病毒純化與分析課件

1、Virus purification and analysis using OptiPrep - competitive mediaSucroseGlycerolCaesium chloride第1頁，共21頁。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

2、preparation times and sterilizationCsCl is expensive第2頁，共21頁。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 dial

3、yzed prior to re-infection of cellsSolutions ionic and corrosive: samples must be dialyzed before electrophoresis or HPLC第3頁，共21頁。SUCROSE PROBLEMS (I)Solutions are hyperosmotic: virus density in sucrose usually higher than in iodixanolSolutions are viscous: slow sedimentation of virus particlesHighl

4、y toxic to cells: must be dialyzed prior to re-infection of cells第4頁，共21頁。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 an

5、d electrophoresis and HPLC performed without dialysis第5頁，共21頁。Virus banding density VirusCsClSucroseIodixanolEpstein-Barr1.261.14Semliki Forest1.261.181.18Poliovirus1.341.261.22Measles1.211.181.16第6頁，共21頁。Kivela et al (1999)Virol. 262, 364-3740102030405060% iodixanol, sucrose, glycerol101110121.11.3

6、1.51.7CsCl g/mlIodixanolGlycerolCsClSucroseInfectivityEffect of medium on PM2 infectivity第7頁，共21頁。Sucrose gradients cause loss of virus surface glycoproteins; no loss with iodixanol gradientsInfectivity recoveries are 10-100 x greater in iodixanol than in sucroseSUCROSE PROBLEMS (II):for enveloped v

7、iruses, particularly retroviruses第8頁，共21頁。15% iodixanolin 1 M NaCl25% iodixanol40% iodixanol54% iodixanolVirus fluidproteinsadenovirusrAAV350,000g 1 hrAAV in discontinuous gradient (V04)Zolotukhin S. et al (1999) Gene Therapy, 6, 973-985第9頁，共21頁。rAAV in continuous gradient (V04)Hermans, W.T.J.M.C. e

8、t al (1999) Human Gene Therapy, 10, 1885-1891Opti-PrepVirusfluidBGradient Master第10頁，共21頁。Gradient Master profiles from 10% and 40% iodixanol at 80 and 20 rpm: effect of timeDensity (g/ml)Fraction Number135791.071.091.111.132 min2.5 min3.0 min第11頁，共21頁。3 Formats for separation of particles according

9、to their density123第12頁，共21頁。rAAV in continuous gradient (V04)Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy, 10, 1885-1891348000g3 hrAAVmembranesOptiPrepVirusfluid第13頁，共21頁。Comparison of rAAV in CsCl and OptiPrepPre-gradientGradient % RecovryVP/ICU(NH4)2 SO4CsCl0.1-60250-108(NH4)2 SO4OptiPrep2

10、6-120264-1333Cellufine-SO4OptiPrep25-91385-600Data from Hermans, WTJMC et al (1999) Human GeneTherapy, 10, 1885-1891第14頁，共21頁。Purification of HIV-1 (V06)Dettenhoffer, M & Yu, X-F (1999) J. Virol., 73 1460-1467250,000g for 1.5 h6-18% iodixanolMicrovesiclesVif geneSoluble proteinsRT (cpmx10-5/ml)第15頁，

11、共21頁。Use of self-generated iodixanol gradients in vertical rotors for virus purification and analysis第16頁，共21頁。1345250,000g1h350,000g1-3hVirus purification flow-chart without virus pelleting第17頁，共21頁。Purification of Herpes virus in self-generated 25% iodixanol gradient (V05)Fraction NumberLog infect

12、ivityDensity (g/ml)13579111315012345611.051.11.151.21.251.3Infectivity Density第18頁，共21頁。Fraction numberOD(PERT) x 10-2Density (g/ml)13579111315171905101520253011.051.11.151.21.25MS1533MS1874 DensityPurification of retrovirus: self-generated gradient (V09)Moller-Larsen A & Christensen, T. (1998) J. V

13、irol Meth., 73, 151-161第19頁，共21頁。OptiPrepApplication Sheets for:AdenovirusEbola virusFoamy virusHerpes simplex virusHIV-1Human papillomavirusLassa virusMurine leukemia virusMurine oncornavirusNorwalk virusPolyoma virusrAAVRabies virusRous sarcoma virusRetrovirusesSemliki Forest virusSARS Corona virusVaccinia virus第20頁，共21頁。Publications database on virus