病毒纯化与分析.pptxVIP

病毒纯化与分析第1页/共22页 Virus purification and analysis using OptiPrep? - competitive mediaSucroseGlycerolCaesium chloride第2页/共22页 SOLUTION HANDLINGDensity 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 sterilizationCsCl is expensive第3页/共22页 CsCl PROBLEMS Big losses in viral infectivity; low recoveries; high particle:infectivity ratiosSolutions have very high osmolality: viruses lose water; high virus density; high [CsCl] required for gradientsHighly toxic to cells: must be dialyzed prior to re-infection of cellsSolutions ionic and corrosive: samples must be dialyzed before electrophoresis or HPLC第4页/共22页 SUCROSE PROBLEMS (I)Solutions are hyperosmotic: virus density in sucrose usually higher than in iodixanolSolutions are viscous: slow sedimentation of virus particlesHighly toxic to cells: must be dialyzed prior to re-infection of cells第5页/共22页 OPTIPREP – NO PROBLEMSSolutions are isoosmotic: virus density is lowSolutions have low viscosity: rapid sedimentation of virus particlesIs non-toxic to cellsHas little or no effect on virus infectivityCells can be re-infected and electrophoresis and HPLC performed without dialysis第6页/共22页 Virus banding density VirusCsClSucroseIodixanolEpstein-Barr1.261.14Semliki Forest1.261.181.18Poliovirus1.341.261.22Measles1.211.181.16第7页/共22页 Kivela et al (1999)Virol. 262, 364-3740102030405060% iodixanol, sucrose, glycerol101110121.11.31.51.7CsCl g/mlIodixanolGlycerolCsClSucroseInfectivityEffect of medium on PM2 infectivity第8页/共22页 Sucrose gradients cause loss of virus surface glycoproteins; no loss with iodixanol gradientsInfectivity recoveries are 10-100x greater in iodixanol than in sucroseSUCROSE PROBLEMS (II):for enveloped viruses, particularly retroviruses第9页/共22页 15% iodixanolin 1 M NaCl25% iodixanol40% iodixanol54% iodixanolVirus fluidproteinsadenovirusrAAV350,000g 1 hrAAV in discontinuous gradie