Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins.
Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins.
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DOI：
10.1186/1472-6750-10-50
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发表时间：
2010-07-05
影响因子：
3.5
通讯作者：
Blissard GW
中科院分区：
文献类型：
--
作者：
Hashimoto Y;Zhang S;Blissard GW
昆虫细胞系是在杆状病毒表达系统中生产重组蛋白的关键组分，并且新的细胞系有望提高蛋白生产的数量和质量。70个细胞系的建立，通过单细胞克隆从细胞的原代培养物来自黑巫蛾（Ascalapha odorata;鳞翅目，夜蛾科）的卵。在8个快速生长的细胞系中，基于对AcMNPV感染的敏感性和从杆状病毒表达载体分泌的碱性磷酸酶（SEAP）的产生，选择细胞系38（Ao 38）用于进一步分析。与低传代High Five（BTI-Tn-5 B1 -4）细胞相比，感染的Ao 38细胞产生的β-半乳糖苷酶和SEAP水平高于High Five细胞（分别为153%和150%）。对Ao 38细胞中产生的SEAP的N-聚糖的分析揭示了两个N-糖基化位点和糖基化模式，与High Five和Sf 9细胞中报道的那些相似。糖肽同种型由与天冬酰胺残基连接的N-乙酰葡糖胺上的寡聚甘露糖或寡聚甘露糖组成，含或不含岩藻糖。对Ao 38细胞体积的估计表明，Ao 38细胞比Sf 9细胞大约2.5倍，但仅为High Five细胞大小的约74%。Ao 38细胞对AcMNPV感染高度敏感，与Sf 9细胞的感染性相似。Ao 38细胞产生的感染性AcMNPV芽殖病毒粒子的最高产量约为4.5 × 107 IU/ml，超过了High Five细胞，但低于Sf 9细胞。Ao 38细胞在静止培养中生长迅速，群体倍增时间为20.2小时，Ao 38细胞易于适应无血清培养基（Sf-900 III）和悬浮培养系统。对Ao 38和亲本Ascalapha odorata细胞系的分析表明，这些细胞系不含最近在High Five细胞系中鉴定为外源因子的亚硝酸盐病毒。Ao 38细胞代表了一种高产的新昆虫细胞系，其将用于异源蛋白表达和生物技术中的其它应用。
The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines. Ao38 cells represent a highly productive new insect cell line that will be useful for heterologous protein expression and other applications in biotechnology.
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影响因子：
2.7
作者：
ENDO, T;OHBAYASHI, H;KOBATA, A
通讯作者：
KOBATA, A
影响因子：
2.9
作者：
WICKHAM, TJ;DAVIS, T;WOOD, HA
通讯作者：
WOOD, HA
影响因子：
2.9
作者：
Joshi, L;Davis, TR;Wood, HA
通讯作者：
Wood, HA
影响因子：
3.4
作者：
Lery, X;Fediere, G;Giannotti, J
通讯作者：
Giannotti, J
影响因子：
3.7
作者：
Zhou, Jian;Blissard, Gary W.
通讯作者：
Blissard, Gary W.