Research On A Modified Adenovirus Type5Large-scale Cultivation, Purification And Biological Activity

Objective: To explore the adenovirus massive amplification experimentconditions, the optimal method of cell disruption, the optimal concentration ofammonium sulfate precipitation, ion exchange column hydrophobic column andpurified adenovirus combined with the best technology, and finally with orthogonaldesign method after comparing different formulations designed to select the bestpreservation conditions of adenovirus, adenovirus meet saved. This experimentprovides a method for amplification and purification of adenovirus, the reference maybe provided to other large-scale production and purification of adenoviral gene drugs.Methods: The adenovirus titers, the purity and recovery as a tool to examine thefinal result.The first, a comparison of different MOI values, the titer adenovirus infectiontime, the optimum condition for determining mass amplification.The second, the comparison repeated freezing and thawing method, the titeradenovirus ultrasonic and high pressure osmometry, the best way to determine thecell disruption.The third, the configuration of the different percentages of saturated ammoniumsulfate precipitation adenovirus titer comparison to determine the most suitableconcentration of ammonium sulfate saturation.The fouth, the comparison of different types of water repellent andconcentration, the type of an anion exchange column, to determine the most suitableprocess conditions by adenovirus titer, purity and recovery.The fifth, the using of orthogonal experimental design method to determine themost suitable adenovirus storage conditions.The sixth, by comparing expression of the green fluorescent proteinprepurification and purification of adenovirus in vivo and in cell using Elisa.Comparing the protein expression of infection with adenovirus titer with different titerand time. In this study, referencing to the relevant literature, the design of the ammoniumsulfate precipitation method adenovirus adenovirus supernatant concentrated crudepure, not processed directly memorial of water, another way to ion exchange columnpurification of adenovirus. Finally through comparing protein expression in vitro andin vivo prepurification and purification.Results:The first, the optimal conditions for amplification of adenovirus scale: MOIvalue of10and24hours of infection time, the highest titer adenovirus harvest.The second, the comparison repeated freezing and thawing method, ultrasonicand high pressure osmometry found ultrasonic cell disruption was the best, and therecovery is96%.The third, by ammonium sulfate precipitation was found the most suitableconcentration of60%saturated ammonium sulfate, the recovery can be achieved70%.The fouth, the using of the U.S. company GE Healthcare Sepharose HighPerformance column in the equilibration buffer solution A:2.5M (NH4)2SO4,40mMTris-Hcl (pH=8.0),5%glycerol,1mM MgCl2. Elution buffer solution B:40mMTris-Hcl (pH=8.0),5%glycerol,1mM MgCl2under20column volume gradientelution, recovery of adenovirus is79.6%.The fifth, the using of the anion exchange column Q xl, balancing liquidequilibration buffer A:40mM Tris-Hcl (pH=8.0),5%glycerol,1mM MgCl2;equilibration buffer solution B:1M NaCl,40mM Tris-Hcl (pH=8.0),5%glycerol, in1mM MgCl2, under20column volume gradient elution. The recovery of adenovirus is53.7%. The final purity of the total is91.0%The sixth, the most suitable storage conditions:-80degrees,5%glycerol,1mMMgCl2.The seven, the expression of green fluorescent protein is high thanprepurification.Conclusion: The purity of adenovirus precipitated by ammonium sulfateprecipitation, hydrophobic column and ion exchange column is too similar to the purity of adenovirus obtained by conventional CsCl density gradient centrifugation,but the recovery rate is higher than the latter. Therefore, this method is better than theconventional CsCl density gradient centrifugation. We proved that the purifiedadenovirus is better than prepurification. This experiment provides a method foramplification and purification of adenovirus and the preservation and the bestconditions for injection to be provided in reference to the study of other adenoviralgene drugs.