Determination Of Kinetic Parameters Of Sf9Cells Infected By AcMNPV And Analysis Of Their Factors

The baculovirus expression vector system （BEVS） was one of the mostpopular expression systems in genetic engineering. It had been widely usedfor recombinant protein expression, vaccine production and bio-pesticidesynthesis, etc. However, there were few mathematical models to depictbaculovirus infection reliably, due to the complexity of infection process. Thishad caused great difficulties to the industrialization of BEVS.In order to establish an accurate dynamic model of infection, a precisedetermination of the parameters for viral infection process was needed. In thispaper, SYBR Green â…  was used to dye the DNA of virus and monoclonalantibody AcV1was used to distinguish the infected-cells. Flow cytometry（FCM） was adopted to detect baculovirus titer, concentration ofinfected/uninfected cells and polyhedra. The results were as follows:1.5viruses are equivalent to1TCID₅₀.2. It was obtained that one cell adsorbed50viruses on average, and we defined infection with MOI value greater than50BVs/cell as high infection.3. Cells infected by virus was not instantaneousbut a process lasting6hours, then after6hours the percentage ofinfected-cells was nearly100%. The rate of cell adsorbing virus became lowerthan the rate of virus entering into cell, i.e. endocytosis rate, at3h postinfection.4. Comparing with threshold and subtraction, the match regionsubtraction was most accurate to analyze the percentage of infected-cells.In this paper, the cell growth phase and cell cycle were considered as themost important factors affecting the cell activity and virus infection. MTTassay and FCM were used to analyze the Sf9cell activity and ability toreplicate baculovirus at lag phase, exponential phase and stationary phase. Itcould be concluded that cells at the exponential phase had highest activity andstronger ability to replicate baculovirus than lag phase and stationary phase.In order to further prove the effect of cell cycle phase on cell activity and virus infection, cells were synchronized in G₂/M phase by nocodazoletreatment. Using MTT assay and FCM analysis, we found that Sf9cells at theG1phase had highest activity and stronger ability to replicate baculovirus andcells at S phase had higher activity and stronger ability to replicatebaculovirus than G₂/M phase. In addition, when cell was infected by virus, thecell cycle was eventually arrested at G₂/M.