权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

固定化培养中杆状病毒感染的昆虫细胞高效生产重组蛋白

基本信息

批准号：
11555220
负责人：
FUKUDA Hideki
金额：
$ 6.4万
依托单位：
KOBE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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

在摇瓶中培养用表达β-半乳糖苷酶的重组杆状病毒感染并在感染时悬浮于新鲜培养基中的Sf 9昆虫细胞。通过将β-半乳糖苷酶产量对从感染时间到β-半乳糖苷酶产量达到平台时间的活细胞密度的时间积分作图，定量分析细胞密度和感染复数（MOI）对β-半乳糖苷酶产量的影响。β-半乳糖苷酶产率在每个测试MOI的相当活细胞密度时间积分下具有最大值。由于当培养物达到细胞密度时间积分时葡萄糖和果糖耗尽，因此高细胞密度培养物中的蛋白质生产受到培养基中营养物耗尽的限制。在以低MOI（MOI 1）感染的培养物中，β-半乳糖苷酶的比生产率以及由此的产量随着MOI的降低而降低。 ...更多信息在高MOI（0.01）和细胞密度下感染细胞，使得细胞密度时间积分在蛋白质生产完成时达到活细胞密度时间积分，然后在摇瓶培养中研究使用多孔生物质支持颗粒（BSP）固定化Sf 9细胞和固定化细胞在感染杆状病毒后的β-半乳糖苷酶生产。将Sf 9细胞被动固定在具有60 μm平均孔径的基质的网状聚乙烯醇缩甲醛树脂BSP（2 mm立方体）中。在定期更换培养基的培养物中，BSP中的细胞密度超过5×10^7个细胞/cm^3-BSP。感染杆状病毒后，固定化细胞内的BSP表现出较高的比生产力，相当于在摇瓶培养的非固定化细胞的最大生产力，只要培养基中的营养物质没有耗尽。即使当固定化细胞以5×10^7个细胞/cm ^3-BSP的高密度感染杆状病毒时，通过以适当的间隔更换培养基以避免营养耗尽，也可以以高比生产率有效生产β-半乳糖苷酶。少