Dynamics Of Sf9 Cells Infected With Baculovirus At Early Stages Of Infection

The baculovirus expression vector system(BEVS)is one of the most important eukaryotic expression systems.It has been widely used for recombinant protein production,vaccines production and biopesticides synthesis.However,due to the complex infection process,there are few mathematical models to depict baculovirus infection process accurately at present,which makes it difficult to industrialize the BEVS.A precise determination of the parameters in baculovirus infection process is needed to establish an accurate dynamic model of infection.In this paper,the flow cytometer(FCM)was adopted to titrate the AcMNPV,the fixation,permeabilization and dyeing conditions of virus sample were optimized through orthogonal experiments.The optimal conditions for virus sample preparation is to fix the virus sample with 0.1%paraformaldehyde for 30 min at 4 ? and then stain it with SYBR Green ?at 80 ? for10 min,the virus sample is supposed to cool down to room temperature before detected by FCM.According to the changes of virus titer and cell density during the initial infection process,it was found that each Sf9 cell absorb about 50 AcMNPV BVs on average.The monoclonal antibody AcV1 conjugated with phycoerythrin was used to distinguish Sf9 cells infected with AcMNPV,the threshold method,match region subtraction method and subtraction method was employed to analyze the percentage of infected cells,and the subtraction method turned out to be the most accurate method to calculate the percentage of infected cells.When the Sf9 cells were infected with AcMNPV at different MOI,the percentage of infected cells reached the maximum 4 hours later,thus,we can conclude that the Sf9 cells are fully infected by AcMNPV after 4 hours.