SCREENING OF INSECT CELL-LINES FOR THE PRODUCTION OF RECOMBINANT PROTEINS AND INFECTIOUS VIRUS IN THE BACULOVIRUS EXPRESSION SYSTEM
SCREENING OF INSECT CELL-LINES FOR THE PRODUCTION OF RECOMBINANT PROTEINS AND INFECTIOUS VIRUS IN THE BACULOVIRUS EXPRESSION SYSTEM
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DOI：
10.1021/bp00017a003
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发表时间：
1992-09-01
影响因子：
2.9
通讯作者：
WOOD, HA
中科院分区：
文献类型：
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作者：
WICKHAM, TJ;DAVIS, T;WOOD, HA
用杆状病毒苜蓿银纹夜蛾多核多角体病毒（AcMNPV）感染来自草地贪夜蛾、粉纹夜蛾、甘蓝夜蛾和Estigmene acrea的8个细胞系，评价重组β-半乳糖苷酶和感染性病毒的产生。在特定（每细胞和每微克未感染的细胞蛋白质）和体积（每毫升）基础上评估产量。发现细胞密度是比较细胞系的一个重要因素，因为细胞-细胞接触似乎导致特异性蛋白质和病毒产生的密度依赖性抑制。以低密度感染细胞后，当以接近汇合和汇合的细胞密度接种在组织培养板上时，8个细胞系中的5个细胞系的每个细胞的特异性β-半乳糖苷酶产量将下降3- 6倍。Sf-21和Sf-9细胞对细胞密度的敏感性最低。在考虑细胞密度效应和细胞大小差异后，鉴定了两种细胞系BTI Tn 5 B1 -4和BTI TnM，其在β-半乳糖苷酶生产方面上级其他细胞系，包括Sf 21和Sf 9。在两种细胞系中，Tn 5 B1 -4和TnM细胞的最佳体积和特异性β-半乳糖苷酶产量分别比Sf 9或Sf 21细胞的最佳产量高2倍和5倍。Tn 5 B1 -4细胞系在感染后3天也具有所有细胞系中最高的活力，并且可以适应无血清培养基。考虑到细胞大小差异后，细胞系之间的特异性（每微克细胞蛋白）感染性病毒产量变化小于2倍。重组蛋白和NOV生产之间没有发现相关性，但发现两者都随着细胞密度的增加而类似地降低。
Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media. Specific (per microgram of cellular protein) infectious virus production between cell lines varied by less than 2-fold between cell lines after cell size differences were taken into account. No correlation was found between recombinant protein and NOV production although both were found to decrease similarly with increasing cell density.