiTrap™ Blue column chromatography 13 1.1.5.2 Cross filtration by ÄKTA™ flux s 13 1.1.5.3 Aqueous two-phase system 14 1.2 Objective 14 1.3 Scope of study 14 PART 2: MATERIALS AND METHODS 15 iii n 2.1 Equipments and Materials 15 2.1.1 Equipments 15 2.1.2 Materials 15 2.2 Methods 17 2.2.1 Collection of Dengue VLP from Sf9-Dg stable cells 17 2.2.2 Purification of dengue Virus-Like-Particle by HiTrap™ Blue column chromatography (Affinity chromatography) 17 2.2.3 Purification of Dengue Virus-Like-Particle by ÄKTA™ flux 18 2.2.4 Purification of Dengue Virus-Like-Particle by Aqueous two-phase system (ATPS) 18 2.2.5 Concentration of Dengue VLP by 100 KDa MWCO Amicon® Ultra-4 Centrifugal Filter Devices for volumes up to mL 19 2.2.6 Concentration of Dengue VLP by 35% PEG precipitation 19 2.2.7 Concentration of Dengue VLP by Acetone precipitation 20 2.2.8 Western Blot Analysis of Dengue VLP 20 2.2.9 Commassie Blue Staining Analysis 21 2.2.10 Silver Staining Analysis 21 2.2.11 Dot Blot Analysis 21 PART 3: RESULTS AND DISCUSSIONS 23 3.1 Purification of dengue Virus- Like- Particle by HiTrap™ BLUE column chromatography 23 3.2 Purification of Dengue Virus Like Particle (VLP) by ÄKTA™ flux 28 3.2 Purification of Dengue Virus Like Particle by Aqueous two-phase system (ATPS) 31 iv n PART 4: CONCLUSIONS AND SUGGESTIONS 36 4.1 CONCLUSIONS 36 4.2 SUGGESTIONS 36 REFERENCES 37 APPENDIX 41 v n LIST OF TABLE TABLE PAGE Technique of purification protein base on different property vi n 10 LIST OF FIGURES Figure 1.1: Time course of clinical signs and symptoms Figure 1.2: Structure of Dengue virus Figure 1.3: Dengue virus life cycle (website: http://www.denguevirusnet.com/denguevirus.html, 2016) Figure 1.4: Model of antibody-dependent enhancement of dengue infection Figure 1.5: Types of dengue vaccines Figure 3.1: Characterization of Dengue VLP from TNMFH + 10% FBS culture after purification by HiTrap™BLUE affinity chromatography column The samples were run SDS-PAGE and were stained by Coomassie blue staining after purification by HiTrap™BLUE affinity chromatography column 25 Figure 3.2: Characterization of Dengue VLP from TNMFH+10% FBS culture after purificationby HiTrap™ BLUE affinity chromatography column The samples were run SDS-PAGE and were stained by Silver staining after purification by HiTrap™BLUE affinity chromatography column 26 Figure 3.3: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by HiTrap™ BLUE affinity chromatography column The samples were run SDS-PAGE and were run by Western Blot after purification by HiTrap™BLUE affinity chromatography column 27 Figure 3.4: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by 10 kDa MWCO ÄKTA™ flux 29 Figure 3.5: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by 50 kDa MWCO ÄKTA™ flux 30 Figure 3.6: Result of purification of Dengue Virus-Like-Particle by Aqueous two-phase system (ATPS) with three phases: PEG-rich phase, intermediate phase (interface) and bottom phase (Salt- rich phase) 32 vi n Figure 3.7: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by Aqueous two-phase system (ATPS) The samples were run SDS-PAGE and were stained by Comassie blue staining analysis (12% resolving gel electrophoresis) of Dg-VLP after purification by Aqueous two-phase system 33 Figure 3.8: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by Aqueous two-phase system (ATPS) The samples were run SDS-PAGE and were stained by Silver staining analysis (12% resolving gel electrophoresis) of DgVLP after purification by Aqueous two-phase system 