腺病毒构建以及培养体系的建立.doc

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1、腺病毒构建以及培养体系的建立LargescaleAdenovirusproduction,purificationandamplificationFollowingprotocolisdesignedtoprovide~3-5X10Ellpurifiedvirusparticles.AlladenovirusinfectionandprocessingofinfectedcellsshouldbedoneinBL-2Hoodunderstandardoperatingconditionsrequiredbythel

2、aw.Useproperdecontaminationprotocol.1.Seed5X10E5HEK293cells(QBICat#AES0503)in60x150mmplatesusingDMEMmedia(LifetechCat#12430-054)containing5%FBS.Incubate@37Cinatissuecultureincubatoruntiltheyare70to80%confluent(itisimportantnottoletthemgrowtoomuch).Usuallyplate

3、willbereadyforinfectionin2to3days.2.Weroutinelyinfectthecells@1to15M.O.I.Dilute0.5ulpurifiedvirusor2-3ulofthecrudevirallysate(CVL)in2・mlinfectionmediaperplate.WeroutinelychecktheCVLforoptimalinfectivity.Theamountvirususedforinfectiondependsonthevirustiter.3.Do

4、notletthecellsdryduringtheinfection.Thereforeaspiratethemediaonlywheneverythingisreadyfortheinfection.The293cellsarealsoverysensitivetothetemperaturealwaysusemediapreheatedto37C.Donotaddthemediarightinthemiddleoftheplatetoavoidthedetachmentofcellsfromtheplate.

5、Addthe2-mlinfectionmedia・containingvirusfromstep2gentlyatthecorneroftheplate.Incubatetheplateforanhourtiltingtheplatesevery15min(verygentlyandsothatthemediacoversalltheplate)orsettingarockerintheincubator(PGCcat#75-7355-10rock@speed3).Aftertheincubationadd15to

6、20mlofDMEMmediacontaining5%FBStotheplates.1.Incubatetheplatesuntiltheystartshowingcyto-pathiceffects(CPE).TypicallyCPEstarttobeevidentafter24hrsandarefullydevelopedafter36hrs.IfcellshavedevelopedCPEafterovernightincubation(<24hrs)suspectanothercausedifferen

7、tfromadenovirusinfection(discardthepreparation).2.Weusuallyharvestthecellsbetween36to48hoursafterinfection(cellswhen20to30%infected?cellpopulationbecomesroundandstartfloating).Detachthecellspipettingupanddownwiththemedia.Pooltherecoveredcellsalongwiththesupern

8、atantina50mlconicaltubeandspinthemfor10minutes@2500RPM(lOOOg).Poolthepelletandresuspendthepelletin20mlsterilePBS(fortotalof60plates).Storethepellet@-20C.3.Breakopenthecellsbyfreezi