ウィルス精製
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This document describes the advantages of using OptiPrep density gradient medium over CsCl and sucrose solutions for virus purification and analysis. OptiPrep forms isoosmotic solutions that do not affect virus infectivity or require dialysis before cell infection. It allows rapid sedimentation of virus particles and purification of many types of viruses without loss of surface proteins or infectivity. The document provides examples of gradient solutions and protocols that have been used to purify viruses such as adenovirus, HIV, hepatitis C virus, and herpes simplex virus using OptiPrep.
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ウィルス精製
- 2. SOLUTION HANDLING • Density solutions prepared by diluting OptiPrep™ with culture medium or any balanced salt solution • Sterile solution • CsCl/sucrose solutions require lengthy preparation times and sterilization • CsCl is expensive
- 3. CsCl PROBLEMS • Big losses in viral infectivity; low recoveries; high particle:infectivity ratios • Solutions have very high osmolality: viruses lose water; high virus density; high [CsCl] required for gradients • Highly toxic to cells: must be dialyzed prior to re-infection of cells • Solutions ionic and corrosive: samples must be dialyzed before electrophoresis or HPLC
- 4. SUCROSE PROBLEMS • Solutions are hyperosmotic: virus density in sucrose usually higher than in iodixanol • Solutions are viscous: slow sedimentation of virus particles and loss of surface proteins from enveloped viruses • Highly toxic to cells: must be dialyzed prior to re-infection of cells
- 5. OPTIPREP – NO PROBLEMS • Solutions are isoosmotic: virus density is low • Solutions have low viscosity: rapid sedimentation of virus particles • Is non-toxic to cells • Has little or no effect on virus infectivity • Cells can be re-infected and electrophoresis and HPLC performed without dialysis • Only electron microscopy requires removal
- 6. Kivela et al (1999)Virol. 262, 364-374 0 10 20 30 40 50 60 % iodixanol, sucrose, glycerol 1011 1012 1.1 1.3 1.5 1.7 CsCl g/ml Iodixanol Glycerol CcCl Sucrose Infectivity Effect of medium on PM2 infectivity
- 7. Virus banding density Virus CsCl Sucrose Iodixanol Epstein-Barr 1.26 1.14 Semliki Forest 1.26 1.18 1.15 Poliovirus 1.34 1.26 1.22 Measles 1.21 1.18 1.14
- 8. OptiPrep™Application Sheets for: • Adenovirus • Ebola virus • Foamy virus • Herpes simplex virus • HIV-1 • Human papillomavirus • Lassa virus • Murine leukemia virus • Murine oncornavirus • Hepatitis C virus • Norwalk virus • Polyoma virus • rAAV • Rabies virus • Rous sarcoma virus • Retroviruses • Semliki Forest virus • SARS Corona virus • Vaccinia virus • Plant viruses
- 9. top bottom density Diffusion of a discontinuous gradient
- 10. rAAV in continuous gradient (V04) Hermens, W.T.J.M.C. et al (1999) Human Gene Therapy, 10, 1885-1891 B Gradient Master Opti- Prep
- 11. rAAV in continuous gradient Hermens, W.T.J.M.C. et al (1999) Human Gene Therapy, 10, 1885-1891 Gradient Master 348000g 3 h NVt rotor rAAV Cell fragments OptiPrep Virus fluid
- 12. Comparison of virus product in CsCl and OptiPrep™ Pre-gradient Gradient % Recov’ry VP/ICU (NH4)2 SO4 CsCl 0.1-60 1000-108 (NH4)2 SO4 OptiPrep 26-120 364-1333 Cellufine-SO4 OptiPrep 25-91 385-600 Data from Hermens, WTJMC et al (1999) Human Gene Therapy, 10, 1885-1891
- 13. 15% iodixanol in 1 M NaCl 25% iodixanol 40% iodixanol 54% iodixanol Virus fluid proteins adenovirus rAAV 350,000g 1 h rAAV in discontinuous gradient Zolotukhin S. et al (1999) Gene Therapy, 6, 973-985
- 14. Purification of HIV by sedimentation velocity Dettenhoffer, M & Yu, X-F (1999) J. Virol., 73 1460-1467 200,000g for 1.5 h 6-18% iodixanol Microvesicles Vif gene Soluble proteins RT (cpmx10-5 /ml)
- 15. 2 50,000g 1h 5 350,000g 3h 4 Virus purification flow-chart – without virus pelleting 1 30 ml 6 ml 50% iodixanol 3 Discard 24 ml
- 16. Purification of Herpes simplex virus in self- generated 25% iodixanol gradient Fraction Number Loginfectivity Density(g/ml) 1 3 5 7 9 11 13 15 0 1 2 3 4 5 6 1 1.05 1.1 1.15 1.2 1.25 1.3 Infectivity Density
- 17. Purification of hepatitis C virus Adair, R., Patel, A.H., Corless, L., Griffin, S., Rowlands, D.J. and McCormick, C.J. (2009) J. Gen. Virol., 90, 833–842 12 h 90,000g Virus/ saline Virus/ 40% iodixanol 12 h
- 18. 100,000g 1h 10-20% 10-40% discontinuous or continuous Hepatitis C virus purification in iodixanol gradients 200,000g/6h 50%
- 19. Purification of hepatitis C virus Merz, A. et al (2011) J. Biol. Chem,, 286, 3018-3032 20 h 96,000g 3 ml 48% iodixanol 20 h 96,000g Aspirate bottom 6 ml (24% iodixanol) 12% 36% 48%
Editor's Notes
- Sucrose solutions cannot be sterilized by autoclaving – unless you like caramel. Filter sterilization is the common tedious alternative.
- Dialysis adds an overnight step to the purification.
- Sucrose is less of a problem than CsCl, but still not an ideal medium for viruses.
- PM2 is a bacteriophage (bacterial virus) which is unusually sensitive to sucrose, for most viruses the sucrose infectivity inhibition is usually in between CsCl and glycerol. Note the ordinate scale is logarithmic.
- If a discontinuous gradient of say 3 ml each of 10%, 20%, 30% and 40% iodixanol in a 14 ml tube is allowed to stand for several hours at room temperature or overnight at 4°C, then the sharp discontinuities of the stepped density profile become smoothed out by diffusion of the solute across each interface and eventually the gradient will become smooth and continuous. This is a commonly used strategy to make a continuous gradient. A linear gradient will be produced if the density increment between each step is the same and the volume of each step is the same. If either the volume of one or more of the steps is changed or the density interval of each step is changed the shape of the gradient can be made concave or convex or irregular in order to accentuate the separation of particular particles. If the diffusion time and temperature are well controlled the gradient shapes are highly reproducible.
- An alternative method for the purification of rAAV is a pre-formed continuous gradient. This illustrates one of the advantages of using the Gradient Master, or other similar device for the creation of a continuous gradient when handling any type of virus. The virus fluid is simply layered over the same volume of OptiPrep (or 50% iodixanol); the tube sealed and the gradient created by controlled mixing in 2-3 min. It is thus a very safe method particularly for potentially infective viruses.
- The column on right is essentially virus particle number/infectivity (sort of reciprocal specific infectivity) – so the higher the number the lower the infectivity.