34 Figure 3.9: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by Aqueous two-phase system (ATPS) The samples were run SDS-PAGE and were run Dot Blot analysis of Dg-VLP after purification by Aqueous two-phase system 35 vii n The result from both purification methods using 10 kDa MWCO and 50 kDa molecular weight cut off (MWCO) showed that VLP supposed to exist in retentated and washed samples as shown in dot blot result Coomassie staining (3.4A, 3.5A) result showed that the albumin band were still appear in all samples indicated that the membrane cut off used in the system were not big enough to separate between serum and dengue VLP target protein Filtration using bigger MWCO might be used further to purify Dengue VLP from insect cell culture 3.3 Purification of Dengue Virus Like Particle by Aqueous two-phase system (ATPS) Another approach to purify dengue VLP is Aqueous-two-phase system (ATPS) The system which composed from liquid-liquid interaction gives mild condition that can support better recovery toward the desired protein product In the study, 15 g ATPS system was performed in 50 ml centrifuge tube PEG 6000 was used and the composition of the systems was: sample, PEG 6000 (10% w/w), Sodium citrate (13.3% w/w) and Sodium chloride (13.3%) All component of the system were mixed and vortexed until dissolve completely, pH adjusted to 7.0 and 8.0 and centrifuged at 6,000 xg for 15 minutes (2.2.4) Three phases were appeared after centrifugation (Figure 3.6) and were collected for further characterized by coomassie blue staining analysis (2.2.9), silver staining analysis (2.2.10) and dot blot analysis (2.2.11) 31 n Figure3.6: Result of purification of Dengue Virus-Like-Particle by Aqueous two-phase system (ATPS) with three phases: PEG-rich phase, intermediate phase (interface) and bottom phase (Salt- rich phase) The characterization results by coomassie blue (2.2.9) and silver staining (2.2.10) (Figure 3.7 and 3.8) These figures showed that ATPS might be able to separate Dengue VLP from serum albumin as the biggest protein impurities in culture medium Based on its character, dengue VLP were expected to bind to PEG polymer therefore it should be located in the PEG phase-upper phase As it shown in the figures, the E proteins that indicated the VLP were not detected due to the very low concentration of sample But the albumins as a band near 90 kDa were found mostly in the interphase phase and bottom phase 32 n Figure 3.7: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by Aqueous two-phase system (ATPS) The samples were run SDSPAGE and were stained by Coomassie blue staining analysis (12% resolving gel electrophoresis) of Dg-VLP after purification by Aqueous two-phase system Lane 1: Pierce Protein Marker, Lane 2: 3X concentrated Dg-VLP from TNMFH by 100 kDa Amicon, Lane 3: TNMFH + 10% FBS medium only, Lane 4: Top PEG phase/NaCl pH = 7.0, Lane 5: Intermediate phase/Nacl pH = 7.0, Lane 6: Bottom phase/Nacl pH =7.0, Lane 7: Top PEG phase/NaCl pH = 8.0, Lane 8: Intermediate phase/Nacl pH = 8.0, Lane 9: Bottom phase/Nacl pH =8.0, Lane 10: Pierce Protein Marker 33 n Figure 3.8: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by Aqueous two-phase system (ATPS) The samples were run SDSPAGE and were stained by Silver staining analysis (12% resolving gel electrophoresis) of Dg-VLP after purification by Aqueous two-phase system Lane 1: Pierce Protein Marker, Lane 2: 3X concentrated Dg-VLP from TNMFH by 100 kDa Amicon, Lane 3: TNMFH + 10% FBS medium only, Lane 4: Top