Efficient production of recombinant protein by baculovirus-infected insect cells in immobilzed cultur

用表达β-半乳糖苷酶的重组杆状病毒感染Sf9昆虫细胞，并在感染时悬浮在新鲜培养基中，在摇瓶中培养。通过绘制β-半乳糖苷酶产量与活细胞密度时间积分的关系图，定量分析了细胞密度和感染复数(MOI)对β-半乳糖苷酶产量的影响。β-半乳糖苷酶产量在每个被测试的MOI具有可比的活细胞密度时间积分时具有最大值。由于葡萄糖和果糖在达到细胞密度时间积分时耗尽，高细胞密度培养中的蛋白质生产受到培养基中营养物质消耗的限制。在低MOI(≦1)条件下，随着MOI的降低，β-半乳糖苷酶的比产量和产量也随之下降。因此，如果采用…，重组蛋白的产量可以得到优化。更多的细胞在高MOI(≧1)和一定的细胞密度下感染，使细胞密度时间积分达到活细胞密度时间积分，就像蛋白质生产完成一样。然后在摇瓶培养中研究了用多孔生物支持颗粒(BSP)固定化SF9细胞和固定化细胞感染杆状病毒后产生β-半乳糖苷酶的情况。在摇瓶中，将SF9细胞被动固定在平均孔径为60μm的网状聚乙烯醇缩甲醛BSP(2 mm立方体)中。在定期更换培养液的情况下，BSP中的细胞密度可达5×107个/cm~3-BSP以上。在感染杆状病毒后，BSP内的固定化细胞显示出较高的比生产率，可与未固定化细胞的摇瓶培养的最大生产率相媲美，只要培养液中的营养物质不被耗尽。即使当固定化细胞以5×107个/cm3-BSP的高密度感染杆状病毒时，通过适当的间隔更换培养基以避免营养耗竭，也可以高效地生产比产量高的β-半乳糖苷酶。较少

Sf9 insect cells infected with a recombinant baculovirus expressing β-galactosidase and suspended in fresh medium at the time of infection were cultured in shake flasks. The effects of cell density and multiplicity of infection (MOI) on β-galactosidase production were quantitatively analyzed by plotting the β-galactosidase yield against the time integral of the viable cell density from the time of infection to the time when the β-galactosidase production reached a plateau. The β-galactosidase yield had a maximum value at a comparable viable cell density time integral for each MOI tested. Since glucose and fructose were exhausted when the culture reached the cell density time integral, protein production in a high-cell-density culture was limited by nutrient depletion in the culture medium. In cultures infected at a low MOI (≦1), the specific productivity, and thereby the yield, of β-galactosidase declined with decreasing MOI.Therefore, recombinant protein production can be optimized if … More the cells are infected at a high MOI (≧1) and at a cell density such that the cell density time integral reaches the viable cell density time integral just as the protein production is completed.Immobilization of Sf9 cells using porous biomass support particles (BSPs) and production of β-galactosidase by the immobilized cells after infection with the baculovirus were then investigated in a shake-flask culture. Sf9 cells were passively immobilized in reticulated polyvinyl formal resin BSPs (2 mm cubes) with matrices of 60 μm mean pore diameter in situ in shake-flasks. The cell density in the BSPs was over 5×10^7 cells/cm^3-BSP in cultures with regular replacement of the culture medium. After infection with the baculovirus, immobilized cells within the BSPs showed a high specific productivity, comparable to the maximum productivity in shake-flask cultures of non-immobilized cells, as long as nutrients in the medium were not depleted. Even when immobilized cells at a high density of 5×10^7 cells/cm^3-BSP were infected with the baculovirus, efficient β-galactosidase production with a high specific productivity was possible by replacing the medium at appropriate intervals to avoid nutrient depletion. Less