《病毒纯化与分析》PPT课件.ppt

Virus purification and analysis using OptiPrep - competitive media,Sucrose Glycerol Caesium chloride,SOLUTION HANDLING,Density solutions prepared by diluting OptiPrep with culture medium or any balanced salt solution (Application Sheet V01) Sterile solution CsCl/sucrose solutions require lengthy preparation times and sterilization CsCl is expensive,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,SUCROSE PROBLEMS (I),Solutions are hyperosmotic: virus density in sucrose usually higher than in iodixanol Solutions are viscous: slow sedimentation of virus particles Highly toxic to cells: must be dialyzed prior to re-infection of cells,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,Virus banding density,Effect of medium on PM2 infectivity,Sucrose gradients cause loss of virus surface glycoproteins; no loss with iodixanol gradients Infectivity recoveries are 10-100x greater in iodixanol than in sucrose,SUCROSE PROBLEMS (II): for enveloped viruses, particularly retroviruses,350,000g 1 h,rAAV in discontinuous gradient (V04) Zolotukhin S. et al (1999) Gene Therapy, 6, 973-985,rAAV in continuous gradient (V04) Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy, 10, 1885-1891,Gradient Master profiles from 10% and 40% iodixanol at 80 and 20 rpm: effect of time,3 Formats for separation of particles according to their density,rAAV in continuous gradient (V04) Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy, 10, 1885-1891,Comparison of rAAV in CsCl and OptiPrep,Data from Hermans, WTJMC et al (1999) Human Gene Therapy, 10, 1885-1891,Use of self-generated iodixanol gradients in vertical rotors for virus purification and analysis,Virus purification flow-chart without virus pelleting,Purification of Herpes virus in self-generated 25% iodixanol gradient (V05),Purification of retrovirus: self-generated gradient (V09) Moller-Larsen A & Christensen, T. (1998) J. Virol Meth., 73, 151-161,OptiPrepApplication Sheets for:,Adenovirus Ebola virus Foamy virus Herpes simplex virus HIV-1 Human papillomavirus Lassa virus Murine leukemia virus Murine oncornavirus,Norwalk virus Polyoma virus rAAV Rabies virus Rous sarcoma virus Retroviruses Semliki Forest virus SARS Corona virus Vaccinia virus,Publications database on viruses,OptiPrep (since 1994) over 600 Nycodenz (since 1984) approx 70 Using either the Applicati