PEG phase/NaCl pH = 7.0, Lane 5: Intermediate phase/Nacl pH = 7.0, Lane 6: Bottom phase/Nacl pH =7.0, Lane 7: Top PEG phase/NaCl pH = 8.0, Lane 8: Intermediate phase/Nacl pH = 8.0, Lane 9: Bottom phase/Nacl pH =8.0, Lane 10: Pierce Protein Marker To support the idea, dot blot analysis was conducted (Figure 3.9) and the result showed the same suggestion as the staining analysis in which the E protein was detected strongly in upper phase in which ATPS with pH 8.0 showed clearer separations With pH 8.0, dengue VLP can be expected to be driven toward the PEG34 n rich phase, and oppositely, the albumin toward the intermediate and bottom phase The system optimization by considering the salt and PEG concentration and also the pH might be done to improve the yield Figure 3.9: Characterization of Dengue VLP from TNMFH+10% FBS culture after purification by Aqueous two-phase system (ATPS) The samples were run SDS-PAGE and were run Dot Blot analysis of Dg-VLP after purification by Aqueous two-phase system Lane B: Dg-VLP from Bacvector medium, Lane 2: 3X concentrated Dg-VLP from TNMFH by 100 kDa Amicon, Lane 3: TNMFH + 10% FBS medium only, Lane 4: Top PEG phase/NaCl pH = 7.0, Lane 5: Intermediate phase/Nacl pH = 7.0, Lane 6: Bottom phase/Nacl pH =7.0, Lane 7: Top PEG phase/NaCl pH = 8.0, Lane 8: Intermediate phase/Nacl pH = 8.0, Lane 9: Bottom phase/Nacl pH =8.0 Lane B: Dg-VLP from Bacvector medium 35 n PART CONCLUSIONS AND SUGGESTIONS 4.1 CONCLUSIONS HiTrap™ Blue Column for the first method: In this sudy, HiTrap™ Blue column could not remove all albumin from Dengue VLP sample due to the binding capacity ÄKTA™ flux s for the second method: Both proteins were still found in the sample Thus, the membrane 10 kDa and 50 kDa MWCO used in AKTA flux s were too small for both albumin and Dengue VLP Aqueous two-phase system (ATPS) for the third method: ATPS was a fast and easy system that remove albumin from Dengue VLP sample Dengue VLP were found in PEG-phase and interphase of the system in this study ATPS with NaCl at pH 8.0 gave the best condition for Dengue VLP purification 4.2 SUGGESTIONS − pH of both binding buffer and elution buffer need to be optimized for HiTrap™ BLUE purification and the Dengue purification from culture with less concentration of serum might be done in consideration of the column capacity in binding albumin 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Amsterdam: IOS Press p 235 ISBN 978-1-60750-582-2 28 Staropoli I, Frenkiel MP, Megret F, et al (1997) Affinity-purified dengue-2 virus envelope glycoprotein induces neutralizing antibodies and protective immunity in mice Vaccine 1997, 15:1946-54 39 n 29 Yorgo Modis, Steven Ogata, David Clements, Stephen C Harrison (2004) Structure of the dengue virus envelope protein after membrane fusion Nature 427, 313-319 30 Zhang Y, Zhang W, Ogata S, Clements D, Strauss JH, Baker TS,Kuhn RJ, Rossmann MG (2004) Conformational changes of the flavivirus E glycoprotein Structure 12:1607-1618 31 Zhang, S., Liang, M., Gu, W., Li, C., Miao, F., Wang, X., Jin, C., Zhang, L., Zhang, F., Zhang, Q., Jiang, L., Li, M., Li, D (2011) Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice Virol J 8, 333 Internet resource Dengue vaccine Initiative (2016) (http://www.denguevaccines.org/live-attenuatedvaccines ) (Accessed 2016) Dengue Virus Net (2016) (http://www.denguevirusnet.com/vaccine-research.html ) (Accessed 2016) Dengue Virus Net (2016) ( http://www.denguevirusnet.com/dengue-virus.html) (Accessed 2016) Nature Education (2014) http://www.nature.com/scitable/content/types-of-denguevirus-vaccines-22405302) (Accessed 2014) UKHealthCentre (2016) (http://www.healthcentre.org.uk/vaccine/types-ofvaccine.html ) (Accessed 27 August 2016) WHO Dengue and dengue haemorrhagic fever Factsheet No 117, revised May 2008 Geneva, World Health Organization, 2008 (http://www.who.int/mediacentre/factsheets/fs117/en/ ) (Acceessed May 2008) WHO Dengue Guidelines for Diagnosis, Treatment, Prevention and Control Geneva: World Health Organization (2009) (http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf?ua=1 ) (Accessed Feb 24, 2014) 40 n APPENDIX 0.5M Tris-HCl (100ml) • Tris: 6.05g • Dissolve in 70ml distilled water • Adjust the pH to 6.8 with HCl and make up to 100ml • Store at 4°C 1.5M Tris-HCl 100ml) • 18.15g • Dissolve in 70ml distilled water • Adjust the pH to 8.8 with HCl and make up to 100ml • Store at 4°C 10% Amino persulphate (10% APS) (1ml) • Ammonium persulphate: 100mg • Dissolve in 70ml distilled water (prepare fresh everytime) 10% resolving (2gel) • 30% Bis-Acrylamide: 6.075ml ã 1.5M Tris-HCl (pH=8.8): 4.95ml ã 10% SDS: 150àl • DI water: 3.75ml • TEMED: 7.5 µl • 10% APS: 75 àl 10% SDS (100ml) ã SDS: 10g • O : 90ml 41 n 12% resolving • 30% Bis-Acrylamide: 5.94ml • 1.5M Tris-HCl (pH=8.8): 3.75ml • 10% SDS: 150àl ã DI water: 5.085ml ã TEMED: 7.5 àl • 10% APS: 75 µl 4% stocking • 30% Bis-Acrylamide: 0.99ml • 1.5M Tris-HCl (pH=8.8): 1.89ml • 10% SDS: 75àl ã DI water: 4.5ml ã TEMED: 7.5 àl ã 10% APS: 37.5 µl Binding buffer (1L) Make 20mM sodium phosphate • 0.2M di-sodium phosphate ( ) (100ml) di-sodium phosphate : 3.56g Dissolve in 100ml distilled water • 0.2M O (100ml) mono-sodium phosphate : 2.76g Dissolve in 100ml distilled water • Adjust pH by mixing mono-sodium phosphate and di-sodium phosphate • Mix 100ml Sodium phosphate and 900ml distilled water 42 n Blocking buffer 3% BSA (50ml) • BSA: 1.5g • Add 30ml PBST • Mix well up to 50ml 10 Blocking buffer 5% skim milk (50 ml) • Skim milk: 2.5g • Add PBST and mix well up to 50ml 11 Destanining Solution (500ml) • Methanol: 200ml • Acetic acid: 35ml • Up to 500ml with distilled water 12 Developeing buffer (5ml) (AP conjuagate substrate kit) • Development buffer: 200ml • Reagent A: 50µl • Reagent B: 50àl ã DI water: 4.7ml 13 Developing solution (100ml) • 500µl of 1% citric (0.01g/1ml DI) • 50µl 37% formaldehyde • Up to 100ml with DI water 14 Elution buffer (1L) Make 20mM sodium phosphate • 0.2M di-sodium phosphate ( di-sodium phosphate : 3.56g Dissolve in 100ml distilled water 43 n ) (100ml) • 0.2M O (100ml) mono-sodium phosphate : 2.76g Dissolve in 100ml distilled water • Adjust pH by mixing mono-sodium phosphate and di-sodium phosphate • Mix 100ml Sodium phosphate and 900ml distilled water Mix 100ml Sodium phosphate with 2M NaCl and up to 1L with distilled water 15 PBS 10X (250ml, pH=7.4) • NaCl: 20g • KCl: 0.5g • : 0.6g • : 3.6g • Dissolve in 100ml distilled water and adjust pH by NaOH up to 7.4 16 PBST (500ml) • 1X PBS: 497.5ml • 0.5% Tween 20: 2.5ml 17 Running buffer 10X (500ml, pH=8.3) • 250mM Tris: 15.15g • 1.92M glycerin: 72g • 1% SDS: 2.5g • Dissolve in distilled water and up to 500ml 18 Silver nitrate solution • 0.8g Ag / 4ml DI water • 0.36% NaOH: 21ml • OH: 1.4ml • Up to 100ml with DI water 44 n 19 Staining solution (500ml) • Commassie blue: 0.125g • Methanol: 200ml • Acetic acid: 35ml • Adjust volume by distilled water 20 Transfer buffer (500ml) • Methanol: 100ml • Tris base: 7.21g • Glycine: 1.52g • Up to 500ml with distilled water 45 n